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Objective: Gene expression profiles are associated with the clinical heterogeneity of systemic lupus erythematosus (SLE) but are not well studied as biomarkers for therapy. We studied gene expression and response to rituximab in a multiethnic UK cohort who were refractory to standard therapy. Methods: We evaluated baseline expression levels of transcripts known to associate with clinical features of SLE using a 96‐probe TaqMan array and whole blood samples from 213 patients with active SLE who had been prospectively enrolled in the British Isles Lupus Assessment Group (BILAG) Biologics Register. We measured autoantibodies using immunoprecipitation and enzyme‐linked immunosorbent assays. We determined responses to first‐cycle rituximab at 6 months from treatment start in 110 SLE patients by assessing BILAG 2004 disease activity. Results: Interferon gene expression scores were lower in patients of European ancestry than in all other ancestry groups. The relationship between blood interferon gene expression scores and scores annotated to plasmablasts, neutrophils, myeloid lineage, inflammation, and erythropoiesis differed between patients of European and non‐European ancestries. Hierarchical clustering revealed 3 distinct non‐European ancestry patient subsets with stratified responses to rituximab that were not explained by sociodemographic and clinical variables, with responses lowest in an interferon‐low, neutrophil‐high cluster and highest in a cluster with high expression levels across all signatures (P < 0.001). Clusters in European ancestry patients did not predict response to rituximab but segregated patients by global disease activity and renal involvement. In both ancestral groups, interferon‐high clusters were associated with U1 RNP/Sm antibodies. Conclusion: Ancestry appears central to the immunologic and clinical heterogeneity in SLE. These results suggest that ancestry, disease activity, and transcriptional signatures could each assist in predicting the effectiveness of B cell depletion therapies. [ABSTRACT FROM AUTHOR]

Background: Thymic squamous cell carcinoma and type B3 thymoma are primary neoplasms of the anterior mediastinum that are sometimes difficult to differentiate from one another histologically. However, only a few immunohistochemical markers are available for the differential diagnosis. The purpose of this study was to discover a novel marker for differentiating between thymic squamous cell carcinoma and type B3 thymoma. Methods: We used histological samples of thymic carcinomas (n = 26) and type B3 thymomas (n = 38) which were resected between 1986 and 2017. To search for candidates of differential markers, gene expression levels were evaluated in samples using promoter analysis by cap analysis of gene expression (CAGE) sequencing. Results: Promoter level expression of CALML5 genes was significantly higher in thymic carcinomas than in type B3 thymomas. We further validated the results of the CAGE analysis in all 26 thymic carcinomas and 38 type B3 thymomas by immunohistochemistry (IHC). CALML5 was strongly expressed in the cytoplasm in 19 of 26 cases with thymic carcinoma, whereas positivity at the protein level was shown in two of 38 type B3 thymomas. Thus, the sensitivity (73.1%) and specificity (94.7%) of CALML5 as markers for immunohistochemical diagnosis of thymic carcinoma were extremely high. Conclusion: We identified CALML5 as a potential marker for differentiating thymic squamous cell carcinoma from type B3 thymoma. It is assumed that future clinical use of CALML5 may improve the diagnostic accuracy of differentiating between these two diseases. [ABSTRACT FROM AUTHOR]

Islet transplantation from organ donors can considerably improve glucose homeostasis and well‐being in individuals with type 1 diabetes, where the beta cells are destroyed by the autoimmune attack, but there are insufficient donor islets to make this a widespread therapy. Strategies are therefore being developed to generate unlimited amounts of insulin‐producing beta cells from pluripotent stem cells, with the aim that they will be transplanted to treat diabetes. Whilst much progress has been made in recent years in the directed differentiation of pluripotent stem cells to beta‐like cells, essential gaps still exist in generating stem cell‐derived beta cells that are fully functional in vitro. This short review provides details of recent multi‐'omics' studies of the human fetal pancreas, which are revealing granular information on the various cell types in the developing pancreas. It is anticipated that this fine mapping of the pancreatic cells at single‐cell resolution will provide additional insights that can be utilised to reproducibly produce human beta cells in vitro that have the functional characteristics of beta cells within native human islets. [ABSTRACT FROM AUTHOR]

This case study about an infant with epidermolysis bullosa and his fourteen‐year‐old mother involves pediatric genomic testing and concerns about the adolescent parent's decision‐making capacity. Four commentaries explore ethics issues in the case, including the following challenges: The current ethics guidelines regarding informed decision‐making for genomic testing and return of results have been relatively silent on how to involve parents who are minors in decision‐making on behalf of their children. This lack of guidance can be particularly difficult for physicians when genetic test results revealing consanguinity raise concerns about sexual abuse of a minor, provoking questions about mandatory reporting requirements. Finally, medical teams may find themselves having to evaluate an adolescent parent's capacity in making medical decisions for her child while questioning the role and relationship of her support person. [ABSTRACT FROM AUTHOR]

Background: The recent introduction of microarrays for genetic analyses has allowed higher etiological diagnostic rates in patient with intellectual disability (ID), autism spectrum disorders (ASD), epilepsy and multiple congenital anomalies (MCA), because of its resolution. This approach still results of high complexity and some limitations have been reported. In fact, it discloses several variants of unknown significance (VOUS) or incidental findings. In all cases, a massive amount of data is generated, because of this, the analysis and the interpretation is very difficult and often without a definitive conclusion. Method: We analysed an Italian cohort of 343 patients with ID, MCA and ASD by array‐comparative genomic hybridization. The purpose of this work was to consider the proportion of the chromosomal abnormalities in such cohort and to assess the distribution of the different type of the chromosomal abnormalities concerning their pathogenic significance, their origin and their correlation to these clinical phenotypes. Results: Array‐comparative genomic hybridization analysis revealed 76 positive results. Abnormalities were detected in 27.8% of patients with ID, 11.1% with ASD, 10.7% with epilepsy and 19.4% with multiple congenital anomalies. The anomalies were classified in three major groups: group 1 (27 patients) with pathogenic alterations (P group); group 2 (34 patients) with VOUS potentially pathogenic (PP group); and group 3 (13 patients) with VOUS potentially benign (PB group). As expected, comparing the diagnostic groups, we observed a greater number of deletions in the P group and that all the abnormalities of the PB group were inherited. Conclusions: Our retrospective study resulted in confirming the high detection rate of microarrays. CNV classification remains a complex procedure. The difficulty in CNV classification points out the importance of the patient selection, helping the interpretation of the molecular cytogenetic results. [ABSTRACT FROM AUTHOR]

Quantification of DNA methylation in neoplastic cells is crucial both from mechanistic and diagnostic perspectives. However, such measurements are prone to different experimental biases. Polymerase chain reaction (PCR) bias results in an unequal recovery of methylated and unmethylated alleles at the sample preparation step. Post‐PCR biases get introduced additionally by the readout processes. Correcting the biases is more practicable than optimising experimental conditions, as demonstrated previously. However, utilisation of our earlier developed algorithm strongly necessitates automation. Here, we present two R packages: rBiasCorrection, the core algorithms to correct biases; and BiasCorrector, its web‐based graphical user interface frontend. The software detects and analyses experimental biases in calibration DNA samples at a single base resolution by using cubic polynomial and hyperbolic regression. The correction coefficients from the best regression type are employed to compensate for the bias. Three common technologies—bisulphite pyrosequencing, next‐generation sequencing and oligonucleotide microarrays—were used to comprehensively test BiasCorrector. We demonstrate the accuracy of BiasCorrector's performance and reveal technology‐specific PCR‐ and post‐PCR biases. BiasCorrector effectively eliminates biases regardless of their nature, locus, the number of interrogated methylation sites and the detection method, thus representing a user‐friendly tool for producing accurate epigenetic results. [ABSTRACT FROM AUTHOR]

BACKGROUND Fabricating oligonucleotide microarrays with high immobilization efficiency and sensitivity by a simple surface modification route remains highly desirable in the biochip field. Here we develop a facile method to prepare a 3D crosslinking thin hydrogel film on glass slides for high‐density immobilization of DNA probes. RESULTS: Thin hydrogel film using poly (ethylene glycol) diacrylate (PEGDA) as crosslinker and glycidyl methacrylate (GMA) as functional monomer was attached on vinyl group‐modified glass slides by a photopolymerization technique. The effect of monomer ratios (PEGDA:GMA) on the surface hydrophilicity of the hydrogel layer and the immobilization density of DNA probes were investigated and an optimal monomer ratio of 10:1 and te maximum immobilization density of 1.4 pmol cm–2 were found. Adjusting the hydrophilicity of the hydrogel film effectively suppressed its swelling and produced a typical dense but not porous structure with a flat surface favorable for printing microarrays. The maximum immobilization density and efficiency of oligonucleotide probes on the 3D hydrogel film were 1.4 pmol cm–2 and 83%, respectively, which are superior values to those on a 1D support. The microarray had a wide detection range of target nucleotides from 15.3 pmol L–1 to 62.5 nmol L–1. In addition, the hybridization assay demonstrated that the microarray showed high sensitivity with a limit of detection of 30.5 pmol L–1. CONCLUSION: The poly (PEGDA‐co‐GMA) hydrogel film could be grafted on the glass slide by the photopolymerization technique and the DNA probe was effectively immobilized on the thin hydrogel by reacting with the epoxy groups. This strategy of attaching a thin cross‐linked poly(PEGDA‐co‐GMA) hydrogel layer on glass slides has promising prospects in the fields of bioanalysis and microarrays. © 2021 Society of Chemical Industry (SCI). [ABSTRACT FROM AUTHOR]

Background and Aim: Direct‐acting antiviral (DAA) therapies have been proven to be highly effective for the eradication of hepatitis C virus (HCV) without resistance‐associated substitutions (RASs). However, even in cases with no detected RASs, treatment sometimes fails, suggestive of the existence of some host‐related factors involved in HCV eradication by DAAs. To explore such factors, we analyzed the serum microRNAs (miRNAs) of patients who received DAA treatment. Methods: The serum miRNA expression levels of 39 patients with chronic HCV infection without any detectable RASs, who achieved sustained virological response with asunaprevir/daclatasvir or grazoprevir/elbasvir therapy, were investigated cyclopedically, using oligonucleotide microarrays. The effects of specific miRNAs on the replication of HCV were measured in the HCV genomic replicon containing Huh‐7 hepatoma cells. Results: Along with the disappearance of HCV, the expression quantiles of 16 miRNAs in the asunaprevir/daclatasvir group and 18 miRNAs in the grazoprevir/elbasvir group showed a tendency to increase or decrease. Among these molecules, adjustments for multiple testing yielded a significant differential expression at a false discovery rate of less than 5% for only one molecule, hsa‐miR‐762. Its expression quantile increased after HCV exclusion in all patients who had achieved sustained virological response. Quantitative polymerase chain reaction analysis validated a significant increase in the serum hsa‐miR‐762 after disappearance of HCV. On the contrary, hsa‐miR‐762 was decreased in the relapse and breakthrough of HCV in DAA failures. Transfection of hsa‐miR‐762 into cultured HCV‐infected hepatocytes significantly decreased HCV‐RNA replication. Conclusion: These data suggest that hsa‐miR‐762 is one of the host factors participating in HCV exclusion by DAA therapy. [ABSTRACT FROM AUTHOR]

Aim: Cardiac aging, which causes cardiac diastolic dysfunction, frequently occurs in older people. The role of autophagy in cardiac aging is the subject of intensive research. Autophagy comprises steps called the autophagosome formation and autophagosome–lysosome fusion. Caloric restriction (CR) is the gold standard used to induce autophagosome formation, and autophagosome–lysosome fusion is reduced by aging. However, few studies are available that survey and compare signaling during CR (autophagosome formation induced status) and old (potentially autophagosome–lysosome fusion‐reduced status). Here we aimed to identify the rate‐limiting step of autophagic disorders during cardiac aging. Methods: We employed bioinformatics to analyze publicly available DNA microarray datasets. The first dataset compared the hearts of young and old C57BL6 mice (OLD). The second dataset compared the hearts of young C57BL6 mice fed a normal diet with those of young C57BL6 mice subjected to CR. Results: We analyzed OLD‐upregulated genes that were significantly associated with the Gene Ontogeny term "Autophagy," indicating that autophagic genes were upregulated in OLD mice. The autophagy‐related gene Atg5 and Atg5‐related genes were upregulated in OLD and CR mice. The identified hub and bottleneck genes are autophagic autophagosome formation suppressors such as Sirt2, Ilk and Islr, as well as the autophagosome–lysosome fusion inducer Snapin. Conclusions: Autophagosome formation genes were upregulated in aging mice subjected to CR, indicating that an upregulated autophagosome formation is not a change specific to cardiac aging. However, autophagosome–lysosome fusion genes, particularly the lysosome transportation‐related gene Snapin, were downregulated in aging, indicating that autophagosome–lysosome fusion may cause autophagic disorders in cardiac aging. Geriatr Gerontol Int 2021; 21: 108–115. [ABSTRACT FROM AUTHOR]

Introduction: MicroRNAs (miRNAs) play a critical role in orchestrating T cell differentiation and activation and may thus play a vital role in acquired aplastic anemia (aAA). The study aimed to evaluate the differential expression of selected miRNAs and their relevant target genes in bone marrow samples of aAA patients. Methods: Differential expression of 8 miRNAs viz; hsa‐miR‐126‐3p, miR‐145‐5p, miR‐155‐5p, miR‐150‐5p, miR‐146b‐5p, miR‐34a, miR‐29a, and miR‐29b was evaluated in the bone marrow mononuclear cells of aAA patients. TaqMan microRNA assay was performed for preparing the cDNA of specific miRNA, followed by expression analysis using qRT‐PCR. Data were normalized using two endogenous controls, RNU6B and RNU48. Delta‐delta CT method was used to calculate the fold change (FC) of miRNA expression in individual samples, and a FC of >1.5 was taken as significant. Target genes of these miRNAs were evaluated by qRT‐PCR. Results: Thirty five samples of aAA patients and 20 controls were evaluated. Irrespective of the disease severity, five miRNAs were found to be deregulated; miR‐126 (FC‐0.348; P‐value‐.0001) and miR‐145 (FC‐0.31; P‐value‐.0001) were downregulated, while miR‐155 (FC‐3.50; P‐value‐.0067), miR‐146 (FC‐3.13; P‐value‐.0105), and miR‐150 (FC‐5.78; P‐value‐.0001) were upregulated. Target gene study revealed an upregulation of PIK3R2, MYC, SOCS1, and TRAF‐6, and downregulation of MYB. Conclusion: This is the first study from the Indian subcontinent demonstrating the presence of altered miRNA expression in the bone marrow samples of aAA patients, suggesting their role in the pathogenesis of the disease. A comprehensive study focusing on the effect of these miRNA‐mRNA interactions is likely to open new avenues of management. [ABSTRACT FROM AUTHOR]

Background: In chronic lymphocytic leukemia, a better understanding of leukemic cell characteristics after treatment would help to design specific therapeutic approaches aimed at preventing clinical relapse. Gene arrays have become a powerful approach to perform gene expression profiling; nevertheless, to work with residual cells entails an intensive labor. The aim of this study was to set forth an effective gene expression approach to analyze residual leukemic cells. Methods: Leukocytes from CLL patient's samples were sorted by flow cytometry using a 6‐color panel. The quality and quantity of RNA isolated from different inputs of cells were compared by two silica column protocols: RNeasy Micro and RNeasy Mini. RNA amplifications were carried out according to two manufacturer's protocols: Ovation Pico SL and Ovation Pico WTA. A total of 3.5 μg of cDNA was labeled and hybridized to Human Gene 2.0 ST arrays. Results: RNA extracted from low number of input cells by RNeasy Micro showed similar RNA integrity number to that obtained from RNeasy Mini; however, the RNA quantity was higher using the RNeasy Micro Kit. In addition, those RNA samples obtained with RNeasy Micro and amplified with Ovation Pico WTA showed good quality to proceed for a gene array study, independently of the number of input cells (range: 1 × 104‐5 × 105 cells). Conclusions: We observed that this workflow is a feasible approach to obtain genomic material extracted from leukemic cells as little as 1 × 104 cells and it can be useful to carry out gene expression profile experiments to characterize residual leukemic cells in chronic lymphocytic leukemia. [ABSTRACT FROM AUTHOR]

Background: In patients with squamous cell carcinoma of the cervix (SCCC), a squamous cell carcinoma (SqCC) in the lung represents either a second primary tumor or metastasis. This distinction between second primary tumors and lung metastases in patients with SCCC significantly influences patient prognosis and therapy. Here, we aimed to differentiate second primary tumors from lung metastases in patients with SCCC by exploring the HPV status in SqCCs involving the lung within a large cohort. Methods: P16 expression was assessed using immunohistochemistry on tissue microarrays including a total of 415 primary lung SqCCs and 21 lung SqCCs with prior SCCC. Following this, we performed HPV DNA typing and the sensitive RNAscope in situ method to screen all the cases for HPV E6/E7 expression, which is a more reliable indicator of transcriptively active HPV in tumor cells. Results: The p16 positive expression rate was 13.7% (57/415) in primary lung SqCCs, but HPV DNA was not detected in any of the 57 primary lung SqCC cases that positively expressed p16. In contrast, HPV DNA was detected in all cases (21/21) with prior SCCC. Consistently, all 21 lung SqCCs with prior SCCC (21/21) showed extensive HPV16 E6/E7 expression. In striking contrast, none of the primary lung SqCCs (0/415) had a detectable RNAscope signal. Conclusions: HPV does not seem to play a role in the development of primary lung SqCCs. HPV detection may be helpful in distinguishing second primary tumors from lung metastases in patients with SCCC. [ABSTRACT FROM AUTHOR]

HIV resistance genotyping detects drug resistance mutations (DRMs) in ≥20% of circulating virus within an infected individual (high‐abundance DRMs). Deep sequencing also detects DRMs in smaller viral subpopulations (low‐abundance DRMs), although these are of uncertain importance. In this retrospective analysis of 292 treatment‐naïve patients, high‐abundance DRMs were present in 30/292 (10%) patients, but only one (0.3%) had resistance to first‐line anti‐retrovirals. Low‐abundance DRMs were present in 36/247 (15%) patients, but none who received anti‐retrovirals for which these were present had virologic failure. These findings demonstrate that starting first‐line therapy in treatment‐naïve patients need not be delayed while awaiting resistance testing. [ABSTRACT FROM AUTHOR]

Background: Mitochondria play a fundamental role in the pathogenesis of alcoholic liver disease (ALD). The preservation of functional mitochondria during toxic alcohol insults is essential for cell survival and is maintained by key processes known as mitochondrial dynamics, including fragmentation and fusion, which are regulated by mitochondria‐shaping proteins (MSP). We have shown mitochondrial dynamics to be distorted by alcohol in cellular and animal models, but the effect in humans remains unknown. Methods: Hepatic gene expression of the main MSP involved in the mitochondrial fusion and fragmentation pathways was evaluated in patients with alcoholic hepatitis (AH) by DNA microarray (n = 15) and Reverse Transcription Polymerase Chain Reaction (n = 32). The activation of dynamin‐1‐like protein (Drp1) was also investigated in mitochondria isolated from liver biopsies of ALD patients (n = 8). The effects of alcohol on mitochondrial dynamics and on MSP protein expression were studied in human precision‐cut liver slices (PCLS) exposed for 24 hours to increasing doses of ethanol (EtOH; 50 to 250 mM). Results: A profound hyperactivation of the fragmentation pathway was observed in AH patients, with a significant increase in the expression of Drp1 and its adapters/receptors. The translocation of Drp1 to the mitochondria was also induced in patients with severe ALD and was affected in the PCLS with short‐term exposure to EtOH but only mildly. The fusion pathway was not altered in ALD, and this was confirmed in the PCLS model. Conclusions: The present study reveals the role of mitochondrial dynamics in human ALD, confirming our previous observations in animal and cell culture models of ALD. Taken together, we show that alcohol has a significant impact on the fragmentation pathway, and we confirm Drp1 as a potential therapeutic target in severe ALD. [ABSTRACT FROM AUTHOR]

Objective: IgG4‐related disease (IgG4‐RD) is a unique inflammatory disorder in which Th2 cytokines promote IgG4 production. In addition, recent studies have implicated the Toll‐like receptor (TLR) pathway. This study was undertaken to examine the expression of TLRs in salivary glands (SGs) from patients with IgG4‐RD. Methods: SGs from 15 patients with IgG4‐RD, 15 patients with Sjögren's syndrome (SS), 10 patients with chronic sialadenitis, and 10 healthy controls were examined histologically. TLR family gene expression (TLR‐1 through TLR‐10) was analyzed by DNA microarray in the submandibular glands (SMGs). Up‐regulation of TLRs was confirmed in SGs from patients with IgG4‐RD. Finally, the phenotype of human TLR‐7 (huTLR‐7)–transgenic C57BL/6 mice was assessed before and after stimulation with TLR agonist. Results: In patients with IgG4‐RD, TLR‐4, TLR‐7, TLR‐8, and TLR‐9 were overexpressed. Polymerase chain reaction validated the up‐regulation of TLR‐7 in IgG4‐RD compared with the other groups. Immunohistochemical analysis confirmed strong infiltration of TLR‐7–positive cells in the SGs of patients with IgG4‐RD. Double immunohistochemical staining showed that TLR‐7 expression colocalized with CD163+ M2 macrophages. After in vitro stimulation with a TLR‐7 agonist, CD163+ M2 macrophages produced higher levels of interleukin‐33 (IL‐33), which is a Th2‐activating cytokine. In huTLR‐7–transgenic mice, the focus and fibrosis scores in SMGs, pancreas, and lungs were significantly higher than those in wild‐type mice (P < 0.05). Moreover, the concentration of serum IgG, IgG1, and IL‐33 in huTLR‐7–transgenic mice was distinctly increased upon stimulation with a TLR‐7 agonist (P < 0.05). Conclusion: TLR‐7–expressing M2 macrophages may promote the activation of Th2 immune responses via IL‐33 secretion in IgG4‐RD. [ABSTRACT FROM AUTHOR]

Background: Global influenza surveillance in humans and animals is a critical component of pandemic preparedness. The FluChip‐8G Insight assay was developed to subtype both seasonal and potentially pandemic influenza viruses in a single assay with a same day result. FluChip‐8G Insight uses whole gene segment RT‐PCR‐based amplification to provide robustness against genetic drift and subsequent microarray detection with artificial neural network‐based data interpretation. Objectives: The objective of this study was to verify and validate the performance of the FluChip‐8G Insight assay for the detection and positive identification of human and animal origin non‐seasonal influenza A specimens. Methods: We evaluated the ability of the FluChip‐8G Insight technology to type and HA and NA subtype a sample set consisting of 297 results from 180 unique non‐seasonal influenza A strains (49 unique subtypes). Results: FluChip‐8G Insight demonstrated a positive percent agreement ≥93% for 5 targeted HA and 5 targeted NA subtypes except for H9 (88%), and negative percent agreement exceeding 95% for all targeted subtypes. Conclusions: The FluChip‐8G Insight neural network‐based algorithm used for virus identification performed well over a data set of 297 naïve sample results, and can be easily updated to improve performance on emerging strains without changing the underlying assay chemistry. [ABSTRACT FROM AUTHOR]

Objective: To assess the prevalence of the MYD88 L265P mutation and variants within NLRP3 and evaluate the status of oligoclonal hematopoiesis in 30 patients with Schnitzler syndrome (SchS). Methods: Thirty patients with SchS were recruited from 3 clinical centers. Six patients with known acquired cryopyrin‐associated periodic syndromes (aCAPS) were included as controls. Allele‐specific oligonucleotide–polymerase chain reaction was used for the detection of the MYD88 L265P variant, next‐generation sequencing was applied to analyze NLRP3 and 28 genes associated with myelodysplastic syndrome, and gene scanning was performed for the detection of X chromosome inactivation. Results: Activating NLRP3 mutations were not present in 11 SchS patients who had not been sequenced for this gene previously. The MYD88 L265P variant was present in 9 of 30 SchS patients, and somatic mutations associated with clonal hematopoiesis were identified in 1 of 30 patients with SchS and 1 of 6 patients with aCAPS. Evidence of nonrandom X chromosome inactivation was detected in 1 female patient with SchS and 1 female patient with aCAPS. Conclusion: A shared molecular mechanism accounting for the pathogenesis of inflammation in SchS remains elusive. Clonal hematopoiesis is not associated with other somatic mutations found in individuals with SchS or aCAPS. [ABSTRACT FROM AUTHOR]

Background: The chromosomal microarray analysis (CMA) is recommended as a first‐tier test for individuals with developmental delay (DD)/intellectual disability (ID) and/or multiple congenital anomalies. However, owing to high costs, this technique is not widely performed for diagnostic purposes in several countries. The aim of this study was to identify clinical features that could favour the hypothesis of genomic imbalances (GIs) in individuals with DD/ID. Methods: The sample consisted of 63 individuals, and all of them underwent a detailed evaluation by a clinical geneticist and were investigated by the CMA. They were divided into two groups. Group A composed of 20 individuals with pathogenic copy number variants (CNVs); and group B composed of 43 individuals with normal CMA results or variants of uncertain clinical significance (VUS). Results: Pathogenic GIs were found in 20 cases (32%), including 11 individuals with an abnormal karyotype, VUS was found in five individuals (8%) and the results were normal in 38 individuals (60%). Major anomalies were found in 15/20 (75%) individuals in group A against 35/43 (81%) in group B. Dysmorphisms (≥5) were found in 17/20 (85%) in group A and 41/43 (95%) in group B. The most frequent major anomalies detected in group A were congenital heart disease, epilepsy and renal malformation; and in group B, they were malformations of central nervous system, congenital heart disease, microcephaly, epilepsy and hearing impairment. There was no significant statistical difference among the frequencies in groups A and B. Conclusions: Evidences point that every individual with DD/ID, with no specific clinical suspicion, should have screening for GIs as a first‐tier test, regardless of the presence or absence of additional major anomalies or dysmorphisms. Future studies with a similar design would be helpful, especially in countries where the access to new technologies is still limited. [ABSTRACT FROM AUTHOR]

Objective: High‐throughput gene expression profiling of tissue samples from patients with systemic sclerosis (SSc) has identified 4 "intrinsic" gene expression subsets: inflammatory, fibroproliferative, normal‐like, and limited. Prior methods required agglomerative clustering of many samples. In order to classify individual patients in clinical trials or for diagnostic purposes, supervised methods that can assign single samples to molecular subsets are required. We undertook this study to introduce a novel machine learning classifier as a robust accurate intrinsic subset predictor. Methods: Three independent gene expression cohorts were curated and merged to create a data set covering 297 skin biopsy samples from 102 unique patients and controls, which was used to train a machine learning algorithm. We performed external validation using 3 independent SSc cohorts, including a gene expression data set generated by an independent laboratory on a different microarray platform. In total, 413 skin biopsy samples from 213 individuals were analyzed in the training and testing cohorts. Results: Repeated cross‐fold validation identified consistent and discriminative markers using multinomial elastic net, performing with an average classification accuracy of 87.1% with high sensitivity and specificity. In external validation, the classifier achieved an average accuracy of 85.4%. Reanalyzing data from a previous study, we identified subsets of patients that represent the canonical inflammatory, fibroproliferative, and normal‐like subsets. Conclusion: We developed a highly accurate classifier for SSc molecular subsets for individual patient samples. The method can be used in SSc clinical trials to identify an intrinsic subset on individual samples. Our method provides a robust data‐driven approach to aid clinical decision‐making and interpretation of heterogeneous molecular information in SSc patients. [ABSTRACT FROM AUTHOR]

Background: Hazardous alcohol consumption has significant adverse medical consequences. These effects may be mediated, in part, by alterations in DNA methylation. Thus, DNA methylation signatures in peripheral cells may provide biomarkers of the medical impact of alcohol use and the risk for future alcohol consumption. Method: Using a high‐density methylation array, we characterized epigenome‐wide DNA methylation in saliva cells with respect to alcohol consumption in a large cohort of male European American veterans. In this study, DNA methylation of over 870,000 CpG DNA sites was profiled in 1,135 European American men. Alcohol consumption was assessed using the Alcohol Use Disorder Identification Test‐Consumption (AUDIT‐C). Linear regression was applied in an epigenome‐wide association study (EWAS), adjusted for confounders. Gene set enrichment analysis was performed in the KEGG database with a correction for gene length. Results: We found that a total of 70 CpG sites reached EWAS‐corrected significance (p < 6E‐08) with small effects on alcohol consumption for individual CpG sites, including 64 new CpG sites and 6 CpG sites that were previously reported as associated with alcohol use disorder, liver function, body mass index, and lipid metabolism. The most significant CpG site was located in SLC7A11 (t = −11.34, p = 2.66E‐28), a gene involved specifically in cysteine and glutamate transportation. The 70 significant CpG sites were located on 44 genes, including genes involved in amino acid transport and metabolism systems. We identified 68 pathways with a false discovery rate < 0.05. Conclusions: We identified novel DNA methylation sites associated with alcohol consumption. Results may shed light on peripheral mechanisms of alcohol consumption on adverse health outcomes among heavy drinkers. [ABSTRACT FROM AUTHOR]

Aim: Chromosome analysis of prenatal samples and products of conception (POC) has conventionally been done by karyotyping (KT). Shortcomings of KT like high turnaround time and culture failure led to technology innovations, such as the bacterial artificial chromosomes (BAC)s‐on‐Beads (BoBs)‐based tests, Prenatal BoBs (prenatal samples) and KaryoLite BoBs (POC samples). In the present study, we validated and evaluated the utility of each test on prenatal, POC and blood samples. Methods: Study A (n = 305; 259 prenatal + 46 blood/POC) and Study B (n = 176; 146 POC/chorionic vill + 30 blood/amniotic fluid) samples were analyzed using Prenatal and KaryoLite BoBs kits, respectively. KT, array‐based Comparative Genomic Hybridization (arrayCGH) and fluorescence in situ hybridization (FISH) were used for comparison of results. Ability of KaryoLite BoBs to identify ring chromosomes was tested. Results: Prenatal BoBs had zero test failure rate and results of all samples were concordant with KT results. Totally four microdeletions were identified by Prenatal BoBs but not by KT. In Study B, all but two POC samples (one triploid and one tetraploid) were concordant with KT and arrayCGH. Partial chromosomal imbalance detection rate was ~64% and KaryoLite BoBs indicated the presence of a ring chromosome in all four cases. The failure rate of KaryoLite BoBs was 3%. Conclusion: We conclude that Prenatal BoBs (common aneuploidies and nine microdeletions) together with KT constitutes more comprehensive prenatal testing compared to FISH and KT. KaryoLite BoBs for aneuploidies of all chromosomes is highly successful in POC analysis and the ability to indicate presence of ring chromosomes improves its clinical sensitivity. Both tests are robust and could also be used for different specimens. [ABSTRACT FROM AUTHOR]

Objective: Orthognathic surgery dramatically changes morphology of the maxillofacial deformity and improves the malocclusion morphologically and functionally. We investigated the influence of orthognathic surgery on genomewide DNA methylation in saliva. Methods: Saliva was obtained from nine patients undergoing orthognathic surgery and two healthy reference individuals before and 3 months after orthognathic surgery. Genomewide DNA methylation profiling of saliva (341,482 CpG dinucleotides) was conducted using Infinium HumanMethylation450 BeadChips. Results: Comparison between pre‐ and postsurgery saliva samples revealed significant changes in DNA methylation patterns at 2,381 CpG sites (p < 0.01) with suggestive significance. The differentially methylated probe sets were significantly associated with the cancer pathway (p = 2.8 × 10−7; a false discovery rate q‐value = 3.7 × 10−4) and PI3K‐Akt signalling pathway (p = 2.4 × 10−5; a false discovery rate q‐value = 3.1 × 10−2). Conclusion: Pathway enrichment analysis of genes with suggestive significance demonstrated that altered DNA methylation in saliva of patients undergoing orthognathic surgery, possibly as a response to surgical stress or bone injury. Further studies with a large sample size and long‐term observation are needed to validate the phenomena identified in this study. [ABSTRACT FROM AUTHOR]

Objectives: MicroRNAs (miRNAs) may play an important role in inflammatory response. However, the involvement of miRNAs in the pathogenesis of periodontitis is unclear. The present study aimed to compare the miRNA expression profiles in individuals with chronic (CP) or aggressive (AP) periodontitis. Materials and methods: Eighteen non‐smoker individuals (CP = 9 and AP = 9) without any history of systemic diseases or previous periodontal therapies were selected at the Clinics of Periodontology from the Federal University of Minas Gerais. Gingival tissue samples were collected during the initial periodontal therapy. miRNAs were isolated, and expression patterns of 754 miRNAs were assessed with a quantitative miRNA PCR array. miRNAs expression profiles were compared between CP and AP groups. Results: There were no differences observed in the miRNAs expression profiles between CP and AP (p > 0.05). According to the microarray analyses, the most expressed miRNAs in both groups were hsa‐miR‐1274b, hsa‐let‐7b‐5p, hsa‐miR‐24‐3p, hsa‐miR‐19b‐3p, hsa‐miR‐720, hsa‐miR‐126‐3p, hsa‐miR‐17‐3p and hsa‐miR‐21‐3p. Conclusion: Findings suggested no differences in miRNAs expression profiles between chronic and aggressive forms of periodontitis. The overexpression of specific miRNAs could provide insights into the pathogenesis of both forms of the disease. [ABSTRACT FROM AUTHOR]

The global physiological function of specifically expressed genes of mitoxantrone (MTX)‐resistant prostate cancer (PCa) is unclear. In this study, gene expression pattern from microarray data was investigated for identifying differentially expressed genes (DEGs) in MTX‐resistant PCa xenografts. Human PCa cell lines DU145 and PC3 were cultured in vitro and xenografted into severe combined immunodeficiency (SCID) mice, treated with MTX intragastrically, three times a week until all mice relapsed. Gene expression profiles of the xenografts from castrated mice were performed with Affymetrix human whole genomic oligonucleotide microarray. The Cytoscape software was used to investigate the relationship between proteins and the signalling transduction network. A total of 355 overlapping genes were differentially expressed in MTX‐resistant DU145R and PC3R xenografts. Of these, 16 genes were selected to be validated by quantitative real‐time PCR (qRT‐PCR) in these xenografts, and further tested in a set of formalin‐fixed, paraffin‐embedded and optimal cutting temperature (OCT) clinical tumour samples. Functional and pathway enrichment analyses revealed that these DEGs were closely related to cellular activity, androgen synthesis, DNA damage and repair, also involved in the ERK/MAPK, PI3K/serine‐threonine protein kinase, also known as protein kinase B, PKB (AKT) and apoptosis signalling pathways. This exploratory analysis provides information about potential candidate genes and may bring new insights into the molecular cascade involvement in MTX‐resistant PCa. [ABSTRACT FROM AUTHOR]

Objective: To determine the frequency and nature of copy number variants (CNVs) identified by chromosomal microarray analysis (CMA) in a large cohort of fetuses with isolated increased nuchal translucency thickness (NT) ≥ 3.5 mm.Methods: This was a retrospective, multicenter study, including 11 French hospitals, of data from the period between April 2012 and December 2015. In total, 720 fetuses were analyzed by rapid aneuploidy test and the fetuses identified as euploid underwent CMA. CNVs detected were evaluated for clinical significance and classified into five groups: pathogenic CNVs; benign CNVs; CNVs predisposing to neurodevelopmental disorders; variants of uncertain significance (VOUS); and CNVs not related to the phenotype (i.e. incidental findings).Results: In 121 (16.8%) fetuses, an aneuploidy involving chromosome 13, 18 or 21 was detected by rapid aneuploidy test and the remaining 599 fetuses were euploid. Among these, 53 (8.8%) had a CNV detected by CMA: 16/599 (2.7%) were considered to be pathogenic, including 11/599 (1.8%) that were cryptic (not visible by karyotyping); 7/599 (1.2%) were CNVs predisposing to neurodevelopmental disorders; and 8/599 (1.3%) were VOUS. Additionally, there was one (0.2%) CNV that was unrelated to the reason for referral diagnosis (i.e. an incidental finding) and the remaining 21 were benign CNVs, without clinical consequence. Interestingly, we identified five genomic imbalances of the 1q21.1 or 15q11.2 regions known to be associated with congenital heart defects.Conclusion: Our study demonstrates the benefit of CMA in the etiological diagnosis of fetuses with isolated increased NT. It is worth noting that most (69%) of the detected pathogenic CNVs were cryptic. Copyright © 2017 ISUOG. Published by John Wiley & Sons Ltd. [ABSTRACT FROM AUTHOR]

Objective: T cells from systemic lupus erythematosus (SLE) patients have reduced protein levels of RasGRP1, a guanine nucleotide exchange factor for Ras, and increased transcript of alternatively spliced (AS) forms lacking exon 11. Serine/arginine‐rich splicing factor 1 (SRSF1) binds pre–messenger RNA (pre‐mRNA) to regulate AS forms of several genes, including CD3ζ in SLE T cells. This study was undertaken to assess whether SRSF1 controls the expression of RasGRP1 in T cells from patients with SLE. Methods: We studied T cells from 45 SLE patients and 18 healthy subjects. Expression levels of SRSF1, wild‐type (WT) RasGRP1, and DNA methyltransferase 1 (DNMT1) were assessed by quantitative polymerase chain reaction. Direct binding of SRSF1 to exon 11 of RasGRP1 mRNA was evaluated with an oligonucleotide–protein pulldown assay. Healthy T cells and SLE T cells were treated with SRSF1‐specific small interfering RNA or SRSF1 expression vector, respectively, and then evaluated for mRNA/protein expression. Results: SRSF1 expression levels were significantly lower in T cells from SLE patients compared to those from healthy subjects, and correlated inversely with disease activity and positively with levels of RasGRP1‐WT and DNMT1. SRSF1 bound directly to exon 11 of RasGRP1 mRNA. Silencing of SRSF1 in human T cells led to increased ratios of RasGRP1‐AS to RasGRP1‐WT and decreased levels of RasGRP1 protein, whereas overexpression of SRSF1 in SLE T cells caused recovery of RasGRP1, which in turn induced DNMT1/interleukin‐2 expression. Conclusion: SRSF1 controls the alternative splicing of RasGRP1 and subsequent protein expression. Our findings extend evidence that alternative splicing plays a central role in the aberrant T cell function in patients with SLE by controlling the expression of multiple genes. [ABSTRACT FROM AUTHOR]

Objective: To determine whether abnormal levels of first-trimester maternal serum free β-hCG and PAPP-A are associated with significant copy number variants (CNVs) on chromosomal microarray analysis (CMA).Methods: Retrospective cohort of singleton prenatal CMA studies (n = 2880). Cases with an abnormal karyotype, benign familial or de novo variants, and absence of heterozygosity were excluded. The prevalence of abnormal serum analytes was compared between patients with significant CNVs (n = 56) and those with normal CMA (n = 884). Odds ratios (ORs) and 95% confidence intervals (CI) were calculated using Fisher's exact test. Mantel-Haenszel method was utilized to adjust ORs for prenatal diagnostic procedure type and indications for testing. Statistical significance was determined as P value < 0.05.Results: Abnormally low serum free β-hCG (≤0.45 MoM) was associated with an increased risk of significant CNVs (OR 3.53, 95% CI, 1.25-8.66, P < 0.01). This association remained significant after adjusting for abnormal nuchal translucency and advanced maternal age (AMA) (adjusted OR 3.04, 95% CI, 1.05-7.48, P < 0.05) or procedure type and AMA (adjusted OR 3.21, 95% CI 1.13-8.16, P < 0.05). The associations of abnormally high serum free β-hCG, low PAPP-A, and high PAPP-A with significant CNVs were not statistically significant.Conclusion: Low first-trimester serum β-hCG is associated with an increased risk of significant CNVs on CMA. [ABSTRACT FROM AUTHOR]

We present three cases of oral hairy leukoplakia (OHL) in whom the diagnosis was established by EBV DNA detection in whole saliva. Three HIV‐infected patients came to the Oral Medicine Clinic with similar chief complaints of asymptomatic white lesions on the tongue. All patients were diagnosed with suspected OHL and oral thrush also in the first patient. A multiplex PCR DNA microarray was performed to detect EBV DNA in saliva collected by spitting method. All saliva samples showed positive results for EBV DNA, and the definitive diagnosis of OHL was made. Resolution of lesions was found at 1‐ to 2‐month follow‐up after treatment with application of acyclovir 5% cream 5 times daily. Additionally, anti‐fungal treatment was given to the first patient and anti‐retroviral treatment to the first and second patients. EBV is mostly transmitted by asymptomatic shedding into saliva. Therefore, the detection of salivary EBV DNA is useful in establishing a definitive diagnosis of OHL allowing more effective treatment for both HIV‐infected patients receiving ART and treatment‐naïve patients at any CD4 + count. [ABSTRACT FROM AUTHOR]

The article mentions the comparison of real‐time polymerase chain reaction (PCR) and PCR with fragment length analysis for the detection of CALR gene mutations in suspected myeloproliferative neoplasms. It mentions that the diversity of CALR mutations presents methodological challenges for routine diagnostic laboratories.

Abstract: Oligonucleotide‐based microarrays are ideal tools for genetic testing and diagnostics due to their stability, ease of synthesis, and high specificity for the target of interest. This study reports on the effectiveness of several coupling strategies to covalently attach single‐stranded nucleic acids to surfaces, focusing on the robustness of the attachment in various environmental conditions, such as pH and temperature. Various characteristics of DNA microarrays produced using amine‐conjugated DNA probes on five different functional surfaces, commonly used for immobilization of biomolecules, namely, epoxy, carboxyl, amine, aldehyde, and N‐hydroxysuccinimide‐coated substrates, are investigated. Immobilization efficiency, changes in surface energy, as well as the stability of the conjugated DNA upon exposure to various environmental conditions are measured. Finally, in order to study the postimmobilization viability of the developed biosensors, microarrays of synthetic RNA cleaving probes (DNAzyme) are immobilized onto these surfaces and their functionality is evaluated through their ability to detect Escherichia coli at various temperatures. Results show that epoxy‐coated plastic surfaces are most ideal for the creation of DNA‐based biosensing chips. This study provides a guideline for producing oligonucleotide‐based microarrays on glass and plastic substrates and can be used for developing microarray‐based biosensors, suitable for long‐term storage. [ABSTRACT FROM AUTHOR]

Background: Increasing evidence has demonstrated that circular RNAs (circRNAs) may play an important role in oncogenesis and tumor development; however, their role in lung adenocarcinoma (LUAD) remains unclear. We identified the differentially expressed circRNAs in LUAD and investigated the potential mechanisms for cancer progression. Methods: We examined differentially expressed circRNAs in LUAD and paired normal tissues using downloaded circRNA microarrays from the Gene Expression Omnibus. We constructed gene co‐expression networks based on the degree of Pearson correlation to predict the critical circRNA in LUAD. Gene Ontology analysis was performed on the genes in the network. We observed one novel circRNA upregulated in LUAD, hsa_circ_0000792, as well as its potential sponged microRNA, miR‐375. Subsequent real‐time quantitative PCR was used to verify the bioinformatics analysis. Results: Several circRNAs showed significantly different expression levels in LUAD tissues. Real‐time quantitative PCR and further co‐expression network analysis of 42 matched tissue samples showed a significant difference in expression between LUAD and normal tissues in hsa_circ_0000792 (P < 0.001). We built a network of hsa_circ_0000792‐targeted miRNA gene interactions, including miR‐375 and the corresponding messenger RNAs. Gene Ontology analysis revealed that hsa_circ_0000792 could participate in signal transduction and cell communication during LUAD development. Larger area under the curve by receiver operating characteristic curve analysis of hsa_circ_0000792 and miR‐375 (0.815 and 0.772, respectively) in LUAD indicated greater potential as biomarkers. Conclusions: We identified hsa_circ_0000792 as a potential LUAD biomarker; however, further studies are required to determine the mechanism of this circRNA in LUAD development. [ABSTRACT FROM AUTHOR]

Abstract: Aim: This study tests the hypothesis that salivary extracellular RNA (exRNA) biomarkers can be developed for gingivitis detection and monitoring disease regression. Materials and Methods: Salivary exRNA biomarker candidates were developed from a total of 100 gingivitis and non‐gingivitis individuals using Affymetrix's expression microarrays. The top 10 differentially expressed exRNAs were tested in a clinical cohort to determine whether the discovered salivary exRNA markers for gingivitis were associated with clinical gingivitis and disease regression. For this purpose, unstimulated saliva was collected from 30 randomly selected gingivitis subjects, the gingival and plaque indexes scores were taken at baseline, 3 and 6 weeks and salivary exRNAs were assayed by means of reverse transcription quantitative polymerase chain reaction. Results: Eight salivary exRNA biomarkers developed for gingivitis were statistically significantly changed over time, consistent with disease regression. A panel of four salivary exRNAs [SPRR1A, lnc‐TET3‐2:1, FAM25A, CRCT1] can detect gingivitis with a clinical performance of 0.91 area under the curve, with 71% sensitivity and 100% specificity. Conclusions: The clinical values of the developed salivary exRNA biomarkers are associated with gingivitis regression. They offer strong potential to be advanced for definitive validation and clinical laboratory development test. [ABSTRACT FROM AUTHOR]

Objective: Juvenile idiopathic arthritis (JIA) comprises 7 heterogeneous categories of chronic childhood arthritides. Approximately 5% of children with JIA have rheumatoid factor (RF)–positive arthritis, which phenotypically resembles adult rheumatoid arthritis (RA). Our objective was to compare and contrast the genetics of RF‐positive polyarticular JIA with those of RA and selected other JIA categories, to more fully understand the pathophysiologic relationships of inflammatory arthropathies. Methods: Patients with RF‐positive polyarticular JIA (n = 340) and controls (n = 14,412) were genotyped using the Immunochip array. Single‐nucleotide polymorphisms were tested for association using a logistic regression model adjusting for admixture proportions. We calculated weighted genetic risk scores (wGRS) of reported RA and JIA risk loci, and we compared the ability of these wGRS to predict RF‐positive polyarticular JIA. Results: As expected, the HLA region was strongly associated with RF‐positive polyarticular JIA (P = 5.51 × 10−31). Nineteen of 44 RA risk loci and 6 of 27 oligoarticular/RF‐negative polyarticular JIA risk loci were associated with RF‐positive polyarticular JIA (P < 0.05). The RA wGRS predicted RF‐positive polyarticular JIA (area under the curve [AUC] 0.71) better than did the oligoarticular/RF‐negative polyarticular JIA wGRS (AUC 0.59). The genetic profile of patients with RF‐positive polyarticular JIA was more similar to that of RA patients with age at onset 16–29 years than to that of RA patients with age at onset ≥70 years. Conclusion: RF‐positive polyarticular JIA is genetically more similar to adult RA than to the most common JIA categories and thus appears to be a childhood‐onset presentation of autoantibody‐positive RA. These findings suggest common disease mechanisms, which could lead to novel therapeutic targets and shared treatment strategies. [ABSTRACT FROM AUTHOR]

Introduction: Mutations in gap junction protein beta 1 (GJB1) on the X chromosome represent one of the most common causes of hereditary neuropathy. We assessed manifestations associated with a rare 3' untranslated region mutation (UTR) of GJB1 in a large family with X-linked Charcot-Marie-Tooth disease (CMTX).Methods: Clinical, electrophysiological, and molecular genetic analyses were performed on an 8-generation family with CMTX.Results: There were 22 affected males and 19 symptomatic females, including an 83-year-old woman followed for 40 years. Electrophysiological studies showed a primarily axonal neuropathy. The c.*15C>T mutation in the GJB1 3' UTR was identified in 4 branches of the family with a log of odds (LOD) of 4.91. This created a BstE II enzyme recognition site that enabled detection by restriction digestion.Discussion: The c.*15C>T mutation in the GJB1 3' UTR segregates with CMTX1 in 8 generations. Penetrance in males and females is essentially complete. A straightforward genetic method to detect this mutation is described. Muscle Nerve 57: 859-862, 2018. [ABSTRACT FROM AUTHOR]

Objective: To evaluate the performance of high-resolution chromosomal microarray (CMA) as the standard diagnostic approach for genomic imbalances in pregnancies with increased risk based on combined first-trimester screening (cFTS).Methods: This was a retrospective study of genomic findings in a cohort of 575 consecutive pregnancies undergoing invasive testing because of a cFTS risk ≥ 1:300 on a publicly funded population-based screening program in the Central and Northern Regions of Denmark, between September 2015 and September 2016. Women with fetal nuchal translucency thickness ≥ 3.5 mm or opting for non-invasive prenatal testing (NIPT) were excluded. Comparative genomic hybridization was performed using a 180-K oligonucleotide array on DNA extracted directly from chorionic villus/amniocentesis samples. Genomic outcomes were reported in relation to cFTS findings.Results: Of the 575 pregnancies that underwent invasive testing, CMA detected 22 (3.8% (95% CI, 2.5-5.7%)) cases of trisomies 21, 18 and 13, 14 (2.4% (95% CI, 1.4-4.0%)) cases of other types of aneuploidy and 15 (2.6% (95% CI, 1.5-4.3%)) cases with a pathogenic or probably pathogenic copy number variant (CNV). Of the 15 CNVs, three were > 10 Mb and would probably have been detected by chromosomal analysis, but the other 12 would most probably not have been detected using conventional cytogenetic techniques; therefore, the overall detection rate of CMA (8.9% (95% CI, 6.8-11.5%)) was significantly higher than that estimated for conventional cytogenetic analysis (6.8% (95% CI, 5.0-9.1%)) (P = 0.0049). Reducing the cFTS risk threshold for invasive diagnostic testing to 1 in 100 or 1 in 50 would have led, respectively, to 60% or 100% of the pathogenic CNVs being missed.Conclusions: CMA is a valuable diagnostic technique that can identify an increased number of genomic aberrations in pregnancies at increased risk on cFTS. Limiting diagnostic testing to pregnancies with a risk above 1 in 100 or 1 in 50, as proposed in contingent NIPT/invasive testing models, would lead to a significant proportion of pathogenic CNVs being missed at first-trimester screening. Copyright © 2017 ISUOG. Published by John Wiley & Sons Ltd. [ABSTRACT FROM AUTHOR]

Abstract: Mutations in small heat shock protein beta‐1 (HspB1) have been linked to Charcot‐Marie‐Tooth (CMT) disease type 2F and distal hereditary motor neuropathy type 2B. Only four cases with HSPB1 mutations have been reported to date in Japan. In this study between April 2007 and October 2014, we conducted gene panel sequencing in a case series of 1,030 patients with inherited peripheral neuropathies (IPNs) using DNA microarray, targeted resequencing, and whole‐exome sequencing. We identified HSPB1 variants in 1.3% (13 of 1,030) of the patients with IPNs, who exhibited a male predominance. Based on neurological and electrophysiological findings, seven patients were diagnosed with CMT disease type 2F, whereas the remaining six patients were diagnosed with distal hereditary motor neuropathy type 2B. P39L, R127W, S135C, R140G, K141Q, T151I, and P182A mutations identified in 12 patients were described previously, whereas a novel K123* variant with unknown significance was found in 1 patient. Diabetes and impaired glucose tolerance were detected in 6 of the 13 patients. Our findings suggest that HSPB1 mutations result in two phenotypes of inherited neuropathies and extend the phenotypic spectrum of HSPB1‐related disorders. [ABSTRACT FROM AUTHOR]

Plants live in a world where they are challenged by abiotic and biotic stresses. In response to unfavorable conditions or an acute challenge like a pathogen attack, plants use various signaling pathways that regulate expression of defense genes and other mechanisms to provide resistance or stress adaptation. Identification of the regulatory steps in defense signaling has seen much progress in recent years. Many of the identified signaling pathways show interactions with each other, exemplified by the modulation of the jasmonic acid response by salicylic acid. Accordingly, defense regulation is more appropriately thought of as a web of interactions, rather than linear pathways. Here we describe various regulatory components and how they interact to provide an appropriate defense response. One of the common assays to monitor the output of defense signaling, as well as interaction between signaling pathways, is the measurement of altered gene expression. We illustrate that, while this is a suitable assay to monitor defense regulation, it can also inadvertently provide overstated conclusions about interaction among signaling pathways. [ABSTRACT FROM AUTHOR]

Abstract: Aim: We investigated whether recombinant mouse interleukin‐6 (IL‐6) affects the development of preimplantation embryos and induces the ‐signal transducers and activators of transcription (JAK–STAT) signaling pathway by binding IL‐6 signal transducer (IL‐6st) and regulating Fos and Jun gene expression, thereby accounting for the negative effect of superovulation on embryo development. Methods: We compared rates of blastocyst formation from embryos cultured with different concentrations of IL‐6 or/and anti‐interleukin 6 receptor antibody (anti‐IL‐6RAb) in superovulated experimental and normal control groups. IL‐6 expression in preimplantation embryos was determined by immunofluorescence identification. Fos, Jun and IL‐6st messenger RNA expression was detected by PCR and microarray experiments. Results: Rates of blastocyst formation significantly decreased in superovulated embryos, whether or not they were incubated in 0.1, 1, 25 or 50 pg/mL IL‐6, (P < 0.01) compared to embryos from naturally ovulated controls, whereas incubation in 5 and 10 pg/mL IL‐6 reversed the negative effects of superovulation. The addition of anti‐IL‐6RAb to naturally ovulated embryos reduced blastocyst rates to those of superovulated embryos. Gene chip analysis indicated that the JAK–STAT signaling pathway contained differentially expressed IL‐6, IL‐6st, Jun and Fos genes. Both anti‐IL‐6RAb or ovarian stimulation downregulated IL‐6st, Jun, and Fos messenger RNA expression, but expression of the same three genes increased in 5 pg/mL IL‐6. Conclusion: Ovarian stimulation negatively impacts the development of preimplantation embryos by reducing IL‐6 release. IL‐6 affects the rate of development of zygotes to blastocyst by regulating IL‐6st, Fos and Jun expression in the JAK–STAT signaling pathway. [ABSTRACT FROM AUTHOR]

In honey bees, continuous foraging at an artificial feeder induced a sustained upregulation of the immediate early genes early growth response protein 1 ( Egr-1) and hormone receptor 38 ( Hr38). This gene expression response was accompanied by an upregulation of several Egr-1 candidate downstream genes: ecdysone receptor ( EcR), dopamine/ecdysteroid receptor ( DopEcR), dopamine decarboxylase and dopamine receptor 2. Hr38, EcR and DopEcR are components of the ecdysteroid signalling pathway, which is highly probably involved in learning and memory processes in honey bees and other insects. Time-trained foragers still showed an upregulation of Egr-1 when the feeder was presented at an earlier time of the day, suggesting that the genomic response is more dependent on the food reward than training time. However, presentation of the feeder at the training time without food was still capable of inducing a transient increase in Egr-1 expression. Thus, learnt feeder cues, or even training time, probably affect Egr-1 expression. In contrast, whole brain Egr-1 expression changes did not differ between dancing and nondancing foragers. On the basis of our results we propose that food reward induced continuous foraging ultimately elicits a genomic response involving Egr-1 and Hr38 and their downstream genes. Furthermore this genomic response is highly probably involved in foraging-related learning and memory responses. [ABSTRACT FROM AUTHOR]

Objective: The American College of Obstetricians and Gynecologists (ACOG) and Society for Maternal-Fetal Medicine (SMFM) recommend chromosomal microarray analysis (CMA) for prenatal diagnosis in cases with 1 or more fetal structural abnormalities. For patients who elect prenatal diagnosis and have a structurally normal fetus, either microarray or karyotype is recommended. This study evaluates the frequency of clinically significant chromosomal abnormalities (CSCA) that would have been missed if all patients offered the choice between CMA and karyotyping chose karyotyping.Methods: A total of 3223 prenatal samples undergoing CMA were evaluated. Cases were categorized into 2 groups: those that met ACOG guidelines for CMA versus those that met ACOG guidelines for either CMA or karyotype.Results: Of the 3223 cases, 1475 (45.8%) met ACOG recommendations for CMA, and 1748 (54.2%) met recommendations for either CMA or karyotype. In patients who could have elected either CMA or karyotype, 2.5% had CSCA that would have been missed if the patient had elected to pursue karyotype.Conclusion: This study suggests that 2.5% of patients will have a CSCA that may be missed if the guidelines continue to suggest that CMA and karyotyping have equivalent diagnostic value for patients without a fetal structural abnormality. [ABSTRACT FROM AUTHOR]

Background and Objective Aggregatibacter actinomycetemcomitans is involved in oral and systemic infections, and is associated with, eg aggressive forms of periodontitis and with endocarditis. The cagE gene encodes a ≈39 kDa putative exotoxin expressed by A. actinomycetemcomitans. The level of conservation of cagE, and its possible significance in periodontal disease, has not yet been thoroughly investigated. In the present study, the role of the cagE gene as a diagnostic marker has been investigated. Material and Methods We have used conventional polymerase chain reaction ( PCR), quantitative PCR and whole genome sequencing data to determine the prevalence of cagE in A. actinomycetemcomitans based on analysis of: (i) 249 isolates, collected and cultivated in a Ghanaian longitudinal cohort study; (ii) a serotype b collection of 19 strains; and (iii) the 36 A. actinomycetemcomitans genomes available in the NCBI database. Results Whereas cagE was absent in the other serotypes, our data support that this gene sequence is linked to a virulent and highly leukotoxic group of serotype b strains, including both JP2 and non- JP2 genotypes of A. actinomycetemcomitans. Conclusion We propose that cagE has the potential to be used as a PCR-based gene marker for the identification of a virulent and highly leukotoxic group of serotype b strains, including both JP2 and non- JP2 genotypes. This finding might be of importance in the risk assessment of the development of periodontal attachment loss in young individuals and hence suggested to be a relevant discovery in future development of new diagnostic tools and/or treatment strategies. [ABSTRACT FROM AUTHOR]

To assess the prevalence of CYP3A5 genomic variances and their impact on tacrolimus (TAC) dosing requirements among kidney transplant recipients in eastern North Carolina, we conducted a single-center retrospective cohort study at a large tertiary care medical center. A total of 162 adults who received a kidney transplant between March 1, 2013, and February 28, 2015, and received oral TAC as part of their maintenance immunosuppression were enrolled. Of these patients, 85 patients expressed a genotype with a CYP3A5*1 variant ( C YP 3A5*1 group), and 77 patients expressed genotypes with other CYP3A5 variants (nonexpressor group). All patients were followed for 1 year posttransplantation. The primary end point was the TAC total daily dose ( TDD) required to achieve the first therapeutic trough level based on the presence or absence of the CYP3A5*1 variant. The prevalence of different CYP3A5 variants across race/ethnicities in the study cohort was determined by CYP3A5 genotyping for each patient. The C YP 3A5*1 and nonexpressor groups did not differ significantly with respect to sex, mean age, or mean weight. The CYP3A5*1 group was largely African American (93%, p≤0.0005) compared with other race/ethnicities. Among the CYP3A5*1 expressors compared with nonexpressors, the mean TAC TDD in milligrams at the first therapeutic TAC level was significantly higher (12 vs 8 mg/day, p≤0.001). Similarly, the mean TAC TDD in milligrams/kilogram was 50% greater among CYP3A5*1 expressors (0.15 vs 0.1 mg/kg/day, p≤0.0005). The predominant genotypic variants were CYP3A5*3/*3 (33%), CYP3A5*1/*3 (20%), and CYP3A5*1/*1 (19%). This study illustrates the prevalence of the CYP3A5*1 variant among African-American kidney transplant recipients and the effect of this gene expression on the TAC TDD. Patients with the CYP3A5*1 variant require higher TAC doses, on average, to achieve desirable drug levels. In addition, this study provides transplant clinicians with insight and support to dose TAC more aggressively in African-American kidney transplant recipients who may be at higher risk for both toxicities as well as poor clinical outcomes related to inadequate immunosuppression. [ABSTRACT FROM AUTHOR]

Objectives The objective of this study was to seek correlates of immune protection in HIV infection. We sought to elucidate the association between the presence of human leucocyte antigen ( HLA) alleles, as well as killer immunoglobulin receptor ( KIR) genotypes, and the susceptibility to HIV infection in a Spanish cohort of HIV-exposed seronegative ( HESN) individuals. Methods A total of 152 individuals were evaluated: 29 HESN individuals in stable heterosexual relationships with an HIV-infected partner admitting high-risk sexual intercourse for at least 12 months prior to inclusion in the study, 61 HIV-infected patients and 62 healthy controls. HLA class I and II alleles and KIR genotypes were assessed in genomic DNA from all individuals in the study by polymerase chain reaction−sequence-specific oligonucleotide ( PCR- SSO) using bead array technology. Results HESN individuals showed a higher prevalence of HLA-A3 (62%) and HLA-B44 (83%) supertypes compared with HIV-infected individuals (42% and 66%, respectively). Regarding specific HLA alleles, HESN individuals had a higher prevalence of HLA-A*33:01, DRB1*04 and DQB1*03:02 alleles (14%, 34% and 31%, respectively) and a lower prevalence of the HLA-A*02:01 allele (27%) than HIV-infected patients (3%, 15%, 11% and 52%, respectively; P < 0.05). Interestingly, in a multivariate analysis, only the presence of DQB1*03:02 and the absence of A*02:01 alleles were independently associated with HESN status [odds ratio ( OR) 3.4 (95% confidence interval ( CI) 1.1-10.5) and 0.4 (95% CI: 0.1-0.9), respectively; P < 0.05]. No KIR genotype was associated with susceptibility to HIV infection. Conclusions Our data showed that the presence of the HLA class II allele DQB1*03:02 was a correlate of immune protection against HIV infection, while the presence of the HLA class I allele A*02:01 was associated with being infected with HIV. [ABSTRACT FROM AUTHOR]

Quahog parasite unknown ( QPX) is a thraustochytrid protist that infects the hard clam, Mercenaria mercenaria, causing significant economic losses along the northeastern coast of North America. Previous investigations noted differences in growth dynamics and virulence in QPX cells from different geographic locations. In order to probe the molecular determinants for these variations, we investigated the transcriptomic profiles of five geographically distinct QPX isolates using custom 15k 60-mer oligonucleotide arrays. A total of 1,263 transcripts were differentially expressed ( DE) among the five QPX isolates. The hierarchical clustering of gene expression profiles showed that the QPX isolates from Raritan Bay ( RB, NY) and from Provincetown Harbor ( MA) were more similar to each other and diverged from QPX isolates from Peconic Bay ( PB, NY) and Old Plantation Creek ( VA), which had more similar gene expression profiles. The most prominent difference was based on 78 transcripts coding for heat shock proteins DE between the five QPX isolates. The study generated contrasting transcriptomic profiles for QPX isolated from northern ( MA) and deeper ( RB, NY) locations as compared to southern ( VA) and shallower ( PB, NY) areas, suggesting the adaptation of the parasite to local environmental, in particular temperature, conditions. [ABSTRACT FROM AUTHOR]

Background: Suppressor of cytokine signaling 3 (SOCS3) has been identified as an inhibitor of JAK/STAT pathway that plays a significant role in carcinogenesis. SOCS3 and JAK2 polymorphisms may influence the gene expression or function, contributing to the disease susceptibility; however, such effect has not been evaluated in head and neck squamous cell carcinoma (HNSCC).Methods: A case-control study was performed to test the associations of SOCS3 and JAK2 polymorphisms with risk of HNSCC in 576 cases and 1552 cancer-free controls from China. Seven potentially functional polymorphisms predicted by bioinformatics tools were genotyped using Infinium BeadChip platform. The association between genotypes and HNSCC risk was estimated by computing odds ratios (ORs) and 95% confidence intervals (CIs) in univariate and multivariate logistic regression models.Results: We found that rs2280148 located at 3'-untranslated region of SOCS3 was significantly associated with an increased risk of HNSCC (additive model: adjusted OR = 1.21, 95% CI = 1.03-1.43, P = 0.021). Moreover, rs8064821 located in the promoter region of SOCS3 was linked with a decreased risk of the cancer (additive model: adjusted OR = 0.83, 95% CI = 0.71-0.97, P = 0.022). Combined analysis of these variants by the number of risk alleles showed a significant locus-dosage effect on the risk of HNSCC (Ptrend = 0.006).Conclusions: We provided the first evidence that SOCS3 polymorphisms may influence the risk of HNSCC, which could be applied as novel biomarkers to identify individuals at high risk of the disease. [ABSTRACT FROM AUTHOR]

Seasonal coat colour change is an important adaptation to seasonally changing environments but the evolution of this and other circannual traits remains poorly understood. In this study, we use gene expression to understand seasonal coat colour moulting in wild snowshoe hares ( Lepus americanus). We used hair colour to follow the progression of the moult, simultaneously sampling skin from three moulting stages in hares collected during the peak of the spring moult from white winter to brown summer pelage. Using RNA sequencing, we tested whether patterns of expression were consistent with predictions based on the established phases of the hair growth cycle. We found functionally consistent clustering across skin types, with 766 genes differentially expressed between moult stages. 'White' pelage showed more differentially expressed genes that were upregulated relative to other skin types, involved in the transition between late telogen (quiescent stage) and the onset of anagen (proliferative stage). Skin samples from transitional 'intermediate' and 'brown' pelage were transcriptionally similar and resembled the regressive transition to catagen (regressive stage). We also detected differential expression of several key circadian clock and pigmentation genes, providing important means to dissect the bases of alternate seasonal colour morphs. Our results reveal that pelage colour is a useful biomarker for seasonal change but that there is a consistent lag between the main gene expression waves and change in visible coat colour. These experiments establish that developmental sampling from natural populations of nonmodel organisms can provide a crucial resource to dissect the genetic basis and evolution of complex seasonally changing traits. [ABSTRACT FROM AUTHOR]

Herbivores regularly ingest natural toxins produced by plants as a defence against herbivory. Recent work suggests that compound toxicity is exacerbated at higher ambient temperatures. This phenomenon, known as temperature-dependent toxicity ( TDT), is the likely result of decreased liver function at warmer temperatures; however, the underlying cause of TDT remains speculative. In the present study, we compared the effects of temperature and dietary plant toxins on differential gene expression in the liver of an herbivorous rodent ( Neotoma lepida), using species-specific microarrays. Expression profiles revealed a greater number of differentially expressed genes at an ambient temperature below the thermal neutral zone for N. lepida (22°C) compared to one within (27°C). Genes and pathways upregulated at 22°C were related to growth and biosynthesis, whereas those upregulated at 27°C were associated with gluconeogenesis, apoptosis and protein misfolding, suggestive of a stressed state for the liver. Additionally, few genes associated with xenobiotic metabolism were induced when woodrats ingested plant toxins compared to nontoxic diets, regardless of temperature. Taken together, the results highlight the important role of ambient temperature on gene expression profiles in the desert woodrat. Temperatures just below the thermal neutral zone might be a favourable state for liver metabolism. Furthermore, the reduction in the number of genes expressed at a temperature within the thermal neutral zone indicates that liver function may be reduced at temperatures that are not typically considered as thermally stressful. Understanding how herbivorous mammals will respond to ambient temperature is imperative to accurately predict the impacts of climate change. [ABSTRACT FROM AUTHOR]

The light environment influences an animal's ability to forage, evade predators, and find mates, and consequently is known to drive local adaptation of visual systems. However, the light environment may also vary over fine spatial scales at which genetic adaptation is difficult. For instance, in aquatic systems, the available wavelengths of light change over a few metres depth. Do animals plastically adjust their visual system to such small-scale environmental light variation? Here, we show that in three-spine stickleback ( Gasterosteus aculeatus), opsin gene expression (an important determinant of colour vision) changes over a 2-m vertical gradient in nest depth. By experimentally altering the light environment using light filters to cover enclosures in a lake, we found that opsin expression can be adjusted on a short time frame (weeks) in response to the local light environment. This is to our knowledge the smallest spatial scale on which visual adjustments through opsin expression have been recorded in a natural setting along a continuously changing light environment. [ABSTRACT FROM AUTHOR]

Variation in gene expression can provide insights into organismal responses to environmental stress and physiological mechanisms mediating adaptation to habitats with contrasting environmental conditions. We performed an RNA-sequencing experiment to quantify gene expression patterns in fish adapted to habitats with different combinations of environmental stressors, including the presence of toxic hydrogen sulphide (H2S) and the absence of light in caves. We specifically asked how gene expression varies among populations living in different habitats, whether population differences were consistent among organs, and whether there is evidence for shared expression responses in populations exposed to the same stressors. We analysed organ-specific transcriptome-wide data from four ecotypes of Poecilia mexicana (nonsulphidic surface, sulphidic surface, nonsulphidic cave and sulphidic cave). The majority of variation in gene expression was correlated with organ type, and the presence of specific environmental stressors elicited unique expression differences among organs. Shared patterns of gene expression between populations exposed to the same environmental stressors increased with levels of organismal organization (from transcript to gene to physiological pathway). In addition, shared patterns of gene expression were more common between populations from sulphidic than populations from cave habitats, potentially indicating that physiochemical stressors with clear biochemical consequences can constrain the diversity of adaptive solutions that mitigate their adverse effects. Overall, our analyses provided insights into transcriptional variation in a unique system, in which adaptation to H2S and darkness coincide. Functional annotations of differentially expressed genes provide a springboard for investigating physiological mechanisms putatively underlying adaptation to extreme environments. [ABSTRACT FROM AUTHOR]

Objective: Maternal cell contamination (MCC) is known to increase the risk of misdiagnosis in prenatal diagnosis as well as in diagnostic tests for the products of conception (POC) from miscarriages. We aimed to develop a data correction method to salvage fetal karyotype information from single-nucleotide polymorphism (SNP) array data for POC with MCC when parental genotype data are available.Methods: We obtained SNP array data from mixed genomic DNAs of a mother-child pair and used the dataset to assess the accuracy of data correction. We subsequently applied our method to miscarriage specimens with MCC.Results: We adopted a linear interpolation model as a data correction method and implemented the method in an R package, snpsal. We successfully determined the fetal karyotypes of two miscarriage specimens that were previously undiagnosed due to MCC to be normal in one case and trisomy 16 in the other case using snpsal.Conclusion: The R package, snpsal, developed in this study facilitates rapid and accurate estimation of the fetal karyotype from SNP array data for POC with MCC. © 2017 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]