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Congenital diaphragmatic hernia (CDH) can occur in isolation or in conjunction with other birth defects (CDH+). A molecular etiology can only be identified in a subset of CDH cases. This is due, in part, to an incomplete understanding of the genes that contribute to diaphragm development. Here, we used clinical and molecular data from 36 individuals with CDH+ who are cataloged in the DECIPHER database to identify genes that may play a role in diaphragm development and to discover new phenotypic expansions. Among this group, we identified individuals who carried putatively deleterious sequence or copy number variants affecting CREBBP, SMARCA4, UBA2, and USP9X. The role of these genes in diaphragm development was supported by their expression in the developing mouse diaphragm, their similarity to known CDH genes using data from a previously published and validated machine learning algorithm, and/or the presence of CDH in other individuals with their associated genetic disorders. Our results demonstrate how data from DECIPHER, and other public databases, can be used to identify new phenotypic expansions and suggest that CREBBP, SMARCA4, UBA2, and USP9X play a role in diaphragm development.

OBJECTIVE To determine the incremental yield of exome sequencing (ES) over chromosomal microarray analysis (CMA) or karyotyping in prenatally diagnosed non-immune hydrops fetalis (NIHF). METHODS A prospective cohort study (comprising an extended group of the Prenatal Assessment of Genomes and Exomes (PAGE) study) was performed which included 28 cases of prenatally diagnosed NIHF undergoing trio ES following negative CMA or karyotyping. These cases were combined with data from a systematic review of the literature. MEDLINE, EMBASE, CINAHL and ClinicalTrials.gov databases were searched electronically (January 2000 to October 2020) for studies reporting on the incremental yield of ES over CMA or karyotyping in fetuses with prenatally detected NIHF. Inclusion criteria for the systematic review were: (i) at least two cases of NIHF undergoing sequencing; (ii) testing initiated based on prenatal ultrasound-based phenotype; and (iii) negative CMA or karyotyping result. The incremental diagnostic yield of ES was assessed in: (i) all cases of NIHF; (ii) isolated NIHF; (iii) NIHF associated with an additional fetal structural anomaly; and (iv) NIHF according to severity (i.e. two vs three or more cavities affected). RESULTS In the extended PAGE study cohort, the additional diagnostic yield of ES over CMA or karyotyping was 25.0% (7/28) in all NIHF cases, 21.4% (3/14) in those with isolated NIHF and 28.6% (4/14) in those with non-isolated NIHF. In the meta-analysis, the pooled incremental yield based on 21 studies (306 cases) was 29% (95% CI, 24-34%; P

OBJECTIVE To evaluate the utility of prenatal exome sequencing (ES) for isolated increased nuchal translucency (NT) and to investigate factors that increase diagnostic yield. DESIGN Retrospective analysis of data from two prospective cohort studies. SETTING Fetal medicine centres in the UK and USA. POPULATION Fetuses with increased NT ≥3.5 mm at 11-14 weeks of gestation recruited to the Prenatal Assessment of Genomes and Exomes (PAGE) and Columbia fetal whole exome sequencing studies (n = 213). METHODS We grouped cases based on (1) the presence of additional structural abnormalities at presentation in the first trimester or later in pregnancy, and (2) NT measurement at presentation. We compared diagnostic rates between groups using Fisher exact test. MAIN OUTCOME MEASURES Detection of diagnostic genetic variants considered to have caused the observed fetal structural anomaly. RESULTS Diagnostic variants were detected in 12 (22.2%) of 54 fetuses presenting with non-isolated increased NT, 12 (32.4%) of 37 fetuses with isolated increased NT in the first trimester and additional abnormalities later in pregnancy, and 2 (1.8%) of 111 fetuses with isolated increased NT in the first trimester and no other abnormalities on subsequent scans. Diagnostic rate also increased with increasing size of NT. CONCLUSIONS The diagnostic yield of prenatal ES is low for fetuses with isolated increased NT but significantly higher where there are additional structural anomalies. Prenatal ES may not be appropriate for truly isolated increased NT but timely, careful ultrasound scanning to identify other anomalies emerging later can direct testing to focus where there is a higher likelihood of diagnosis.

Introduction: The objective was to evaluate: (i) the proportion of prenatally diagnosed congenital heart disease (CHD) associated with an abnormal quantitative fluorescence-PCR (QF-PCR), chromosome microarray (CMA), and exome sequencing (ES) result; and (ii) the diagnostic yield of these technologies based on CHD category and presence of extra-cardiac anomalies (ECAs). Methods: This prospective cohort study was set across 12 UK foetal medicine centres. All cases underwent QF-PCR, CMA, and ES, and the diagnostic yield in n = 147 cases of prenatally diagnosed CHD was assessed. Results: In 34.7% (n = 51/147), a genetic diagnosis was obtained. Using a stepwise testing strategy, the diagnostic yield for QF-PCR, CMA, and ES was 15.6% (n = 23/147), 13.7% (n = 17/124), and 10.2% (n = 11/107), respectively. Abnormal QF-PCR/shunt (septal) defects 31.4% (n = 11/35), p = 0.046, and abnormal CMA/conotruncal anomalies 22.7% (n = 10/44), p = 0.04, had significant associations. Monogenic variants were commonest in complex CHD 36.4% (n = 4/11). Multisystem CHD had a greater diagnostic yield overall compared to isolated OR 2.41 (95% CI, 1.1–5.1), particularly in association with brain and gastrointestinal tract anomalies. The proportion of variants of uncertain significance was 4.7% (n = 5/107) with ES, with none in the CMA group. Conclusion: In the era of prenatal ES, there remains an important role for QF-PCR and CMA. Identification of monogenic pathologic variants further allows delineation of prognosis in CHD.

OBJECTIVE To determine the incremental yield of antenatal exome sequencing (ES) over chromosomal microarray analysis (CMA) or conventional karyotyping in prenatally diagnosed congenital heart disease (CHD). METHODS A prospective cohort study of 197 trios undergoing ES following CMA or karyotyping owing to CHD identified prenatally and a systematic review of the literature were performed. MEDLINE, EMBASE, CINAHL and ClinicalTrials.gov (January 2000 to October 2019) databases were searched electronically for studies reporting on the diagnostic yield of ES in prenatally diagnosed CHD. Selected studies included those with more than three cases, with initiation of testing based upon prenatal phenotype only and that included cases in which CMA or karyotyping was negative. The incremental diagnostic yield of ES was assessed in: (1) all cases of CHD; (2) isolated CHD; (3) CHD associated with extracardiac anomaly (ECA); and (4) CHD according to phenotypic subgroup. RESULTS In our cohort, ES had an additional diagnostic yield in all CHD, isolated CHD and CHD associated with ECA of 12.7% (25/197), 11.5% (14/122) and 14.7% (11/75), respectively (P = 0.81). The corresponding pooled incremental yields from 18 studies (encompassing 636 CHD cases) included in the systematic review were 21% (95% CI, 15-27%), 11% (95% CI, 7-15%) and 37% (95% CI, 18-56%), respectively. The results did not differ significantly when subanalysis was limited to studies including more than 20 cases, except for CHD associated with ECA, in which the incremental yield was greater (49% (95% CI, 17-80%)). In cases of CHD associated with ECA in the primary analysis, the most common extracardiac anomalies associated with a pathogenic variant were those affecting the genitourinary system (23/52 (44.2%)). The greatest incremental yield was in cardiac shunt lesions (41% (95% CI, 19-63%)), followed by right-sided lesions (26% (95% CI, 9-43%)). In the majority (68/96 (70.8%)) of instances, pathogenic variants occurred de novo and in autosomal dominant (monoallelic) disease genes. The most common (19/96 (19.8%)) monogenic syndrome identified was Kabuki syndrome. CONCLUSIONS There is an apparent incremental yield of prenatal ES in CHD. While the greatest yield is in CHD associated with ECA, consideration could also be given to performing ES in the presence of an isolated cardiac abnormality. A policy of routine application of ES would require the adoption of robust bioinformatic, clinical and ethical pathways. Copyright © 2020 ISUOG. Published by John Wiley & Sons Ltd.

Nager syndrome is a rare, complex malformation syndrome, for which there is limited information on prenatal genetic testing. Clinical diagnosis of Nager syndrome, which can be caused by deletions encompassing SF3B4 gene, is possible prenatally. Prenatal chromosomal microarray can aid genotype‐phenotype correlation in pregnancies with structural abnormalities seen on ultrasound.

Structural differences (congenital anomalies) in the makeup of the baby's heart, brain and other organs are found on antenatal ultrasound scans in up to 3% of pregnancies. These often have a genetic cause, arising because of changes in the chromosomes (which store our genetic material) or the DNA code that make up the genes. The more differences a baby has the more likely the risk of underlying genetic disease. If a structural difference is found, parents are usually offered a genetic test, which may be carried out on cells taken either from the placenta (chorionic villous sampling) or the fluid surrounding the baby (amniocentesis). At the moment, these cells are only tested for changes in the chromosomes and are only able to reveal the underlying cause in about 40% of unborn babies. Prenatal exome sequencing (ES) is a new genetic test, which, when combined with testing the DNA of both parents can find changes in the baby's genetic code. If a DNA change is found that can explain the structural changes seen on ultrasound, specific information about the underlying diagnosis can be given to the parents. Having this information can help parents make important decisions about their ongoing pregnancy, as well as help doctors to care for the mother and baby. Finding a genetic change can also help to understand how the condition has arisen and whether it might happen again in another pregnancy. It may also be possible to test for the genetic condition in future pregnancies. Although prenatal ES is an exciting new way to improve diagnosis rates for structural differences, it has some challenges. While the test is very detailed, it may not always find a genetic explanation and sometimes the results are difficult to interpret. For example, genetic changes can be found where their significance for the pregnancy is unclear. More recently, two studies have now shown that prenatal ES can find a genetic diagnosis in at least 10% of pregnancies with structural differences where standard chromosome testing has been negative. This paper reviews these studies, along with earlier evidence on ES and provides clinicians with guidance for future practice.

Background Fetal structural anomalies, which are detected by ultrasonography, have a range of genetic causes, including chromosomal aneuploidy, copy number variations (CNVs; which are detectable by chromosomal microarrays), and pathogenic sequence variants in developmental genes. Testing for aneuploidy and CNVs is routine during the investigation of fetal structural anomalies, but there is little information on the clinical usefulness of genome-wide next-generation sequencing in the prenatal setting. We therefore aimed to evaluate the proportion of fetuses with structural abnormalities that had identifiable variants in genes associated with developmental disorders when assessed with whole-exome sequencing (WES). Methods In this prospective cohort study, two groups in Birmingham and London recruited patients from 34 fetal medicine units in England and Scotland. We used whole-exome sequencing (WES) to evaluate the presence of genetic variants in developmental disorder genes (diagnostic genetic variants) in a cohort of fetuses with structural anomalies and samples from their parents, after exclusion of aneuploidy and large CNVs. Women were eligible for inclusion if they were undergoing invasive testing for identified nuchal translucency or structural anomalies in their fetus, as detected by ultrasound after 11 weeks of gestation. The partners of these women also had to consent to participate. Sequencing results were interpreted with a targeted virtual gene panel for developmental disorders that comprised 1628 genes. Genetic results related to fetal structural anomaly phenotypes were then validated and reported postnatally. The primary endpoint, which was assessed in all fetuses, was the detection of diagnostic genetic variants considered to have caused the fetal developmental anomaly. Findings The cohort was recruited between Oct 22, 2014, and June 29, 2017, and clinical data were collected until March 31, 2018. After exclusion of fetuses with aneuploidy and CNVs, 610 fetuses with structural anomalies and 1202 matched parental samples (analysed as 596 fetus-parental trios, including two sets of twins, and 14 fetus-parent dyads) were analysed by WES. After bioinformatic filtering and prioritisation according to allele frequency and effect on protein and inheritance pattern, 321 genetic variants (representing 255 potential diagnoses) were selected as potentially pathogenic genetic variants (diagnostic genetic variants), and these variants were reviewed by a multidisciplinary clinical review panel. A diagnostic genetic variant was identified in 52 (8·5%; 95% CI 6·4–11·0) of 610 fetuses assessed and an additional 24 (3·9%) fetuses had a variant of uncertain significance that had potential clinical usefulness. Detection of diagnostic genetic variants enabled us to distinguish between syndromic and non-syndromic fetal anomalies (eg, congenital heart disease only vs a syndrome with congenital heart disease and learning disability). Diagnostic genetic variants were present in 22 (15·4%) of 143 fetuses with multisystem anomalies (ie, more than one fetal structural anomaly), nine (11·1%) of 81 fetuses with cardiac anomalies, and ten (15·4%) of 65 fetuses with skeletal anomalies; these phenotypes were most commonly associated with diagnostic variants. However, diagnostic genetic variants were least common in fetuses with isolated increased nuchal translucency (≥4·0 mm) in the first trimester (in three [3·2%] of 93 fetuses). Interpretation WES facilitates genetic diagnosis of fetal structural anomalies, which enables more accurate predictions of fetal prognosis and risk of recurrence in future pregnancies. However, the overall detection of diagnostic genetic variants in a prospectively ascertained cohort with a broad range of fetal structural anomalies is lower than that suggested by previous smaller-scale studies of fewer phenotypes. WES improved the identification of genetic disorders in fetuses with structural abnormalities; however, before clinical implementation, careful consideration should be given to case selection to maximise clinical usefulness. Funding UK Department of Health and Social Care and The Wellcome Trust.

International audience; GJA8 encodes connexin 50 (Cx50), a transmembrane protein involved in the formation of lens gap junctions. GJA8 mutations have been linked to early onset cataracts in humans and animal models. In mice, missense mutations and homozygous Gja8 deletions lead to smaller lenses and microphthalmia in addition to cataract, suggesting that Gja8 may play a role in both lens development and ocular growth. Following screening of GJA8 in a cohort of 426 individuals with severe congenital eye anomalies, primarily anophthalmia, microphthalmia and coloboma, we identified four known [p.(Thr39Arg), p.(Trp45Leu), p.(Asp51Asn), and p.(Gly94Arg)] and two novel [p.(Phe70Leu) and p.(Val97Gly)] likely pathogenic variants in seven families. Five of these co-segregated with cataracts and microphthalmia, whereas the variant p.(Gly94Arg) was identified in an individual with congenital aphakia, sclerocornea, microphthalmia and coloboma. Four missense variants of unknown or unlikely clinical significance were also identified. Furthermore, the screening of GJA8 structural variants in a subgroup of 188 individuals identified heterozygous 1q21 microdeletions in five families with coloboma and other ocular and/or extraocular findings. However, the exact genotype-phenotype correlation of these structural variants remains to be established. Our data expand the spectrum of GJA8 variants and associated phenotypes, confirming the importance of this gene in early eye development.

Contains fulltext : 206572.pdf (Publisher’s version ) (Open Access) We delineate a KMT2E-related neurodevelopmental disorder on the basis of 38 individuals in 36 families. This study includes 31 distinct heterozygous variants in KMT2E (28 ascertained from Matchmaker Exchange and three previously reported), and four individuals with chromosome 7q22.2-22.23 microdeletions encompassing KMT2E (one previously reported). Almost all variants occurred de novo, and most were truncating. Most affected individuals with protein-truncating variants presented with mild intellectual disability. One-quarter of individuals met criteria for autism. Additional common features include macrocephaly, hypotonia, functional gastrointestinal abnormalities, and a subtle facial gestalt. Epilepsy was present in about one-fifth of individuals with truncating variants and was responsive to treatment with anti-epileptic medications in almost all. More than 70% of the individuals were male, and expressivity was variable by sex; epilepsy was more common in females and autism more common in males. The four individuals with microdeletions encompassing KMT2E generally presented similarly to those with truncating variants, but the degree of developmental delay was greater. The group of four individuals with missense variants in KMT2E presented with the most severe developmental delays. Epilepsy was present in all individuals with missense variants, often manifesting as treatment-resistant infantile epileptic encephalopathy. Microcephaly was also common in this group. Haploinsufficiency versus gain-of-function or dominant-negative effects specific to these missense variants in KMT2E might explain this divergence in phenotype, but requires independent validation. Disruptive variants in KMT2E are an under-recognized cause of neurodevelopmental abnormalities.

Objective Evaluate the diagnostic yield of prenatal submicroscopic chromosome anomalies using prenatal array comparative genomic hybridisation (aCGH). Method Prospective cohort study conducted between March 2013 and June 2017 including fetuses where an elevated nuchal translucency (NT) or structural anomaly was identified on ultrasound and common aneuploidy testing was negative. aCGH was performed using an 8-plex oligonucleotide platform with a genome wide backbone resolution of greater than 200 kb and interpretation in line with American College of Medical Genetics guidance. Results One thousand one hundred twenty-nine fetuses were included; 371 fetuses with an increased NT (32.9%) and 758 with a structural anomaly (67.1%). The rate of pathogenic copy number variants (CNVs) and variant of uncertain significance (VUS) was 5.9% (n = 22) and 0.5% (n = 2) in the elevated NT group and 7.3% (n = 55) and 0.8% (n = 6) in the mid-trimester anomaly group. No pathogenic CNVs were identified in fetuses with an NT less than 4.0 mm. Multisystem and cardiac anomalies had the greatest yield of pathogenic CNV with a 22q11.2 microdeletion present in 40% (12/30). Conclusion Prenatal aCGH is a useful diagnostic tool in the investigation of fetuses with a significantly elevated NT or structural anomaly. With time and experience, rates of pathogenic CNVs have increased, and VUS have reduced, supporting the prenatal application of increasingly high resolution aCGH platforms.

Introduction: In light of the prospective Prenatal Assessment of Genomes and Exomes (PAGE) study, this paper aimed to determine the additional costs of using exome sequencing (ES) alongside or in place of chromosomal microarray (CMA) in a fetus with an identified congenital anomaly. Methods: A decision tree was populated using data from a prospective cohort of women undergoing invasive diagnostic testing. Four testing strategies were evaluated: CMA, ES, CMA followed by ES (“stepwise”); CMA and ES combined. Results: When ES is priced at GBP 2,100 (EUR 2,407/USD 2,694), performing ES alone prenatally would cost a further GBP 31,410 (EUR 36,001/USD 40,289) per additional genetic diagnosis, whereas the stepwise would cost a further GBP 24,657 (EUR 28,261/USD 31,627) per additional genetic diagnosis. When ES is priced at GBP 966 (EUR 1,107/USD 1,239), performing ES alone prenatally would cost a further GBP 11,532 (EUR 13,217/USD 14,792) per additional genetic diagnosis, whereas the stepwise would cost a further additional GBP 11,639 (EUR 13,340/USD 14,929) per additional genetic diagnosis. The sub-group analysis suggests that performing stepwise on cases indicative of multiple anomalies at ultrasound scan (USS) compared to cases indicative of a single anomaly, is more cost-effective compared to using ES alone. Discussion/Conclusion: Performing ES alongside CMA is more cost-effective than ES alone, which can potentially lead to improvements in pregnancy management. The direct effects of test results on pregnancy outcomes were not examined; therefore, further research is recommended to examine changes on the projected incremental cost-effectiveness ratios.

Anophthalmia, microphthalmia, and coloboma are a genetically heterogeneous spectrum of developmental eye disorders and affect around 30 per 100,000 live births. OLFM2 encodes a secreted glycoprotein belonging to the noelin family of olfactomedin domain-containing proteins that modulate the timing of neuronal differentiation during development. OLFM2 SNPs have been associated with open angle glaucoma in a case-control study, and knockdown of Olfm2 in zebrafish results in reduced eye size. From a cohort of 258 individuals with developmental eye anomalies, we identified two with heterozygous variants in OLFM2: an individual with bilateral microphthalmia carrying a de novo 19p13.2 microdeletion involving OLFM2 and a second individual with unilateral microphthalmia and contralateral coloboma who had a novel single base change in the 5' untranslated region. Dual luciferase assays demonstrated that the latter variant causes a significant decrease in expression of OLFM2. Furthermore, RNA in situ hybridisation experiments using human developmental tissue revealed expression in relevant structures, including the lens vesicle and optic cup. Our study indicates that OLFM2 is likely to be important in mammalian eye development and disease and should be considered as a gene for human ocular anomalies.

Objective Focus groups were conducted with individuals involved in prenatal diagnosis to determine their opinions relating to WES in fetuses with structural anomalies. Method Five representatives of patient groups/charities (PRGs) and eight clinical professionals (CPs) participated. Three focus groups occurred (the two groups separately and then combined). Framework analysis was performed to elicit themes. A thematic coding frame was identified based on emerging themes. Results Seven main themes (consent, analysis, interpretation/reinterpretation of results, prenatal issues, uncertainty, incidental findings, and information access) with sub-themes emerged. The main themes were raised by both groups, apart from ‘analysis’ which was raised by CPs only. Some subthemes were raised by PRGs and CPs (with different perspectives). Others were raised either by PRGs or CPs, showing differences in patient/clinician agendas. Conclusions Prenatal consent for WES is not a ‘perfect’ process but consent takers should be fully educated regarding the test. PRGs highlighted issues involving access to results feeling that women want to know all information. PRGs also felt that patients want re-interpretation of results over time whilst CPs felt that interpretation should be performed at the point of testing only.

© 2019 American Society of Human Genetics The occurrence of non-epileptic hyperkinetic movements in the context of developmental epileptic encephalopathies is an increasingly recognized phenomenon. Identification of causative mutations provides an important insight into common pathogenic mechanisms that cause both seizures and abnormal motor control. We report bi-allelic loss-of-function CACNA1B variants in six children from three unrelated families whose affected members present with a complex and progressive neurological syndrome. All affected individuals presented with epileptic encephalopathy, severe neurodevelopmental delay (often with regression), and a hyperkinetic movement disorder. Additional neurological features included postnatal microcephaly and hypotonia. Five children died in childhood or adolescence (mean age of death: 9 years), mainly as a result of secondary respiratory complications. CACNA1B encodes the pore-forming subunit of the pre-synaptic neuronal voltage-gated calcium channel Cav2.2/N-type, crucial for SNARE-mediated neurotransmission, particularly in the early postnatal period. Bi-allelic loss-of-function variants in CACNA1B are predicted to cause disruption of Ca2+ influx, leading to impaired synaptic neurotransmission. The resultant effect on neuronal function is likely to be important in the development of involuntary movements and epilepsy. Overall, our findings provide further evidence for the key role of Cav2.2 in normal human neurodevelopment.

Clinical testing with chromosomal microarray (CMA) is most commonly undertaken for clinical indications such as intellectual disability, dysmorphic features and/or congenital abnormalities. Identification of a structural aberration (SA) involving a cancer susceptibility gene (CSG) constitutes a type of incidental or secondary finding. Laboratory reporting, risk communication and clinical management of these structural aberrations with secondary implications (SASIs) is currently inconsistent. We undertake meta-analysis of 18 622 instances of CMA performed for unrelated indications in which 106 SASIs are identified involving in total 40 different CSGs. Here we present the recommendations of a joint UK working group representing the British Society of Genomic Medicine, UK Cancer Genetics Group and UK Association for Clinical Genomic Science. SASIs are categorised into four groups, defined by the type of SA and the cancer risk. For each group, recommendations are provided regarding reflex parental testing and cancer risk management.

Discovery of most autosomal recessive disease-associated genes has involved analysis of large, often consanguineous multiplex families or small cohorts of unrelated individuals with a well-defined clinical condition. Discovery of new dominant causes of rare, genetically heterogeneous developmental disorders has been revolutionized by exome analysis of large cohorts of phenotypically diverse parent-offspring trios. Here we analyzed 4,125 families with diverse, rare and genetically heterogeneous developmental disorders and identified four new autosomal recessive disorders. These four disorders were identified by integrating Mendelian filtering (selecting probands with rare, biallelic and putatively damaging variants in the same gene) with statistical assessments of (i) the likelihood of sampling the observed genotypes from the general population and (ii) the phenotypic similarity of patients with recessive variants in the same candidate gene. This new paradigm promises to catalyze the discovery of novel recessive disorders, especially those with less consistent or nonspecific clinical presentations and those caused predominantly by compound heterozygous genotypes.

Histone lysine methyltransferases (KMTs) and demethylases (KDMs) underpin gene regulation. Here we demonstrate that variants causing haploinsufficiency of KMTs and KDMs are frequently encountered in individuals with developmental disorders. Using a combination of human variation databases and existing animal models, we determine 22 KMTs and KDMs as additional candidates for dominantly inherited developmental disorders. We show that KMTs and KDMs that are associated with, or are candidates for, dominant developmental disorders tend to have a higher level of transcription, longer canonical transcripts, more interactors, and a higher number and more types of post-translational modifications than other KMT and KDMs. We provide evidence to firmly associate KMT2C, ASH1L, and KMT5B haploinsufficiency with dominant developmental disorders. Whereas KMT2C or ASH1L haploinsufficiency results in a predominantly neurodevelopmental phenotype with occasional physical anomalies, KMT5B mutations cause an overgrowth syndrome with intellectual disability. We further expand the phenotypic spectrum of KMT2B-related disorders and show that some individuals can have severe developmental delay without dystonia at least until mid-childhood. Additionally, we describe a recessive histone lysine-methylation defect caused by homozygous or compound heterozygous KDM5B variants and resulting in a recognizable syndrome with developmental delay, facial dysmorphism, and camptodactyly. Collectively, these results emphasize the significance of histone lysine methylation in normal human development and the importance of this process in human developmental disorders. Our results demonstrate that systematic clinically oriented pathway-based analysis of genomic data can accelerate the discovery of rare genetic disorders.

BackgroundIdentification of CNVs through chromosomal microarray (CMA) testing is the first-line investigation in individuals with learning difficulties/congenital abnormalities. Although recognised that CMA testing may identify CNVs encompassing a cancer predisposition gene (CPG), limited information is available on the frequency and nature of such results.MethodsWe investigated CNV gains and losses affecting 39 CPGs in 3366 pilot index case individuals undergoing CMA testing, and then studied an extended cohort (n=10 454) for CNV losses at 105 CPGs and CNV gains at 9 proto-oncogenes implicated in inherited cancer susceptibility.ResultsIn the pilot cohort, 31/3366 (0.92%) individuals had a CNV involving one or more of 16/39 CPGs. 30/31 CNVs involved a tumour suppressor gene (TSG), and 1/30 a proto-oncogene (gain of MET). BMPR1A, TSC2 and TMEM127 were affected in multiple cases. In the second stage analysis, 49/10 454 (0.47%) individuals in the extended cohort had 50 CNVs involving 24/105 CPGs. 43/50 CNVs involved a TSG and 7/50 a proto-oncogene (4 gains, 3 deletions). The most frequently involved genes, FLCN (n=10) and SDHA (n=7), map to the Smith-Magenis and cri-du-chat regions, respectively.ConclusionIncidental identification of a CNV involving a CPG is not rare and poses challenges for future cancer risk estimation. Prospective data collection from CPG-CNV cohorts ascertained incidentally and through syndromic presentations is required to determine the risks posed by specific CNVs. In particular, ascertainment and investigation of adults with CPG-CNVs and adults with learning disability and cancer, could provide important information to guide clinical management and surveillance.

The genetic etiology of non-aneuploid fetal structural abnormalities is typically investigated by karyotyping and array-based detection of microscopically detectable rearrangements, and submicroscopic copy-number variants (CNVs), which collectively yield a pathogenic finding in up to 10% of cases. We propose that exome sequencing may substantially increase the identification of underlying etiologies. We performed exome sequencing on a cohort of 30 non-aneuploid fetuses and neonates (along with their parents) with diverse structural abnormalities first identified by prenatal ultrasound. We identified candidate pathogenic variants with a range of inheritance models, and evaluated these in the context of detailed phenotypic information. We identified 35 de novo single-nucleotide variants (SNVs), small indels, deletions or duplications, of which three (accounting for 10% of the cohort) are highly likely to be causative. These are de novo missense variants in FGFR3 and COL2A1, and a de novo 16.8 kb deletion that includes most of OFD1. In five further cases (17%) we identified de novo or inherited recessive or X-linked variants in plausible candidate genes, which require additional validation to determine pathogenicity. Our diagnostic yield of 10% is comparable to, and supplementary to, the diagnostic yield of existing microarray testing for large chromosomal rearrangements and targeted CNV detection. The de novo nature of these events could enable couples to be counseled as to their low recurrence risk. This study outlines the way for a substantial improvement in the diagnostic yield of prenatal genetic abnormalities through the application of next-generation sequencing.

Objectives Chromosomal microarray analysis (CMA) is utilized in prenatal diagnosis to detect chromosomal abnormalities not visible by conventional karyotyping. A prospective cohort of women undergoing fetal CMA and karyotyping following abnormal prenatal ultrasound findings is presented in the context of a systematic review and meta-analysis of the literature describing detection rates by CMA and karyotyping. Methods We performed a prospective cohort study of 243 women undergoing CMA alongside karyotyping when a structural abnormality was detected on prenatal ultrasound. A systematic review of the literature was also performed. MEDLINE (1970–Dec 2012), EMBASE (1980–Dec 2012) and CINAHL (1982–June 2012) databases were searched electronically. Selected studies included > 10 cases and prenatal CMA in addition to karyotyping. The search yielded 560 citations. Full papers were retrieved for 86, and 25 primary studies were included in the systematic review. Results Our cohort study found an excess detection rate of abnormalities by CMA of 4.1% over conventional karyotyping when the clinical indication for testing was an abnormal fetal ultrasound finding; this was lower than the detection rate of 10% (95% CI, 8–13%) by meta-analysis. The rate of detection for variants of unknown significance (VOUS) was 2.1% (95% CI, 1.3–3.3%) when the indication for CMA was an abnormal scan finding. The VOUS detection rate was lower (1.4%; 95% CI, 0.5–3.7%) when any indication for prenatal CMA was meta-analyzed. Conclusion We present evidence for a higher detection rate by CMA than by karyotyping not just in the case of abnormal ultrasound findings but also in cases of other indications for invasive testing. It is likely that CMA will replace karyotyping in high-risk pregnancies. Copyright © 2013 ISUOG. Published by John Wiley & Sons Ltd.

Key content The results of prenatal fetal chromosomal analysis may inform long term prognosis. Recent advances in chromosome microarray (CMA) are enabling genome-wide evaluation of submicroscopic chromosomal changes. Such technological advances promise increased sensitivity and specificity. They also potentially pose challenges of interpretation and clinical management. Learning objectives To systematically review the current literature and explain the benefit and pitfalls of CMAs and their relevance to the practising obstetrician in the NHS. Comment on the future of CMA and its cost effectiveness. Ethical issues Difficulties of informed consent with genome-wide prenatal testing. Difficulties surround results of unknown significance.

Urothelial bladder cancers (UBCs) have heterogeneous clinical characteristics that are mirrored in their diverse genomic profiles. Genomic profiling of UBCs has the potential to benefit routine clinical practice by providing prognostic utility above and beyond conventional clinicopathological factors, and allowing for prediction and surveillance of treatment responses. Urinary DNAs representative of the tumour genome provide a promising resource as a liquid biopsy for non-invasive genomic profiling of UBCs. We compared the genomic profiles of urinary cellular DNA and cell-free DNA (cfDNA) from the urine with matched diagnostic formalin-fixed paraffin-embedded tumour DNAs for 23 well-characterised UBC patients. Our data show urinary DNAs to be highly representative of patient tumours, allowing for detection of recurrent clinically actionable genomic aberrations. Furthermore, a greater aberrant load (indicative of tumour genome) was observed in cfDNA over cellular DNA (P

Objective To map the current testing being undertaken following pregnancy loss across the UK and to examine the clinical utility in terms of identifying a cause for the loss and in identifying couples at risk of an unbalanced liveborn child. Design Retrospective audit. Setting UK, for the year 2014. Population An audit of 6465 referrals for genetic testing of tissue samples following pregnancy loss. Methods Data were obtained by questionnaire from 15 UK regional genetics laboratories. Main outcome measures Data were analysed with respect to gestational age, the presence of identified fetal anomalies, methodologies used, abnormality rates and the presence of a parental balanced rearrangement. Results Of 6465 referrals a genetic cause was identified in 22% of cases (before 12 weeks’ gestation, in 47%; at 12–24 weeks, in 14%; after 24 weeks, in 6%). In 0.4% of cases a balanced parental rearrangement was identified where there was a risk of an affected liveborn child in a future pregnancy. Eighty percent of genetic imbalances identified were aneuploidy or triploidy and could be identified by quantitative fluorescence polymerase chain reaction alone. There was significant variation across the UK in acceptance criteria, testing strategies and thus level of resolution of testing. Conclusions Genetic testing of tissues following pregnancy loss identifies a probable cause of fetal demise in 22% of cases, but it is of low clinical utility in identifying couples at risk of a future unbalanced liveborn child. A comprehensive multidisciplinary review is needed to develop proposals for an affordable and equitable service. Tweetable abstract UK audit of genetic testing of fetal loss shows variation in access to and resolution of analysis.

G-band chromosomal karyotyping of fetal cells obtained by invasive prenatal testing has been used since the 1960s to identify structural chromosomal anomalies. Prenatal testing is usually performed in response to parental request, increased risk of fetal chromosomal abnormality associated with advanced maternal age, a high-risk screening test and/or the presence of a congenital malformation identified by ultrasonography. The results of karyotyping may inform the long-term prognosis (e.g. aneuploidy being associated with a poor outcome or microscopic chromosomal anomalies predicting global neurodevelopmental morbidity). Relatively recent advances in microarray technology are now enabling high-resolution genome-wide evaluation for DNA copy number abnormalities (e.g. deletions or duplications). While such technological advances promise increased sensitivity and specificity they can also pose difficult challenges of interpretation and clinical management. This review aims to give interested clinicians without an extensive prior knowledge of microarray technology, an overview of its use in prenatal diagnosis, the literature to date, advantages, potential pitfalls and experience from our own tertiary center.

Deletions of 17q12 are associated with renal cysts and maturity onset diabetes of the young, and have also been identified in women with reproductive tract anomalies due to Mullerian aplasia. Although initially identified in patients with normal cognitive ability, some patients with this recurrent microdeletion syndrome have learning problems. We identified a 17q12 microdeletion in three patients with renal cystic disease by array comparative genomic hybridization and the phenotypic spectrum of the 17q12 microdeletion syndrome is illustrated by the description of these patients. Of two patients who are old enough to be assessed, one has significant speech delay, autism spectrum disorder, and mild learning difficulty, while the other patient has only mild speech delay. This highlights the variability of cognitive involvement in this condition. The third patient presented with Alagille syndrome-like features in the neonatal period. All three patients had transient hypercalcemia in the neonatal period, a finding that has not previously been described in this condition. Moreover, two patients have mild or no dysmorphism, while one displays striking facial dysmorphism in addition to minor congenital anomalies. We suggest that while patients with 17q12 microdeletion syndrome can present with type 2 diabetes or renal cysts without any dysmorphic features, a subgroup may have dysmorphic features or present with neonatal cholestasis. Transient neonatal hypercalcemia may be a feature of this microdeletion syndrome.

Genome-wide arrays are rapidly replacing conventional karyotyping in postnatal cytogenetic diagnostics and there is a growing request for arrays in the prenatal setting. Several studies have documented 1-3% additional abnormal findings in prenatal diagnosis with arrays compared to conventional karyotyping. A recent meta-analysis demonstrated that 5.2% extra diagnoses can be expected in fetuses with ultrasound abnormalities. However, no consensus exists as to whether the use of genome-wide arrays should be restricted to pregnancies with ultrasound abnormalities, performed in all women undergoing invasive prenatal testing or offered to all pregnant women. Moreover, the interpretation of array results in the prenatal situation is challenging due to the large numbers of copy number variants with no major phenotypic effect. This also raises the question of what, or what not to report, for example, how to deal with unsolicited findings. These issues were discussed at a working group meeting that preceded the European Society of Human Genetics 2011 Conference in Amsterdam. This article is the result of this meeting and explores the introduction of genome-wide arrays into routine prenatal diagnosis. We aim to give some general recommendations on how to develop practical guidelines that can be implemented in the local setting and that are consistent with the emerging international consensus. Hum Mutat 33: 923-929, 2012. (C) 2012 Wiley Periodicals, Inc.

Wolf–Hirschhorn syndrome is caused by anomalies of the short arm of chromosome 4. About 55% of cases are due to de novo terminal deletions, 40% from unbalanced translocations and 5% from other abnormalities. The facial phenotype is characterized by hypertelorism, protruding eyes, prominent glabella, broad nasal bridge and short philtrum. We used dense surface modelling and pattern recognition techniques to delineate the milder facial phenotype of individuals with a small terminal deletion (breakpoint within 4p16.3) compared to those with a large deletion (breakpoint more proximal than 4p16.3). Further, fine-grained facial analysis of several individuals with an atypical genotype and/or phenotype suggests that multiple genes contiguously contribute to the characteristic Wolf–Hirschhorn syndrome facial phenotype.

Germline mutations in the VHL tumor suppressor gene cause von Hippel-Lindau (VHL) disease and somatic VHL mutations occur in the majority of clear cell renal cell carcinoma (cRCC). To compare copy number abnormalities (CNAs) between cRCC from VHL patients and sporadic cRCC cases without detectable somatic VHL mutations, we analyzed 34 cRCC with Affymetrix 250K arrays. To increase the power of the study, we then combined our results with those of a previously published study and compared CNAs in VHL and non-VHL related cRCC using the genomic identification of significant targets in cancer (GISTIC) program. In VHL, cRCC GISTIC analysis identified four statistically significant regions of copy number gain and four statistically significant regions of deletion that occurred in >10% of tumors analyzed. Sporadic cRCC without detectable VHL mutations had, on average, more copy number abnormalities than VHL cRCC though the most common regions of loss/gain (e.g., 3p and 14q loss and 5q gain) were present in both tumor sets. However, CNAs on chromosome arms 7p (gain) and 8p (loss) were only detected in VHL RCC. Although individual copy number abnormality peaks contained clear candidate cancer genes in some cases (e.g., the 3p loss peak in VHL cRCC contained only six genes including VHL), most peaks contained many genes. To date, only a minority of the candidate genes included in these peaks have been analyzed for mutation or epigenetic inactivation in cRCC but TNFRSF10C and DUSP4 map to the 8p region deleted in VHL cRCC and TP53 and HIF2A (EPAS1) mapped to CNA loss and gain peaks (chromosomes 17 and 2, respectively) detected in sporadic VHL wild-type cRCC.

Gilles de la Tourette syndrome is a complex neuropsychiatric disorder with a strong genetic basis. We identified a male patient with Tourette syndrome-like tics and an apparently balanced de novo translocation [46,XY,t(2;7)(p24.2;q31)]. Further analysis using array comparative genomic hybridisation (CGH) revealed a cryptic deletion at 7q31.1-7q31.2. Breakpoints disrupting this region have been reported in one isolated and one familial case of Tourette syndrome. In our case, IMMP2L, a gene coding for a human homologue of the yeast inner mitochondrial membrane peptidase subunit 2, was disrupted by the breakpoint on 7q31.1, with deletion of exons 1-3 of the gene. The IMMP2L gene has previously been proposed as a candidate gene for Tourette syndrome, and our case provides further evidence of its possible role in the pathogenesis. The deleted region (7q31.1-7q31.2) of 7.2 Mb of genomic DNA also encompasses numerous genes, including FOXP2, associated with verbal dyspraxia, and the CFTR gene.

Objective Array comparative genomic hybridization (CGH) is transforming clinical cytogenetics with its ability to interrogate the human genome at increasingly high resolution. The aim of this study was to determine whether array CGH testing in the prenatal population provides diagnostic information over conventional karyotyping. Methods MEDLINE (1970 to December 2009), EMBASE (1980 to December 2009) and CINAHL (1982 to December 2009) databases were searched electronically. Studies were selected if array CGH was used on prenatal samples or if array CGH was used on postnatal samples following termination of pregnancy for structural abnormalities that were detected on an ultrasound scan. Of the 135 potential articles, 10 were included in this systematic review and eight were included in the meta-analysis. The pooled rate of extra information detected by array CGH when the prenatal karyotype was normal was meta-analyzed using a random-effects model. The pooled rate of receiving an array CGH result of unknown significance was also meta-analyzed. Results Array CGH detected 3.6% (95% CI, 1.5–8.5) additional genomic imbalances when conventional karyo-typing was ‘normal’, regardless of referral indication. This increased to 5.2% (95% CI, 1.9–13.9) more than karyotyping when the referral indication was a structural malformation on ultrasound. Conclusions There appears to be an increased detection rate of chromosomal imbalances, compared with conventional karyotyping, when array CGH techniques are employed in the prenatal population. However, some are copy number imbalances that are not clinically significant. This carries implications for prenatal counseling and maternal anxiety. Copyright © 2010 ISUOG. Published by John Wiley & Sons, Ltd.

The molecular genetic diagnosis of inherited metabolic disorders is challenging. The diseases are rare, and most show locus heterogeneity. Hence, testing of the genes associated with IMDs is time consuming and often not easily available. We report a resequencing array that allows the simultaneous resequencing of up to 92 genes associated with IMDs. To validate the array, DNA samples from 51 patients with 52 different known variants (including point variants, small insertion, and deletions [indels]) in seven genes (C140RF133, GAA, NPC1, NPC2, VPS33B, WFS1, and SLC19A2) were amplified by PCR and hybridized to the array. A further patient cohort with 48 different mutations in NPCI. were analyzed blind. Out of 76 point variants, 73 were identified using automated software analysis followed by manual review. Ten insertion and deletion variants were detected in the extra tiling using mutation specific probes, with 11 heterozygous deletions and 3 heterozygous insertions. In summary, we identified 96% (95% confidence interval [CI] 89-99%) of point variants added to the array, but the pickup rate reduced to 83% (95% CI 75-89%) when insertions/deletions were included. Although the methodology has strengths and weaknesses, application of this technique could expedite diagnosis in most patients with multilocus IMDs. Hum Mutat 31:858-865,2010. (C) 2010 Wiley-Liss, Inc.

Distal deletion of chromosome 3p25-pter (3p- syndrome) produces a distinct clinical syndrome characterized by low birth weight, mental retardation, telecanthus, ptosis, and micrognathia. Congenital heart disease (CHD), typically atrioventricular septal defect (AVSD) occurs in about a third of patients. Previously we reported on an association between the presence of CHD and the proximal extent of the deletion such that a CHD susceptibility gene was mapped between D3S1263 and D3S3594. In addition, we and others have suggested several candidate genes for the psychomotor retardation usually seen with constitutional 3p25 deletions. In order to further investigate genotype-phenotype correlations in 3p- syndrome we analyzed 14 patients with cytogenetically detectable deletions of 3p25 (including one patient with a normal phenotype) using Affymetrix 250K SNP microarrays. Deletion size varied from approximately 6 to 12 Mb. Assuming complete penetrance, a candidate critical region for a CHD susceptibility gene was refined to approximately 200 kb and a candidate critical region for mental retardation was mapped to an approximately 1 Mb interval containing SRGAP3 but other 3p neurodevelopmental genes including CHL1, CNTN4, LRRN1, and ITPR1 mapped outside the candidate critical interval. We suggest that current evidence suggests that SRGAP3 is the major determinant of mental retardation in distal 3p deletions.

We report a case of anaplastic large cell lymphoma (ALCL) showing aberrant expression of β subunit of human chorionic gonadotrophin (β-HCG). The patient was a 14-year-old boy who presented with a right inguinal mass and a raised serum β-HCG level. Biopsy of the mass revealed a malignant neoplasm composed of large, pleomorphic cells with prominent nucleoli. These malignant cells showed positive staining with CD30, ALK, epithelial membrane antigen, and β-HCG. Chromosomal analysis showed t(2;5)(p23;q35) translocation, and polymerase chain reaction demonstrated T-cell receptor gene rearrangement. The patient did not respond well to chemotherapy, and he died 8 months after the diagnosis. To the best of our knowledge, this is the 1st case of ALCL showing aberrant expression of β-HCG and associated with a raised serum level of β-HCG. We report this case to bring awareness of this presumably rare occurrence to avoid the risk of misdiagnosis.

Copy number variations are a common cause of intellectual disability (ID). Determining the contribution of copy number variants (CNVs), particularly gains, to disease remains challenging. Here, we report four males with ID with sub-microscopic duplications at Xp11.2 and review the few cases with overlapping duplications reported to date. We established the extent of the duplicated regions in each case encompassing a minimum of three known disease genes TSPYL2, KDM5C and IQSEC2 with one case also duplicating the known disease gene HUWE1. Patients with a duplication encompassing TSPYL2, KDM5C and IQSEC2 without gains of nearby SMC1A and HUWE1 genes have not been reported thus far. All cases presented with ID and significant deficits of speech development. Some patients also manifested behavioral disturbances such as hyperactivity and attention-deficit/hyperactivity disorder. Lymphoblastic cell lines from patients show markedly elevated levels of TSPYL2, KDM5C and SMC1A, transcripts consistent with the extent of their CNVs. The duplicated region in our patients contains several genes known to escape X-inactivation, including KDM5C, IQSEC2 and SMC1A. In silico analysis of expression data in selected gene expression omnibus series indicates that dosage of these genes, especially IQSEC2, is similar in males and females despite the fact they escape from X-inactivation in females. Taken together, the data suggest that gains in Xp11.22 including IQSEC2 cause ID and are associated with hyperactivity and attention-deficit/hyperactivity disorder, and are likely to be dosage-sensitive in males.

Neuroblastoma is the most common extracranial solid malignancy in children. The disease possesses a broad range of clinical phenotypes with widely varying prognoses. Numerous studies have sought to identify the associated genetic abnormalities in the tumour, resulting in the identification of useful prognostic markers. In particular, the presence of multiple copies of the MYCN oncogene (referred to as MYCN amplification) has been found to confer a poor prognosis. However, the molecular pathways involved are as yet poorly defined. Metabolite profiles generated by in vitro (1)H MRS provide a means of investigating the downstream metabolic consequences of genetic alterations and can identify potential targets for new agents. Thirteen neuroblastoma cell lines possessing multiple genetic alterations were investigated; seven were MYCN amplified and six MYCN non-amplified. In vitro magic angle spinning (1)H MRS was performed on cell suspensions, and the spectra analysed to obtain metabolite concentration ratios relative to total choline (tCho). A principal component analysis using these concentration ratios showed that MYCN-amplified and non-amplified cell lines form separate classes according to their metabolite profiles. Phosphocholine/tCho and taurine/tCho were found to be significantly raised (p < 0.05) and glycerophosphocholine/tCho significantly reduced (p < 0.05) in the MYCN-amplified compared with the MYCN non-amplified cell lines (two-tailed t test). (1)H MRS of the SH-EP1 cell line and an isogenic cell line transfected with the MYCN oncogene also showed that MYCN oncogene over-expression causes alterations in phosphocholine, glycerophosphocholine and taurine concentrations. Molecular pathways of choline and taurine metabolism are potential targets for new agents tailored to MYCN-amplified neuroblastoma.

Background Adoption of new technology in both basic research and clinical settings requires rigorous validation of analytical performance. The OncoScan® FFPE Assay is a multiplexing tool that offers genome-wide copy number and loss of heterozygosity detection, as well as identification of frequently tested somatic mutations. Methods In this study, 162 formalin fixed paraffin embedded samples, representing six different tumour types, were profiled in triplicate across three independent laboratories. OncoScan® formalin fixed paraffin embedded assay data was then analysed for reproducibility of genome-wide copy number, loss of heterozygosity and somatic mutations. Where available, somatic mutation data was compared to data from orthogonal technologies (pyro/sanger sequencing). Results Cross site comparisons of genome-wide copy number and loss of heterozygosity profiles showed greater than 95% average agreement between sites. Somatic mutations pre-validated by orthogonal technologies showed greater than 90% agreement with OncoScan® somatic mutation calls and somatic mutation concordance between sites averaged 97%. Conclusions Reproducibility of whole-genome copy number, loss of heterozygosity and somatic mutation data using the OncoScan® assay has been demonstrated with comparatively low DNA inputs from a range of highly degraded formalin fixed paraffin embedded samples. In addition, our data shows examples of clinically-relevant aberrations that demonstrate the potential utility of the OncoScan® assay as a robust clinical tool for guiding tumour therapy. Electronic supplementary material The online version of this article (doi:10.1186/s12920-015-0079-z) contains supplementary material, which is available to authorized users.

Neuroblastoma is a heterogeneous tumour with a variety of clinical phenotypes, ranging from a localised tumour with excellent outcome (stage 1) to a metastatic, usually fatal malignancy (stage 4). In order to investigate the genetic relationship between these tumour subtypes, a loss of heterozygosity (LOH) analysis was carried out. Composite LOH allelotypes incorporating data from 96 loci on 5 chromosomes (1p, 3p, 4p, 11q, 14q), were constructed for 62 neuroblastomas. Neuroblastomas with similar allelotypes were clustered into groups and allelotype patterns correlated with clinical features. Three distinct genetic subgroups of neuroblastoma were observed. The largest group (50% of tumours) was characterised by specific allelotype patterns indicative of a stepwise accumulation of genetic alterations (11q LOH → 1p, 4p, and/or 14q LOH → 3p LOH), associated with progression from low to high stage disease. These tumours are distinct from MYCN amplified neuroblastomas which have a more rapid and aggressive disease course, and also a proportion of low stage tumours, often ganglioneuromas or ganglioneuroblastomas, with restricted growth potential.

Prenatal diagnosis aids parental decision making. Diagnostic testing in pregnancies with abnormal ultrasound scan findings is rapidly evolving from karyotyping to chromosomal microarray analysis (C...

Background After allogeneic hematopoietic stem cell transplantation, donor T cells interact with an antigen-presenting cell environment that is distorted in number, level of activation, and origin. The role of antigen presentation in the development of chronic graft-versus host disease (cGVHD) is unknown. Methods The number and origin of peripheral blood immature myeloid (CD19- CD1c+) and plasmacytoid (BDCA-2+) dendritic cells (DCs) was determined in 30 patients at more than 100 days after allogeneic hematopoietic stem cell transplantation. Results Patients with cGVHD had significantly higher plasmacytoid DC numbers than individuals without this complication (9.1+/-2.0 x 10(6)/L versus 3.8+/-0.6 x 10(6)/L, =0.025). Chimerism studies demonstrated that DCs in patients with cGVHD were exclusively of donor origin, whereas persistence of host DCs was observed in some control patients. Conclusions The antigen-presenting cell environment in patients with cGVHD, as represented by immature blood DCs, is of donor origin but distorted in terms of subset distribution.

Axenfeld-Rieger syndrome (ARS) is an autosomal dominant disorder with variable expressivity. It is characterized by dysgenesis of the anterior segment of the eye together with dental, cardiac, and umbilical anomalies. There is a high incidence of secondary high tension glaucoma. It is a genetically heterogeneous condition due to deletion or mutations of FOXC1 (6p25) or PITX2 (4q25). We report on four unrelated patients with overlapping microdeletions encompassing PITX2 at 4q25. We compare the genotypes and phenotypes of these newly described ARS patients and discuss the involvement of contiguous genes. Patients 1, 2, and 3 had mild learning difficulties, not typically seen in patients with ARS. We implicate the adjacent neuronally expressed genes; NEUROG2, UGT8, NDST3, and PRSS12 as potentially causal. Our findings support the use of microarray analysis in ARS patients for full prognostic information in infants presenting with ARS-like phenotypes.

Chromosomal Microarray Analysis (CMA) has a higher detection rate of pathogenic chromosome abnormalities over conventional (G-band) karyotyping. The optimum resolution of CMA in the prenatal setting remains debatable. Our objective was to determine if an increased detection rate was obtained by performing differing resolution of CMA on the same fetal samples and whether this resulted in an increase in variants of uncertain clinical significance (VOUS).Sixty-two fetal cases initially underwent a 1 Mb targeted BAC microarray within a clinical diagnostic setting in addition to conventional karyotyping. At the conclusion of pregnancy, a higher resolution 60 K oligonucleotide microarray was performed.The 1 Mb BAC analysis demonstrated a detection rate of pathogenic copy number variations (CNVs) in 4.1% (95% CI 2.1-7.6) of cases and a variation of unknown significance (VOUS) rate of 0.4% (95% CI 0.07-2.2) over conventional G-band karyotyping. The 60 K array had an additional pathogenic detection rate of 4.8% (95% CI 1.6-13.3) over the BAC array but also detected an additional 8% (95% CI 1.3-14.8) VOUS.As the CMA platform resolution increases detection rates increase but are associated with an increase in VOUS rates. Our findings support the need for further large scale studies to inform the national consensus on the resolution required and on reporting of VOUS in the antenatal setting.

Background Approximately 50% of spontaneous miscarriages are associated with chromosome abnormalities. Identification of these karyotypic abnormalities helps to estimate recurrence risks in future pregnancies. Chromosomal microarray analysis (CMA) is transforming clinical cytogenetic practice with its ability to examine the human genome at increasingly high resolution. Objectives The aim of this study was to determine whether CMA testing on the products of conception following miscarriage provides better diagnostic information compared with conventional karyotyping. Search strategy MEDLINE (from 1996 to December 2012), EMBASE (from 1974 to December 2012), and CINAHL (from 1996 to December 2012) databases were searched electronically. Selection criteria Studies were selected if CMA was used on products of conception following miscarriage, alongside conventional karyotyping. Data collection and analysis Nine papers were included in the systematic review and meta-analysis. All statistical analyses were performed using stata 11.0 (Stata Corp., College Station, TX, USA). Main results There was agreement between CMA and karyotyping in 86.0% of cases (95% CI 77.0–96.0%). CMA detected 13% (95% CI 8.0–21.0) additional chromosome abnormalities over conventional full karyotyping. In addition, traditional, full karyotyping detected 3% (95% CI 1.0–10.0%) additional abnormalities over CMA. The incidence of a variant of unknown significance (VOUS) being detected was 2% (95% CI 1.0–10.0%). Author's conclusions Compared with karyotyping, there appears to be an increased detection rate of chromosomal abnormalities when CMA is used to analyse the products of conception; however, some of these abnormalities are VOUS, and this information should be provided when counselling women following miscarriage and when taking consent for the analysis of miscarriage products by CMA.

Holt–Oram syndrome (HOS) is a rare autosomal dominant heart–hand syndrome due to mutations in the TBX5 transcription factor. Affected individuals can have structural cardiac defects and/or conduction abnormalities, and exclusively upper limb defects (typically bilateral, asymmetrical radial ray defects). TBX5 mutations reported include nonsense, missense, splicing mutations and exon deletions. Most result in a null allele and haploinsufficiency, but some impair nuclear localisation of TBX5 protein or disrupt its interaction with co-factors and downstream targets. We present a five generation family of nine affected individuals with an atypical HOS phenotype, consisting of ulnar ray defects (ulnar hypoplasia, short fifth fingers with clinodactyly) and very mild radial ray defects (short thumbs, bowing of the radius and dislocation of the radial head). The cardiac defects seen are those more rarely reported in HOS (atrioventricular septal defect, hypoplastic left heart syndrome, mitral valve disease and pulmonary stenosis). Conduction abnormalities include atrial fibrillation, atrial flutter and sick sinus syndrome. TBX5 mutation screening (exons 3–10) identified no mutations. Array comparative genomic hybridisation (CGH) revealed a 48 kb duplication at 12q24.21, encompassing exons 2–9 of the TBX5 gene, with breakpoints within introns 1–2 and 9–10. The duplication segregates with the phenotype in the family, and is likely to be pathogenic. This is the first known report of an intragenic duplication of TBX5 and its clinical effects; an atypical HOS phenotype. Further functional studies are needed to establish the effects of the duplication and pathogenic mechanism. All typical/atypical HOS cases should be screened for TBX5 exon duplications.

A proportion of cytogenetic abnormalities in myelodysplastic syndromes (MDS) and acute myeloid leukaemia (AML) may escape detection by high-resolution genomic technologies, but can be identified by conventional cytogenetic and molecular analysis. Here, we report the detection of a reciprocal translocation t(7;21)(p22;q22) in the marrow of two adults with MDS and AML, using conventional cytogenetic analysis and fluorescence-in situ-hybridization (FISH). Reverse-transcription polymerase chain reaction (RT-PCR) and sequence analysis identified a fusion between RUNX1 and the gene encoding ubiquitin specific peptidase-42 (USP42), with splice-variants and variable break-points within RUNX1. Combined cytomorphology and FISH studies in MDS marrow revealed abnormal RUNX1 signals within megakaryocytes, suggesting that the acquisition of t(7;21)(p22;q22) does not confer complete differentiation arrest and may represent an early genetic event in leukaemogenesis. Single nucleotide polymorphism-arrays failed to detect additional sub-microscopic genomic changes predisposing to or associated with t(7;21). Molecular analysis of 100 MDS and AML marrow specimens by RT-PCR did not reveal new cases with the RUNX1-USP42 fusion. Thus, our studies have identified t(7;21)(p22;q22) as a rare but recurrent abnormality in MDS/AML, with the existence of alternative spliced forms of the RUNX1-USP42 transcript in different patients. Further studies are required to identify the potential contribution of these splice-variants to disease heterogeneity.

Objectives Copy number (CN) and somatic mutation (SM) analysis in tumors is rapidly gaining importance in cancer management as a tool for differential diagnosis, determination of prognosis, and selection of therapeutic. Genome-wide copy number and LOH detection as well as a panel of frequently tested somatic mutations can be detected with OncoScan® FFPE Assay Kit. We report on a validation study of OncoScan FFPE Assay Kit CN and SM data by orthogonal technologies (FISH and NGS, respectively) to estimate the sensitivity and specificity parameters of the platform. In addition, we assessed the reproducibility of the platform across three different sites in the UK: Leeds Institute of Cancer and Pathology (LICP), West Midlands Regional Genetics Laboratory (WMRGL), and Almac Diagnostics (Almac). Methods Validation of CN data was performed on a cohort of cancer samples identified as Her2 positive/ambiguous by FISH. The panel of SMs available was validated by a custom targeted amplicon NGS panel on a diverse collection of samples derived from multiple cohorts across several tumor types sourced from both LICP and WMRGL. For the reproducibility study, 162 samples encompassing six different tumor types (breast, colorectal, lung, melanoma, prostate, and ovarian) were collected from LICP and WMRGL. DNA was extracted and plated in triplicate and distributed to the three testing sites: LICP, WMRGL, and Almac. Data from all sites was analyzed for reproducibility of genome-wide CN/LOH calls and SM calls. Results Cross-site comparisons of genome-wide CN and LOH profiles on 162 FFPE solid tumor samples showed greater than 95% average agreement between three sites (LICP, WMGRL, and Almac), while SM classification concordance between the sites averaged 97%. Initial orthogonal validation of Her2 amplification by FISH showed greater than 90% concordance, as did initial test samples used for validating OncoScan SM calls by a targeted amplicon NGS panel. Conclusion In this study we validated both CN and SM calls using OncoScan FFPE Assay Kit and demonstrated a high degree of agreement with orthogonal methods in all aspects. Reproducibility of whole-genome CN, LOH, and SM data using OncoScan FFPE Assay Kit has also been demonstrated for a range of FFPE samples, including highly degraded samples. This study is a step forward in evaluating the potential clinical utility of a platform combining genome-wide copy number and somatic mutation calls within the national health service of the UK. Citation Format: Joseph M. Foster, Assa Oumie, Fiona S. Togneri, Morag Taylor, Sofia Alyas, Paula Wojtowicz, Henry Wood, Emma Tinkler-Hundal, Katie Southward, Dominic McMullan, Phil Quirke, Katherine E. Keating, Mike Griffiths, Karen G. Spink, Fiona Brew, Eric Fung, Jeanette Schmidt. Cross-site reproducibility and orthogonal validation of copy number and somatic mutation calls of OncoScan® FFPE Assay Kit in solid tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 626. doi:10.1158/1538-7445.AM2015-626

Ewing's tumors are rare pediatric neoplasms that are characterized by specific chromosomal translocations and gene rearrangements. All of the fusion genes reported to date in Ewing's tumors juxtapose the EWS gene at 22q12 to an ETS-related gene, the most common of which are FLI1 at 11q24 and ERG at 21q22. We present here four cases of Ewing's tumor, which showed no evidence of a EWS gene rearrangement, but instead contained translocations involving 16p11 and 21q22. A rearrangement involving the same chromosome bands, t(16;21)(p11;q22), is found in rare cases of acute myeloid leukemia and fuses the FUS gene at 16p11 to the ERG gene at 21q22. In two of our Ewing's tumor cases, we were able to show at the sequence level that the translocation between chromosomes 16 and 21 similarly results in a FUS/ERG fusion. In one case, exons 1-5 and most of exon 6 of FUS were fused in-frame to exon 9 of ERG; in the other case, FUS exons 1-7 were fused in-frame to ERG exons 8-9. The functional fusion transcript is expected to be expressed from the der(21)t(16;21) derivative. In the two other t(16;21)-positive Ewing's cases, we performed bacterial artificial chromosome fluorescence in situ hybridization analysis on metaphases and interphase nuclei to demonstrate colocalization of bacterial artificial chromosomes containing FUS and ERG genes, also highly suggestive of fusion gene formation. These represent the first four cases where FUS, rather than EWS, is rearranged with an ETS-family transcription factor in Ewing's tumors. Our data provide additional evidence that the transactivation domains of the TET family of RNA-binding proteins (such as EWS and FUS) are interchangeable, and suggests a novel mechanism of oncogenesis in Ewing's tumors.