Search

Background: There is variation in the outcomes reported in clinical studies of basal cell carcinoma. This can prevent effective meta-analyses from answering important clinical questions. Objective: To identify a recommended minimum set of core outcomes for basal cell carcinoma clinical trials. Methods: Patient and professional Delphi process to cull a long list, culminating in a consensus meeting. To be provisionally accepted, outcomes needed to be deemed important (score, 7-9, with 9 being the maximum) by 70% of each stakeholder group. Results: Two hundred thirty-five candidate outcomes identified via a systematic literature review and survey of key stakeholders were reduced to 74 that were rated by 100 health care professionals and patients in 2 Delphi rounds. Twenty-seven outcomes were provisionally accepted. The final core set of 5 agreed-upon outcomes after the consensus meeting included complete response; persistent or serious adverse events; recurrence-free survival; quality of life; and patient satisfaction, including cosmetic outcome. Limitations: English-speaking patients and professionals rated outcomes extracted from English language studies. Conclusion: A core outcome set for basal cell carcinoma has been developed. The use of relevant measures may improve the utility of clinical research and the quality of therapeutic guidance available to clinicians.

A short history of the establishment of kindergartens in British Columbia prefaces this study of 22,000 public school children in grades one, two, and three (who had or had not attended kindergarten) in School District 39 of Vancouver and District 61 of Victoria. The effect of kindergarten attendance was evaluated as it related to (1) report card ratings, (2) adaptation to school, (3) intelligence, (4) academic achievement in grade 2 (Victoria only), and (5) retardation and acceleration in grades one, two, and three. Somewhat less than half the pupils had attended kindergarten. A "blind study" was conducted, which obtained teacher ratings on individuals on a descending scale from outstanding to unsatisfactory on the five effects under investigation. Results revealed that (1) report card ratings (for work and health habits and behavior) were generally higher for children who attended private kindergartens; (2) school adaptation seemed to be related to kindergarten attendance; (3) IQ's were highest for those who had attended private kindergartens, and nonkindergartners ranked lowest; (4) kindergarten attendance was related to higher achievement scores in reading comprehension, word meaning, spelling, and arithmetic for grade two; and (5) very little acceleration was found, but that in evidence was related to private kindergarten attendance. (DO)

Skin cancer is the most common malignancy affecting solid organ transplant recipients (SOTR), and SOTR experience increased skin cancer-associated morbidity and mortality. There are no formal multidisciplinary guidelines for skin cancer screening after transplant, and current practices are widely variable. We conducted three rounds of Delphi method surveys with a panel of 84 U.S. dermatologists and transplant physicians to establish skin cancer screening recommendations for SOTR. The transplant team should risk stratify SOTR for screening, and dermatologists should perform skin cancer screening by full-body skin examination. SOTR with a history of skin cancer should continue regular follow-up with dermatology for skin cancer surveillance. High-risk transplant patients include thoracic organ recipients, SOTR age 50 and above, and male SOTR. High-risk Caucasian patients should be screened within 2 years after transplant, all Caucasian, Asian, Hispanic, and high-risk African American patients should be screened within 5 years after transplant. No consensus was reached regarding screening for low-risk African American SOTR. We propose a standardized approach to skin cancer screening in SOTR based on multidisciplinary expert consensus. These guidelines prioritize and emphasize the need for screening for SOTR at greatest risk for skin cancer.

Myocyte enhancer factor 2 C (MEF2C) is a transcription factor that plays a central role regulating cell differentiation, proliferation, survival and apoptosis. MEF2C has been implicated in each of the most recent GWAS of cognitive ability (CA) and educational attainment (EA). Animal studies have indicated that knockout of Mef2c interferes with healthy development of brain regions associated with cognitive function, e.g. hippocampal dentate gyrus, neocortex. Furthermore, mutation/deletion of MEF2C can cause severe intellectual and developmental disability. We therefore hypothesised that genes regulated by MEF2C would be associated with cognitive function. We created a set of differentially expressed genes (DEGs) based on an RNA-seq study that captured the transcriptional changes in mouse adult brain that result from early embryonic deletion of Mef2c in cortical and hippocampal excitatory neurons. This mouse DEG list was converted to human orthologues (n=1052) and tested for enrichment of genes associated with 1) CA, and 2) EA, using MAGMA and recent GWAS summary statistics for each phenotype. We also performed hypergeometric tests to investigate if the DEGs were enriched for current primary intellectual disability (ID), autism, and loss-of-function (LoF) intolerant (i.e. highly constrained) genes. We then used Ingenuity Pathway Analysis (IPA) to explore functional pathways implicated by the MEF2C DEGs. The DEGs were significantly enriched for CA (p=1.08e-07) and EA (p=9.88e-09) genes; along with ID (p=0.008), autism (p=0.001) and LoF intolerant (p=5.55e-21) genes. The top functions IPA predicted to be decreased from these DEGs are ‘development of neurons’ (p=5.41e-38, z-score=-2.0) and ‘formation of cellular protrusions’ (p=1.02e-28, z-score=-2.1). These findings indicate that genes influenced by MEF2C are highly constrained and contribute to cognitive function and neurodevelopmental disorders with severe cognitive deficits., Nearly 50% of all colorectal cancer patients progress to develop metastatic lesions (mCRC) and despite ongoing efforts the survival rates for these patients remains significantly low (90% CRC-specific enhancer regions, which was subsequently integrated with RNAseq derived gene expression in order to identify gene-enhancer pairs. Applying motif and transcription factor identification algorithms to the methylation signature, showed intricate networks of disease-associated transcription factors whose binding sites are significantly impacted as a result of the altered methylation within these enhancer regions. Utilization of deep machine learning approaches to the methylation data, demonstrates specific methylation patterns that allow stratification of patients independent of their clinical features. Finally, we show that two methylation derived patient clusters overlap significantly with expression derived consensus molecular subtype (CMS) -2 (WNT-p53 cluster) and CMS-4 (EMT-like). This study for the first time presents a critical insight into an enhancer driven epigenomic landscapes, which potentially regulates disease-associated phenotype within mCRC., Depression is a common and disabling disorder, representing a major social and economic health issue. Moreover, depression is associated with the progression of diseases with an inflammatory aetiology including many inflammatory-related disorders. At the molecular level, the mechanisms by which depression might promote the onset of these diseases and associated immune-dysfunction are not well understood. In this study we assessed genome-wide patterns of DNA methylation in whole blood-derived DNA obtained from individuals with a self-reported history of depression (n=100) and individuals without a history of depression (n=100) using the Illumina 450K microarray. Our analysis identified 6 significant (Sidak corrected P < 0.05) depression-associated differentially methylated regions (DMRs); the top-ranked DMR was located in exon 1 of the LTB4R2 gene (Sidak corrected P = 1.27 x 10-14). Polygenic risk scores (PRS) for depression were generated and known biological markers of inflammation, telomere length (TL) and IL-6, were measured in DNA and serum samples respectively. Next, we employed a systems-level approach to identify networks of co-methylated loci associated with a history of depression, in addition to depression PRS, TL and IL-6 levels. Our analysis identified one depression-associated co-methylation module (P = 0.04). Interestingly, the depression-associated module was highly enriched for pathways related to immune function and was also associated with TL and IL-6 cytokine levels. In summary, our genome-wide DNA methylation analysis of individuals with and without a self-reported history of depression identified several candidate DMRs of potential relevance to the pathogenesis of depression and its associated immune-dysfunction phenotype., Mutations in RP2 are responsible for approximately 15% of X-linked Retinitis Pigmentosa cases. RP2 is ubiquitously expressed and involved in ciliary trafficking of lipid-modified proteins. A patient harbouring the most common nonsense RP2 mutation, R120X, was identified through the Target 5000 programme. This enabled the generation of a patient-derived primary fibroblast disease model. The aims of this study were (i) to identify a vector capable of effectively transducing primary fibroblasts, (ii) to rescue RP2 expression in the R120X cell model and (iii) to explore potential assays for evaluating rescue of RP2 function in these cells. Transduction efficiencies were determined by treating normal fibroblasts with a CAG.EGFP construct packaged in AAV2/2, 2/5 and 2/8 capsids. The results were 55.5% ± 2.5, 17.5% ± 15.4 and 2.2% ± 1.0, respectively. The expression level of RP2 mRNA in untreated R120X fibroblasts was 7.5 fold ± 3.2 lower than that of wild type fibroblasts, while RP2 protein was absent in R120X cells. Transduction of mutant cells with AAV2/2.CAG.RP2 resulted in overexpression of RP2 protein by 1.19 fold ± 0.67. The R120X cell line was evaluated for phenotypes associated with absence of RP2, including Golgi fragmentation and mislocalisation of an intraflagellar trafficking protein, IFT20.The areas of both GM130 and IFT20 were significantly larger in mutant fibroblasts compared to control cells. Treatment with AAV2/2.CAG.RP2 was beneficial in reversing Golgi fragmentation, as the Golgi area in transduced R120X fibroblasts was reduced by 1.5 fold ± 0.5 when compared to untreated cells (p < 0.0001)., Background: Genome-wide association studies (GWAS) identify associations of individual SNPs with cancer risk but usually only explain a fraction of the inherited variability. Pathway analysis of genetic variants is a powerful tool to identify networks of susceptibility genes. Methods: we conducted a large agnostic pathway-based meta-analysis of GWAS data using the summary-based adaptive rank truncated product (sARTP) method to identify gene sets and pathways associated with pancreatic ductal adenocarcinoma (PDAC) in 9,040 cases and 12,496 controls. We performed expression quantitative trait loci (eQTL) analysis and functional annotation of the top SNPs in genes contributing to the top associated pathways and gene sets. Results: We identified 14 pathways and gene sets associated with PDAC at FDR < 0.05. After Bonferroni correction (P-value ≤ 1.3x10-5), the strongest associations were detected in five pathways and gene sets, including maturity onset diabetes of the young (MODY), regulation of beta cell development, role of epidermal growth factor (EGF) receptor transactivation by G-protein-coupled receptors in cardiac hypertrophy pathways, and the Nikolsky breast cancer chr17q11-q21 amplicon and Pujana ATM Pearson correlation coefficient (PCC) network gene sets. We identified and validated rs876493 and three correlating SNPs (PGAP3) and rs3124737 (CASP7) from the Pujana ATM PCC gene set as eQTLs in two normal derived pancreas tissue datasets. Conclusion: Our agnostic pathway and gene set analysis integrated with functional annotation, eQTL analysis and experimental validation provides insight into genes and pathways that maybe biologically relevant for risk of PDAC, including those not previously identified., We carried out a detailed genetic study of the population structure, local migration rates and population changes across in the Netherlands using cutting edge methods. Our dataset couples genome wide SNP data and geographic information (N=1422), which together allow us to investigate the interplay between genetics and local geography. To interrogate fine scale population structure we applied the haplotype-based method Chromo Painter/fineSTRUCTURE, which partitions data based on patterns of haplotype sharing. FineSTRUCTURE identified 16 genetic clusters which correlate closely with regional geography. At the finest level, this clustering has the resolution to distinguish subtly different eastern and western genetic groups within the North-Brabant province. At the coarsest level, clustering delineates a clear north/ south split in the Netherlands, reflecting deeper differences. We investigated whether our clustering reflects barriers to gene flow using the “Estimating Effective Migration Surfaces” (EEMS) method, and observed a strong migrational cold spot splitting the country, broadly overlapping the course of the Rhine. We also estimated recent changes in the effective population size (Ne) using the IBDNe method, observing super-exponential population growth across the past 50 generations. This expansion rapidly increases in rate from ~1650 CE onwards, potentially driven by the Dutch Golden age of the 17th Century. Notably our Ne estimates are systematically lower in northern populations than southern suggesting lower diversity in the north, which is consistent with reported ROH and IBD analysis. Combined our results paint a picture of the dynamic population genetics of the Netherlands that are strongly linked to geography., We present here a demographic scaffold for Irish prehistory based on the palaeogenomic analysis of 93 ancient individuals from all major periods of the island’s human occupation, sequenced to a median of 1X coverage. ADMIXTURE and principal component analysis identify three ancestrally distinct Irish populations, whose inhabitation of the island corresponds closely to the Mesolithic, Neolithic and Chalcolithic/Early Bronze Age eras. Large scale migrations into the island are implied during the transitionary periods carrying with them ancestry ultimately derived from Anatolia and later the Russian steppe. Patterns of haplotypic-sharing and Y chromosome analysis demonstrate strong continuity between the Early Bronze Age and modern Irish populations, suggesting no major population replacement has occurred on the island since this point in time. We further dissect the genetic affinities of each Irish population with reference to wider palaeogenomic datasets, using both allele and haplotype-sharing methods, the latter made possible through genotype imputation., Background: Rare diseases (RDs) affect at a minimum 5 per 10,000 people. Although individually rare and under-recognised in healthcare systems, collectively RDs are common with up to 8,000 diseases now described. The National Plan for RDs (2014), recommended the need for epidemiological studies, highlighting the requirement for RD coding to identify RD patients and thereby improve both cost efficiencies and care of patients with RDs. Objectives: To derive an estimate of the number of childhood onset RDs through analysis of records held at TSCUH & OLCHC. Methods: Reports of patients born in the year 2000 were extracted from: the National Paediatric Mortality Registry office; clinical, cytogenetics and molecular genetics databases, and the Hospital In-Patient Enquiry system (HIPE) TSCUH/OLCHC. RD cases were identified using electronic/manual results and assigned orpha-codes. Results: 54, 7893 livebirths, census 2000. National Paediatric Mortality Register, 73 deaths of children born in year 2000 of these 60 had a RD (82%). Clinical, cytogenetic and molecular genetics from TSCUH/OLCHC identified 603, 121 and 77 cases of RD respectively. HIPE TSCUH/OLCHC searches to-date have identified 202 and 242 cases of RD respectively. Conclusions: RD epidemiological data is difficult to acquire in the current structure of the Irish health service, requiring multiple sources and an inordinate amount of time accessing manual records. This study to-date has identified over 1,000 RD patients presenting by age 17 to OLCHC/TSCUH giving a minimum incidence of 2% for paediatric RDs. In the coming year records from TSCUH specialties will be accessed for inclusion in the study., Background & Aims: Newborn screening for Cystic Fibrosis (CF) commenced in the Republic of Ireland in July 2011. The aim of this study was to do a comprehensive review of the first five years, focusing on those who had CFTR genetic testing following an elevated IRT. Methods: This study included all neonates screened from July 2011 to June 2016. Data was expanded by cross-referencing patient charts, clinical and lab databases with the Non-NBS database to track down cascade tested relatives. Results: In this period a total of 342,424 infants were screened. 141 CF and 19 CF-SPID cases were identified in addition to 238 healthy carriers. 2 babies died from unrelated illnesses, before their Sweat Test. A total of 300/400 (75%) couples with a CF/CF-SPID/Carrier child were seen by a Genetic Counsellor. Phe508del was the most common mutation (79.9%) followed by Gly551Asp (8.7%). Consequently, 185/238 Carrier parents (78%) underwent genetic testing, identifying 1 carrier couple. 101/160 (63%) CF/CF-SPID parents were tested. 255 additional relatives came forward for cascade testing - 184 from 68/162 CF affected/CF-SPID/RIP families (42%), resulting in 3 new CF cases, 3 new CF-SPID cases and 64 additional carriers. Two cases were siblings born prior to NBS. One case was missed though NBS. 71 relatives from 33/238 Carrier families (14%) came forward for cascade testing, identifying a further 18 carriers. Conclusion: Through early detection of CFTR mutations, NBS provides the opportunity of early intervention and complication prevention as well as improvements in prenatal diagnoses and availability of cascade testing., Background: Multi-gene testing is useful in genetically heterogeneous conditions, including inherited cardiac pathologies. Extended panels increased diagnostic yield of variants where pathogenicity is certain (class 5), likely (class 4) and uncertain (class 3). Concerns exist regarding management of class 3 and 4 variants in conditions of oligogenic inheritance or variable expressivity. Aim: 1. To review diagnostic yield of genetic tests performed in families with inherited cardiac pathologies 2. To assess management of different classes of variants by clinicians internationally. Methods: A retrospective cohort analysis was undertaken. Patients in whom “cardiac” genetic tests were requested between 2015 and 2017 were identified from a prospectively maintained departmental patient database. Data regarding indication for testing, diagnostic yield, and classification of variants were retrieved by manual chart review. An electronic survey regarding clinical management of variants (http://www.surveymonkey.com/r/cardiacvariants) was distributed to colleagues internationally via professional bodies and direct email. Results: 636 tests (630 patients) were performed between 2015 and 2017 in our centre (183 diagnostic; 453 predictive). At least one variant was identified in 71(39%) patients (28(15%) class 5; 9(5%) class 4; 38(21%) class 3. 135 respondents (23 countries) completed the survey. Considering class 4 variants, 110(81%) counselled patients about the possibility of variant reclassification. In the case of a negative predictive test, 17(13%) were fully reassuring that the patient would not develop the familial phenotype. Conclusion: Considerable variability in management of class 3 and 4 variants exists. Decision-making relies on interpretation of the phenotype, family history and genotype. Close multi-disciplinary working between cardiology and clinical/molecular genetics teams is critical., Familial hypercholesterolaemia (FH) is an autosomal dominant disorder due primarily to mutations in LDLR, APOB and PCSK9, which causes marked increases in LDL cholesterol levels and predisposes to premature CVD. Given a prevalence of 1:250, there are approximately 23,000 FH sufferers in the Republic of Ireland, most of whom are as yet undiagnosed. The most cost-effective strategy for identifying FH is genetic cascade screening in kindreds with an identified proband. We report interim outcomes of a FH genetic diagnostic service configured around an initial screen of 40 known FH variants followed by either a confirmatory analysis or a full variant scan using PCR and direct nucleotide sequencing, in positive and negative screens respectively. To date our service has genetically diagnosed 69 patients with FH, including 50 index cases and 19 positive cascade screens. In total, 30 disease-associated variants in LDLR and APOB have been identified including four due to copy number variation using MLPA. Based on phenotypic classification by Dutch Lipid Clinic Network scoring 75% of those designated “Definite/Probable FH” were genetically confirmed compared with, Hereditary Breast & Ovarian Cancer syndrome (HBOC) is caused by mutations in BRCA1/2 genes and is associated with a high life time risk of breast cancer and ovarian cancer. Ovarian tumours with inherited (germline) or acquired (somatic) BRCA1/2 mutations respond to drugs that inhibit poly ADP-ribose polymerase (PARPi). Currently, mutation screening for HBOC patients are ‘sent away’ to the UK, with a predictive (pre-symptomatic) service for known familial mutations offered at DCG. At this time, there is no service for tumour BRCA testing for potential PARPi treatment. Supported by the National Cancer Control Programme, DCG & CMD have collaborated to assess next generation sequencing BRCA gene panels & platforms to establish a pathway for germline & tumour mutation analysis and validate an optimal clinical testing method for diagnostic and therapeutic use. The ThermoFisher Oncomine panel with the Ion Torrent PGM/S5 was used to target and sequence 64 unique germline samples with a wide range of known BRCA mutations. These were analysed using JSI SeqNext software and others. After optimisation, 99.84% (624/625) variants were detected at some level. However, there were 117 false positive calls, all in homopolymer regions. Distinguishing false positives from some true positives with a low variant fraction was challenging. Subsequently, the Nimagen EasySeq kit (employing single molecule Molecular Inversion Probes, smMIPs) with the Illumina MiSeq was used for 32 samples. There was a 100% variant call rate (376/376) with no false positive calls. Initial tumour results are also very convincing. Now proceeding to a full clinical validation., Establishing the pathogenicity of missense variants detected in Lynch syndrome / Hereditary Non Polyposis Colorectal Cancer (HNPCC) families is a challenge for diagnostic laboratories. Here we consider two families from Northern Ireland who meet Amsterdam II Criteria for HNPCC, in whom the presence of a PMS2 gene missense variant c.137G>T p.(Ser46Ile) rs121434629 has been shown. This variant occurs within a conserved ADP/ATP binding region of the PMS2 protein. Disruption of this domain is predicted to result in reduced mismatch repair efficiency and has previously been reported in the literature as a recurrent and founder variant in the PMS2 gene. However, no co-segregation data has been published for this variant. Adoption of the ACMG Standards and guidelines (Richards et al Genetics in Medicine 2015) along with the release of ACGS best practice guidelines for the interpretation of sequence variants has initiated a review of the classification of variants detected within the region against these standards. Bioinformatic analysis and evidence available in the published press, had led to a classification of likely pathogenic for this variant. However, addition of the co-segregation evidence provided by local families, at the strong level, enables the variant to be re-classified as pathogenic. In conclusion, we have shown co-segregation of the PMS2 c.137G>T p.(Ser46Ile) variant with Lynch syndrome associated phenotype to a Path P1 strong level of significance through family studies., The C677T polymorphism in the folate metabolising enzyme methylenetetrahydrofolate reductase (MTHFR) is associated with hypertension. Riboflavin is a cofactor for MTHFR in one-carbon metabolism, for generating methyl groups important in DNA methylation. Supplementation with riboflavin has been shown to lower blood pressure in MTHFR 677TT genotype individuals. The mechanism regulating this gene-nutrient interaction is currently unknown but may involve aberrant DNA methylation also implicated in hypertension. This study examined DNA methylation of hypertension-related genes in adults stratified by MTHFR genotype and the effect of riboflavin supplementation on methylation of these genes in the MTHFR 677TT genotype group. We measured DNA methylation using pyrosequencing in a set of candidate genes associated with hypertension including angiotensin II receptor type 1 (AGTR1), G nucleotide binding-protein subunit alpha 12 (GNA12), insulin-like growth factor 2 (IGF2) and nitric oxide synthase 3 (NOS3). Stored leukocyte samples from participants with the MTHFR C677T genotype who had participated in targeted RCTs (1.6mg/d for 16wks) at Ulster University were accessed for this analysis (n=120). Baseline methylation differed between MTHFR C677T genotype groups at NOS3 (p=0.026) and AGTR1 (p=0.045). Riboflavin supplementation in the MTHFR 677TT genotype group resulted in altered average methylation at IGF2 (p=0.025) and CpG site specific alterations at the AGTR1 and GNA12 loci. This study demonstrates an interaction between DNA methylation of hypertension-related genes and riboflavin supplementation in adults with the MTHFR 677TT genotype. Further work using a genome-wide approach is required to better understand the role of riboflavin in altering DNA methylation in these genetically at-risk individuals., Chronic kidney disease (CKD) is considered a major public health problem, affecting approximately 10% of the global population. While a comprehensive review of known CKD biomarkers yielded many results, it also highlighted a lack of research in chromosome Y. Single nucleotide polymorphisms (SNPs) on chromosome Y have previously been associated with a 50% increase in risk of developing coronary artery disease, a condition with close links to CKD. Therefore, Y chromosome SNPs may also impart increased risk of developing CKD. Individuals from the Genetics of Nephropathy: an International Effort (GENIE) consortium (n=791) and the Northern Ireland Cohort for the Longitudinal Study of Aging (NICOLA; n=1241) were genotyped using the Illumina HumanOmni1-Quad array and the Illumina CoreExome-24 array, respectively, to determine if any association exists between Y chromosome SNPs and CKD, or estimated glomerular filtration rate (eGFR), a measure of kidney function. However, poor coverage of chromosome Y resulted in only 3 SNPs in the GENIE cohort and 421 SNPs in the NICOLA cohort passing quality control. Association analysis of both datasets did not reveal any significant associations. Due to limitations of this study, further analysis is required to determine whether SNPs on chromosome Y are associated with CKD and/or eGFR. An array with greater Y chromosome coverage will be selected and be used to re-genotype these individuals, and individuals from additional cohorts, allowing greater SNP coverage and direct comparison of SNPs between these cohorts. Increased SNP coverage and increased participant numbers will allow meta-analysis to be performed with sufficient power., Background: Tumour hypoxia is a major driver of prostate cancer progression and metastasis. miR-21 is a microRNA which has been previously linked to hypoxia, but this relationship remains poorly characterised in a prostate cancer setting. Therefore, in this study, we investigate the link between hypoxia and miR-21 in prostate cancer cells. Methods: We have used 2D and 3D cell prostate cell models of hypoxia to investigate the functionality of miR-21. Expression levels of miR-21 have been measured by qPCR and functional bioassays used to examine its effect on prostate cell behaviour. Target genes have been identified and bioinformatic analysis has been employed to investigate a clinical significance for miR-21 in prostate cancer. Results: miR-21 is induced by hypoxia in prostate cancer cell-lines. Over-expression of miR-21 impacts upon target genes which in turn affects cell behaviour. Data-mining of online repositories of clinical data and bioinformatic analysis of miR-21 cellular networks reveal that miR-21 exerts a wide influence on several important cell processes, the dysregulation of which can lead to development of prostate cancer. Conclusions: We propose that miR-21 could be an important microRNA in the pathogenesis of prostate cancer and has potential as a biomarker in this disease., The Neolithic period begins in Europe around 8500 years before present (BP) and is characterized by the adoption of farming and domestication of various types of animal. In our project we focus on the structure of the Maltese population during the latter part of the Neolithic period. Nine individuals, from 4900 to 4350 years BP, collected from the Xaghra Circle site in the island of Gozo, were sampled. DNA was extracted from both teeth and the inner part of petrous bones giving an average endogenous DNA respectively of: 1.7% for 4 teeth and of 21% for 5 petrous bones. We then used a median of 363,579 SNPs from the Human Origin dataset to compare our samples with 37 ancient individuals from Neolithic and Bronze Age period and 604 present-day European individuals already published. PCA analysis shows, for the 5 high coverage samples, places the Maltese individuals with the early European farmers (EEF) from Germany and Hungary. Further analysis with D-statistics depict that the Maltese population do not resemble any hunter-gatherer population from Caucasus or Eastern Europe, while they show a higher affinity with Western European hunter gather individuals (WHG)., According to the WHO, glaucoma is the second leading cause of blindness in the world and is the leading cause of irreversible blindness. The total number of suspected cases of glaucoma is estimated to be over 60 million worldwide, increasing to 79.6 million by 2020. Commonly, glaucoma is treated using eye drops containing prostaglandin analogs, including latanoprost and bimatoprost. However, these treatments come with ocular adverse reactions including ocular surface irritation, acute iritis, conjunctival hyperemia, thickening and elongation of eyelashes, induced iris darkening as well as periocular skin pigmentation. Patient compliance has been shown to be affected by these side-affects including non-compliance for cosmetic reasons with thickening and lengthened eyelashes and the occurrence of pigmentation. This study aimed to identify whether there are differences in gene expression between those prostaglandin treatments containing preservatives and those without preservatives. Primary human trabecular meshwork cells were stained with phalloidin to determine morphology. The cells were treated with prostaglandins either with or without preservatives, gene expression analysis was performed by PCR to determine differences between preservative containing and preservative free treatments. Differences in gene expression were shown at different time-points after treatment. Differences were also shown between treatments which were preservative free and those treatments which contained preservative. With the significant differences in gene expression levels between prostaglandins containing preservatives and those without preservatives, it indicates that prostaglandins without preservatives are likely to produce less side effects in glaucoma patients., For the majority of its history the field of ancient population genetics was restricted to non-human samples due to the difficulties with modern contamination and the nature of ancient DNA (aDNA) sequences: short, highly degraded, chemically modified and present in low concentrations with high concentrations of microbial contamination. The development of efficient extraction techniques, the discovery that the petrous part of the temporal bone is a rich reservoir for aDNA and the development of high-throughput next-generation sequencing (NGS), have resulted in the rapid expansion of the field, with sequences from over 1000 ancient individuals published to date. Portugal occupies a unique position in Europe; facing both the Atlantic and the Mediterranean it was connected to two major maritime trade and migration routes, as well as experiencing influx from central mainland Europe throughout its prehistory. Many open questions remain about population changes in the Iberian Peninsula at major transition periods in European prehistory, such as the transition to the Bronze Age involving migrations from the Pontic Steppe, the source for the R1b Y-chromosome haplotype now dominant in European populations. In this study we present high quality whole genome sequences (0.052.9X, 13 samples at ~1X) from 25 ancient Portuguese individuals, covering a period of over 3000 years, to examine the demographic and selection processes acting on prehistoric Portuguese populations. We use principal component analysis (PCA), outgroup f3 statistics, Patterson’s D-statistic and ADMIXTURE analysis to investigate questions such as hunter-gatherer admixture in the Neolithic and Steppe introgression in the Bronze Age., Background: Epilepsy is a neurological condition affecting an estimated 50 million people worldwide and roughly 40,000 people in Ireland. Levetiracetam (LEV) is an effective anti-epileptic drug, but 10-20% of patients exposed to LEV report behavioural side-effects and up to 1% of those treated experience acute psychosis. We set out to determine contribution of common genetic variation to these adverse drug responses (ADRs). Methods: Individuals from the EpiPGX study cohort were screened for European ancestry and matched to predefined phenotypic criteria. Controls were exposed to LEV, but without any adverse reactions. GWAS were carried out on patients who experienced behavioural disorders (n=149), acute psychosis (n=19), or any affective symptoms in response to LEV treatment (n=90). After identification of a genome-wide significant hit in the affective disorder analysis, a further GWAS was performed in a replication cohort (n=68). Following this, polygenic risk scores (PRS) for all cases and controls were calculated using the results from the Psychiatric Genomics Consortium’s GWASes of Schizophrenia (SCZ) and Bipolar Disorder (BIP). Results: A genome-wide significant result was found in SNP rs7500119 in the CALB2 gene. Upon replication the SNP lost genome-wide significance but maintained nominal significance. PRS analysis for both SCZ and BIP were predictive of LEV-induced psychosis. Discussion: The univariate analysis did not identify a genome-wide significant signal for neurological ADRs to LEV that survived replication in an independent cohort. Further work with larger sample sizes may identify such variants. Increased PRS for SCZ and BIP are associated with LEV-induced psychosis, this analysis will also benefit from a larger sample, The impact of natural selection on beneficial alleles can be observed in modern human genetic variation; however deciphering the origins of these alleles is complicated by the vast complexity of human history, in which many population splits and admixture events have occurred. Here we describe a new statistical framework of Approximate Bayesian Computation (ABC) that can detect which ancestral group an allele undergoing selection first appeared. We assume a specific model in which a source population splits into two groups that later undergo admixture to form the lineage leading to the contemporary population and simulate the origin of beneficial alleles at different stages of the population’s history. Using genetic variation observed at the allele at the present time, as well as the knowledge we have of the timing of demographic changes and admixture events, we test if our approach can accurately predict the time the allele arose, and in which ancestral population it first emerged in. In this presentation, we will show preliminary results from our simulation study and discuss a potential application of the method for whole-genome data from an admixed human population., There are over 12000 people in Northern Ireland living with rheumatoid arthritis (RA); a painful, systemic autoimmune disease, causing swelling, stiffness, loss-of-function in joints, disability and significantly lowering ones quality of life. Various medication options are available; low-dose (10 to 25 mg/wk.) methotrexate (MTX), a small-molecule disease-modifying anti-rheumatic drug (DMARD), is a first-line therapy, due to its affordability, cost-effectiveness and efficacy. Other DMARDs used in RA are sulfasalazine, chloroquine, hydroxychloroquine, azathioprine, and leflunomide. However, there is significant person-to-person variability in treatment responses with nearly 50% of patients indicating poor or no-response to any of these medications. Serum drug metabolite concentration of 100 RA patients treated with DMARDs were determined using tandem mass-spectrometry. Allelic discrimination analysis using Taqman probes was performed on the following SNPs; rs246240 (ABCC1), rs1476413 (MTHFR), rs2231142 (ABCG2), rs3740065 (ABCC2), rs4149081 (SLCO1B1), rs4846051 (MTHFR), rs10280623 (ABCB1), rs16853826 (ATIC), rs17421511 (MTHFR) and rs717620 (ABCC2). Demographic analysis, clinical parameters and disease scores (e.g. DAS28) were also recorded. These SNPs are located within the genes involved in the metabolism of DMARDS and anecdotal evidence has been reported in the literature of their participation in modulating normal metabolism and function of DMARDs. Correlation statistics was used to determine if the genetic profiles associate with the emergence of drug metabolites responsible for poor or non-response to DMARDs. Our findings suggest that genetic-profiling studies may help predict future treatment responses of patients to certain DMARDs. A stratified medicine strategy can help prioritise treatments to those patients most likely to respond while avoiding ineffective treatments. Abbreviations: single nucleotide polymorphisms (SNP); rheumatoid arthritis (RA), disease-modifying anti-rheumatic drug (DMARD), methotrexate (MTX), Rare mutations in genes that encode centrosomal or ciliary proteins cause disorders that present with severe cognitive deficits and variable neuropsychiatric phenotypes. We set out to explore the involvement of centrosomal/ciliary genes in schizophrenia, a neuropsychiatric disorder that affects 1% of adults and is a major global health issue. Our analysis of publicly-available genome-wide association study (GWAS) data revealed that seven schizophrenia risk genes encode proteins with centrosomal functions. Of these, SDCCAG8 is also associated with educational attainment. To analyse the molecular function of SDCCAG8, we used genome editing to ablate it in SHSY5Y neuronal and hTERT-RPE1 retinal epithelial cells. Loss of SDCCAG8 impairs cells’ ability to make primary cilia and the signalling capacity of residual cilia, although centrosome structure appears normal by immunofluorescence microscopy. Recent RNA-Seq analysis on RPE1 SDCCAG8 deficient cells compared to wildtype cells revealed a large number of differentially expressed genes (DEGs; n=2,045) in the absence of SDCCAG8. Pathway analysis of DEGs revealed that there is enrichment in axonal guidance signalling (p=2.51-15). There were also significant enrichments for several pathways that are involved in the production and turnover of extracellular matrix (ECM). Previously, many components of the ECM have been shown to be perturbed in patients with schizophrenia. Using MAGMA gene-set analysis, we found that set of DEGs were enriched for genes associated with schizophrenia (p=0.03) and cognitive ability (p=0.03). This study shows that a combination of gene editing and genomic analyses can help uncover the processes that implicate centrosome/ciliary genes in neurodevelopmental phenotypes., Scotland and Ireland are separated in places by less than 20 kilometres of sea. They share the Gaelic language and similar frequencies of particular alleles and phenotypes, hinting at shared ancestry. The population structure within England and Ireland have recently been described. However, the extent of structure within the majority of Scotland, its surrounding islands, and their links to Ireland are currently unknown. We present an analysis of the British Isles and Ireland using a combined and comprehensive sample (n=2,556) of all major regions – expanding coverage in mainland Scotland (n=567), the Hebrides (n=57), the Isle of Man (n=40), Orkney (n=111) and Shetland (n=172). By analysing individuals with extended ancestry from specific regions, we demonstrate extensive structure in all regions of the British Isles and Ireland, as well as some of the finest scale structure observed worldwide within Orkney. We resolve the shared genetic history between Ireland and Mainland Scotland, confirm the strongest differentiation of Orkney and Shetland from other populations, show the major differentiation in Mainland Scotland is between the south-west and the north-east, and reveal the distinctiveness of the Hebrides and the Isle of Man. We additionally show decreasing cline of Norwegian ancestries across northern Britain, following the spread of the Norse Vikings. Our work represents a comprehensive description of genetic structure in the British Isles and Ireland and greatly expands the knowledge of genetic stratification within the north of the British Isles, informing on the study of rare genetic variants and genetic trait associations in these populations., The Savage et al. (in press) GWAS meta-analysis of intelligence of healthy controls supports increasing findings on variability in intelligence and evidence of overlap with schizophrenia. Utilising convenience sample of pre-existing Irish dataset of broad psychosis cases (916 cases and 330 controls), wherein the controls participated in the Savage et al. (in press) meta-analysis, the present study functioned as secondary analysis of said meta-analysis findings regarding the broad psychosis cases. With the five most significant single nucleotide polymorphisms (SNPs) as identified by Savage et al. (in press) and patient diagnosis as independent variables, this statistical regression analysis focused on the extent to which these genetic variances were of importance in a clinical population by examining the effects in schizophrenia of previously identified genetic variation associated with intelligence (IQ) in healthy controls. Further objective was to extend the Savage et al. (in press) findings to investigate the effects in schizophrenia of genetic variation on memory (working memory and episodic memory). As hypothesized the present study observed nominal trend association for SNP rs2726491 with decreased errors in performance IQ, and a nominally significant association with decreased errors in working memory for rs2726491 across both healthy and clinical population samples. These nominal associations would be suggestive of stronger effects in psychosis, however, the present study was underpowered to observe an association at the corrected level. Nevertheless, future research building on these suggestive findings could further our understanding of the biological psychopathology of schizophrenia, and crucially bring about improved cognitive function in schizophrenia patients., We present a model, algorithm, and results for multiway admixture events. This is where two or more genetically differentiated groups come together. Data from such events can inform us of the demographic history of a species, carry signatures of natural selection, and may increase the power of genome wide association studies. Our model is based on Li and Stephens style haplotype copying and delivers accurate local ancestry estimation along the genome for each admixed individual. Unlike existing methods that return local ancestry, we do not assume knowledge of the relationship between sub-groups of donor reference haplotypes and the unseen mixing ancestral populations. Instead, our approach infers these in terms of conditional copying probabilities. We also infer admixing proportions, timings, and recombination rates. Furthermore, we can estimate drift between modern reference populations and the unseen mixing groups using a version of Fst that is computed on putative partial genomes derived by assignment of chromosome segments to ancestral backgrounds. We demonstrate compelling results using the Human Genome Diversity Panel, including replication of some known admixture events, and we detail novel findings such as a recent 4-way admixture in San-Khomani individuals. Keywords: Population Genetics, admixture, demography, local ancestry estimation., Sibling transplant pairs have better transplant outcomes than unrelated donor-recipient (DR) pairs suggesting shared genetic ancestry between donors and recipients has potential for predicting transplant outcome. We set out to evaluate methods to detect and quantify shared ancestry using GWAS data, to see which could best predict renal-transplant outcome. We tested three different methods for estimating shared genetic ancestry on deceased donor DR pairs of European ancestry. Method 1 calculated identity by descent (IBD) which was then used to estimate the degree of relationship. Method 2 calculated genetic distance using identity by state which examines the number of shared alleles across the genome. Method 3 created a mosaic of an individual’s genome from the haplotypes of the other individuals in the dataset. The similarity of mosaic genomes in a given DR pair was used as a measure of shared ancestry. These measures were then tested against estimated glomerular filtration rate (eGFR) at 1 year (DR pairs, n=1,450) and 5 years (DR pairs, n=1,309) post-kidney transplant, change in eGFR between 1 and 5 years (Δ eGFR; DR pairs, n=982) and time to graft failure (DR pairs, n = 1,806). We did not find significant correlations between any of the measures of shared ancestry in the European ancestry deceased-donor DR pairs and graft function. The genetic relationship between the vast majority of our donor-recipient pairs was distant, and not detectable via IBD. The effect size of shared ancestry at the genomic level on eGFR is limited, and not detectable in our analysis., Background: The treatment of comorbidities remains costly and represents a major priority in Evidence Based Medicine (EBM). Determining genetically the molecular-subclasses of pro-inflammatory comorbid conditions is important to stratify patients that may more effectively respond to specific treatment interventions. The objective of this study is to develop a Machine Learning (ML) based classifier to stratify patients with Type-2-Diabetes and different comorbidities. Methods: A preliminary dataset of samples from 254 people with Type-2-Diabetes recruited at NICSM were genotyped with an Affymetrix UKBioBank Axiom Array. SNP results for 80 patient samples of class DCM1 (i.e. Type-2 Diabetes associated with comorbidities of circulatory system) and 90 patient samples of class DCM2 (i.e. Type-2-Diabetes associated with comorbidities of digestive system) were filtered through feature selection using ANOVA, Chi-square and Fast Correlation Based Filter. The top-10 SNPs along with information from Electronic Care Records (ECR), were selected for building 5 ML binary classifiers, using Support Vector Machine, Random Forest, Artificial Neural Network, Decision Tree and Naive Bayes algorithms, and their performances were tested with a 10-fold cross validation. Results: Of the 5 classifiers, the Naive Bayes algorithm outperformed all others with an Area under the Curve score of 0.681, overall Classification Accuracy of 65.68% and Mathews Correlation Coefficient of 0.316. Conclusion: Further improvement in the performance of our ML classifier is currently in-progress. With the inclusion of further data from ECR, as well as data from public repositories, we hope to build a better classifier., This project aims to investigate the relationship between folate status and the accumulation of mutations within the human mitochondrial genome. Folate is an essential B vitamin that is required for DNA synthesis, methylation reactions and is a major contributor to NADPH production through the folate one-carbon metabolism (FOCM) pathway. As a diet with a suboptimal level of folate can impact on DNA precursor availability, there is a strong biological plausibility that this will cause an increased occurrence of mutations within a cell’s genome due to errors in DNA replication. Mitochondrial dysfunction has been linked to many age-related conditions such as cardiac myopathies, neurological disorders and muscular wastage. The accumulation of mutations within the mitochondria over one’s lifetime may increase the level of mitochondrial dysfunction thus increasing the likelihood of developing such diseases. This project will look at the potential relationship between folate-status and the frequency of mutations occurring within the mitochondrial genome using a combination of both cell line and animal models plus a human cohort with known folate status and age ranges., Common variants associated with schizophrenia are enriched among highly constrained (HC) genes. As schizophrenia and cognition are genetically correlated, we hypothesized that genes associated with cognitive function are enriched for HC genes. Using MAGMA to perform gene set analysis of the largest available GWAS datasets, we found that HC genes (n=3,230 (loss-of-function intolerant)) are strongly enriched for genes associated with educational attainment(EA; p=1.27E-09) and cognitive ability(CA; p=5.64E-09) in comparison to genes under lesser or weak constraint (p>0.05 for both EA and CA). This signal remained significant following conditional analysis to co-vary for ‘brain-expressed’ (n=14,243) and ‘brain-specific’ (n=1,424) gene-sets. In schizophrenia, evidence shows that common variants are likely to persist in the population due to background selection (BGS) mechanisms. BGS refers to the phenomenon by which selection against deleterious variants reduces genetic diversity, impairing the overall efficiency of selection and allowing alleles with small effects to rise in frequency by drift. We ran a stratified linkage disequilibrium score regression (LDSR) analysis to test for heritability enrichment in EA and CA for SNPs within genomic regions that are under various types of selection. The heritability of EA and CA is enriched for SNPs in regions under background selection(p=0.028 for EA and p=0.002 for CA) and depleted for SNPs in regions under positive selection. Recent studies suggest that natural selection is acting against phenotypes such as EA or CA. This study suggests a mechanism by which variants contributing to these phenotypes are not removed by negative selection and are maintained in the population., Mutations in the photoreceptor-specific tubby-like protein 1 (TULP1) are associated with recessive retinitis pigmentosa 14 and Leber congenital amaurosis 15; severe, early-onset forms of retinal degeneration. We have explored an adeno-associated virus (AAV)-mediated gene replacement therapy in a murine model carrying a targeted disruption of the Tulp1 gene (Tulp1 -/- mice). The human TULP1 cDNA driven by the chicken beta-actin promoter (CBA) promoter was generated in an AAV serotype 5 (AAV-CBAP-TULP1). 1x10e11 vg of AAV-CBAP-TULP1 (+1:600 of an AAV-EGFP vector for tracing) was delivered to TULP1-/- mice at postnatal day 2 via sub retinal injection. Immunoblotting and qPCR demonstrated that the replacement TULP1 protein had the correct molecular weight and that the level of expression of protein achieved was ~55 % (n=8; p, Background: Diabetic kidney disease (DKD) is the most frequent cause of end stage renal disease. There is a need for improved biomarkers for the early detection of DKD. MicroRNAs (miRNAs) are short, non-coding regulatory RNA molecules commonly found in urinary exosomes that may be differentially expressed during renal dysfunction. Therefore, we profiled urinary exosomal miRNA expression in type 2 DKD (T2DKD). Methods: Qiagen Human Urine Exosome Focus miRNA Panel was used to profile 87 miRNAs in a discovery cohort of 14 T2DKD and 15 age and gender matched type 2 diabetic patients with normal renal function (T2NC). Differentially expressed miRNAs were validated in a second cohort of 22 T2DKD, 18 non-diabetic patients with poor renal function (CKD), and 22 T2NC. Results: Three urinary miRNAs (miR-21-5p, let-7e-5p and miR-23b-3p) were significantly upregulated (P, Werner syndrome (WS) is a rare genetic disorder due to mutations in the WRN or LMNA genes, with an estimated global incidence of 1 in 1,000,000 - 10,000,000. It is a segmental progeroid disorder characterised by an array of clinical features consistent with accelerated aging. We report the case of a 28 year old female patient, the offspring of a consanguineous union, who was referred to our metabolic clinic for review. She reported a history of vocal cord paralysis aged 19 years and subcapsular cataracts aged 24 years. Moreover, she had been diagnosed with primary hypothyroidism, primary hyperparathyroidism and subfertility despite normal menstruation. Further diagnoses included NAFLD with mild fibrosis. On examination, she had skin atrophy, hyperkeratosis, a loud S2, scalp alopecia, axillary acanthosis nigricans, and marked visceral adiposity with lipodsytrophic upper and lower limbs. Echocardiography confirmed trace regurgitation in aortic, mitral and tricuspid valves and DEXA confirmed osteoporosis. HOMA score was > 11 confirming severe insulin resistance and AMH levels were low. Phenotypically the patient had a diagnosis of definite WS but genetic confirmation was sought. Analysis of LMNA did not identify pathogenic variants. An RT-PCR method with direct sequencing was developed in-house to examine the extensive coding region of WRN. This revealed a homozygous genotype for the nonsense variant g.129, 248C>T, c.3961C>T, p.Arg132Ter. To our knowledge this is the first reported case of WS in the Republic of Ireland. In cases with multiple early-onset morbidities a genetic basis should be considered, particularly if there is a risk of consanguinity., Hyperlucent zones within areas of pulmonary consolidations may represent cavitatory lung lesions on CT imaging, from multi-factorial causes such as TB, pulmonary infarction, pyogenic lung abscess, pneumocystis pneumonia, Klebsiella pneumonia and less frequently due to necrotic processes from fungi. We were presented with this clinical conundrum in a patient against a background of refractory asthma, chronic cough, worsening dyspnoea, poor spirometry results and becoming progressively unwell. Due to a strong history of cancer in the family, EBUS-TBNA was carried out to obtain lung-biopsy samples. Laboratory histological analysis and ROSE revealed hyphae and fungal spores within the tissue samples biopsied, no malignant cells were recovered from the lymph node biopsy samples in all stations. We initiated anti-fungal treatment; itraconazole, 200mg once daily for 2 days after which the patient began to show signs of improvement. Seven family members with prior history of fungal-lung disease had developed lung-cancer later in life, and anecdotal prior research had shown that a premature stop-codon mutation at the tyrosine-238 residue of the dectin-1 gene in a Dutch family had predisposed patients to risks of contracting fungal-lung disease and subsequently developing lung-cancers in the long-term. We carried out Sanger-sequencing of all the exons of the dectin-1 gene as well as whole-exome sequencing on the HiSeq (Illumina) platform to identify candidate markers that may explain the heritability in this Kent family of Irish descent. We highlight the results of this study in this presentation. Abbreviations: endo-bronchial ultra-sound transbroncial-needle-aspiration; EBUS-TBNA, Rapid-OnSite-Examination; ROSE, tuberculosis; TB, Lynch syndrome (LS) (previously Hereditary Non-Polyposis Colorectal Cancer syndrome) is a cancer predisposition syndrome conferring variable risks of endometrial, colorectal, upper gastrointestinal, urinary and biliary tract cancers. Lynch syndrome is a dominantly inherited trait, caused by pathogenic germline variants in one of the mismatch repair (MMR) genes MLH1, MSH2, MSH6, or PMS2; and more rarely by deletions in EPCAM causing hypermethylation of the MSH2promoter. A recent report suggested that germline variants in MSH6 or PMS2 are associated with an increased incidence of breast cancer. Other data with respect to this association is conflicting, and prospective studies have not shown evidence for this association. Here, we report a case of a 37-year old female patient with multifocal breast cancer demonstrating defective MMR, associated with a germline variant in MSH2. This prompted us to undertake a respective cohort study to assess the prevalence of breast cancer in patients with Lynch syndrome managed in our centre. We report on 60 consecutive patients (including the case described here above) tested and found to carry germline pathogenic/likely pathogenic variants in MMR genes were identified from a prospectively maintained departmental database. Pedigrees from these patients were analysed, and number of breast cancers in probands and first and second degree relatives were recorded. Age at diagnosis, phenotypic data and genotype were noted., Amyotrophic lateral sclerosis (ALS), usually23 known as a motor neuron disease, is a fatal neurodegenerative disorder which causes death of neurons controlling voluntary muscles. ALS has no cure, and its underlying cause is mostly unknown, although a strong genetic component is known to play a role. The gene ATXN2 normally has a repeat structure of around 22-23 triplets encoding for glutamine (CAG) within the reading frame of the gene encoding the ataxin two protein. Studies have shown that harbouring more than 40 repeats causes spinocerebellar ataxia type 2 (SCA2). Recently, it was discovered that intermediate-length repeat expansions (27-33 repeats) in ATXN2 are significantly associated with the risk of ALS. The aim of this study is to genotype the ATXN2 gene in a cohort of controls and patients from the Irish ALS bank in order to assess the association between this genotype and ALS. The most common alleles in this cohort were 22, 23, and 27 repeats, at frequencies (cases and control combined) of 87.0%, 8.3% and 1.9%. Trinucleotide repeat counts ≥27, ≥29 and ≥30 for the larger allele were significantly associated with ALS (p < 3.6×10-3, corresponding to α = 0.05) and the odds ratio for ALS in the established ALS risk range was 1.90 (95% CI 1.03-3.51). This study further exemplifies the correlation between this gene and ALS in the Irish population, contributing to the research of causative genes for this devastating disease. Currently, our research is assessing the length of repeat expansions in other ataxia-associated genes, including ATXN1., Introduction: Huntington’s disease (HD) is a progressive, incurable, autosomal dominant, neurodegenerative disease. Genetic testing for HD has been available in the Department of Clinical Genetics since 1995. This clinic employs the gold-standard multistep approach to genetic testing, involving pre-test counselling, two blood draws and psychiatric review, allowing patients time to consider the consequences of testing and to withdraw at any time. Aims: To establish the uptake of predictive testing among first-degree relatives of patients diagnosed with HD. Methods: Families with at least one relative referred for genetic counselling between 2014 and 2016 were identified from a prospectively maintained departmental database. Familial pedigrees were analysed to identify at-risk relatives. Data was collected by retrospective chart review regarding number of first-degree relatives of the family proband attending clinical genetics for predictive testing, number who completed testing, diagnostic yield and patient demographics. Results: 241 asymptomatic adult first-degree relatives of the proband in 35 families were identified. 125 of these were children of the proband and 106 were siblings. 41 (17.4%) self-referred for predictive testing and 26 (10.8%) completed testing (9 positive; 17 negative). The median age for those seeking genetic testing was 36y (23-69). Patients completing testing were younger than those withdrawing from process (median 35 (23-55)-vs-40 (33-69y)). Conclusion: Uptake of genetic testing among relatives of patients affected by HD is currently low, in-keeping with rates reported in international literature. However, this may change in time with increasing advent of therapy. Decision-making in an incurable disorder is complex and may explain this low figure., Introduction: Over recent decades the life expectancy of those with Down Syndrome (DS) has increased dramatically. Much of this improvement can been attributed to early intervention, and the research which supports these interventions. Despite medical advancements, individuals with DS still have a greater mortality and morbidity compared with individuals from the general population and those with other forms of intellectual disability. Demonstrably there is a need for ongoing research to improve the quality and duration of life for those with DS. In modern academia there have been significant developments in the prenatal diagnosis of DS (e.g. Non-Invasive Prenatal Testing). Some of these developments have been met with controversy from members the DS community. Methods: A structured PubMed search was performed utilising comprehensive terms to identify publications focusing on DS, childhood and the prenatal period. This was compared to the total number of publications available on PubMed per year (1990-2017). Results: Since 1990, there are has been a general increase in the number of publications focusing on DS. However, the proportion of publications focusing on DS, compared to total PubMed publications, has decreased. Among those publications focusing on DS there has been a decline in the proportion of studies focusing on childhood and a proportionate increase in those focusing on the prenatal period. Conclusion: The results of this preliminary review of the literature suggest a general decline in the proportion of academic publications focusing on DS and a shift in focus away from childhood and towards prenatal studies, Introduction: Interstitial deletions of 12q are rare with around 6 cases including 12q21 deletions described in the literature. We identified a male infant with 12q21.1-q21.33 deletion with phenotypic features including wide sandal gap and longitudinal plantar creases, short upturned nose, low set ears, feeding difficulties and delayed development. Methods: Array-CGH using the Agilent (ISCA*v2) 8x60K oligo array (genome assembly Build GRCh37) was undertaken on a chorionic villus sample at 13 weeks gestation due to raised nuchal translucency, and confirmed on venous blood after birth. A comparison of a-CGH microarray profiles was undertaken on the existing described cases. Results: Array-CGH confirmed a ~16Mb deletion containing nine OMIMMorbid genes ALX1 (OMIM *601527), BBS10 (OMIM *610148), CEP290 (OMIM *610142), DUSP6 (OMIM *602748), KITLG (OMIM *184745), MYF6 (OMIM *159991), OTOGL (OMIM *614925), PTPRQ (OMIM *603317) and TMTC3 (OMIM *617218). Using overlapping features of different 12q21 cases allowed microarray profiles to confirm a common deletion region including a non-morbid gene LIN7A. Its role encodes a scaffold protein within the CASK pathway which is important in synaptic function and is a possible responsible gene for the intellectual disability and cortical development present in all described cases. Parental a-CGH was normal confirming our case is de-novo. Conclusion: We delineate a 12q21 deletion syndrome with characteristic phenotypic features. LIN7A is a consistent deleted gene in this region and may be responsible for the intellectual disability due to cortical maldevelopment in this syndrome., Trisomy 18 (T18) is a relatively common chromosomal disorder with a prenatal prevalence of ~1/2,500. Features associated with T18 include congenital heart defects (CHD), microcephaly, overriding fingers and rocker bottom feet. Radial ray anomalies (RRA) occur in ~ 1/10,000 pregnancies. RRA are associated with prenatal teratogen exposure, abnormal glycaemic control in pregnant women and syndromic disorders. To date there are few reported cases of T18 and bilateral RRA in the literature. We describe two cases of T18 with bilateral RRA: Case A: Male infant who passed shortly after delivery at 31 weeks gestation to 37 year old mother with a history of Crohn’s disease. PM identified CHD, significant growth restriction, overlapping fingers, bilateral talipes equinovarus and bi-lateral absent radii and thumbs. Case B: Male infant born at 16+4 weeks gestation to a 44 year old mother. PM examination identified significant growth restriction, an omphalocele, absent left radius, dysplastic right radius and absent thumbs, among other anomalies. For Case A and B karyotype and FISH analysis performed at post mortem confirmed T18. In both cases the diagnosis of T18 was not made antenatally. Here we discuss the importance of antenatal assessment which combines the use of ultrasound, clinical, genetic, cytogenetic and molecular testing in order to obtain the correct diagnosis from a wide spectrum of differentials. Foetal karyotype analysis should be considered in cases of RRA, especially if other malformations are detected. Cases with bilateral lesions have a significantly higher association with aneuploidy, in particular T18., Background: Clinical Genetics services provide a diagnostic, counselling and genetic testing service for children and adults affected by, or at risk of, a genetic condition, most of which are rare, or genetically heterogeneous. Appropriate triage of referrals is crucial to ensure the most urgent referrals are seen as quickly as possible, without negatively impacting the waiting times of less urgent cases. Aim: To examine triage practice in 6 Clinical Genetic centres across the UK and Ireland. Method: Thirteen simulated referrals were drafted based on common referrals to Clinical Genetics. Copies of each referral were forwarded to each centre, where 10 nominated clinicians were asked to triage each referral. Triaged referrals were returned to the coordinating author for analysis. An electronic questionnaire was contemporaneously completed by clinical leads in each unit to gather local demographic details and local operating procedures relevant to triage. Results: Widespread inconsistencies were noted both within and between units, with respect to acceptance of referrals to services, prioritisation, and designated clinic type. Referral rates, staffing levels, and waiting lists varied widely between units. Conclusion: Inconsistencies observed between units are likely influenced by a number of factors including; staffing levels, referral rates, and average family size. Inconsistency within units likely reflects the complex nature of many Clinical Genetic referrals and triage guidelines should help improve decision making in this setting., Ireland’s breast cancer(BC) incidence is 122.6/100,000. 3% of BCs are attributed to variants in BRCA1/BRCA2. Knowledge of pathogenic variants drastically changes the risk management of patients. Variants in other genes(CHEK2, ATM) confer moderate-risk; up to 50% of inherited BC risk is unexplained. Analysing multiple genes in a cost-effective manner is possible through next-generation sequencing(NGS). We aimed to identify variants contributing to Irish BC susceptibility using NGS. A custom gene-panel was designed; genes were primarily selected from clinical panels (BC, BC and ovarian cancer, broad cancer) and candidate genes identified through GWAS. Captured libraries from 90 BCs and 77 controls were sequenced using Illumina’s NextSeq. Variant calling was performed following GATK best practices. Following variant annotation (VEP, ANNOVAR, SnpEff), loss-of-function(LOF) and missense variants were analysed. Missense deleteriousness prediction scores were obtained from five sources. Clinvar reports were considered. Frequencies were obtained from ExAC/gnomAD. LOF variants were identified in BCs/controls in known BC risk genes BRCA1, ATM, CHEK2, and MSH6(candidate risk gene). A splice-region LOF variant in PBRM1 was identified (4 BCs:1 control). 22 novel LOF variants were identified. Deleteriousness prediction tools unanimously scored 40 missense variants “damaging”; three in BRCA1, BRCA2, ATM had opposing Clinvar reports. Rare missense variants were identified in FANCD2, SFN, ARID1B. Novel missense variants were identified in genes appearing on clinical panels(XPC, FANCA) and reported in GWAS(PTGS2, NOTH2, CYP1B1). These results demonstrate the challenges of accurately predicting variant pathogenicity, and highlights the need for caution when considering the use of broad panel testing on an unselected population., Background: Autism Spectrum Disorder (ASD) is a neurodevelopmental disorder with a population frequency of ~1 in 88, frequently co-occurring with other psychiatric disorders. While it is accepted that ASD is a highly heritable disorder (h2 >0.8), much of the effect of genetic variation on autism remains unclear. A major search is currently underway to seek out the variation underpinning this disorder. Methods and Results: A family was enrolled comprised of unaffected parents and 4 ASD-affected offspring. DNA was extracted from saliva samples using Perkin Elmer Prepito D cyto kit. All six samples were sequenced using SOPHiA GENETICS Whole Exome Panel covering 26,000 genes, run on HiSeq 4000 (2x250). QC was performed as standard. Data analysis was carried out using SOPHiA DDM. The identification and annotation of variants implicated in ASD will be reported. Discussion: This study will contribute to the autism genomics field with the most up to date technology in a clinically relevant family based study. The genes identified will add to those already associated with ASD, giving a deeper understanding of the genomics of the disorder. In turn, this genomic understanding will bring a clearer picture of the mechanism of disease, both on an individual level and on a global level. This gives the opportunity to develop personalised therapies and management strategies, improving patient outcomes. Genomics is certain to play a crucial role in the diagnosis and intervention of ASD in the future., Background: Little is known of the true epidemiological burden or character of mitochondrial disease in Ireland. Yet such information is important for provision/planning of evidence-based health policies/future services. Aim of study: 1) to characterise the cohort of patients with mitochondrial disease attending the National Centre for Inherited Metabolic Disease(NCIMD)/Adult Metabolic Service in reference to phenotype (clinical and biochemical), genotype, treatments/management and outcomes. Methods: A retrospective study was conducted on all patients attending the NCIMD/Adult Metabolic Service with a diagnosis of mitochondrial disease. Results: Fifty five patients (33/55 (60%) male and 22/55 (40%) female) have a mitochondrial disease diagnosis. Pathogenic variants were identified in 39/55 (71%), testing pending in 5/55 (9%) and no pathogenic variants were identified in 11/55 (20%). 31/55 (57%) patients have MELAS; 2/55 (4%) have Kearns-Sayre syndrome and 1/55 (2%) have leber hereditary optic neuropathy or pyruvate dehydrogenase deficiency (PDD) deficiency or neuropathy, ataxia and retinitis pigmentosa. 19/55 patients (34%) have another mitochondrial disorder with only 9/19 (47%) having a confirmed genetic diagnosis. Conclusions: MELAS, due to m.3243A>G, is the most common mitochondrial disorder which is in keeping with international studies. 30% of patients have a mitochondrial diagnosis due an abnormal biochemistry. Mitochondrial disease criteria (Wolf NI et al., 2002) will be applied to identify those for further genetic testing. Low numbers of patients suggest there is a large cohort of mitochondrial patients not yet captured by this clinic. The study will be expanded to calculate the prevalence of adult mitochondrial disease in the Irish population., Abnormal methylation affecting allele-specific expression of the H19, IGF2, KCNQ1 and CDKN1C genes at the 11p15.5 locus are variably associated with congenital disorders of growth including Beckwith Weidemann syndrome (BWS), Silver Russell syndrome (SRS), and isolated lateralizing overgrowth. Methylation defects causing isolated hemi-hypertrophy commonly overlap with those causing BWS. At the 11p15.5 locus, hypomethylation of the H19 DMR (differentially methylated region) (IC1) on the paternal allele, or hypermethylation of the KCNQ1OT1: TSS – DMR (IC2) on the maternal allele are mechanisms underlying SRS. We present atypical cases related to SRS methylation abnormalities at the 11p15.5 locus. Patient 1 is a 2y-old girl with leg-length discrepancy, and asymmetric facies. Relatively small at birth (5lb 4oz), post-natal growth velocity was normal. Patient 2 is a 16y-old boy measuring over 6ft with isolated hemi-hypertrophy. In both cases, hypomethylation at H19 was reported. Patient 3 is a 2y-old boy with history of IUGR, speech delay and short stature. Investigations identified a maternally inherited duplication of KCNQ1OT1: TSS – DMR. His mother inherited the same duplication from her mother, and was mildly affected, with final adult height of 4’ 11”, without growth hormone treatment, and no issues with development or feeding. The Netchine-Harbison Clinical Scoring system outlines diagnostic criteria for SRS, including pre- and post-natal growth restriction, feeding issues, and characteristic facies. None of these cases would fulfil these criteria and yet have molecular defects consistent with SRS. A low threshold for investigation of methylation abnormalities should be adopted in cases of short stature or isolated hemi-hypertrophy., SHOX deficiency is characterised by a clinical spectrum from idiopathic short stature to Leri Weill dyschondroestosis with triad of disproportionate short stature, Madelung deformity and mesomelia. Heterozygous mutations or deletions of the SHOX gene located in terminal Pseudo-Autosomal pairing region (PAR1) of either Yp11.2 or Xp22.33, cause this condition in both sexes. This disorder behaves as an autosomal dominant disorder, (rather than X linked) due to its location within the pseudo-autosomal region. Case: The proband was seen by clinical geneticist due to a co-incidental paternally inherited chromosome deletion in her son. The proband was noted to be short (143cm, 7cm below 3rd centile) and has shortened and bowed forearms. Analysis by aCGH showed an atypical Xp chromosome deletion of 881kb that included the SHOX gene. (Typical deletion involving SHOX is about 1.5Mb). In addition, she had gain of Yq11.221-q12 chromosomal material, which was inserted onto the distal region of Xp. She and her elder sister attended paediatric endocrinologist 25 years ago for their short stature. Her sister responded to growth hormone therapy, pre-treatment height (10cm below the 3rd centile) improved to above 3rd centile, height 10cm > than the proband who was not treated. Their parents heights were both, Malan syndrome, also known as Sotos 2 syndrome as it clinically resembles Sotos syndrome, is a recently described overgrowth syndrome. It is associated with deletions or mutations affecting the N terminal DNA binding site and dimerization domain (exons 2 and 3) in the Nuclear Factor I type X encoding gene (NFIX) on chromosome 19p13. Other mutations within the donor splice site of exon 6 of NFIX are known to cause the distinct clinical entity Marshall Smith syndrome. Typical clinical features are tall stature, macrocephaly, craniofacial features such as narrow and long face with high forehead, developmental delay, intellectual disability and behavioural abnormalities such as autistic traits and anxiety. Musculoskeletal abnormalities such as advanced bone age and scoliosis are also well described. Here we report a case of Malan syndrome with typical and atypical features, thus expanding the known phenotype, who was originally treated and referred as clinically suspected Marfan’s syndrome. She presented to the Department of Clinical Genetics at 13 years of age having been referred by her General Paediatrician. She was tall and slim, macrocephaly, with mild intellectual disability who showed a mildly dilated aortic root for which she was prescribed a beta-blocker. Subsequent to genetic and biochemical investigation, a pathogenic mutation was identified in the NFIX gene. This case emphasises the need to consider NFIX gene analysis in FBN1 negative Marfanoid appearing patients presenting with an atypical history and features such as intellectual disability, joints contractures, and dilated aortic root. Moreover, screening Malan syndrome patients for aortic root dilatation may help further understanding of the possible involvement in vasculature development of the NFIX gene function., Guidelines published by the Institute of cancer research (2013) and NICE (2017) recommend testing all women diagnosed with high grade serous ovarian carcinoma (HGSOC) for germline pathogenic variants in the BRCA1 and BRCA2 genes. It is predicted that using these guidelines that 10% of cases in this cohort harbour a pathogenic variant. We have carried out a retrospective study on 2years of data (April 2016-March 2018) from genetic screening of BRCA1 and BRCA2 genes on HGSOC patients. The aim of this audit was to establish the number and incidence of germline BRCA1 and BRCA2 pathogenic variants identified within this cohort in Northern Ireland and to explore the contributing factors to these results. During this period, 155 women with ovarian cancer were screened for germline mutations in the BRCA1 and BRCA2 genes by fluorescent sequence analysis of the coding sequence and associated splice sites and screening for whole exon deletion/duplication variants. The clinical details and family history of these patients were reviewed in light of existing screening guidelines and amendments to local testing protocols considered., The rapidly emerging field of Genomics promises improved diagnosis and personalised medicine at the front line of patient care. Genetic counsellors (GCs) bring essential skills and knowledge for delivering genomic information to patients and in education of healthcare professionals. In the Republic of Ireland there are 13 Genetic Counsellors (GC) working across different hospital sites with a variety of clinical roles. The majority have attained professional registration through the UK Genetic Counselling Registration Board (GCRB) or the European Board of Medical Genetics (EBMG) and/or an MSc in Genetic Counselling. The number of GCs falls significantly below recommendations for the Irish population as compared to other European countries. We are in the process of setting up a professional body called the Irish Association of Genetic Counsellors (IAGC) to represent the profession in Ireland. To achieve this two working groups have been established: Professional body: this working group has developed a constitution detailing membership, council roles and setting out the aims for the organisation - advocating for the profession, development of CPD opportunities and education of allied health professionals. Regulation: Given the significant implications associated with mishandling of genomic information this working group will aim to achieve consideration for the statutory regulation of the Genetic Counselling profession. Initial steps include direct approach to CORU - Ireland’s health and social care professional regulator. Our goal is to promote high standards of professional conduct, education, training and competency in the Genetic Counselling profession., Immunohistochemistry (IHC) performed on tumour tissue to detect loss of mis-match repair (MMR) protein expression is used to screen individuals at risk of Lynch Syndrome (HNPCC). Germline mutation analysis for HNPCC is guided by loss of expression of MMR proteins on IHC and it has been local practice to arrange MLH1 mutation analysis for isolated loss of MLH1/PMS2 protein expression for all cases without testing the tumour tissue for BRAF or promoter hypermethylation as recommended by NICE guidelines due to lack of access to BRAF/promoter hypermethylation testing locally. Presence of BRAF and/or presence of methylation of MLH1 promoter region suggest sporadic cancer and therefore molecular testing for HNPCC is not indicated in these cases. It is likely that sporadic bowel cancer is being tested for HNPCC based on IHC results alone as per existing practice. This audit would help us to quantify the issue and will help us in creating a testing pathway incorporating BRAF/ promoter hypermethylation testing for better diagnostic yield. This would avoid unnecessary genetic testing and would be a cost saving measure for the service helping us to utilize our resources efficiently, Mutations in the LMX1B gene cause nail-patella syndrome, a rare autosomal dominant disorder which is characterized by abnormalities of the nails, knees, elbows, and pelvis. The features of nail-patella syndrome vary in severity between affected individuals, even among members of the same family. Other areas of the body that can be affected in this condition are eyes (glaucoma) and kidneys where progressive disease can cause renal failure. The LMX1B gene provides instructions for producing a protein that binds to specific regions of DNA and regulates the activity of other genes. On the basis of this role, the LMX1B protein is called a transcription factor. The LMX1B protein appears to be particularly important during early embryonic development of the limbs, kidneys, and eyes. Mutations in the LMX1B gene lead to the production of an abnormally short, nonfunctional protein or affect the protein’s ability to bind to DNA. It is unclear how mutations in the LMX1B gene lead to the signs and symptoms of nail-patella syndrome. We describe a family with significant history of kidney failure and no systemic manifestations of nail-patella syndrome Molecular studies identified a pathogenic variant in one allele of LMX1B c.737G>A missense p.Arg246Gln predicted to result in an arginine to glutamine substitution at amino acid position 246. This variant has been described previously in multiple unrelated families who presented with autosomal dominant nephropathy without nail and patellar abnormalities, which suggest this variant mutation is phenotype specific. This case reports adds to a growing evidence of LMX1B-associated nephropathy without nail and skeletal manifestations seen in classical nail-patella syndrome., ICR guidelines recommended testing all women diagnosed with triple negative breast cancer (i.e. negative for the oestrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2)) under 50 years old should be offered genetic testing for BRCA 1 and 2 pathogenic mutations. It was predicted that testing in this population should identify pathogenic mutations in around 10% of this cohort. This retrospective study will analyse 2 years (April 2016 – March 2018) of BRCA 1 and 2 testing in women diagnosed with triple negative breast cancer under 50 years of age. The main aim would be to find out if BRCA 1 and 2 mutations are accurately represented for our population and to explore the contributing factors to these results. For example, establish true pathology of the triple negative referrals tested and the strength of the family history of cancer in these cases. The hope is to identify whether tighter departmental guidelines for testing and developing a testing criteria proforma for mainstreaming could be beneficial for better mutation pick up rate., Background: Polycystic kidney disease, the most common inherited renal disease, is characterized by renal cysts and progressive reduction in kidney function. Although it is well established that autosomal dominant (ADPKD) is primarily caused by mutations in PKD1 and PKD2, sequencing of PKD1 is difficult due to multiple pseudogenes. Further, there is considerable unexplained variance in the age-of-onset of PKD even within families. Aims: Firstly, to apply NGS technologies for the molecular diagnosis of ADPKD. Secondly, to identify, using genomic and clinical data, large PKD ‘super-families’ to facilitate investigation of genetic modifiers of age-of-onset. Methods: NGS sequencing was performed using a custom Roche NimbleGen SeqCap targeted panel on the Illumina platform. Bioinformatics was performed using a custom, in-house pipeline based on GATK best practices. Copy number variants were identified from NGS data. Whole exome sequencing was performed on selected families using Roche NimbleGen library preparation. Pathogenicity was assigned to variants using ACMG pathogenicity guidelines. Results: 73 ADPKD patients were sequenced and a molecular diagnosis was obtained in 63% (41/73) indicating that NGS technologies were successful for variant identification in difficult to sequence PKD1 regions. We identified five pairs of individuals recorded as unrelated who shared rare PKD1 variants and have inflated genomic relatedness (IBD) scores. Conclusions: NGS with specific capture methods is suitable for the sequencing of renal disease genes including PKD1. We identified one large ADPKD pedigree chart using genomic data for the generation of Irish ADPKD ‘super-families’. Sequencing of additional ADPKD patients (underway) will facilitate expansion of ‘super-families’ concept., Approximately 80% of breast cancers overexpress the estrogen receptor α (ERα) and depend on this key transcriptional regulator for growth. The discovery of novel mechanisms controlling ERα function represents major advances in our understanding of breast cancer progression and potentially offers new therapeutic opportunities. Here, we investigated the role of deubiquitinating enzymes (DUBs), which remove ubiquitin moieties from proteins, in regulating ERα in breast cancer. We performed an RNAi loss-of-function screen using a library of shRNA vectors targeting all 108 known or putative human DUB genes. Suppression of a number of DUBs repressed or enhanced the activity of an estrogen-response-element (ERE) luciferase reporter. Interestingly, suppression of the BRCA2-associated DUB, USP11, was found to downregulate ERα transcriptional activity. Subsequent validation using two individual siRNAs targeted to USP11 revealed a reduction in expression of endogenous ERα target genes in ZR-75-1 cells, as quantified using qRT-PCR. Estradiol (E2) stimulation enhanced USP11 expression in the cell nucleus, while proteomic analysis by mass spectrometry revealed a significant change to the proteome in USP11 knockdown cells in the presence of E2 only. Furthermore, USP11 expression was found to be upregulated in LCC1 breast cancer cells when compared to other cell lines. RNA-seq in LCC1 USP11 knockdown revealed a downregulation of several putative ERα target genes and many cell cycle-associated genes. To support the prognostic relevance of USP11, immunohistochemical staining of a breast cancer tissue microarray (103 ERα+ patients) was performed. Kaplan-Meier analysis of this cohort revealed a significant association between high USP11 expression and poor overall (p=0.030) and breast cancer-specific survival (p=0.041). These results suggest a role for USP11 in ERα transcriptional activity and identify USP11 as a potential therapeutic target in ERα+ breast cancer.

Basal cell carcinomas (BCCs) account for majority of skin malignancies in the United States. The incidence of BCCs is strongly associated with exposure of ultraviolet (UV) radiation. Nucleotide-binding domain, leucine-rich-repeat-containing family, pyrin domain-containing 3 (NLRP3) inflammasome plays an important role in innate immune responses. Different stimuli such as toxins, microorganisms and particles released from injured cells activate the NLRP3 inflammasome. Activated NLRP3 results in activation of caspase-1, which cleaves pro-IL-1β to active IL-1β. In this study, we have shown that NLRP3 is expressed in human basal cell carcinomas. The proximal steps in activation of NLRP3 inflammasome are not well understood. Here, we have attempted to elucidate a critical role for Ca2+ mobilization in activation of the NLRP3 inflammasome by UVB exposure using HaCaT keratinocytes. We have demonstrated that UVB exposure blocks Ca2+ mobilization by downregulating the expression of sarco/endoplasmic reticulum Ca2+ -ATPase (SERCA2), a component of store-operated Ca2+ entry that leads to activation of the NLRP3 inflammasome.

Behcet’s disease (BD) is a complex, multifactorial rare disease, which is poorly understood. Genetic and environmental factors contribute to BD, but the process of diagnosis is challenging with inconsistent clinical manifestations. A recent survey of individuals living with rare disease(s) in Northern Ireland revealed ~50% of individuals receive ≥1 misdiagnosis with 1/20 seeing >10 doctors. Individuals with BD report a range of symptoms, which are variable in onset, severity, and frequency for this systemic vasculitis. Patients describe prolonged journeys to diagnosis with multiple healthcare professionals and medical specialties; there is no BD specialist in Northern Ireland. Using invitations via social media, voluntary groups, and direct contact we are using surveys incorporating micro-narratives, one-to-one semi-structured interviews, and focus groups to collect detailed family histories and stressor information to help characterise recurrent features in patients living with BD and their relatives in Northern Ireland. BD is most often reported in populations along the Silk Road. The highest prevalence is reported in Turkey at 20-420/100,000, compared 1.5/100,000 individuals in the UK. Mapping through general practitioners revealed a much higher than expected prevalence of 12.6/100,000 in the Northern Ireland population. Clusters were observed in Co. Down and Co. Antrim and plotted with social-demographic information. This high ‘UK’ prevalence and the identification of several families with multiple members diagnosed makes NI ideal to explore genetic and epigenetic risk factors for BD. This project involves deep phenotyping and strategies to improve recognition of Behcet’s disease, build collaborative partnerships, improve data collection, enhance training, and information sharing., The National Rare Disease Office (NRDO), initiated in June 2015, collates and disseminates Irish rare disease (RD) information. The prevalent nature of RD (1 in 16 of the population, approximately 80% of which has a genetic basis) and the burden to the health care system is under-recognised and a neglected public health issue. Awareness of rare diseases is a challenge, especially for GPs who each care for > 90 RD patients. The NRDO has made 58 presentations, lectures and publications and received numerous enquiries (58% of contacts from patients/ families, 25% from health care professionals and 4% researchers). Mapping Irish RD clinical and research expertise is developing through Orphanet Ireland. Enrolment of clinical expert centres has increased by 50%,but only, Aims To assess the tumour surveillance advice given to patients in Northern Ireland with confirmed PTEN hamartoma tumour syndrome (HTS). Methods We used the surveillance advice laid out by the Pan Thames Cancer Genetics Group in 2014 to benchmark our patients against. A coding search was carried out on our regional information management system to identify all patients with a confirmed diagnosis. The written/ electronic notes of these patients were reviewed. We adhered to the National PTEN audit inclusion criteria of including patients older than16 years, those with a pathogenic/likely pathogenic PTEN mutation or at 50% risk and those who had received advice between 01/08/10 - 01/08/2015. Results 21 patients were identified. All patients had a pathogenic PTEN mutation. 6 children were excluded. 1 adult was excluded due to lack of documented advice. 6 patients had a cancer diagnosis. 9 patients had a positive family history of cancer. Annual breast screening was recommended for 67% of patients which involved mammography in 83% and MRI in 17%. Annual thyroid USS and TFTs were recommended for 54% and 31% of patients respectively. 16% of female patients had gynaecology referrals completed. An annual dermatological review was recommended for 23% of patients. Widely variable colonoscopy and renal USS screening was recommended for 77% and 65% of patients respectively. No cases of Lhermitte-Duclos disease were identified vs 12% in the national UK audit. Conclusions There is a need for regional PTEN tumour surveillance guidelines to be produced and implemented through a regional PTEN specialist clinic., Catastrophic genomic alterations can drive unusually aggressive cancer phenotypes. We describe a diagnostically challenging rapidly fatal case of medullary thyroid carcinoma (MTC) occurring in a young, morbidly obese man presenting with diffuse bone marrow involvement and disseminated intravascular coagulation. Whole-exome sequencing and shallow whole-genome sequencing was carried out for the primary tumour and multiple metastases. We identified three germline SNP’s within the RET proto-oncogene which remained undetected using routine hospital genetic testing procedures. Indeed, one of the variants identified (L769L) has been previously reported in literature to be associated aggressive MTC presentation, yet remains untested for in the routine diagnosis of MTC. Supported by findings from shallow whole genome sequencing, we report for the first time in thyroid cancer, the occurrence of a catastrophic “chromothripsis-like pattern” (CTLP) event, which involved shattering of chromosome 4 leading to complete abrogation of normal chromosomal function, in addition to dramatic wide-spread copy number aberrations (CNA), across both primary tumour and bone marrow samples. We further describe the presence of loss-of-heterozygosity (LOH) in key genes involved in DNA repair mechanism pathways such as ATM, which possibly facilitated the CTLP event, in addition to LOH in other disease-associated genes such as ALK and NOTCH1 as key drivers of the aggressive and rapidly fatal clinical course in this patient and unresponsiveness to the standard-of-care targeted agent chosen. Given a possible rapid generation of tumor neo-antigens as a result of the CTLP event, immunotherapy may have been more suitable as a treatment option. Moreover, the presence of disease-associated SNP’s within the RET proto-oncogene, support their inclusion as part of routine RET genetic testing for aggressive MTC cases. These results provide a rationale for application of comprehensive genomic analysis of cancers presenting with unusually aggressive behavior to facilitate more appropriate therapeutic options and diagnoses., The Target 5000 research project aims to provide genetic testing for the estimated 5,000 people in Ireland who have an inherited retinal condition. Many clinical trials are available for patients with sight loss, however, many such trials require patients to have their causative mutation identified in order to enter the trial. The objective of the study is to genetically characterise patients with inherited retinal degenerations (IRDs) in Ireland and in principle to make clinical trials more accessible to some Irish people suffering from sight loss. The study also seeks to identify previously undiscovered pathological mutations in a panel of known retinopathy genes evaluated utilizing target capture next generation sequencing (NGS). Thus far in the study, as part of Target 5000 roughly 10% of the Irish IRD population has been sequenced and the results obtained are encouraging. Target 5000 offers not only a chance to discover new causative mutations, but is vital in giving patients access to information regarding the pathogenesis of their disease. Over 50 novel mutations have been discovered, as well as some previously ambiguous phenotypes resolved. More precise matching of genotype with phenotype from this study and similar studies globally should start to enable clinicians to better formulate accurate future diagnoses and at times prognoses., MicroRNAs are understood to play a functional role within the establishment of epigenetic marks and are in turn under epigenetic control. Emerging evidence suggests microRNAs are vital for both kidney development and renal function. This study aimed to identify differential methylation affecting microRNAs in patients with end-stage renal disease (ESRD). Methylation status was determined for 485,577 unique CpG sites in 105 individuals with ESRD and 52 donor controls with no evidence of renal disease using the HumanMethylation450K BeadChip array (Illumina). Statistically significant associations (P, Amyotrophic lateral sclerosis (ALS) is a complex neurodegenerative disease characterized by rapid-onset loss of upper and lower motor neurones, resulting in progressive paralysis and death from respiratory failure. Schizophrenia is a neuropsychiatric disease with positive symptoms, negative symptoms and impairment over a range of cognitive abilities. We have recently shown that schizophrenia occurs more frequently than expected in the pedigrees of ALS patients, suggesting an aetiological relationship between both diseases. Using linkage disequilibrium score regression with summary statistics for GWAS of ALS and schizophrenia comprising over 100,000 unique individuals, we estimated the genetic correlation between ALS and schizophrenia to be 14.3% (95% CI 7.05-21.6; p = 1×10-4). Up to 0.12% of the variance in ALS was explained by schizophrenia polygenic risk scores (p = 8.4×10). We leveraged the apparent pleiotropic relationship between ALS and schizophrenia to identify five potential novel ALS-associated genomic loci at conditional false discovery rate < 0.01. Diagnostic misclassification in the schizophrenia cohort did not contribute significantly to our observations (BUHMBOX p = 0.94) and we estimated that 4.86% (2.47-7.13%) of ALS cases would need to be misdiagnosed as schizophrenia to observe our genetic correlation estimate under a true genetic correlation of 0%. Our results indicate that the lifetime risk for comorbid ALS and schizophrenia increases from 1 in 40,000 to 1 in 34,336, which would require an incident cohort of 16,488 ALS patients to observe epidemiologically. Our findings suggest shared underlying biology between ALS and schizophrenia which will direct novel approaches in research and therapeutic development., Background Imprinted genes are autosomal, but only expressed from one parental allele and are often clustered in small groups. They play an important role in the regulation of normal mammalian development. Differentially methylated regions (DMR) on each allele are important in regulating the genes, with marks being characterised as primary or secondary DMRs, depending on whether they are inherited from the germ cells or arise later, respectively. Imprinting disorders such as Prader-Willi Syndome (PWS) and Beckwith-Weidemann Syndrome (BWS) arise either from uniparental disomy or faulty DNA methylation. We wished to determine 1) which of the loci are most sensitive to loss of methylation 2) to more precisely define the sensitive regions and 3) determine what happens at primary versus secondary imprints. Methods Stable knockdowns of the maintenance methyltransferase DNMT1 were generated in hTERT-immortalised adult fibroblasts using shRNA. Genome wide methylation levels were assayed using the Illumina 450k BeadChip array and analysed using bioinformatic approaches. Results We found that 1) the imprinted loci varied extensively in their sensitivity to loss of methylation 2) the extended locus involved in PWS was particularly sensitive 3) that loss of methylation at primary DMR appears to drive gains in methylation at secondary DMR. Conclusion Our results point to a mechanistic link between primary and secondary DMR which may explain why imprints are difficult to reprogram in somatic tissues., Osteoarthritis (OA) is a degenerative joint disease that affects millions of people globally with no disease-modifying strategies yet available. Our understanding of the pathology of OA is inadequate and this impedes investigation of efficient diagnosis and treatment. To expand our understanding of the underlying cellular pathology of OA, we studied a monogenic condition, familial osteochondritis dissecans (FOCD), associated with a known mutation in the ACAN gene. Patients with FOCD develop early onset OA with multiple joint involvement. The objectives of the project were to investigate the cellular pathogenesis of FOCD by studying (a) chondrogenesis of patient-derived bone marrow-mesenchymal stem cells (BM-MSCs) and (b) induced pluripotent stem cells (iPSCs) generated from patient fibroblasts. Our findings revealed that the mutation resulted in a misfolded or unfolded aggrecan protein, which accumulated in the rough endoplasmic reticulum (rER) during protein production. The consistent accumulation resulted in ER stress throughout chondrogenesis. Moreover, the rER stress caused abnormal or disregulated global extracellular matrix (ECM) production and assembly. Importantly, ECM composition analysis indicated that the patient chondrocytes produced abundant amounts of OA-associated markers. Using patient-specific stem cell models, we have discovered a cellular pathogenesis of FOCD involving abnormal cell function and defective tissue formation, contributing to the OA phenotype., Aims The Irish Travellers are a nomadic population primarily found within Ireland and the UK. Consanguineous unions are common, and as a population they are socially and genetically isolated from the surrounding, “settled” Irish population. Previous low-resolution genetic analyses suggested a common Irish origin between the settled and the Traveller populations. It is not known, however, what is the extent of population structure within the Irish Traveller population, the time of divergence from the general Irish population, and the extent of autozygosity. Methods We recruited Irish Travellers from across Ireland and the UK. For inclusion, a participant had to have had at least three grandparents with a surname associated with the Irish Travellers. DNA was extracted from saliva samples, and genotypes were generated using the Illumina OmniExpress SNP genotyping platform. With this data, we investigated population structure using fineStructure, quantified the levels of autozygosity with PLINK, and estimated a time of divergence using a method based on Identity by Descent (IBD) segment sharing. Results We merged, cleaned, and analysed data from 42 Irish Travellers, 2232 settled Irish, 2039 British, 143 Roma Gypsies, and 931 individuals from 57 world-wide populations. We confirm an Irish origin for the Irish Travellers, demonstrate evidence for population substructure within the population, confirm high levels of autozygosity consistent with a consanguineous population, and for the first time provide estimates for a date of divergence between the Irish Travellers and settled Irish. Conclusion Our findings have implications for disease mapping within Ireland, and they additionally inform on the social history of the Irish Traveller population., Copy number variants at 16p13.11 have been described in association with a variety of neurodevelopmental disorders. While deletions of this region are perhaps better described, the clinical significance of the reciprocal duplication is less clearly defined. Phenotypes reported in association with the duplication include developmental delay, speech delay, behavioural difficulties and neurodevelopmental phenotype such as autism, schizophrenia and ADHD. However, the region appears to be subject to variable expressivity and incomplete penetrance. To date we have detected duplications of 16p13.11 in 5 probands using oligonucleotide array CGH. Of these patients 3 showed duplications within the typical ~1.5Mb duplication region while 2 patients had a larger ~2.8Mb duplication, encompassing all of the above region. The clinical phenotype of these patients will be described. Two of these patients have inherited the duplication from their mothers, one was a de novo finding and the inheritance of the others is currently unknown. One of the maternal duplication carriers are also known to have a phenotype. Our data provides further clinical information on the phenotypic features of patients with this syndrome and provides more evidence for the pathogenic nature of this duplication., Introduction Patients referred to the NI Regional Cancer Genetics Service for genetic counselling were sent a questionnaire to evaluate patient satisfaction. The questionnaire focused on satisfaction surrounding the referral process, waiting times and communication during and after the appointment. Method One hundred patients, whose episode of care was completed between November 2015 and June 2016, were sent an anonymised structured questionnaire by post. Patients were seen by a genetic counsellor for assessment of their family history of cancer, predictive testing and genetic mutation screening Results To date (23/06/2016) the questionnaire response rate is 34%. So far 91% have expressed satisfaction with the service that they received. Useful comments and observations have been feedback in the questionnaire to aid service improvement. Data collection will be completed imminently to allow for complete analysis. Discussion Useful data has been collected which reinforces the service currently being delivered by genetic counsellors whilst also highlighting areas of service development., Leber’s Hereditary Optic Neuropathy (LHON) is one of the most commonly inherited optic neuropathies and results in significant visual morbidity among young adults. 95% of LHON patients will present with one of three primary mitochondrial mutations; G3460A, G11778A and T14484C. We describe a novel real time diagnostic test to detect the three common mutations leading to LHON. The test uses a combination of multiplex allele specific PCR (ARMS PCR) in combination with high resolution melt curve analysis to detect the presence of the G3460A, G11778A and T14484C mutations. PCR primer sets were designed to produce a control PCR product and PCR products only in the presence of the 3460A, 11778A and 14484C mutations in a multiplex single tube format. Products produce discrete well separated melt curves allowing clear detection of the mutations. The test has proved to be robust, cost and time effective with the real time closed tube system taking approximately 1 hour to complete. This test provides a simple, robust, easy to read output that is both cost and time effective, thus providing an alternative method to individual endpoint PCR – RFLP, PCR followed by Sanger / pyrosequencing and next generation sequencing. It will also allow diagnostic laboratories to detect 95% of LHON causing mutations in a single tube assay allowing diagnostic laboratories to avoid costly NGS assays for the vast majority of LHON patients, thus allowing resources to be focussed on patients with unknown mutations requiring further analysis., Atherosclerotic coronary artery disease (CAD) is a progressive chronic inflammatory condition that can lead to Major Adverse Cardiac Events (MACE) such as heart attacks. Currently there is no definitive test to predict MACE risk. Tumour necrosis factor alpha converting enzyme (TACE), also known as A Disintegrin And Metalloproteinase 17 (ADAM17) is a membrane-anchored protein responsible for the ectodomain shedding of a variety of transmembrane proteins such as cytokines, chemokines, growth factors and their receptors. TACE has been linked to several major acute and chronic inflammatory diseases including atherosclerosis. The aim of this study was to investigate if TACE may be a valuable predictive biomarker for CAD and MACE risk. TACE levels were measured in the plasma of CAD patients including those with acute coronary syndrome (ACS) and elective patients attending the catheterisation laboratory for coronary angiogram. TACE levels were measured using ELISA and quantitative real time PCR. Levels were compared with control samples collected from apparently healthy individuals and a subset of patients with no CAD as evidenced by coronary angiogram. Other factors that might affect TACE detection were also measured including sample type and storage time. To date 207 consecutive CAD patients and 40 controls have been recruited to the study. Results demonstrate that CAD patients have higher levels of plasma TACE in comparison to controls. TACE protein levels were especially highest in those ACS and elective patients with a previous history of MACE. Results to date indicate that TACE may be a useful marker to predict disease progression and recurrent MACE in CAD patients., Introduction NRG1 (neuregulin1) is a candidate tumour suppressor gene. NRG1 encodes ligands for members of the ERBB family, and has been shown to be silenced by methylation in breast cancer1. Breast and thyroid cancers share some genetic loci (e.g. PTEN, STK11), and an increased risk of thyroid cancer has been noted in survivors of breast cancer2. A single nucleotide variant (C>G) in NRG1 (rs2439302), has been associated with increased risk of non-medullary thyroid cancer3. Aim Our aim was to investigate the association between rs2439302 in NRG1 and predisposition to thyroid and breast cancers in an Irish population. Methods A two-arm case-control study was undertaken. Patients with mutations in high-risk cancer susceptibility genes were excluded. Controls included adults with no personal or familial history of breast or thyroid cancers. Male controls were included in thyroid case- control analysis only. DNA was extracted from whole blood/buccal swabs by ethanol precipitation. Genotyping was performed using Taqman-based PCR. Results 257 patients with thyroid cancer, 518 with breast cancer and 367 unaffected controls were genotyped. Homozygous carriers of the variant were found to have an increased risk of thyroid cancer (OR1.89 (1.21-2.95), p=0.005), but risk for mono-allelic carriers was not significantly increased (OR1.27 (0.87-1.84), p=0.21). The presence of the variant was not associated significantly with breast malignancy for mono-allelic (OR1.31 (0.95-1.8), p=0.095) or biallelic mutation carriers (OR1.15 (0.76-1.73), p=0.51). Conclusion Homozygous carriers of the G allele were found to be at increased risk of thyroid cancer, but no association was observed between the variant and breast cancer., The UGT1A gene family encode (UGT) activity that facilitate the transfer of glucuronic acid to a range of xenogenous and endogenous substrates, the polar end products of which are better suited for elimination through urine and bile. UGT1A genes exhibit an inducible pattern of expression regulated through the activities of such nuclear receptors (NRs) as pregnane X receptor (PXR) farensoid X receptor (FXR) and liver X receptor (LXR) that form a complex interactive network of ‘sensors’ to facilitate the elimination of potentially harmful metabolites and exogenous toxins. We have previously reported that activation of vitamin D receptor (VDR) through both synthetic agonists and nutritionally derived ligands, can induce the expression of both phase I metabolic (CYP3A) and phase III transporter (ABCA1) genes. Little is known however, as to how activated VDR may impact upon the regulation of phase II genes such as UGT1A1. In this study we demonstrate that ligand-activated VDR can significantly enhance the expression of several members of the UGT1A gene family. With particular respect to UGT1A1, we identify within the proximal promoter region of this gene a functional vitamin D response element (VDRE) also recognized by PXR but distinct from previously established regulatory elements that mediate FXR and LXR signalling. Based upon our data, we propose a model for VDR and circulating levels of vitamin D as maintaining stable expression of phase II and functionally related genes as a means to provide baseline protection against the effects of toxic xeno and endobiotic metabolites., Depression is a complex disorder with multiple symptoms, including a persistent low mood, anhedonia and cognitive impairments, and is currently the third leading cause of global disability. The underlying pathophysiology of depression is poorly understood but a growing body of evidence supports an important role for the microbiome in the aetiology of depression and other psychiatric disorders. While much interest is currently focused on the role of the microbiome-gut-brain axis in brain physiology and neurochemistry, the importance of the oral microbiome has received little attention. The aim of this study is to characterise the oral microbiome in adults with severe depression versus matched controls with no history of the disease. To achieve this, participants were asked to complete an online validated mental health survey and to provide a saliva sample. We identified 46 individuals who met the DSM-V criteria for severe depression and 46 age and sex-matched controls with no history of depression. Bacterial DNA was extracted from the saliva samples and 16S rRNA surveys were conducted using next generation sequencing. Differences in the bacterial community composition of the oral microbiota between patients and controls were determined. Metagenomic analyses were conducted using machine learning and computational intelligence algorithms using the 16S RNA data to generate inferred metagenome feature sets. Charting the oral microbiome in depressed patients could therefore provide new insights into the development of the condition, and lead to the identification of novel diagnostic and therapeutic response biomarkers., The nuclear receptors (NRs) pregnane X receptor (PXR) and constitutive androstane receptor (CAR) modulate transcriptional networks that dictate the bioavailability of many endogenous and exogenous compounds such as steroid hormones and therapeutic drug compounds. Elucidating those factors that invoke PXR/ CAR activity has been important for understanding the genetic basis for both metabolic disease and inter-individual variations in drug response. PXR is most closely related to Vitamin D receptor (VDR) for which there is relatively little is known for how this NR may impact upon these same physiological processes. In this study, we employed enteric cell models and ex-vivo based human colon explants to examine how activated VDR may impact upon the expression of genes of a metabolism and transporter function. We find that in relation to PXR and other evaluated NRs, VDR is the most efficient and dominant receptor for induced expression of CYP2B6, CYP3A4/5 and ABCA1. We note that upon activation with the synthetic agonist EB1089, VDR will achieve striking and sustained elevated expression of CYP3A4 at mRNA, protein and enzymatic level suggesting the potential for selective metabolic gene targeting through ligand design. In addition, we report members of the UGT1A gene family to be novel VDR regulated genes, thus extending the known metabolic effects of vitamin D to also encompass expression of phase II (conjugating) genes. This study intimates that systemic vitamin D status and/or activating VDR ligands may have pharmacokinetic relevance to co-administered drug regimes., Ancient genomes are often typically analysed with regard to ancestry and physical phenotype. Less common is examination and identification of genetic diseases, primarily due to the very low numbers of samples sequenced and poor level of sequencing related to the difficulties in sequencing from ancient DNA. Here we present the results of analysing 21 ancient Irish genomes. The data were screened for a wide range of pathogenic genotypes and markers. Giving information for the potential effects and prevalence of certain conditions as well as the earliest known confirmation of their presence. Using records of the remains, we also examined if any displayed phenotypes correlated to identified diseases., Coronary Artery Disease is the largest contributor of CVD, the leading cause of death worldwide. It is caused by atherosclerosis, a build-up of cholesterol in the blood vessels and chronic inflammation. The NLRP3 inflammasome plays a critical role in the secretion of IL-1β, and there is significant evidence that it is involved in the pathogenesis of a number of inflammatory diseases including atherosclerosis. Recent studies demonstrate that particular cell surface receptors namely the scavenger receptor CD36 and the endocannabinoid receptor CB1 are involved in the activation and regulation of the NLRP3 inflammasome and they have also been implicated in the pathogenesis of atherosclerosis. The present study aimed to investigate expression and activation levels of the NLRP3 inflammasome, the CD36 and CB1 receptors in blood samples obtained from patients with atherosclerosis at very high risk of a Major Adverse Cardiac Event (MACE) such as a heart attack. The cell signalling processes involved in NLRP3 inflammasome activation were also investigated in a THP1 in vitro model of atherosclerosis. Results to date indicate increased expression of NLRP3 in patients at very high risk of MACE and also demonstrate that THP1 macrophages require both the CD36 and CB1 receptors for optimal NLRP3 expression in response to oxidized LDL. These preliminary findings provide an insight into the mechanism of action of the NLRP3 inflammasome in atherosclerosis and prompt further exploration of this protein complex and its regulatory receptors as potential targets for prognostic and or therapeutic development in the strive towards a more personalised approach to the management of coronary artery disease., Approximately 30% of patients with epilepsy are refractory to anti-epileptic drug (AED) treatment and continue to have debilitating seizures that severely impact upon their quality of life. Exome sequencing in encephs etc has illustrated the importance of de-novo variants in the pathogenesis of rare neurological disorders. However, the contribution of de-novo mutations to pharmacoresistance in adult epilepsy is uncertain. In this study we investigated whether a trio whole exome sequencing paradigm could be applied to identify genetic causes of chronic, refractory epilepsy. We selected adult patients (n=5) with onset of seizures after 5 years of age, had failed ≥6 AEDs and were still experiencing >4 disabling seizures per month. Patients were excluded if they had a potentially ‘explanatory’ lesion on MRI. Parents were exome sequenced to identify de-novo mutations and these were assessed bioinformatically for pathogenicity. We confirmed the presence of coding de-novo mutations that were bioinformatically predicted to be functional and damaging in 3/5 patients. One of these occurred in the gene DNM1L, which was recently implicated in pharmacoresistant epilepsy (Vanstone et al. EJHG, 2015;Nov 25). This represents a potential diagnostic yield of 20% however more data is required and more trios are currently being sequenced. We have demonstrated the potential diagnostic yield of whole exome sequencing in a small number of adult patients with chronic refractory epilepsy. Identifying genetic mutations underpinning this disorder may provide new insight into the underlying biology and offers the potential for therapeutic intervention in the form of precision medicine., IgA nephropathy (IgAN) is the most common form of glomerular nephritis worldwide1. Difference in incidences between ethnicities and familial inheritance patterns indicate this is a genetic disorder. An IgAN locus on chromosome 6q22-23 was identified via linkage analysis; however the causal gene remains elusive2. We set out to identify mutations underlying familial IgAN using whole exome sequencing. DNA was collected on 25 (unaffected and affected) individuals across 6 families with IgAN. Families were chosen on the basis of having at least 2 affected members with IgAN. We carried out full exome sequencing on 12 of the affected members from these families. Depending on the pattern of inheritance in a given family, mutations that fitted a dominant, recessive or compound heterozygote model of inheritance were screened for. These variants were then filtered based on being shared between affected individuals within a family, their minor allele frequency, region, function and predicted deleterious nature. We identified a number of potential candidate mutations in these families and including a mutation in the gene COL4A5 which was previously described as pathogenic3. Mutations in COL4A5 have previously been found in individuals with Alport syndrome, a disease which is often mistaken for IgAN. We are currently working to confirm these candidate mutations via Sanger sequencing and will be screening for segregation., Atherosclerosis is a chronic inflammatory disorder that is responsible for approximately 71% of incidents of cardiovascular disease. A mathematical model of atherosclerosis has been developed, capturing the cell types and proteins involved in atheroma formation and describing the dynamics of disease progression. This is the first model of this type to be developed using open systems biology standards. We have predicted tertiary protein structures for all the proteins involved in this atherosclerosis model and all of their recorded mutations, using phase 3 sequence data obtained from the 1000 Genomes Project. By comparing the electrostatic potentials of these tertiary structures, we predict how the dynamics of atherosclerosis stratifies across population subgroups., The aim of this pilot study was to test the feasibility of carrying out a large scale study using this design to investigate whether methylation of the oxytocin receptor (OXTR) can serve as a potential biomarker for response to oxytocin administration in women during and after labour. Background Oxytocin is a nine-amino acid peptide with hormonal and neurotransmitter functions during labour and lactation. We hypothesised that a difference in methylation levels of the oxytocin receptor (OXTR) gene may impact the woman’s ability to become established in labour and her response to oxytocin administration. Method Blood samples were taken pre-birth and postnatally from 21 women and subjected to DNA methylation analysis of the OXTR gene by pyrosequencing. Methylation status of CpG sites -924 and -934 upstream from the initiation transcription site (ITS) of the OXTR gene was determined. Expression of the OXTR gene before and after birth was measured using qPCR. Global methylation levels were examined using Luminometric Methylation Assay (LUMA). Results We found both hypo and hypermethylation of OXTR promoter at CpG sites -924 and -934 in individual samples, however we observed no profound changes in overall OXTR methylation levels within the patient cohort at these CpG sites. We found a strong correlation between OXTR promoter methylation levels found in whole blood and those found in matched PMBC samples. Global methylation analysis using Luminometric Methylation Assay (LUMA) revealed no significant differences between whole blood and PMBC. Conclusions A larger sample is required to determine whether OXTR methylation status is predictive of response to oxytocin administration. Whole blood sampling is a suitable alternative for OXTR methylation analysis in a larger cohort of women undergoing labour., Background Cardiovascular disease (CVD) is the leading cause globally of morbidity and mortality. microRNAs (miRNAs) are small, non-coding RNAs which have a fundamental role in the pathology of various diseases including CVD. Circulating serum levels of miRNAs have been proposed as potentially valuable markers of heart failure, stroke, myocardial infarction and arterial hypertension, but the specific miRNAs involved and their function remains unclear. Therefore, this pilot study aims to profile miRNA expression in premature CVD patients to identify which miRNAs correlate best with hypertension. Methods The Multiplex Circulating miRNA Assay with Firefly™ Particle Technologies was used to profile 68 miRNAs on a cardiology focus panel in serum samples from 170 premature CVD patients recruited from Altnagelvin Area Hospital and screened for the C677T polymorphism in methylenetetrahydrofolate reductase, a risk factor for hypertension. Samples were collected at baseline and following intervention with riboflavin, a co-factor for MTHFR, which significantly lowers blood pressure specifically in adults with this polymorphism. Statistical analysis was used to correlate miRNA expression with blood pressure, MTHFR genotype and other relevant clinical data. Results The assay successfully measured miRNA expression in the sample set. miRNAs which expressed differentially between MTHFR genotype groups were highlighted and the functional significance of these miRNAs was assessed using bioinformatics to identify target genes involved in CVD. Conclusions The data provides further evidence that using specific miRNAs as serum markers could aid early prediction of CVD and may lead to better diagnostic modalities and therapeutic regimes., Gene-environment interactions, particularly in genes related to regulation of serotonin and neuronal function, have been implicated in the aetiology of depression. Allelic variations in the 5’ flanking transcriptional region of the serotonin transporter gene (5-HTTLPR) and higher levels of promoter DNA methylation are associated with depression. Brain derived neurotrophic factor (BDNF) plays an important role in neuronal differentiation and survival, and is also involved in regulation of serotonin. A single nucleotide polymorphism in the BDNF gene, leading to a valine to methionine substitution at codon 66 (Val66Met), and increased methylation of the BDNF promoter have also been associated with depression. The goal of this study is to determine whether length of the 5-HTTLPR, prevalence of the Val66Met polymorphism of the BDNF gene and DNA methylation in both 5-HTT and BDNF promoter regions are associated with depression in the student population. First year students provided a saliva sample for genetic analysis and completed an online mental health survey. Presence and severity of depression was determined from survey responses based on DSM-IV criteria. Length of the 5-HTTLPR was determined by PCR and gel electrophoresis and presence of the SNP at BDNF rs6265 and examined using restriction fragment length polymorphism analysis. Bisulphite-treated DNA was amplified by PCR and pyrosequencing assays used to determine methylation patterns of BDNF and SERT. Our preliminary findings suggest that genetic and epigenetic variation in the 5HTT and BDNF genes are associated with depression in the student population and may be candidate biological markers to assist in diagnosis., BACKGROUND Prostate cancer is the most common male cancer in the UK, where it kills approximately 11,000 men annually. There has been growing interest in the role played by the anaerobic bacterium Propionibacterium acnes, an important component if the skin microflora, in the aetiology of the condition via a chronic, asymptomatic infection of the prostate leading to oncogenesis. METHODS A quantitative real-time PCR (qRT-PRC) assay for retrospective detection of P. acnes in formalin-fixed paraffin embedded sections from archived prostate samples was developed. An in vitro infection model of prostate infection with P. acnes is being optimised, which should allow us to get insight into the dysregulation P. acnes infection causes in prostate epithelial cells. RESULTS A total of 81 biopsy samples, representing one or both prostate lobes, were examined from 53 patients with prostate carcinoma, versus 111 samples from 60 patients whose biopsies were histologically normal, and the assay revealed that 35% of cancerous prostate samples were positive for the presence of P. acnes, compared with only 8% of the disease-free samples (p, Oral squamous cell carcinoma (OSCC) is one of the top ten most prevalent cancers in the world. Prognosis is poor and quality of life is commonly reduced for patients who survive. OSCC is thought to progress via a premalignant stage called dysplasia. Effective treatment of dysplasia prior to malignant transformation, or the ability to more accurately predict the 10-20% of dysplasias that will progress to OSCC, is an unmet clinical need. With the aim to better understand the biology of OSCC development, and attempt to identify potential markers of early disease and therapeutic targets, we performed parallel whole exome sequencing and total RNA sequencing on 16 micro-dissected formalin-fixed paraffin embedded dysplasia and their associated OSCC. These are the largest omic analyses on matched patient samples from the oral cavity in non-HPV infected patients where all dysplasias are associated with progression to OSCC, that has been performed to date. Whole exome analysis revealed that every OSCC and adjacent associated dysplasia sample did have a common clonal ancestor, with many shared potential drivers of progression, but that there is also considerable genomic heterogeneity between associated pre-invasive and invasive disease, as seen in a previous study1. RNAseq analysis revealed differences in the immune cell signatures present at different disease stages, distinguished early events in pathogenesis from later events and identified several novel coding and non-coding candidates with potential involvement in oral dysplasia development and malignant transformation. These findings merit further investigation in a larger retrospective longitudinal study of patients with oral dysplasia., Many disorders involving tissues, which have significant energy requirements, involve mitochondrial dysfunction often due to mutations affecting the mitochondrial genome. Some such mutations can involve genes coding for subunits of complex I of the electron transport chain leading to a complex I deficiency in disorders such as Leber Hereditary Optic Neuropathy (LHON) amongst others. Mitochondrial dysfunction leads to a lack of energy production and ultimately the death of the cell. In disorders such as LHON, retinal ganglion cells (RGCs) are affected, leading to retinal dysfunction. These observations have prompted interest in exploring innovative therapeutics to modulate mitochondrial disorders involving complex I deficiency. The Farrar laboratory has explored candidate gene therapies for complex I deficiency using Ndi1, a yeast gene which is a complex I homologue. In order to test the efficacy of candidate therapies, we have developed a robust, empirical assay of mitochondrial function. Previous assays measured the level of NADH oxidation in a sample, both before and after rotenone as a measure of complex I activity. To optimally distinguish between the activity of complex I and the potential therapeutic, the assay was modified with the addition of a second inhibitor which allowed specific measurement of the therapeutic, such as Ndi1. Given that this is an in vitro assay, it enables large-scale screening of potential therapeutics and ensures only those that show strong evidence of efficacy are then tested in vivo. In combination with other quantitative assays such as Reactive Oxygen Species (ROS) generation this allows detailed evaluation of the health of mitochondria within a sample., Schizophrenia is an adult-onset mental illness with that impacts cognitive function. The largest GWAS has revealed 108 loci associated with schizophrenia risk but how variation affects genes and impacts brain function to increase risk is largely unknown. The centrosome is the microtubule organising centre of the cell and seeds the growth of the primary cilium. The disproportionate number of brain disorders associated with centrosomal genes suggests the organelle underlies normal brain and cognitive development. Schizophrenia is neurodevelopmental and cognitive deficits are a core element of the disorder. We hypothesise that some of the newly identified risk genes for schizophrenia will function in the centrosome and variants in these genes will be associated with cognitive deficits. Cross-referencing genes with centrosomal functions with genes from schizophrenia GWAS, identified six candidate genes; SDCCAG8, MAD1L1, GIGYF2, MPHOSPH9, PRKD1 and MAPK3. The effect of risk SNPs on cognition was examined using an Irish dataset of psychosis cases and controls (n=1,236) using linear regression. Among the associations identified, the SDCCAG8 risk SNP was shown to affect attribution style, a measure of social cognition (P=0.001). The MAD1L1 risk SNP was associated with poorer performance on episodic memory tasks (P=0.003). A suitable replication dataset was not available for social cognition measures. We attempted replication for episodic memory results in UK and German samples but results were non-significant. Overall, we have identified a number of schizophrenia risk genes that function in the centrosome but further larger datasets are required to establish a role for these genes in cognition., Epigenetic mechanisms are an important heritable and dynamic means of regulating various genomic functions, including gene expression to orchestrate brain development. These processes when perturbed are thought to contribute to schizophrenia (SZ). A core feature of SZ is cognitive dysfunction. GWAS have identified 108 genomic loci associated with SZ risk, containing 350 genes. My aim was to identify genes that have epigenetic functions which map to loci associated with SZ, and to test the associated SNPs for association with cognitive deficits. Risk SNPs in 8 genes: BCL11B, CHD7, EP300, EPC2, GATAD2A, KDM3B, RERE and SATB2 were analysed using an Irish dataset of psychosis cases and controls (n=1235) who had completed tests across 5 cognitive domains. Five of the eight variants had significant associations with at least one cognitive task. Strongest associations were for CHD7 (rs6984242) for IQ (p=0.001) and episodic memory (p=0.007). These results did not replicate in independent samples. We link rs6984242 to CHD7 via a long range expression quantitative trait loci (eQTL) and CHD7 has not been previously reported as a candidate risk gene for SZ. To further explore its novel association with SZ, we identified a set of 45 interacting genes and used SNPs across these genes to develop a polygenic risk score for SZ, independent of CHD7 itself. This score was tested for association with cognitive function. Significant associations(p, The relevance of nutrition and other environmental influences on epigenetic modifications including DNA methylation is a topic of considerable interest. Folate One Carbon Metabolism (FOCM) is the principal supplier of the methyl groups required for DNA methylation, giving folate status a strong biological plausibility of having an impact on an individual’s and an offspring’s DNA methylation profile at both the mitotic and meiotic level. We sought to identify DNA methylation sites in the human genome that are sensitive to folate status i.e., Folate-sensitive Differentially Methylated Regions (FS-DMR) using a folic acid intervention trial in pregnant women known as FASSTT (Folic Acid Supplementation in the Second and Third Trimesters). To minimize the amount of DNA methylation ‘noise’ due to non-folate related factors such as other environmental stimuli and individual genetic variation, we compared the DNA methylation profile of the same individual pre- and post- intervention to identify putative FS-DMR. We selected six healthy pregnant women, three from the folic acid intervention arm and three from the placebo arm of the trial. We performed MeDIP (Methylated DNA Immunoprecipitation) on all 12 samples and hybridized to a Roche Nimblegen Delux 2.1M promoter array. While we observed DNA methylation changes pre- and post- folic acid intervention in each individual, the actual DNA methylation sites were not consistent across all three individuals. Of course, it is possible that a more in-depth Next Generation Sequencing approach might yield our elusive FS-DMRs. However, the published literature to date does not appear to support such a promise., The loss of retinal ganglion cells (RGCs) is a hallmark of a number of retinopathies. There are a number of gene therapies being developed that have shown efficacy in preserving RGCs when administered using an AAV vector. Localising expression of any therapeutic to the target cell type (ganglion cell layer, GCL) would represent a significant optimisation of the approach. The packaging capacity of AAV (4.7kb) imposes a limit on the size of promoters and genes relevant for AAV-mediated gene delivery. Few GCL-specific promoter sequences have been defined of a size suitable for use in AAV-guided gene expression. Exploring this, a panel of genes was chosen with GCL-limited expression profiles. A pipeline program was developed that analysed regions upstream of these genes for sequence conservation across placental mammals (as a proxy for putative promoter function), weighted by enriched GCL expression levels. Adopting this strategy, ganglion cell promoter 1 (GCP1), demonstrating the key features outlined above, was identified. To test its function, GCP1 (2.2kb in size) was engineered into an AAV2 virus expressing EGFP. Here we demonstrate the effectiveness of GCP1 in localising EGFP expression to the GCL when administered via intravitreal injection. Furthermore, absence of EGFP expression was demonstrated when targeted towards photoreceptors via subretinal injection, verifying GCP1 tissue-specificity. Expression of AAV2.GCP1-EGFP was compared to expression from a non-specific promoter construct, AAV2.CMV-EGFP. GCP1-EGFP was shown to provide equivalent expression to CMV-EGFP in the GCL. GCP1 thus offers a tissue-specific promoter option, suitable for deployment within AAV vectors without compromising functionality., Purpose Hypoxia is a common hallmark of the tumour microenvironment. Recently we have shown the anti-androgen bicalutamide induces profound hypoxia in prostate tumours in vivo. This resulted in the promotion of epithelial to mesenchymal transition. Here we target tumour hypoxia using a novel unidirectional hypoxia-activated prodrug OCT1002 to enhance the anti-tumour effect of bicalutamide. Experimental Design The effect of OCT1002 treatment on LNCaP-luc cells was measured in normoxia and hypoxia in vitro. In vivo, tumour growth and lung metastases were measured in mice treated with bicalutamide, OCT1002 or a combination. Dorsal skin fold chambers were used to image tumour vasculature in vivo. Longitudinal genetic changes in tumours were analysed using PCR. Results Reduction of OCT1002 to its active form (OCT1001) decreased LNCaP-luc cell viability. In LNCaP-luc spheroids, OCT1002 caused increased apoptosis and decreased clonogenicity. In vivo, treatment with OCT1002 alone or with bicalutamide, showed significantly greater tumour growth control and reduced lung metastases compared to controls. Re-establishment of the tumour vasculature following bicalutamide-induced vascular collapse is inhibited by OCT1002. Significantly, the up-regulation of RUNX2 and its targets caused by bicalutamide alone were also blocked by OCT1002. Conclusions OCT1002 selectively targets hypoxic tumour cells and enhances the anti-tumour efficacy of bicalutamide. Furthermore, bicalutamide causes changing genetic profiles during treatment, with development of a more malignant genotype; OCT1002 can block this effect. This study indicates that more attention should be attached to understanding genetic changes that may occur during treatment. Early targeting of hypoxic cells with OCT1002 can provide a means of inhibiting prostate tumour growth and malignant progression., Background In prostate cancer (PCa), abnormal expression of several microRNAs (miRNAs) has been previously reported. Increasing evidence shows that aberrant epigenetic regulation is a contributing factor to their altered expression in cancer. In this study we investigate whether expression of miR-200c and miR-141 in PCa is related to the DNA methylation status of their promoter. Methods PCR analysis of miR-200c and miR-141, and CpG methylation analysis of their common promoter, was performed in PCa cell-lines and in FFPE prostate biopsy specimens. The functionality of miR-200c and miR-141 expression in prostate cancer cells was assessed by a series of in vitro bioassays. Results miR-200c and miR-141 expression correlates inversely with the methylation status of the miR-200c/miR-141 promoter in PCa cells. In PC3 cells, miR-200c and miR-141 expression is elevated by treatment with the demethylating agents suggesting their expression is linked to methylation. Expression of miR-200c and miR-141 in prostate biopsy tissue was inversely correlated with methylation in CpG sites closest to the miR-200c/miR-141 loci. Over-expression of miR-200c in PC3 cells inhibited growth and clonogenic potential, as well as inducing apoptosis. Expression of the genes DNMT3A and TET1/TET3 were down-regulated by miR-200c and miR-141 respectively. Finally, treatment with the soy isoflavone genistein caused demethylation of the promoter CpG sites closest to the miR-200c/miR-141 loci resulting in increased miR-200c expression. Conclusions Our findings provide evidence that miR-200c and miR-141 are under epigenetic regulation in PCa cells. Profiling their expression and methylation status may have potential in the improved diagnosis and prognosis of PCa., Increasingly accurate surveys of human health throughout the life course has led experts to propose that stresses on the child while still in the mother’s womb can affect the individual’s health much later in life. Such long-term effects on health are thought to be mediated by a semi-permanent trace on the genes of the affected person called an epigenetic mark. Epigenetic mechanisms, such as DNA methylation, are dynamic during pregnancy whereby epigenetic marks are seeded which persist throughout the lifetime of the developing child. It has been suggested that these patterns may be altered by the mother’s diet, particularly folate – a key component in the DNA methylation cycle. Currently, mothers are universally recommended to supplement their diet with 400μg folic acid/day as a preventative measure against neural tube defects in the offspring prior to and during the first trimester. However, there remains no clinical recommendation as to whether mothers should continue supplementation during the final two trimesters and the potentially heritable effects on DNA methylation. Observational studies have suggested that folate-rich maternal diets are associated with changes in DNA methylation of the child during this period of gestation. We present here the results of a randomised control trial (FASSTT study) examining the effects of folic acid supplementation in late gestation (week 12 onwards) on DNA methylation of several gene classes in offspring cord blood samples. We report small but significant sex-specific differences between the two intervention groups. These preliminary results indicate that folic acid supplementation throughout pregnancy may exert significant effects on cord blood DNA methylation., Introduction New-onset diabetes after transplantation (NODAT) is a common complication of kidney transplantation which increases risk of subsequent graft failure, cardiovascular complications and death. NODAT is defined as the new requirement for oral hypoglycaemic agents or insulin as a result of hyperglycaemia after renal transplant. The first genome wide association study (GWAS) for NODAT was published by our group in 2014; seven of the eight top-ranked, common SNPs are implicated in β-cell apoptosis. Methods To further understand the genetic architecture of the NODAT phenotype we used whole exome sequencing for 134renal transplant recipients from a Northern Ireland renal transplant cohort. We sequenced 53 individuals with NODAT (cases)and 81 transplant recipients without NODAT (controls). Library preparation was performed using the Ion TargetSeq™ Exome Kit with samples sequenced on an Ion Torrent Proton sequencer. TheIon OneTouch 2 for emulsion PCR and Ion Enrichment System were used. Association analysis was performed using PLINK Version 1.9 to identify variants associated with NODAT (with age and weight at transplant included in the regression model). Results Following appropriate quality control, initial analysis identified 6 variants nominally associated with NODAT (Ptrend, The Irish Traveller community has a high incidence of autosomal recessive (AR) disorders due to consanguinity. The Division of Molecular Genetics at the DCG offers genetic testing, primarily to members of this community, for five specific pathogenic mutations found in five AR disorders. The pathogenic mutations are detected by bi-directional Sanger sequencing and the service includes: Gene Disorder/ Disease Phenotype LARS (leucyl-tRNA synthetase) Infantile Liver Failure Syndrome 1 (ILFS1) Infantile hepatopathy with failure to thrive (FTT) and developmental delay. MCM4 (minichromosome maintenance 4) Natural Killer Cell & Glucocorticoid Deficiency with DNA Repair Defect (NKGCD) FTT, adrenocorticotropin hormone (ACTH) resistance, familial glucocorticoid deficiency (FGD), mosaic Fanconi anaemia and recurrent infections due to NK cell deficiency. STRA6 (stimulated by retinoic acid 6 gene) Autosomal recessive isolated colobomatous microanopthalmia (MCOPS9) Microphthalmia, anophthalmia, coloboma.Specific STRA6 mutation can also cause the Matthew-Wood syndrome [anophthalmia/ severe microphthalmia, with pulmonary hypoplasia/ aplasia] LEPRE1 (Leucine-and proline-enriched proteoglycan 1)syn. PH31(prolyl-3-hydroxylase-1) Type VIII Osteogenesis Imperfecta, Variable phenoptype of bone fragility, susceptibility to fracture, short stature, bowing of the long bones and can be perinatally lethal. ATP8B1 (ATPase, Class I, Type 8B, Member1) Progressive Familial Intrahepatic Cholestasis type 1 (PFIC1) syn. Byler disease Hepatic and systemic accumulation of bile acids, hepatic fibrosis, end-stage liver disease and growth retardation. This study will detail (1) the service offered to users, (2) an audit of the test requests received over the last two years, (3) the challenges encountered in offering this unique service and (4) some interesting family pedigrees., Background Hypoxia in prostate tumours has been linked with promotion of disease progression and metastasis. miR-210 is a microRNA which is apparently affected by hypoxia, but this relationship has not been extensively studied in a prostate cancer setting. Therefore, in this study, we investigate the link between hypoxia and miR-210 in prostate cancer cells. Methods We have used 2D and 3D cell prostate cell models of hypoxia to investigate the functionality of miR-210. Expression levels of miR-210 have been measured by qPCR and functional bioassays used to examine its effect on prostate cell behaviour. Target genes have been identified and bioinformatic analysis has been employed to investigate a clinical significance for miR-210 in prostate cancer. Results miR-210 is induced by hypoxia in prostate cancer cell-lines. Over-expression of miR-210 impacts upon target genes, including SP1 and TPD52, which in turns affects cell proliferation. Data-mining of online repositories of clinical data and bioinformatic analysis of miR-210 cellular networks reveal that miR-210 plays a key role in a number of important cell processes, the dysregulation of which can lead to development of prostate cancer. Conclusions We propose that miR-210 could be an important microRNA in the pathogenesis of prostate cancer and has potential as a biomarker in this disease.

Background Diffuse large B-cell lymphoma (DLBCL) is the most common aggressive non-Hodgkin lymphoma subtype with a 5-year survival of ~64%. While DLBCL is treated using immunochemotherapy (IC) with curative intent, 20%-40% of patients do not reach remission or relapse post IC. Clonal somatic mutations have been associated with aging, hematologic malignancies (predominately myeloid), and reduced OS in the general population. The objective of this study was to evaluate the association of clonal somatic mutations with event-free survival (EFS) and overall survival (OS). Methods We studied newly diagnosed DLBCL cases treated with IC who were enrolled in the Molecular Epidemiology Resource (MER) of the University of Iowa/Mayo Clinic Lymphoma Specialized Program of Research Excellence (SPORE). Clinical and treatment data were abstracted from medical records and all patients were systematically followed for disease outcomes. Pre-treatment DNA was extracted from matched peripheral blood and paraffin-embedded tumor tissue, and whole exome sequencing was conducted at 100x coverage. From the peripheral blood, allele counts from GVCF files produced by HaplotypeCaller in GATK were extracted for 42 genes commonly associated with clonal hematopoiesis (e.g. DNMT3A, TET2, and ASXL1). Mutations were deemed clonal somatic if the population minor allele frequency was Results The study consisted of 261 DLBCL patients treated with IC. The median age at diagnosis was 65 years (range 20-90) and 56% were male. With a median follow-up time of 5.1 years (range 0.1-15.0), there were 100 events and 80 deaths. A total of 17 (6.5%) patients had clonal somatic mutations, and16 patients were over the age of 60. A total of 8 (of 42) genes had clonal somatic mutations, with SF3B1, ASXL1, and TET2, being the most frequent (4 individuals per gene). VAFs ranged from 0.10 to 0.28 and none of the patients had multiple mutations. Additionally, the clonal somatic variants found in the peripheral blood were abscent in the tumor sample. Of the 17 patients with clonal somatic mutations, 12 had an event while 88 patients without a mutation had an event. In a univariate analysis, clonal somatic mutations were associated with inferior EFS (HR=2.55, 95% CI 1.39-4.68, p=0.002; Figure 1A). After adjusting for age, sex, and IPI, clonal somatic mutations remained associated with inferior EFS (HR=2.02, 95% CI 1.09-3.74, p=0.026). Clonal somatic mutations were also associated with inferior OS in the univariate analysis (HR=2.06, 95% CI 0.99-4.29, p=0.053), which attenuated after multivariate adjustment (HR=1.59, 95% CI: 0.76-3.34, p=0.22, Figure 1B). Although based on small numbers, mutations in SF3B1 were associated with inferior EFS (HR=3.25, 95% CI 1.16-9.12, p=0.025), but did not reach significance for OS (HR=2.56, 95% CI 0.78-8.38, p=0.120). Conclusions In this novel study of newly diagnosed DLBCL patients, clonal somatic mutations were identified in 6.5% of patients and were associated with inferior outcomes. Additional research is required at deeper sequencing to validate these findings and integration with tumor genomics is required to understand the prognosis of DLBCL patients with smaller clonal populations. Disclosures Shah: Dren Bio: Consultancy. Maurer:Celgene / BMS: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees; Morphosys: Membership on an entity's Board of Directors or advisory committees; Kite: Membership on an entity's Board of Directors or advisory committees; Nanostring: Research Funding. Martin:Celgene: Consultancy; Karyopharm: Consultancy, Research Funding; Morphosys: Consultancy; Regeneron: Consultancy; Incyte: Consultancy; Kite: Consultancy; Cellectar: Consultancy; Bayer: Consultancy; Beigene: Consultancy; Sandoz: Consultancy; I-MAB: Consultancy; Janssen: Consultancy; Teneobio: Consultancy. Witzig:AbbVie: Consultancy; Incyte: Consultancy; Acerta: Research Funding; Karyopharm Therapeutics: Research Funding; Immune Design: Research Funding; Spectrum: Consultancy; Celgene: Consultancy, Research Funding; MorphSys: Consultancy. Nowakowski:NanoString: Research Funding; Seattle Genetics: Consultancy; Curis: Consultancy; Kymera: Consultancy; Kite: Consultancy; Ryvu: Consultancy, Membership on an entity's Board of Directors or advisory committees; MorphoSys: Consultancy, Research Funding; Celgene/BMS: Consultancy, Research Funding. Novak:Celgene/BMS: Research Funding. Cerhan:NanoString: Research Funding; BMS/Celgene: Research Funding.

Lipedematous alopecia (LA) and lipedematous scalp (LS) are 2 rare conditions of unknown etiology that are thought by some to be 2 stages on a spectrum of a single scalp disorder.1 LA was first described by Coskey et al2 in 1961, and in 2007, the term spongy scalp was used to describe LA and LS.3 Both LA and LS conditions are characterized by a localized or diffusely thickened, boggy scalp, but LS lacks alopecia. Associated pruritus and soreness can occur with LA, but most LS patients are asymptomatic.4 Both conditions are characterized pathologically by a measurable hyperplasia of the subcutaneous tissue.5

Background: It is frequently assumed that pre-invasive lesions are simpler precursors of cancer, and will contain a limited subset of the genomic changes seen in their associated invasive disease. Driver mutations are thought to occur early, but it is not known how many of these are present in pre-invasive lesions. These assumptions need to be tested with the increasing focus on both personalised cancer treatments, and early detection methodologies. Methods: We examined genomic copy number changes in 256 pre-invasive and invasive samples from 69 oral cancer patients. Forty-eight samples from 16 patients were further examined using exome sequencing. Results: Evidence of a shared ancestor of both dysplasia and carcinoma was seen in all but one patient. One third of dysplasias showed independent copy number events. The remainder had a similar or simpler copy number pattern to the carcinoma. All dysplasias examined contained somatic mutations absent in the related carcinoma. Previously observed copy number changes and TP53 mutations were very frequently observed, and almost always shared between dysplasia and carcinoma. Other gene changes were more sporadic. Pathway analysis confirmed that each patient’s disease developed in a different way. Examining the numbers of shared mutations, and the rate of accumulation of mutations showed evidence that all samples contain a population of sub-clones, with little evidence of selective advantage of a subset of these. Conclusions: These findings suggest that most of the genomic changes driving oral cancer occur in the pre-cancerous state by way of gradual random accumulation rather than a dramatic single event.

Background Indigo naturalis is a Traditional Chinese Medicine (TCM) ingredient long-recognized as a therapy for several inflammatory conditions, including psoriasis. However, its mechanism is unknown due to lack of knowledge about the responsible chemical entity. We took a different approach to this challenge by investigating the molecular profile of Indigo naturalis treatment and impacted pathways. Methods A randomized, double-blind, placebo-controlled clinical study was conducted using Indigo naturalis as topical monotherapy to treat moderate plaque psoriasis in a Chinese cohort (n = 24). Patients were treated with Indigo naturalis ointment (n = 16) or matched placebo (n = 8) twice daily for 8 weeks, with 1 week of follow-up. Results At week 8, significant improvements in Psoriasis Area and Severity Index (PASI) scores from baseline were observed in Indigo naturalis-treated patients (56.3% had 75% improvement [PASI 75] response) compared with placebo (0.0%). A gene expression signature of moderate psoriasis was established from baseline skin biopsies, which included the up-regulation of the interleukin (IL)-17 pathway as a key component; Indigo naturalis treatment resulted in most of these signature genes returning toward normal, including down-regulation of the IL-17 pathway. Using an in vitro keratinocyte assay, an IL-17-inhibitory effect was observed for tryptanthrin, a component of Indigo naturalis. Conclusions This study demonstrated the clinical efficacy of Indigo naturalis in moderate psoriasis, and exemplified a novel experimental medicine approach to understand TCM targeting mechanisms. Trial registration NCT01901705. Electronic supplementary material The online version of this article (10.1186/s12906-017-1947-1) contains supplementary material, which is available to authorized users.

Background: DLBCL has traditionally been classified by cell of origin (COO) subcategories based on tumor gene expression profiles which include Activated B-Cell (ABC) and Germinal Center B-Cell (GCB). Recently, using tumor samples from patients treated with RCHOP, new classification models have focused on DNA alterations. However, a comprehensive integrative approach using a large transcriptomic data set across both newly diagnosed (nd) and relapsed/refractory (r/r) DLBCL is yet to be accomplished. A robust clustering of this type will allow for identification of biologically driven DLBCL patient subgroups and may predict patient outcome and inform treatment approaches. Methods: RNAseq was performed on a total of 882 DLBCL tumor FFPE biopsies from 2 ndDLBCL cohorts (cohort 1 and 2) and 2 r/r DLBCL cohorts (cohorts 3 and 4). Cohort 1 (N=267) was commercially sourced and served as the discovery cohort. Cohort 2 (N=340) was from the Mayo/Iowa Lymphoma SPORE Molecular Epidemiology Resource (MER) and served as the replication cohort. Cohort 3 (N=189) was from the CC-122-ST-001 and CC-122-DLBCL-001 clinical trials (NCT01421524 and NCT02031419), and cohort 4 (N=86) was from r/r patients from the MER. Clustering input consisted of gene expression data, gene set variation analysis (GSVA) scores computed from the hallmark gene sets of MSigDB gene sets, as well as immune cell abundance estimates from a DLBCL-specific deconvolution method. An integrative clustering method iClusterPlus was applied to the input data to identify patient subgroups. A multinomial generalized linear model classifier was trained on the discovery dataset and applied to cohorts 2, 3, and 4 to assess patterns of gene expression and clinical features among the subgroups. Results: Integrative clustering identified 8 subgroups of ndDLBCL patients (DLBCL1-8; D1-D8) in cohort 1. Classifiers trained on cohort 1 were applied to cohort 2 and the same 8 clusters were identified. Among RCHOP treated patients in cohort 2, subgroups D4 (p Within D4, 92% of patients were ABC, representing a high risk subset of ABC patients. The mEFS in D4 ABCs was 38.2 months, while mEFS of non-D4 ABCs was not reached (p2, compared to 33% of non-D4 with IPI>2 (p D8 represented a high-risk subset which was 73% GCB. The mEFS of D8 GCBs was 5.4 months, while mEFS of non-D8 GCBs was not reached (p In the r/r DLBCL setting, D1-D8 were all present, with an increased prevalence of D4 and D8 in Cohort 3 (30% and 17%, respectively) and Cohort 4 (30% and 14%) compared to the newly diagnosed setting. Mutational data for these cohorts has been collected and is being interpreted in the context of the discovered subgroups. Conclusion: A novel integrative clustering of transformed gene expression data revealed 8 biologically homogeneous groups, two of which had inferior outcomes when treated with RCHOP therapy. Furthermore, these two subgroups were more prevalent in r/r DLBCL. This classification allows for the transcriptomic identification of high-risk patients underserved by RCHOP therapy. *Ortiz, Wenzl and Stokes contributed equally **Gandhi and Novak contributed equally Disclosures Ortiz: Celgene Corporation: Employment, Equity Ownership. Stokes:Celgene Corporation: Employment, Equity Ownership. Huang:Celgene Corporation: Employment, Equity Ownership. Maurer:Celgene: Research Funding; Morphosys: Membership on an entity's Board of Directors or advisory committees; Nanostring: Research Funding. Towfic:Celgene Corporation: Employment, Equity Ownership. Hagner:Celgene Corporation: Employment, Equity Ownership, Patents & Royalties. Ratushny:Celgene Corporation: Employment, Equity Ownership. McConnell:Celgene Corporation: Employment, Equity Ownership. Danziger:Celgene Corporation: Employment, Equity Ownership. Stong:Celgene Corporation: Employment, Equity Ownership. Lata:Celgene Corporation: Employment, Equity Ownership. Kamalakaran:Celgene Corporation: Employment, Equity Ownership. Mavrommatis:Celgene Corporation: Employment, Equity Ownership. Trotter:Celgene Corporation: Employment, Equity Ownership. Czuczman:Celgene Corporation: Employment, Equity Ownership. Ansell:Seattle Genetics: Research Funding; Affimed: Research Funding; Regeneron: Research Funding; Trillium: Research Funding; Mayo Clinic Rochester: Employment; Seattle Genetics: Research Funding; Mayo Clinic Rochester: Employment; Regeneron: Research Funding; Trillium: Research Funding; Bristol-Myers Squibb: Research Funding; Bristol-Myers Squibb: Research Funding; LAM Therapeutics: Research Funding; LAM Therapeutics: Research Funding; Affimed: Research Funding. Cerhan:NanoString: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding. Nowakowski:Genentech, Inc.: Research Funding; F. Hoffmann-La Roche Ltd: Research Funding; Curis: Research Funding; Bayer: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Selvita: Membership on an entity's Board of Directors or advisory committees; NanoString: Research Funding; MorphoSys: Consultancy, Research Funding. Gandhi:Celgene Corporation: Employment, Equity Ownership, Patents & Royalties. Novak:Celgene Coorperation: Research Funding.

Background: Diffuse large B-cell lymphoma (DLBCL) is a genetically heterogeneous disease of malignant B cells most often classified by tumor gene expression and/or mutations. DLBCL is also characterized by a tumor microenvironmental influence that has not been well described. Immuno-oncology-targeting agents such as immune checkpoint inhibitors have limited clinical activity in DLBCL, highlighting the need for a better understanding of the DLBCL tumor microenvironment for rational drug development, combinations, and disease stratification. Here, we systematically characterize the immune composition of more than 100 DLBCL tumors using 2 imaging technologies and provide insight into the complexity of DLBCL disease biology. Methods: A total of 110 cases of newly diagnosed DLBCL were analyzed by multiplex immunohistochemistry (IHC; n=70) and multiplexed ion beam imaging (MIBI) (n=40), each with 10 common markers (CD20, CD3, CD8, Foxp3, CD56, CD163, CD11c, CD56, PD-1, PD-L1) and a few platform-specific markers. Both IHC and MIBI images were digitalized to generate marker-positive cell counts (including single, double, or triple positivity), cell density (cell count/mm2), and population fraction (% of total nucleated cells). Marker density was analyzed for the major components of tumor-infiltrating cell types and correlation between any pair of markers. In addition, RNAseq data and fluorescence in situ hybridization (FISH) data on MYC, BCL-2, and BCL-6 translocation were generated. Results: In the IHC cohort, T cells (CD3+), dendritic cells (DCs; by CD11c+), and macrophages (CD163+)were the major immune components, with median population fractions of 22%, 16%, and 2.7%, respectively. Natural killer cells (CD56+CD20−) were a minor component at a median of 0.1%. A significant negative correlation was observed between tumor cells (CD20+) and CD4+ (Spearman ρ = −0.47; P = 1.3 × 10−04), and CD8+ T cells (Spearman ρ = −0.42; P = 1.4 × 10−03) cells, and an unexpectedly negative correlation between DCs (CD11c+) and macrophages (CD163+, r = −0.63; P = 9.8 × 10−06) was found. Similar to follicular lymphoma, 2 PD-1+ T-cell populations were identified: PD-1bright and PD-1dim. The PD-1bright cells co-stained with CXCR5, indicating T follicular helper cells (Tfh). The PD-1dim were expressed on exhausted effector cells that co-stained with Tim3 or Lag3. The median population fraction of Tim3+ or Lag3+ among T cells was 25%, whereas that of PD-1dim among T cells was 0.2%, indicating that PD-1 was not a useful marker for exhausted T cells. PD-L1 was predominantly found on DCs and macrophages, and the median population fraction of PD-L1+ among tumor cells was only 6.2%. By unsupervised hierarchical clustering on marker density, 3 major immune-infiltration patterns (P1, P2, and P3) were identified. The first 2 segments (P1 and P2) were 20% and 25% of the total cases, respectively. Both were characterized by high T-cell, macrophage, and DC infiltration. P1 was enriched for PD-1+ T cells, whereas P2 was void of any PD-1+ cells. The third segment (P3) that comprised 55% of the cases was predominantly tumor cells with low T-cell, macrophage, and DC infiltration. PD-L1+ cells were primarily found in segments P1 and P2 but rare in segment P3. Additional analysis on the associations between the 3 immune-infiltration patterns and prognostic DLBCL molecular features such as cell-of-origin, double-hit gene signature, and double-hit FISH will also be presented. Conclusions: These data show the complexity of DLBCL disease biology and show classification of DLBCL at the immune-infiltration level as 3 distinct patterns. The overall low expression of PD-1+ T cells and the restricted pattern of PD-L1+ tumor cells provide a possible explanation for the lack of clinical activity of PD-1/PD-L1 blockade in DLBCL. We also observed other exhausted T cells expressing Lag3 and Tim3, suggesting alternative therapeutic opportunities in stratified populations. These data also highlight the opportunity to develop rational immuno-oncology-targeted agents based on the immune infiltration pattern of DLBCL and selection of patients who may respond more favorably to particular agents. Disclosures Huang: Celgene Corporation: Employment, Equity Ownership. Nakayama:Celgene Corporation: Employment, Equity Ownership. Stokes:Celgene Corporation: Employment, Equity Ownership. Towfic:Celgene Corporation: Employment, Equity Ownership. Lee:Celgene Corporation: Employment, Equity Ownership. Ren:Celgene Corporation: Employment, Equity Ownership. Marella:Celgene Corporation: Employment, Equity Ownership. Wang:Celgene Corporation: Employment, Equity Ownership. Hagner:Celgene Corporation: Employment, Equity Ownership, Patents & Royalties. Couto:Celgene Corporation: Employment, Equity Ownership, Patents & Royalties. Newhall:Celgene Corporation: Employment, Equity Ownership. Gandhi:Celgene Corporation: Employment, Equity Ownership, Patents & Royalties.

Background: Diffuse large B cell lymphoma (DLBCL) is the most common aggressive lymphoid malignancy in adults. Though standard immunochemotherapy regimens can result in clinical remission and cure in a majority of patients, approximately 30% of patients are primary refractory or eventually relapse, and their prognosis is very poor. Recent progress by large scale genomic and transcriptomic profiling of DLBCL patients has resulted in a deep understanding of disease drivers in newly diagnosed DLBCL (ndDLBCL). However, previous genomic studies of relapsed and/or refractory DLBCL (rrDLBCL) are limited by small sample sizes and much less is known about the genomic landscape or the changes in clonal populations that occur within a patient undergoing R-CHOP therapy. Analysis of a large cohort of rrDLBCL is needed to uncover the importance of known drivers, define clonal shifts that occur in relapsing patients, and discover novel mutations that impact resistance to therapy. Methods: DNA sequencing (whole exome or genome sequencing) was performed on tumor and matching germline samples from two rrDLBCL patient cohorts (N=143). The first rrDLBCL cohort consisted of 68 formalin fixed paraffin embedded (FFPE) tumor samples with matched germline obtained from the Mayo/Iowa Lymphoma SPORE Molecular Epidemiology Resource (MER); including 18 diagnostic/relapse paired samples. The second cohort consisted of 75 FFPE samples from 2 rrDLBCL clinical trials CC-122-ST-001 and CC-122-DLBCL-001 (NCT01421524 and NCT02031419). In addition, a ndDLBCL cohort (n=364) consisting of MER front line samples were also analyzed. CNV and SV calling was performed with Sclust and Manta, respectively. Mutational drivers were identified with cDriver. Cosmic mutational signatures were calculated for all samples. Results: For comparison of overall mutation rates between the ndDLBCL and rrDLBCL we analyzed the frequency of mutations in known driver genes [n=211, Chapuy et al, 2018; Schmitz et al, 2018; Reddy et al 2017]. Of these, the top 5 most mutated genes found in the relapse cohort were KMT2D (34.3%), IGLL5 (25.9%), CREBBP (22.4%), TP53 (21.0%), and HIST1H1E (19.6%) all of which were previously reported as mutated genes in rrDLBCL [Morin et al. 2016; Park et al. 2016;Greenawalt et al. 2017]. None of these frequencies changed significantly from the MER ndDLBCL cohort. However, frequencies of 2 genes NCOR1 and ACTB were increased significantly from 3.3% to 13.3% (p= 5.85 x 10-05) and 0.5% to 6.3% (p=2.56 x 10-04), respectively, in the rrDLBCL cohorts. Analysis of the previously reported driver mutations in 18 paired samples revealed a significant increase in the mean variant allele frequency (VAF) from diagnosis to relapse of mutations in BCL2 and BIRC6 from 18.5% to 28.8%(p=0.016) and 4.3% to 13.7%(p=0.011), respectively. In our analysis of novel drivers in rrDLBCL cohorts, 59 significant driver mutations (FDR 0.1) were identified, 23 were novel driver mutations while the remaining 36 have been previously reported as drivers of newly diagnosed DLBCL. These include novel mutations in genes such as IL4R (8.8%), CDK5R1 (6.7%), LAPTM5 (3.7%) and others whose function in lymphoma are unknown. In a secondary analysis, we performed mutational signature analysis for rrDLBCL and identified groups of patients with COSMIC mutational signatures that are associated with aging, defects in homologous end joining resulting in large insertions/deletions, and with defective mismatch repair often found in microsatellite unstable tumors. Together with the high prevalence of TP53 mutations, these results indicate genetic instability as a hallmark of relapse/refractory DLBCL. Analysis of CNV and SV will also be reported. Conclusion: Here we report to our knowledge the largest genomic dataset in relapsed and /or refractory DLBCL using tumor biopsies. Previously identified mutated genes in rrDLBCL were confirmed and novel drivers identified. Paired samples analysis reveals clonal shifts which occur during treatment and at time of relapse. Whether the novel driver mutations appeared as a function of selection of a rare clone over the course of therapy or a therapy induced mutation will be the focus of ongoing work. Novel variants including SNV, CNV and SV can potentially lead to new insights and deepen our understanding of the mechanisms of resistance in DLBCL. * Wenzl, Ortiz and Stong contributed equally **Novak and Gandhi contributed equally Disclosures Ortiz: Celgene Corporation: Employment, Equity Ownership. Stong:Celgene Corporation: Employment, Equity Ownership. Huang:Celgene Corporation: Employment, Equity Ownership. Maurer:Morphosys: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Nanostring: Research Funding. Towfic:Celgene Corporation: Employment, Equity Ownership. Hagner:Celgene Corporation: Employment, Equity Ownership, Patents & Royalties. Nikbakht:Celgene Corporation: Employment, Equity Ownership. Stokes:Celgene Corporation: Employment, Equity Ownership. Lata:Celgene Corporation: Employment, Equity Ownership. Kamalakaran:Celgene Corporation: Employment, Equity Ownership. Mavrommatis:Celgene Corporation: Employment, Equity Ownership. Trotter:Celgene Corporation: Employment, Equity Ownership. Czuczman:Celgene Corporation: Employment, Equity Ownership. Ansell:Regeneron: Research Funding; LAM Therapeutics: Research Funding; Trillium: Research Funding; Affimed: Research Funding; Bristol-Myers Squibb: Research Funding; Mayo Clinic Rochester: Employment; Affimed: Research Funding; Seattle Genetics: Research Funding; LAM Therapeutics: Research Funding; Mayo Clinic Rochester: Employment; Bristol-Myers Squibb: Research Funding; Trillium: Research Funding; Regeneron: Research Funding; Seattle Genetics: Research Funding. Cerhan:Celgene: Research Funding; NanoString: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees. Nowakowski:Bayer: Consultancy, Research Funding; Curis: Research Funding; Celgene: Consultancy, Research Funding; MorphoSys: Consultancy, Research Funding; Genentech, Inc.: Research Funding; F. Hoffmann-La Roche Ltd: Research Funding; Selvita: Membership on an entity's Board of Directors or advisory committees; NanoString: Research Funding. Novak:Celgene Coorperation: Research Funding. Gandhi:Celgene Corporation: Employment, Equity Ownership, Patents & Royalties.

To assess the feasibility of panitumumab in real-time fluorescent imaging and histologic processing of cutaneous squamous cell carcinoma (cSCC) in mice.A near-infrared (NIR) fluorescent probe (IRDye800CW) was covalently linked to a monoclonal antibody-targeting epidermal growth factor receptor (panitumumab) or nonspecific IgG and injected into mice bearing flank xenografts from a cSCC cell line (SCC-13 or SRB-12; n = 7), human split-thickness skin grafts (STSGs; n = 3), or a human tumor explant (n = 1). The tumor and lymph nodes were imaged and dissected using fluorescence guidance with the SPY imaging system and verified with a charge-coupled NIR system. An NIR scanning device (Odyssey) was used to measure fluorescence intensity in histological sections.Immunodeficient mice.In vivo and in vitro imaging lab.Tumor tissue could be delineated from the human STSG with tumor-to-background ratios of 4.5 (Pearl) and 3.4 (SPY). Tumor detection was substantially improved with panitumumab-IRDye800 compared with IgG-IRDye800. Biopsies positive for fluorescence were assessed by histology and immunohistochemistry (n = 18/18) to confirm the presence of tumor, yielding a 100% sensitivity. Biopsies of nonfluorescent tissue negative for malignancy (n = 18/18) yielded a specificity of 100%. Furthermore, the SPY system was able to detect residual disease as small as 200 µm in diameter. In addition, the Odyssey confirmed fluorescence of microscopic disease (in tumor samples of frozen and paraffin-embedded histologic specimens) but not in adjacent noncancerous tissue.These data suggest panitumumab-IRDye800 may have clinical utility in detection and removal of subclinical cSCC using Food and Drug Administration-approved imaging hardware.

Background An individual patient’s response to a particular drug is influenced by multiple factors, which may include genetic predisposition. Pharmacogenetic studies attempt to discover and estimate the contributions of genetic variants to the variability in response to a drug treatment. The task of identifying the genetic contribution is often complicated by response phenotypes that are based on imprecise or subjective clinical observations. Because the success of a pharmacogenetic study depends on the analysis of a heritable phenotype, it is important to identify phenotypes with a significant heritable component to ensure reliable and reproducible results in subsequent genetic association studies. Methods We retrospectively analyzed data collected from 436 rheumatoid arthritis patients treated with golimumab during the phase III GO-FURTHER study. We investigated the reliability of several potential response outcomes after golimumab treatment. Using whole-genome sequencing of the clinical trial cohort, we estimated the heritability of each potential outcome measure. We further performed a longitudinal analysis of the clinical data to estimate variability of outcome measures over time and the degree to which each response metric could be confounded by placebo response. Results We determined that the high degree of within-patient variation over time makes a single follow-up visit insufficient to assess an individual patient’s response to golimumab treatment. We found that different potential response outcomes had varying degrees of heritability and that averaging across multiple follow-up visits yielded higher heritability estimates than single follow-up estimates. Importantly, we found that the change in swollen and tender joint counts were the most heritable outcome metrics we tested; however, we showed that they are also more likely to be confounded by a placebo response than objective phenotypes like the change in C-reactive protein levels. Conclusions Our rigorous approach to finding robust and heritable response phenotypes could be beneficial to all pharmacogenetic studies and may lead to more reliable and reproducible results. Trial Registration Clinicaltrials.gov NCT00973479. Registered 4 September 2009. Electronic supplementary material The online version of this article (doi:10.1186/s13075-017-1299-8) contains supplementary material, which is available to authorized users.

Background Although genome-wide association studies (GWAS) have identified over 100 genetic loci associated with rheumatoid arthritis (RA), our ability to translate these results into disease understanding and novel therapeutics is limited. Most RA GWAS loci reside outside of protein-coding regions and likely affect distal transcriptional enhancers. Furthermore, GWAS do not identify the cell types where the associated causal gene functions. Thus, mapping the transcriptional regulatory roles of GWAS hits and the relevant cell types will lead to better understanding of RA pathogenesis. Results We combine the whole-genome sequences and blood transcription profiles of 377 RA patients and identify over 6000 unique genes with expression quantitative trait loci (eQTLs). We demonstrate the quality of the identified eQTLs through comparison to non-RA individuals. We integrate the eQTLs with immune cell epigenome maps, RA GWAS risk loci, and adjustment for linkage disequilibrium to propose target genes of immune cell enhancers that overlap RA risk loci. We examine 20 immune cell epigenomes and perform a focused analysis on primary monocytes, B cells, and T cells. Conclusions We highlight cell-specific gene associations with relevance to RA pathogenesis including the identification of FCGR2B in B cells as possessing both intragenic and enhancer regulatory GWAS hits. We show that our RA patient cohort derived eQTL network is more informative for studying RA than that from a healthy cohort. While not experimentally validated here, the reported eQTLs and cell type-specific RA risk associations can prioritize future experiments with the goal of elucidating the regulatory mechanisms behind genetic risk associations. Electronic supplementary material The online version of this article (doi:10.1186/s13059-016-0948-6) contains supplementary material, which is available to authorized users.

Several small studies suggest that the presence of the human leukocyte antigen (HLA)-Cw6 (C*06:02) allele may be a predictor of improved response to ustekinumab. This study was designed to assess the association of the HLA-C*06:02 allele with response to ustekinumab in large cohorts of patients from the phase 3 studies of ustekinumab in moderate-to-severe psoriasis. In this retrospective study, both HLA-C*06:02-positive and -negative patients demonstrated good responses to ustekinumab (86% vs. 76%, respectively, achieved at least a 75% improvement from baseline in Psoriasis Area and Severity Index [PASI 75] at week 24). A modestly higher proportion of HLA-C*06:02-positive than HLA-C*06:02-negative patients achieved PASI 75/90 responses at weeks 12 and 24. The largest response difference between the positive and negative patients (17.9%) was observed for PASI 75 (week 12), with smaller differences noted at later time points for PASI 90 (11.8% at week 24) and PASI 100 (10.2% at week 28). A differential response to ustekinumab has been confirmed in HLA-C*06:02-positive versus HLA-C*06:02-negative patients; however, this difference is modest, particularly at the higher response rate thresholds (PASI 90/100) and later time points (weeks 24/28).

Organ transplant recipients (OTRs) develop multiple aggressive and metastatic non-melanoma skin cancers (NMSCs). Yet, the underlying mechanism remains elusive. Employing a variety of immune-compromised murine models, immunoblotting, immunohistochemical and immunofluorescence techniques, we show that human squamous xenograft tumors in nude mice grow faster and become significantly larger in size following treatment with the immunosuppressive drug, cyclosporine A (CsA). Re-injected tumor cells isolated from CsA-treated xenografts continued to form larger tumors in nude mice than those from vehicle-controls and retained the CsA-signatures of calcineurin signaling inhibition. Similar results were obtained when these tumors were grown in SCID-beige mice or in immuno-competent mice inoculated with syngeinic tumor cells. Consistently, tumors in the CsA group manifested enhanced cellular proliferation and decreased apoptosis. Tumors in CsA-treated animals also showed an augmented epithelial-mesenchymal transition (EMT) characterized by an increased expression of fibronectin, α-SMA, vimentin, N-cadherin, MMP-9/-2, snail and twist with a concomitant decrease in E-cadherin. CsA-treated xenograft tumors manifested increased TGFβ1 expression and TGFβ-dependent signaling characterized by increased nuclear p-Smad 2/3. Our data demonstrate that CsA alters the phenotype of skin SCCs to an invasive and aggressive tumor-type by enhancing expression of proteins regulating EMT acting through the TGFβ1 signaling pathway providing at least one unique mechanism by which multiple aggressive and metastatic NMSCs develop in OTRs.

We have created a stand-alone software tool, ConsensusCluster, for the analysis of high-dimensional single nucleotide polymorphism (SNP) and gene expression microarray data. Our software implements the consensus clustering algorithm and principal component analysis to stratify the data into a given number of robust clusters. The robustness is achieved by combining clustering results from data and sample resampling as well as by averaging over various algorithms and parameter settings to achieve accurate, stable clustering results. We have implemented several different clustering algorithms in the software, including K-Means, Partition Around Medoids, Self-Organizing Map, and Hierarchical clustering methods. After clustering the data, ConsensusCluster generates a consensus matrix heatmap to give a useful visual representation of cluster membership, and automatically generates a log of selected features that distinguish each pair of clusters. ConsensusCluster gives more robust and more reliable clusters than common software packages and, therefore, is a powerful unsupervised learning tool that finds hidden patterns in data that might shed light on its biological interpretation. This software is free and available from http://code.google.com/p/consensus-cluster .

This teacher education self-study examined music teacher and music teacher educator development through the formal and informal interactions of undergraduate and doctoral students designed to facilitate community. Research questions included: (a) how do undergraduate students describe their interactions with music education PhD students; (b) how do music education PhD students describe their interactions with music education undergraduate students; and (c) how can the researchers change their practices to better meet the needs of the undergraduate students? Data included: a questionnaire from sophomore, junior and senior undergraduate students (N = 34); PhD student journals; a teacher education faculty journal; 12 undergraduate student interviews; an undergraduate student focus group; and six "self study" team focus group meetings. Findings suggest that: (a) interactions were positive but it was sometimes difficult to navigate the interactions; (b) undergraduates specifically value the stories of recent teaching experience that the PhD students can provide; and (c) undergraduates seem to change their views of the PhD students as they (the undergraduates) mature as teachers. Other findings address development of the PhD students as teacher educators. Suggestions for teacher education and research conclude the paper.

Studies performed to discover genes overexpressed in inflammatory diseases identified dermokine as being upregulated in such disease conditions. Dermokine is a gene that was first observed as expressed in the differentiated layers of skin. Its two major isoforms, alpha and beta, are transcribed from different promoters of the same locus, with the alpha isoform representing the C terminus of the beta isoform. Recently, additional transcript variants have been identified. Extensive in silico analysis and reverse transcriptase (RT)-PCR cloning has confirmed the existence of these variants in human cells and tissues, identified a new human isoform as well as the gamma isoform in mouse. Recombinant expression and analysis of the C-terminal truncated isoform indicate that the molecule is O-linked glycosylated and forms multimers in solution. In situ hybridization and immunohistochemistry has shown that the gene is differentially expressed in various cells and tissues, other than the skin. These results show that the dermokine gene is expressed in epithelial tissues other than the skin and this expression is transcriptionally and posttranscriptionally complex.

SummaryConstitutive activation of MEK-ERK signaling is often found in melanomas. Here, we identify a mechanism that links ERK with JNK signaling in human melanoma. Constitutively active ERK increases c-Jun transcription and stability, which are mediated by CREB and GSK3, respectively. Subsequently, c-Jun increases transcription of target genes, including RACK1, an adaptor protein that enables PKC to phosphorylate and enhance JNK activity, enforcing a feed-forward mechanism of the JNK-Jun pathway. Activated c-Jun is also responsible for elevated cyclin D1 expression, which is frequently overexpressed in human melanoma. Our data reveal that, in human melanoma, the rewired ERK signaling pathway upregulates JNK and activates the c-Jun oncogene and its downstream targets, including RACK1 and cyclin D1.

Verrucous carcinoma of the oral cavity (OVC) is considered a subtype of classical oral squamous cell carcinoma (OSCC). Diagnosis is problematic, and additional biomarkers are needed to better stratify patients. To investigate their molecular signature, we performed low coverage copy number sequencing on 57 OVC and exome and RNA sequencing on a subset of these and compared the data to the same OSCC parameters. Copy number results showed that OVC lacked any of the classical OSCC patterns such as gain of 3q and loss of 3p and demonstrated considerably fewer genomic rearrangements compared to the OSCC cohort. OVC and OSCC samples could be clearly differentiated. Exome sequencing showed that OVC samples lacked mutations in genes commonly associated with OSCC (TP53, NOTCH1, NOTCH2, CDKN2A and FAT1). RNA sequencing identified genes that were differentially expressed between the groups. In silico functional analysis showed that the mutated and differentially expressed genes in OVC samples were involved in cell adhesion and keratinocyte proliferation, while those in the OSCC cohort were enriched for cell death and apoptosis pathways. This is the largest and most detailed genomic and transcriptomic analysis yet performed on this tumour type, which, as an example of non-metastatic cancer, may shed light on the nature of metastases. These three independent investigations consistently show substantial differences between the cohorts. Taken together they lead to the conclusion that OVC is not a subtype of OSCC, but should be classified as a distinct entity.

The study of the relationships between pre-cancer and cancer and identification of early driver mutations is becoming increasingly important as the value of molecular markers of early disease and personalised drug targets is recognized, especially now the extent of clonal heterogeneity in fully invasive disease is being realized. It has been assumed that pre-cancerous lesions exhibit a fairly passive progression to invasive disease; the degree to which they, too, are heterogeneous is unknown. We performed ultra-deep sequencing of thousands of selected mutations, together with copy number analysis, from multiple, matched pre-invasive lesions, primary tumours and metastases from five patients with oral cancer, some with multiple primary tumours presenting either synchronously or metachronously, totalling 75 samples. This allowed the clonal relationships between the samples to be observed for each patient. We expose for the first time the unexpected variety and complexity of the relationships between this group of oral dysplasias and their associated carcinomas and, ultimately, the diversity of processes by which tumours are initiated, spread and metastasize. Instead of a series of genomic precursors of their adjacent invasive disease, we have shown dysplasia to be a distinct dynamic entity, refuting the belief that pre-cancer and invasive tumours with a close spatial relationship always have linearly related genomes. We show that oral pre-cancer exhibits considerable subclonal heterogeneity in its own right, that mutational changes in pre-cancer do not predict the onset of invasion, and that the genomic pathway to invasion is neither unified nor predictable. Sequence data from this study have been deposited in the European Nucleotide Archive, Accession No. PRJEB6588. Copyright (c) 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.