scholarly journals mRNA Analysis Identifies deep Intronic Splicing Variants Leading to Alport Syndrome and Overcomes the Problem of Negative Results of Exome Sequencing

2021 ◽  
Author(s):  
Xiaoyuan Wang ◽  
Yanqin Zhang ◽  
Jie Ding ◽  
Fang Wang
Keyword(s):  
Exome Sequencing ◽  
Alport Syndrome ◽  
Direct Sequencing ◽  
Genetic Diagnosis ◽  
Skin Fibroblasts ◽  
Negative Results ◽  
Pathogenic Variants ◽  
First Choice ◽  
Splicing Variants ◽  
Mrna Analysis

Abstract Mutations in COL4A3, COL4A4 and COL4A5 genes lead to Alport syndrome (AS). However, pathogenic variants in some AS patients are not detected by exome sequencing. The aim of this study was to identify the underlying genetic causes of five unrelated AS probands with negative NGS test results. Urine COL4A3–5 mRNAs were analyzed in the probands with an uncertain inherited mode of AS, and COL4A5 mRNA of skin fibroblasts was analyzed in the probands with X-linked AS. RT-PCR and direct sequencing were performed to detect mRNA abnormalities. PCR and direct sequencing were used to analyze the exons with flanking intronic sequences corresponding to mRNA abnormalities. Nine novel deep intronic splicing variants in COL4A4 and COL4A5 genes that cannot be captured by exome sequencing were identified in the five AS probands. Skipping of an exon was caused by four intronic variants, and retention of an intron fragment led to the remaining variant. Our results reveal that mRNA analysis for AS genes from either urine or skin fibroblasts can resolve genetic diagnosis in AS patients with negative NGS results. We recommend analyzing COL4A3–5 mRNA from urine as the first choice for these patients because it is feasible and non-invasive.

scholarly journals mRNA analysis identifies deep intronic variants causing Alport syndrome and overcomes the problem of negative results of exome sequencing

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Xiaoyuan Wang ◽  
Yanqin Zhang ◽  
Jie Ding ◽  
Fang Wang
Keyword(s):  
Exome Sequencing ◽  
Alport Syndrome ◽  
Direct Sequencing ◽  
Genetic Diagnosis ◽  
Skin Fibroblasts ◽  
Negative Results ◽  
Dna And Rna ◽  
Pathogenic Variants ◽  
First Choice ◽  
Mrna Analysis

AbstractMutations in COL4A3, COL4A4 and COL4A5 genes lead to Alport syndrome (AS). However, pathogenic variants in some AS patients are not detected by exome sequencing. The aim of this study was to identify the underlying genetic causes of five unrelated AS probands with negative NGS test results. Urine COL4A3–5 mRNAs were analyzed in the probands with an uncertain inherited mode of AS, and COL4A5 mRNA of skin fibroblasts was analyzed in the probands with X-linked AS. RT-PCR and direct sequencing were performed to detect mRNA abnormalities. PCR and direct sequencing were used to analyze the exons with flanking intronic sequences corresponding to mRNA abnormalities. Six novel deep intronic splicing variants in COL4A4 and COL4A5 genes that cannot be captured by exome sequencing were identified in the four AS probands. Skipping of an exon was caused by an intronic variant, and retention of an intron fragment caused by five variants. In the remaining AS proband, COL4A5 variants c.2677 + 646 C > T and r.2678_r.2767del were detected at the DNA and RNA level, respectively, whereas it is unclear whether c.2677 + 646 C > T may not lead to r.2678_r.2767del. Our results reveal that mRNA analysis for AS genes from either urine or skin fibroblasts can resolve genetic diagnosis in AS patients with negative NGS results. We recommend analyzing COL4A3–5 mRNA from urine as the first choice for these patients because it is feasible and non-invasive.

scholarly journals Impact of prenatal exome sequencing for fetal genetic diagnosis on maternal psychological outcomes and decisional conflict in a prospective cohort

2020 ◽  
Author(s):  
Asha Nikesh Talati ◽  
Kelly Gilmore ◽  
Emily Hardisty ◽  
Anne Drapkin Lyerly ◽  
Christine Rini ◽  
...  
Keyword(s):  
Exome Sequencing ◽  
Genetic Diagnosis ◽  
Psychological Adaptation ◽  
Decisional Conflict ◽  
Psychological Outcomes ◽  
Secondary Findings ◽  
Free Response ◽  
Negative Results ◽  
Genomic Knowledge ◽  
Future Pregnancy

Objective: To evaluate associations between prenatal trio exome sequencing (trio-ES) and psychological outcomes among women with an anomalous pregnancy. Methods: Trio-ES study enrolling patients with major fetal anomaly and normal microarray. Women completed self-reported measures and free response interviews at two time points, pre- (1) and post- (2) sequencing. Pre-sequencing responses were compared to post-sequencing responses; post-sequencing responses were stratified by women who received trio-ES results that may explain fetal findings, secondary findings (medically actionable or carrier couple status), or negative results. Free responses were content analyzed. Results: 115 trios were enrolled. Of those, 41/115 (35.7%) received results from trio-ES, including 36 (31.3%) who received results that may explain the fetal phenotype. These women had greater post-sequencing distress compared to women who received negative results, including generalized distress (p=0.03) and test-related distress (p=0.2); they also had worse psychological adaptation to results (p=0.001). Genomic knowledge did not change from pre- to post-sequencing (p=0.51). Major themes from content analyses included closure, future pregnancy, altruism, anxiety, and gratitude. Conclusions: Women show more distress after receiving trio-ES results compared to those who do not.

Leukodystrophies and Genetic Leukoencephalopathies in Children Specified by Exome Sequencing in an Expanded Gene Panel

2020 ◽  
Vol 35 (7) ◽  
pp. 433-441
Author(s):  
Bindu Parayil Sankaran ◽  
Madhu Nagappa ◽  
Shwetha Chiplunkar ◽  
Sonam Kothari ◽  
Periyasamy Govindaraj ◽  
...  
Keyword(s):  
Exome Sequencing ◽  
Genetic Diagnosis ◽  
Gene Panel ◽  
Unknown Etiology ◽  
Mri Findings ◽  
Diagnostic Challenge ◽  
Variants Of Unknown Significance ◽  
Pathogenic Variants ◽  
Genetic Features ◽  
Biopsy Confirmation

The overlapping clinical and neuroimaging phenotypes of leukodystrophies pose a diagnostic challenge to both clinicians and researchers alike. Studies on the application of exome sequencing in the diagnosis of leukodystrophies are emerging. We used targeted gene panel sequencing of 6440 genes to investigate the genetic etiology in a cohort of 50 children with neuroimaging diagnosis of leukodystrophy/genetic leukoencephalopathy of unknown etiology. These 50 patients without a definite biochemical or genetic diagnosis were derived from a cohort of 88 patients seen during a 2.5-year period (2015 January-2017 June). Patients who had diagnosis by biochemical or biopsy confirmation (n = 17) and patients with incomplete data or lack of follow-up (n = 21) were excluded. Exome sequencing identified variants in 30 (60%) patients, which included pathogenic or likely pathogenic variants in 28 and variants of unknown significance in 2. Among the patients with pathogenic or likely pathogenic variants, classic leukodystrophies constituted 13 (26%) and genetic leukoencephalopathies 15 (30%). The clinical and magnetic resonance imaging (MRI) findings and genetic features of the identified disorders are discussed.

scholarly journals Consensus statement on standards and guidelines for the molecular diagnostics of Alport syndrome: refining the ACMG criteria

2021 ◽  
Author(s):  
Judy Savige ◽  
Helen Storey ◽  
Elizabeth Watson ◽  
Jens Michael Hertz ◽  
Constantinos Deltas ◽  
...  
Keyword(s):  
Renal Failure ◽  
Alport Syndrome ◽  
Pathogenic Variants ◽  
Persistent Proteinuria ◽  
Mutational Hotspots ◽  
Splicing Variants ◽  
History Of ◽  
Standards And Guidelines ◽  
Reference Databases ◽  
End Stage

AbstractThe recent Chandos House meeting of the Alport Variant Collaborative extended the indications for screening for pathogenic variants in the COL4A5, COL4A3 and COL4A4 genes beyond the classical Alport phenotype (haematuria, renal failure; family history of haematuria or renal failure) to include persistent proteinuria, steroid-resistant nephrotic syndrome, focal and segmental glomerulosclerosis (FSGS), familial IgA glomerulonephritis and end-stage kidney failure without an obvious cause. The meeting refined the ACMG criteria for variant assessment for the Alport genes (COL4A3–5). It identified ‘mutational hotspots’ (PM1) in the collagen IV α5, α3 and α4 chains including position 1 Glycine residues in the Gly-X-Y repeats in the intermediate collagenous domains; and Cysteine residues in the carboxy non-collagenous domain (PP3). It considered that ‘well-established’ functional assays (PS3, BS3) were still mainly research tools but sequencing and minigene assays were commonly used to confirm splicing variants. It was not possible to define the Minor Allele Frequency (MAF) threshold above which variants were considered Benign (BA1, BS1), because of the different modes of inheritances of Alport syndrome, and the occurrence of hypomorphic variants (often Glycine adjacent to a non-collagenous interruption) and local founder effects. Heterozygous COL4A3 and COL4A4 variants were common ‘incidental’ findings also present in normal reference databases. The recognition and interpretation of hypomorphic variants in the COL4A3–COL4A5 genes remains a challenge.

scholarly journals Detection of pathogenic splicing events from RNA-sequencing data using dasper

2021 ◽  
Author(s):  
David Zhang ◽  
Regina H. Reynolds ◽  
Sonia Garcia-Ruiz ◽  
Emil K Gustavsson ◽  
Sid Sethi ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Diagnostic Yield ◽  
Genetic Diagnosis ◽  
Fibroblast Cell ◽  
Sequencing Data ◽  
Sequencing Technologies ◽  
Pathogenic Variants ◽  
Splicing Variants ◽  
Gene Filter ◽  
Disease Associations

AbstractAlthough next-generation sequencing technologies have accelerated the discovery of novel gene-to-disease associations, many patients with suspected Mendelian diseases still leave the clinic without a genetic diagnosis. An estimated one third of these patients will have disorders caused by mutations impacting splicing. RNA-sequencing has been shown to be a promising diagnostic tool, however few methods have been developed to integrate RNA-sequencing data into the diagnostic pipeline. Here, we introduce dasper, an R/Bioconductor package that improves upon existing tools for detecting aberrant splicing by using machine learning to incorporate disruptions in exon-exon junction counts as well as coverage. dasper is designed for diagnostics, providing a rank-based report of how aberrant each splicing event looks, as well as including visualization functionality to facilitate interpretation. We validate dasper using 16 patient-derived fibroblast cell lines harbouring pathogenic variants known to impact splicing. We find that dasper is able to detect pathogenic splicing events with greater accuracy than existing LeafCutterMD or z-score approaches. Furthermore, by only applying a broad OMIM gene filter (without any variant-level filters), dasper is able to detect pathogenic splicing events within the top 10 most aberrant identified for each patient. Since using publicly available control data minimises costs associated with incorporating RNA-sequencing into diagnostic pipelines, we also investigate the use of 504 GTEx fibroblast samples as controls. We find that dasper leverages publicly available data effectively, ranking pathogenic splicing events in the top 25. Thus, we believe dasper can increase diagnostic yield for a pathogenic splicing variants and enable the efficient implementation of RNA-sequencing for diagnostics in clinical laboratories.

scholarly journals Identification of Two Novel Mutations in the ATM Gene from Patients with Ataxia-Telangiectasia by Whole Exome Sequencing

2020 ◽  
Vol 20 (7) ◽  
pp. 531-534
Author(s):  
Masoud Heidari ◽  
Morteza Soleyman-Nejad ◽  
Mohammad H. Taskhiri ◽  
Javad Shahpouri ◽  
Alireza Isazadeh ◽  
...  
Keyword(s):  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
Ataxia Telangiectasia ◽  
Direct Sequencing ◽  
Genetic Diagnosis ◽  
Cell Cycle Checkpoints ◽  
Causative Mutation ◽  
Novel Mutations ◽  
Whole Exome ◽  
Atm Gene

Background: Ataxia telangiectasia (AT) is one of the most common autosomal recessive hereditary ataxia presenting in childhood. The responsible gene for AT designated ATM (AT, mutated) encodes a protein which is involved in cell cycle checkpoints and other responses to genotoxicity. We describe two novel disease-causing mutations in two unrelated Iranian families with Ataxiatelangiectasia. Methods: The probands including a 6-year-old female and an 18-year-old boy were diagnosed with Ataxia-telangiectasia among two different Iranian families. In this study, Whole-Exome Sequencing (WES) was employed for the detection of genetic changes in probands. The analysis of the cosegregation of the variants with the disease in families was conducted using PCR direct sequencing. Results: Two novel frameshift mutations, (c.4236_4236del p. Pro1412fs) and (c.8907T>G p. Tyr2969Ter) in the ataxia telangiectasia mutated ATM gene were detected using Whole-Exome Sequencing (WES) in the probands. These mutations were observed in two separate A-T families. Conclusion: Next-generation sequencing successfully identified the causative mutation in families with ataxia-telangiectasia. These novel mutations in the ATM gene reported in the present study could assist genetic counseling, Preimplantation Genetic Diagnosis (PGD) and prenatal diagnosis (PND) of AT.

scholarly journals B.06 Whole exome sequencing in genetic ataxias associated with cerebellar atrophy: the Canadian experience

2019 ◽  
Vol 46 (s1) ◽  
pp. S11
Author(s):  
L Gauquelin ◽  
T Hartley ◽  
M Tarnopolsky ◽  
DA Dyment ◽  
B Brais ◽  
...  
Keyword(s):  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
Diagnostic Yield ◽  
Genetic Diseases ◽  
Genetic Diagnosis ◽  
Cerebellar Atrophy ◽  
Clinical Findings ◽  
Disease Genes ◽  
Pathogenic Variants ◽  
Whole Exome

Background: Cerebellar atrophy is characterized by loss of cerebellar tissue, with evidence on brain imaging of enlarged interfolial spaces compared to the foliae. Genetic ataxias associated with cerebellar atrophy are a heterogeneous group of disorders. We investigated the prevalence in Canada and the diagnostic yield of whole exome sequencing (WES) for this group of conditions. Methods: Between 2011 and 2017, WES was performed in 91 participants with cerebellar atrophy as part of one of two national research programs, Finding of Rare Genetic Disease Genes (FORGE) or Enhanced Care for Rare Genetic Diseases in Canada (Care4Rare). Results: A genetic diagnosis was established in 58% of cases (53/91). Pathogenic variants were found in 24 known genes, providing a diagnosis for 46/53 participants (87%), and in four novel genes, accounting for 7/53 cases (13%). 38/91 cases (42%) remained unsolved. The most common diagnoses were channelopathies in 12/53 patients (23%) and mitochondrial disorders in 9/53 (17%). Inheritance was autosomal recessive in the majority of cases. Additional clinical findings provided useful clues to some of the diagnoses. Conclusions: This is the first report on the prevalence of genetic ataxias associated with cerebellar atrophy in Canada, and the utility of WES for this group of conditions.

scholarly journals Genomic profiling of 553 uncharacterized neurodevelopment patients reveals a high proportion of recessive pathogenic variant carriers in an outbred population

2019 ◽  
Author(s):  
Youngha Lee ◽  
Soojin Park ◽  
Jin Sook Lee ◽  
Soo Yeon Kim ◽  
Jaeso Cho ◽  
...  
Keyword(s):  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
Genetic Diagnosis ◽  
Pathogenic Variant ◽  
Mendelian Disease ◽  
Loss Of Function ◽  
Variant Discovery ◽  
Pathogenic Variants ◽  
Whole Exome ◽  
Neurodevelopmental Problems

AbstractBackgroundA substantial portion of Mendelian disease patients suffers from genetic variants that are inherited in a recessive manner. A precise understanding of pathogenic recessive variants in a population would assist in pre-screening births of such patients. However, a systematic understanding of the contribution of recessive variants to Mendelian diseases is still lacking.MethodsGenetic diagnosis and variant discovery of 553 undiagnosed Korean patients with complex neurodevelopmental problems (KND for Korean NeuroDevelopmental cohort) were performed using whole exome sequencing of patients and their parents. Pathogenic variants were selected and evaluated based on a comparison to patient symptoms and genetic properties of the variants were analyzed.ResultsDisease-causing variants, including newly discovered variants, were identified in in 57.5% of the probands of the KND cohort. Of the 553 patients, 47.4% harbored variants that were previously reported as being pathogenic, and 35.1% of the previous reported pathogenic variants were inherited in a recessive manner. Genes that cause recessive disorders tend to be less constrained by loss-of-function variants and enriched in metabolic and mitochondrial pathways. This observation was applied to an estimation that approximately 1 in 17 healthy Korean individuals carry at least one of these pathogenic variants that develop severe neurodevelopmental problems in a recessive manner. Furthermore, the feasibility of these genes for carrier screening was evaluated.ConclusionsWe suggest that the odds are high for healthy individuals carrying a potentially pathogenic variant, and its genetic properties. Our results will serve as a foundation for recessive variant screening to reduce occurrences of rare Mendelian disease patients. Additionally, our results highlight the utility and necessity of whole exome sequencing-based diagnostics for improving patient care in a country with a centralized medical system.

scholarly journals Targeted Exome Sequencing Provided Comprehensive Genetic Diagnosis of Congenital Anomalies of the Kidney and Urinary Tract

2020 ◽  
Vol 9 (3) ◽  
pp. 751 ◽  
Author(s):  
Yo Han Ahn ◽  
Chung Lee ◽  
Nayoung K. D. Kim ◽  
Eujin Park ◽  
Hee Gyung Kang ◽  
...  
Keyword(s):  
Urinary Tract ◽  
Exome Sequencing ◽  
Congenital Anomalies ◽  
Genetic Diagnosis ◽  
Copy Number Variations ◽  
Single Nucleotide Variants ◽  
Korean Children ◽  
Targeted Exome Sequencing ◽  
Genetic Causes ◽  
Pathogenic Variants

Congenital anomalies of the kidney and urinary tract (CAKUT) are the most common cause of chronic kidney disease in children. The search for genetic causes of CAKUT has led to genetic diagnosis in approximately 5–20 % of CAKUT patients from Western countries. In this study, genetic causes of CAKUT in Korean children were sought using targeted exome sequencing (TES) of 60 genes reported to cause CAKUT in human or murine models. We identified genetic causes in 13.8% of the 94 recruited patients. Pathogenic single nucleotide variants of five known disease-causing genes, HNF1B, PAX2, EYA1, UPK3A, and FRAS1 were found in 7 cases. Pathogenic copy number variations of 6 patients were found in HNF1B, EYA1, and CHD1L. Genetic abnormality types did not significantly differ according to CAKUT phenotypes. Patients with pathogenic variants of targeted genes had syndromic features more frequently than those without (p < 0.001). This is the first genetic analysis study of Korean patients with CAKUT. Only one-seventh of patients were found to have pathogenic mutations in known CAKUT-related genes, indicating that there are more CAKUT-causing genes or environmental factors to discover.

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