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A combination of targeted enrichment methodologies for whole-exome sequencing reveals novel pathogenic mutations.

ABSTRACT: Whole-exome sequencing (WES) is a useful method to identify disease-causing mutations, however, often no candidate mutations are identified using commonly available targeted probe sets. In a recent analysis, we also could not find candidate mutations for 20.9% (9/43) of our pedigrees with congenital neurological disorder using pre-designed capture probes (SureSelect V4 or V5). One possible cause for this lack of candidates is that standard WES cannot sequence all protein-coding sequences (CDS) due to capture probe design and regions of low coverage, which account for approximately 10% of all CDS regions. In this study, we combined a selective circularization-based target enrichment method (HaloPlex) with a hybrid capture method (SureSelect V5; WES), and achieved a more complete coverage of CDS regions (~97% of all CDS). We applied this approach to 7 (SureSelect V5) out of 9 pedigrees with no candidates through standard WES analysis and identified novel pathogenic mutations in one pedigree. The application of this effective combination of targeted enrichment methodologies can be expected to aid in the identification of novel pathogenic mutations previously missed by standard WES analysis.

SUBMITTER: Miya F

PROVIDER: S-EPMC4365396 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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A combination of targeted enrichment methodologies for whole-exome sequencing reveals novel pathogenic mutations.

Miya Fuyuki F Kato Mitsuhiro M Shiohama Tadashi T Okamoto Nobuhiko N Saitoh Shinji S Yamasaki Mami M Shigemizu Daichi D Abe Tetsuo T Morizono Takashi T Boroevich Keith A KA Kosaki Kenjiro K Kanemura Yonehiro Y Tsunoda Tatsuhiko T

Scientific reports 20150319

Whole-exome sequencing (WES) is a useful method to identify disease-causing mutations, however, often no candidate mutations are identified using commonly available targeted probe sets. In a recent analysis, we also could not find candidate mutations for 20.9% (9/43) of our pedigrees with congenital neurological disorder using pre-designed capture probes (SureSelect V4 or V5). One possible cause for this lack of candidates is that standard WES cannot sequence all protein-coding sequences (CDS) d ...[more]

PMID: 25786579

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Project description:Lung cancer remains a substantial health challenge, with distinct genetic factors influencing disease susceptibility and progression. This study aimed to decipher the landscape of DNA repair gene mutations in Pakistani lung cancer patients using Whole Exome Sequencing (WES) and to investigate their potential functional implications through downstream analyses. WES analysis of genomic DNA from 15 lung cancer patients identified clinically important pathogenic mutations in 6 DNA repair genes, including, BReast CAncer gene 1 (BRCA1), BReast CAncer gene 2 (BRCA2), Excision Repair Cross Complementing rodent repair deficiency, complementation group 6 (ERCC6), Checkpoint Kinase 1 (CHEK1), mutY DNA glycosylase (MUTYH), and RAD51D (RAD51 Paralog D). Kaplan-Meier (KM) analysis showed that pathogenic mutations in BRCA1, BRCA2, ERCC6, CHEK1, MUTYH, and RAD51D genes were the prognostic biomarkers of worse OS in lung cancer patients. To explore the functional impact of these mutations, we performed Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Immunohistochemistry (IHC) analyses. Our results revealed a down-regulation in the expression of the mutated genes, indicating a potential link between the identified mutations and reduced gene activity. This down-regulation could contribute to compromised DNA repair efficiency, thereby fostering genomic instability in lung cancer cells. Furthermore, targeted bisulfite sequencing analysis was employed to assess the DNA methylation status of the mutated genes. Strikingly, hypermethylation in the promoters of BRCA1, BRCA2, ERCC6, CHEK1, MUTYH, and RAD51D was observed across lung cancer samples harboring pathogenic mutations, suggesting the involvement of epigenetic mechanism underlying the altered gene expression. In conclusion, this study provides insights into the genetic landscape of DNA repair gene mutations in Pakistani lung cancer patients. The observed pathogenic mutations in BRCA1, BRCA2, ERCC6, CHEK1, MUTYH, and RAD51D, coupled with their down-regulation and hypermethylation, suggest a potential convergence of genetic and epigenetic factors driving genomic instability in lung cancer cells. These findings contribute to our understanding of lung cancer susceptibility and highlight potential avenues for targeted therapeutic interventions in Pakistani lung cancer patients.

Dataset Information

A combination of targeted enrichment methodologies for whole-exome sequencing reveals novel pathogenic mutations.

Publications

A combination of targeted enrichment methodologies for whole-exome sequencing reveals novel pathogenic mutations.

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