scholarly journals Genetic Etiology Study of Ten Chinese Families with Nonsyndromic Hearing Loss

2018 ◽  
Vol 2018 ◽  
pp. 1-7 ◽  
Author(s):  
Songqun Hu ◽  
Feifei Sun ◽  
Jie Zhang ◽  
Yan Tang ◽  
Jinhong Qiu ◽  
...  
Keyword(s):  
Hearing Loss ◽  
Next Generation Sequencing ◽  
Chinese Population ◽  
Sanger Sequencing ◽  
Nonsyndromic Hearing Loss ◽  
Genetic Etiology ◽  
Chinese Families ◽  
Pathogenic Variants ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

Nonsyndromic hearing loss has been shown to have high genetic heterogeneity. In this report, we aimed to disclose the genetic causes of the subjects from the ten Chinese deaf families who did not have pathogenic common genes/mutation. Next-generation sequencing (NGS) of 142 known deafness genes was performed in the probands of ten families followed by cosegregation analysis of all family members. We identified novel pathogenic variants in six families including p.D1806E/p.R1588W, p.R964W/p.R1588W, and p.G17C/p.G1449D in CDH23; p.T584M/p.D1939N in LOXHD1; p.P1225L in MYO7A; and p.K612X in EYA4. Sanger sequencing confirmed that these mutations segregated with the hearing loss of each family. In four families, no pathogenic variants were identified. Our study provided better understanding of the mutation spectrum of hearing loss in the Chinese population.

scholarly journals Use of sanger and next-generation sequencing to screen for mosaic and intronic APC variants in unexplained colorectal polyposis patients

2021 ◽  
Author(s):  
Fadwa A. Elsayed ◽  
Carli M. J. Tops ◽  
Maartje Nielsen ◽  
Hans Morreau ◽  
Frederik J. Hes ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Sanger Sequencing ◽  
Colorectal Polyps ◽  
Adenomatous Polyposis ◽  
Next Generation ◽  
Colorectal Polyposis ◽  
Pathogenic Variants ◽  
Predisposing Genes ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

AbstractIn addition to classic germline APC gene variants, APC mosaicism and deep intronic germline APC variants have also been reported to be causes of adenomatous polyposis. In this study, we investigated 80 unexplained colorectal polyposis patients without germline pathogenic variants in known polyposis predisposing genes to detect mosaic and deep intronic APC variants. All patients developed more than 50 colorectal polyps, with adenomas being predominantly observed. To detect APC mosaicism, we performed next-generation sequencing (NGS) in leukocyte DNA. Furthermore, using Sanger sequencing, the cohort was screened for the following previously reported deep intronic pathogenic germline APC variants: c.1408 + 731C > T, p.(Gly471Serfs*55), c.1408 + 735A > T, p.(Gly471Serfs*55), c.1408 + 729A > G, p.(Gly471Serfs*55) and c.532-941G > A, p.(Phe178Argfs*22). We did not detect mosaic or intronic APC variants in the screened unexplained colorectal polyposis patients. The results of this study indicate that the deep intronic APC variants investigated in this study are not a cause of colorectal polyposis in this Dutch population. In addition, NGS did not detect any further mosaic variants in our cohort.

scholarly journals Detection and validation of six pathogenic variants in Chinese inherited heart disease patients using clinical whole-exome sequencing

2021 ◽  
Author(s):  
Lichao Cao ◽  
Fei Ye ◽  
Shuqi Xie ◽  
Ying Ba ◽  
Ying Zeng ◽  
...  
Keyword(s):  
Heart Disease ◽  
Next Generation Sequencing ◽  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
Sanger Sequencing ◽  
Targeted Next Generation Sequencing ◽  
Pathogenic Variants ◽  
Whole Exome ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

The targeted next-generation sequencing (NGS) was employed in detecting the pathogenic mutations in inherited heart disease patients in the present study. Two main methods, the NGS and the classic Sanger sequencing, were used in this study. And, the whole-exome sequencing (WES) was specifically used in this study.

scholarly journals Development and Validation of a Next-Generation Sequencing Panel for Syndromic and Nonsyndromic Hearing Loss

2020 ◽  
Vol 5 (3) ◽  
pp. 467-479 ◽  
Author(s):  
Malinda Butz ◽  
Amber McDonald ◽  
Patrick A Lundquist ◽  
Melanie Meyer ◽  
Sean Harrington ◽  
...  
Keyword(s):  
Hearing Loss ◽  
Next Generation Sequencing ◽  
Copy Number Variants ◽  
Analytical Sensitivity ◽  
Nonsyndromic Hearing Loss ◽  
Next Generation ◽  
Single Nucleotide Variants ◽  
Small Indels ◽  
Syndromic Hearing Loss ◽  
Generation Sequencing

Abstract Background Deafness and hearing loss are common conditions that can be seen independently or as part of a syndrome and are often mediated by genetic causes. We sought to develop and validate a hereditary hearing loss panel (HHLP) to detect single nucleotide variants (SNVs), insertions and deletions (indels), and copy number variants (CNVs) in 166 genes related to nonsyndromic and syndromic hearing loss. Methods We developed a custom-capture next-generation sequencing (NGS) reagent to detect all coding regions, ±10 flanking bp, for the 166 genes related to nonsyndromic and syndromic hearing loss. Our validation consisted of testing 52 samples to establish accuracy, reproducibility, and analytical sensitivity. In addition to NGS, supplementary methods, including multiplex ligation-dependent probe amplification, long-range PCR, and Sanger sequencing, were used to ensure coverage of regions that had high complexity or homology. Results We observed 100% positive and negative percentage agreement for detection of SNVs (n = 362), small indels (1–22 bp, n = 25), and CNVs (gains, n = 8; losses, n = 17). Finally, we showed that this assay was able to detect variants with a variant allele frequency ≥20% for SNVs and indels and ≥30% to 35% for CNVs. Conclusions We validated an HHLP that detects SNVs, indels, and CNVs in 166 genes related to syndromic and nonsyndromic hearing loss. The results of this assay can be utilized to confirm a diagnosis of hearing loss and related syndromic disorders associated with known causal genes.

scholarly journals Next Generation Sequencing Identifies Five Novel Mutations in Lebanese Patients with Bardet–Biedl and Usher Syndromes

Genes ◽  
2019 ◽  
Vol 10 (12) ◽  
pp. 1047 ◽  
Author(s):  
Lama Jaffal ◽  
Wissam H Joumaa ◽  
Alexandre Assi ◽  
Charles Helou ◽  
George Cherfan ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Usher Syndrome ◽  
Bioinformatic Analysis ◽  
Next Generation ◽  
Novel Mutations ◽  
Pathogenic Variants ◽  
Whole Exome ◽  
Targeted Ngs ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

Aim: To identify disease-causing mutations in four Lebanese families: three families with Bardet–Biedl and one family with Usher syndrome (BBS and USH respectively), using next generation sequencing (NGS). Methods: We applied targeted NGS in two families and whole exome sequencing (WES) in two other families. Pathogenicity of candidate mutations was evaluated according to frequency, conservation, in silico prediction tools, segregation with disease, and compatibility with inheritance pattern. The presence of pathogenic variants was confirmed via Sanger sequencing followed by segregation analysis. Results: Most likely disease-causing mutations were identified in all included patients. In BBS patients, we found (M1): c.2258A > T, p. (Glu753Val) in BBS9, (M2): c.68T > C; p. (Leu23Pro) in ARL6, (M3): c.265_266delTT; p. (Leu89Valfs*11) and (M4): c.880T > G; p. (Tyr294Asp) in BBS12. A previously known variant (M5): c.551A > G; p. (Asp184Ser) was also detected in BBS5. In the USH patient, we found (M6): c.188A > C, p. (Tyr63Ser) in CLRN1. M2, M3, M4, and M6 were novel. All of the candidate mutations were shown to be likely disease-causing through our bioinformatic analysis. They also segregated with the corresponding phenotype in available family members. Conclusion: This study expanded the mutational spectrum and showed the genetic diversity of BBS and USH. It also spotlighted the efficiency of NGS techniques in revealing mutations underlying clinically and genetically heterogeneous disorders.

scholarly journals Molekulares Verstehen des Hörens – Was ändert sich für den Patienten?

2018 ◽  
Vol 97 (S 01) ◽  
pp. S214-S230 ◽  
Author(s):  
Tobias Moser
Keyword(s):  
Hearing Loss ◽  
Next Generation Sequencing ◽  
Next Generation ◽  
Kausale Therapie ◽  
Molekulare Analyse ◽  
Hidden Hearing Loss ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing ◽  
Eine Verbesserte

ZusammenfassungDas Innenohr und die Hörbahn mit ihren vergleichsweise geringen Zellzahlen haben sich einigen molekularen Ansätzen bislang beharrlich entzogen. Gleichzeitig vollbringt das Hören Spitzenleistungen, die sehr spezialisierte biologische Mechanismen nahelegen. Dies bedeutet einerseits, dass Analogieschlüsse zur molekularen Anatomie und Physiologie der Zellen des Hörsystems auf der Grundlage von Erkenntnissen aus molekular besser zugänglichen Systemen von beschränktem Nutzen sind. Andererseits legt eine solche Spezialisierung Gendefekte nahe, die von der Evolution toleriert wurden, weil sie nicht zur Fehlfunktion von essentiellen Körperprozessen führen. Technologische Fortschritte in der Humangenetik und der molekularen Analyse des Innenohrs im Tier bestätigen beide Annahmen und beleuchten den faszinierenden Mikrokosmos der Cochlea. Auf kleinstem Raum werden hier in konsequenter Arbeitsteilung herausragende Leistungen im Ionentransport, der Mechanotransduktion, der aktiven Zellmotilität und der synaptischen Verarbeitung erbracht. Einige der zugrundeliegenden molekularen Maschinen, z. B. das Motorprotein Prestin und das an synaptischer Fusion beteiligte Otoferlin, sind ausschließlich im Ohr aktiv. Dementsprechend führen ihre Defekte zu spezifischen nicht-syndromalen Schwerhörigkeiten, wie etwa bei der auditorischen Synaptopathie durch autosomal rezessive Mutationen im Otoferlin-Gen. Andere Mutationen, wie die den cochleären Kalium-Zyklus betreffenden, bedingen einen globalen Funktionsverlust der Cochlea. Viele genetische Defekte führen schließlich zur Degeneration des Innenohrs. Letztlich führt die molekulare Analyse sowohl beim Menschen, als auch im Tier-Innenohr aber auch zu neuen Erkenntnissen für häufige Formen der Schwerhörigkeit. So wurde der immunhistochemische Nachweis des Verlusts von Bandsynapsen der inneren Haarzellen zum Biomarker für „hidden hearing loss“ im Tiermodell. Die moderne Hochdurchsatz-Sequenzierung (sog. Next Generation Sequencing – NGS) bietet Zugang zu bislang nicht bekannten Taubheitsgenen, Mutationsspektren von bekannten Taubheitsgenen und zu einem genetischen Profil der individuellen Schwerhörigkeit, ihre Interpretation erfordert jedoch große humangenetische Expertise und umfangreiche tierexperimentelle Einsichten. Eine kausale Therapie etwa durch viralen Genersatz, der im Tier-Innenohr und bei einzelnen Formen der humanen Blindheit bereits erfolgreich ist, steht für die Schwerhörigkeit in der Klinik noch nicht zur Verfügung. Bereits jetzt ermöglichen molekulare Ansätze aber schon eine verbesserte Beratung von schwerhörigen Patienten.

scholarly journals Using a Next Generation Sequencing Panel to Discover the Obscure Causes of Hereditary Hemolytic Anemias

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2433-2433 ◽  
Author(s):  
Archana M Agarwal ◽  
N. Scott Reading ◽  
Kimberly Frizzell ◽  
Wei Shen ◽  
Shelly Sorrells ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Hemolytic Anemia ◽  
Clinical Laboratory ◽  
Single Gene ◽  
Cytoskeletal Proteins ◽  
Next Generation ◽  
Pathogenic Variants ◽  
Late Morbidity ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

Abstract Hereditary hemolytic anemias are a heterogeneous group of disorders with consequences ranging from non-anemic hemolysis to severe life-threatening anemia. However, the late morbidity in patients without transfusions is often underappreciated because of erythropoietic compensatory stimulation inducing hematopoiesis by erythroferrone/hepcidin axis. Principal causes of hereditary hemolytic anemias are germline mutations of red cell cytoskeleton (e.g. hereditary spherocytosis and elliptocytosis/pyropoikilocytosis) or enzyme deficiencies (e.g. Glucose 6 phosphate dehydrogenase deficiency and pyruvate kinase deficiency). Routine morphological and biochemical analysis may be inconclusive and misleading particularly in transfusion-dependent infants and children. Molecular studies have not been extensively used to diagnose these disorders due to the complex genetic nature of these disorders, and multi-gene disorders. In these cases, patients may undergo multiple rounds of single gene testing, which can be very costly and time consuming. The advent of next generation sequencing (NGS) methods in the clinical laboratory has made diagnosing complex genetic disorders feasible. Our diagnostic panel includes 28 genes encoding cytoskeletal proteins and enzymes, and covers the complete coding region, splice site junctions, and, where appropriate, deep intronic or regulatory regions. Targeted gene capture and library construction for next-generation sequencing (NGS) was performed using Sure Select kit (Agilent Technologies, Santa Clara, USA). Prior to sequencing on the Illumina Next Seq, (Illumina Inc) instrument, indexed samples are quantified using qPCR and then pooled. Samples were sequenced using 2x150 paired end sequencing. We now report the first 68 patients evaluated using our NGS panel. The age of the patients ranged from newborn to 62 years. These patients presented with symptoms ranging from mild lifelong anemia to severe hemolytic anemia with extreme hyperbilirubinemia. Genetic variants were classified using the American College of Medical Genetics (ACMG) guidelines. We identified pathogenic variants in 11 patients and likely pathogenic variants in 12 others, the majority of these were novel. Many variants with unknown significance were also identified that could potentially contribute to disease. The most commonly mutated genes were SPTB and SPTA1, encoding spectrin subunits. Some complex interactions were uncovered i.e. SPTA1 mutations along with alpha LELY leading to hereditary pyropoikilocytosis; Spectrin variants along with Gilbert syndrome causing severe hyperbilirubinemia in neonates; and Spectrin variants in combination with PKLR and G6PD variants. Our results demonstrate that many patients with hemolytic anemia harbor complex combinations of known and novel mutations in RBC cytoskeleton/enzyme genes, but their clinical significance is further augmented by polymorphisms of UGT1A1 gene contributing to severe neonatal hyperbilirubinemia and its consequences. To conclude, next-generation sequencing provides a cost-effective and relatively rapid approach to molecular diagnosis, especially in instances where traditional testing failed. We have used this technology successfully to determine the molecular causes of hemolytic anemia in many cases with no prior family history. Disclosures Yaish: Octapharma: Other: Study investigator.

scholarly journals Molecular profiling of congenital glioblastoma using a next-generation sequencing panel

2021 ◽  
Author(s):  
Débora Cabral de Carvalho Corrêa ◽  
Francine Tesser-Gamba ◽  
Nasjla da Silva ◽  
Andrea Capellano ◽  
Maria Teresa Alves ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Genetic Variants ◽  
Next Generation ◽  
Congenital Brain Tumor ◽  
Molecular Alterations ◽  
Pathogenic Variants ◽  
Pediatric Neoplasms ◽  
Tumor Entity ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

Background Congenital GBM (cGBM), presenting prenatally or within the first months of life, is among the rarest type of congenital brain tumor, with approximately 120 cases reported. Due to its infrequent occurrence, few studies have focused on the molecular and genetic aspects of this tumor, and the mutational events involved in the pathogenesis and progression of cGBM still remains poorly understood. This study aimed to investigate molecular alterations, with a potential prognostic marker and therapeutic target in cGBM using the next-generation sequencing (NGS) strategy. Methods We selected seven tumor samples from patients diagnosed with cGBM and treated at Pediatric Oncology Institute-GRAACC/UNIFESP. NGS was performed to identify somatic genetic variants in tumor samples using the Oncomine Childhood Cancer Research Assay panel, from ThermoFisher Scientific, designed specifically for pediatric neoplasms. Results Of all seven patients analyzed, three patients exhibited tumors with genetic variants, which include two pathogenic variants in NF1 and SUZ12 genes that have not been reported in cGBM yet, an increase in the number of copies of ALK gene, and two gene fusions, PPP1CB-ALK and TPM3-NTRK1. Also, none of the cases showed variants in H3F3A, TP53 and ATRX genes, alterations which are frequently seen in pediatric and adolescent GBM. Conclusions Our results suggest that cGBM may comprise a unique tumor entity and alterations in ALK and NTRK genes provide a potential target for therapy. Therefore, identification of genetic variants in cGBM is highly relevant in order to define prognosis and therapeutic strategies.

scholarly journals 40 minutes RT-qPCR Assay for Screening Spike N501Y and HV69-70del Mutations

2021 ◽  
Author(s):  
Gulay Korukluoglu ◽  
Mustafa Kolukirik ◽  
Fatma Bayrakdar ◽  
Gozde Girgin Ozgumus ◽  
Ayse Basak Altas ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Internal Control ◽  
Sanger Sequencing ◽  
Limit Of Detection ◽  
Qpcr Assay ◽  
Rapid Screening ◽  
Clinical Samples ◽  
One Step ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

ABSTRACTA one-step reverse transcription and real-time PCR (RT-qPCR) test was developed for rapid screening (40 minutes) of the Spike N501Y and HV69-70del mutations in SARS-CoV-2 positive samples. The test also targets a conserved region of SARS-CoV-2 Orf1ab as an internal control. The samples containing both the N501Y and HV69-70del mutations are concluded as VOC-202012/01 positive. Samples suspected to be positive for B.1.351 or P.1 are the N501Y positive and HV69-70del negative cases. Limit of detection (LOD) of the kit for Orf1ab target is 500 copies/mL, while that of the N501, Y501 and HV69-70del targets are 5000 copies/mL. The developed assay was applied to 165 clinical samples containing SARS-CoV-2 from 32 different lineages. The SARS-CoV-2 lineages were determined via the next-generation sequencing (NGS). The RT-qPCR results were in 100% agreement with the NGS results that 19 samples were N501Y and HV69-70del positive, 10 samples were N501Y positive and HV69-70del negative, 1 sample was N501Y negative and HV69-70del positive, and 135 samples were N501Y and HV69-70del negative. All the VOC-202012/01 positive samples were detected in people who have traveled from England to Turkey. The RT-qPCR test and the Sanger sequencing was further applied to 1000 SARS-CoV-2 positive clinical samples collected in Jan2021 from the 81 different provinces of Turkey. The RT-qPCR results were in 100% agreement with the Sanger sequencing results that 32 samples were N501Y positive and HV69-70del negative, 4 samples were N501Y negative and HV69-70del positive, 964 samples were N501Y and HV69-70del negative. The specificity of the 40 minutes RT-qPCR assay relative to the sequencing-based technologies is 100%. The developed assay is an advantageous tool for timely and representative estimation of the N501Y positive variants’ prevalence because it allows testing a much higher portion of the SARS-CoV-2 positives in much lower time compared to the sequencing-based technologies.

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