Next-generation sequencing in the clinical genetic screening of patients with pheochromocytoma and paraganglioma

BackgroundRecent findings have shown that up to 60% of pheochromocytomas (PCCs) and paragangliomas (PGLs) are caused by germline or somatic mutations in one of the 11 hitherto known susceptibility genes: SDHA, SDHB, SDHC, SDHD, SDHAF2, VHL, HIF2A (EPAS1), RET, NF1, TMEM127 and MAX. This list of genes is constantly growing and the 11 genes together consist of 144 exons. A genetic screening test is extensively time consuming and expensive. Hence, we introduce next-generation sequencing (NGS) as a time-efficient and cost-effective alternative.MethodsTumour lesions from three patients with apparently sporadic PCC were subjected to whole exome sequencing utilizing Agilent Sureselect target enrichment system and Illumina Hi seq platform. Bioinformatics analysis was performed in-house using commercially available software. Variants in PCC and PGL susceptibility genes were identified.ResultsWe have identified 16 unique genetic variants in PCC susceptibility loci in three different PCC, spending less than a 30-min hands-on, in-house time. Two patients had one unique variant each that was classified as probably and possibly pathogenic: NF1 Arg304Ter and RET Tyr791Phe. The RET variant was verified by Sanger sequencing.ConclusionsNGS can serve as a fast and cost-effective method in the clinical genetic screening of PCC. The bioinformatics analysis may be performed without expert skills. We identified process optimization, characterization of unknown variants and determination of additive effects of multiple variants as key issues to be addressed by future studies.

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Validation of a semiconductor next-generation sequencing assay for the clinical genetic screening ofCFTR

Molecular Genetics & Genomic Medicine ◽

10.1002/mgg3.149 ◽

2015 ◽

Vol 3 (5) ◽

pp. 396-403 ◽

Cited By ~ 16

Author(s):

Daniel Trujillano ◽

Maximilian E. R. Weiss ◽

Julia Köster ◽

Efstathios B. Papachristos ◽

Martin Werber ◽

...

Keyword(s):

Next Generation Sequencing ◽

Genetic Screening ◽

Next Generation ◽

Clinical Genetic ◽

Generation Sequencing

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Combined use of gap-PCR and next-generation sequencing improves thalassaemia carrier screening among premarital adults in China

Journal of Clinical Pathology ◽

10.1136/jclinpath-2019-206339 ◽

2020 ◽

Vol 73 (8) ◽

pp. 488-492 ◽

Cited By ~ 4

Author(s):

Jianghong Zhao ◽

Jia Li ◽

Qiaohong Lai ◽

Yanping Yu

Keyword(s):

Next Generation Sequencing ◽

Southern China ◽

Cost Effective ◽

Carrier Screening ◽

Lower Sensitivity ◽

Carrier Rate ◽

Next Generation ◽

Cost Effective Method ◽

Next Generation Sequencing Ngs ◽

Generation Sequencing

AimsThalassaemia is one of the most common genetics disorders in the world, especially in southern China. The aim of the present study was to investigate the feasibility of combining the gap-PCR and next-generation sequencing (NGS) for thalassaemia carrier screening in the Chinese population.MethodsBlood samples were obtained from 944 prepregnancy couples; thalassaemia carrier screening was performed by using a routine haematological method and a combination of gap-PCR and NGS method.ResultsWe found that the α thalassaemia carrier rate was 11% (207/1888); the β thalassaemia carrier rate was 3.7% (70/1888); the composite α thalassaemia and β thalassaemia carrier rate was 0.4% (8/1888). We also identified seven novel mutations, including HBA1: c.412A>G, −50 (G>A), HBB: c.*+129T>A, HBB: c.-64G>C, HBB: c.-180G>C, HBB: c.*+5G>A and HBB: c.-113A>G. By comparing the combined gap-PCR and NGS method, the MCV+MCH and HbA2 detection strategy showed a lower sensitivity of 61.05% (105/172) and a higher missed diagnosis ratio of 38.95% (67/172) for α thalassaemia mutations. The sensitivity was improved with the MCV+MCH and HbA2 detection screen when compared with MCV+MCH detection for β thalassaemia (98.51% vs 85.90%).ConclusionsOur study suggests the combined gap-PCR and NGS method is a cost-effective method for the thalassaemia carrier screening, particularly for the α thalassaemia mutation carriers.

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Clinical Use of Next-Generation Sequencing in the Diagnosis of Wilson’s Disease

Gastroenterology Research and Practice ◽

10.1155/2016/4548039 ◽

2016 ◽

Vol 2016 ◽

pp. 1-6 ◽

Cited By ~ 6

Author(s):

Dániel Németh ◽

Kristóf Árvai ◽

Péter Horváth ◽

János Pál Kósa ◽

Bálint Tobiás ◽

...

Keyword(s):

Next Generation Sequencing ◽

Sanger Sequencing ◽

Wilson’S Disease ◽

Cost Effective ◽

Wilson's Disease ◽

Clinical Use ◽

Next Generation ◽

Time Saving ◽

Cost Effective Method ◽

Generation Sequencing

Objective. Wilson’s disease is a disorder of copper metabolism which is fatal without treatment. The great number of disease-causingATP7Bgene mutations and the variable clinical presentation of WD may cause a real diagnostic challenge. The emergence of next-generation sequencing provides a time-saving, cost-effective method for full sequencing of the wholeATP7Bgene compared to the traditional Sanger sequencing. This is the first report on the clinical use of NGS to examineATP7Bgene.Materials and Methods. We used Ion Torrent Personal Genome Machine in four heterozygous patients for the identification of the other mutations and also in two patients with no known mutation. One patient with acute on chronic liver failure was a candidate for acute liver transplantation. The results were validated by Sanger sequencing.Results. In each case, the diagnosis of Wilson’s disease was confirmed by identifying the mutations in both alleles within 48 hours. One novel mutation (p.Ala1270Ile) was found beyond the eight other known ones. The rapid detection of the mutations made possible the prompt diagnosis of WD in a patient with acute liver failure.Conclusions. According to our results we found next-generation sequencing a very useful, reliable, time-saving, and cost-effective method for diagnosing Wilson’s disease in selected cases.

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Germline Pathogenic Variants Identified by Targeted Next-Generation Sequencing of Susceptibility Genes in Pheochromocytoma and Paraganglioma

Journal of Kidney Cancer and VHL ◽

10.15586/jkcvhl.v8i1.171 ◽

2021 ◽

Vol 8 (1) ◽

pp. 19-24

Author(s):

Sinem Yalcintepe ◽

Hakan Gurkan ◽

Fatma Nur Korkmaz ◽

Selma Demir ◽

Engin Atli ◽

...

Keyword(s):

Next Generation Sequencing ◽

Susceptibility Genes ◽

Next Generation ◽

Clinical Genetic ◽

Targeted Next Generation Sequencing ◽

Clinical Genetic Testing ◽

Pathogenic Variants ◽

Diagnosis Rate ◽

Gene Panels ◽

Generation Sequencing

The aim of this study was to evaluate germline variant frequencies of pheochromocytoma and paraganglioma targeted susceptibility genes with next-generation sequencing method. Germline DNA from 75 cases were evaluated with targeted next-generation sequencing on an Illumina NextSeq550 instrument. KIF1B, RET, SDHB, SDHD, TMEM127, and VHL genes were included in the study, and Sanger sequencing was used for verifying the variants. The pathogenic/likely pathogenic variants were in the VHL, RET, SDHB, and SDHD genes, and the diagnosis rate was 24% in this study. Three different novel pathogenic variants were determined in five cases. This is the first study from Turkey, evaluating germline susceptibility genes of pheochromocytoma and paraganglioma with a detection rate of 24% and three novel variants. All patients with pheochromocytoma and paraganglioma need clinical genetic testing with expanded targeted gene panels for higher diagnosis rates.

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An efficient and cost-effective method for primer-induced nucleotide labeling for massive sequencing on next-generation sequencing platforms

Scientific Reports ◽

10.1038/s41598-019-38996-8 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 1

Author(s):

Junjie Guo ◽

Tao Cheng ◽

Han Xu ◽

Yide Li ◽

Jie Zeng

Keyword(s):

Next Generation Sequencing ◽

Cost Effective ◽

Next Generation ◽

Cost Effective Method ◽

Massive Sequencing ◽

Sequencing Platforms ◽

Generation Sequencing

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The Genetic Approach: Next-Generation Sequencing-Based Diagnosis of Congenital and Infantile Myopathies/Muscle Dystrophies

Neuropediatrics ◽

10.1055/s-0037-1602660 ◽

2017 ◽

Vol 48 (04) ◽

pp. 242-246 ◽

Cited By ~ 6

Author(s):

Simone Rost ◽

Konstantin Kolokotronis ◽

Gerhard Meng ◽

Natalie Pluta ◽

Clemens Müller-Reible ◽

...

Keyword(s):

Next Generation Sequencing ◽

Cost Effective ◽

Massive Parallel Sequencing ◽

Next Generation ◽

Juvenile Onset ◽

Parallel Sequencing ◽

Copy Numbers ◽

Cost Effective Method ◽

Generation Sequencing ◽

Variant Identification

AbstractThe practical basis for massive parallel sequencing is described to help clinicians in choosing the most adequate diagnostic approach for childhood myopathies. The key quality feature for massive parallel sequencing is the sequence depth (coverage) as a prerequisite for variant identification and quantification of sequence copy numbers. Our experience with a next-generation sequencing gene panel for the analysis of muscular dystrophies/myopathies with infantile or juvenile onset resulted in the identification of pathogenic or likely pathogenic mutations in approximately 41% (of 141 patients), thus leading to a definitive diagnosis. A subset of patients shows an accumulation of “excess” heterozygous variants that may act as modifiers of the phenotype. Massive parallel sequencing has become a reliable and cost-effective method, but it requires exact clinical, bioptic, and/or radiologic information to evaluate the clinical relevance of possibly pathologic variants.

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Validation of variants using cost effective highresolution melting (HRM) analysis predicted from target re-sequencing in Eucalyptus

Acta Botanica Croatica ◽

10.37427/botcro-2020-019 ◽

2020 ◽

Vol 79 (2) ◽

pp. 105-113

Author(s):

Abdul Bari Muneera Parveen ◽

Divya Lakshmanan ◽

Modhumita Ghosh Dasgupta

Keyword(s):

Next Generation Sequencing ◽

Large Scale ◽

Sequence Data ◽

Cost Effective ◽

Nucleotide Polymorphisms ◽

Next Generation ◽

Time Saving ◽

Hrm Analysis ◽

The Cost ◽

Generation Sequencing

The advent of next-generation sequencing has facilitated large-scale discovery and mapping of genomic variants for high-throughput genotyping. Several research groups working in tree species are presently employing next generation sequencing (NGS) platforms for marker discovery, since it is a cost effective and time saving strategy. However, most trees lack a chromosome level genome map and validation of variants for downstream application becomes obligatory. The cost associated with identifying potential variants from the enormous amount of sequence data is a major limitation. In the present study, high resolution melting (HRM) analysis was optimized for rapid validation of single nucleotide polymorphisms (SNPs), insertions or deletions (InDels) and simple sequence repeats (SSRs) predicted from exome sequencing of parents and hybrids of Eucalyptus tereticornis Sm. ? Eucalyptus grandis Hill ex Maiden generated from controlled hybridization. The cost per data point was less than 0.5 USD, providing great flexibility in terms of cost and sensitivity, when compared to other validation methods. The sensitivity of this technology in variant detection can be extended to other applications including Bar-HRM for species authentication and TILLING for detection of mutants.

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