Whole-exome sequencing of human pancreatic cancers and characterization of genomic instability caused by MLH1 haploinsufficiency and complete deficiency

Abstract

Whole-exome sequencing (Exome-seq) has been successfully applied in several recent studies. We here sequenced the exomes of 15 pancreatic tumor cell lines and their matched normal samples. We captured 162,073 exons of 16,954 genes and sequenced the targeted regions to a mean coverage of 56-fold. This study identified a total of 1517 somatic mutations and validated 934 mutations by transcriptome sequencing. We detected recurrent mutations in 56 genes. Among them, 41 have not been described. The mutation rates varied widely among cell lines. The diversity of the mutation rates was significantly correlated with the distinct MLH1 copy-number status. Exome-seq revealed intensive genomic instability in a cell line with MLH1 homozygous deletion, indicated by a dramatically elevated rate of somatic substitutions, small insertions/deletions (indels), as well as indels in microsatellites. Notably, we found that MLH1 expression was decreased by nearly half in cell lines with an allelic loss of MLH1. While these cell lines were negative in conventional microsatellite instability assay, they showed a 10.5-fold increase in the rate of somatic indels, e.g., truncating indels in TP53 and TGFBR2, indicating MLH1 haploinsufficiency in the correction of DNA indel errors. We further analyzed the exomes of 15 renal cell carcinomas and confirmed MLH1 haploinsufficiency. We observed a much higher rate of indel mutations in the affected cases and identified recurrent truncating indels in several cancer genes such as VHL, PBRM1, and JARID1C. Together, our data suggest that MLH1 hemizygous deletion, through increasing the rate of indel mutations, could drive the development and progression of sporadic cancers.

From human genome to cancer genome: The first decade

“…RNA-seq provides not only gene expression levels, but also aberrant splicing, chimeric gene fusion transcripts characteristic of cancer cells and expressed somatic mutations (Bainbridge et al 2006;Dong et al 2009;Maher et al 2009;Shah et al 2009;Berger et al 2010;Tuch et al 2010;Wang et al 2012). Analysis of chromatin modification is in its infancy as applied to the cancer cell, but the recent reporting of the ENCODE Project Consortium's genome-wide results (The ENCODE Project Consortium 2012) may provide the tools and technologies to enable new approaches.…”

Essential roles of the histone methyltransferase ESET in the epigenetic control of neural progenitor cells during development

“…After amplification for 15 cycles, PCR products were purified with Agencourt AMPure XP magnetic beads (Beckman Coulter), and their size distribution and concentration were evaluated using a DNA 1000 Series II Assay on the 2100 Bioanalyzer. cDNA at 4.2 pM/lane was applied to the flow cell and paired-end 75 nt reads were sequenced on an Illumina GAIIx following the manufacturer's instructions (Wang et al, 2011). RNA sequencing results were presented using Integrative Genomics Viewer (Broad Institute) version 2.0.…”

Epigenetic Alterations in Pancreatic Cancer

“…[1][2][3] Most of the studies have been focused on genetic abnormalities of driver mutations that occur in more than 50% of the pancreatic cancer cases, such as KRAS, TP53, SMAD4, and CDKN2A. The other lower prevalence mutated genes include those involved in Deoxyribonucleic acid (DNA) damage repair, chromatin remodeling, WNT signaling, Transforming Growth Factor (TGF) beta signaling, Hedgehog (Hh) signaling, and cell cycle regulation pathways.…”