Abstract:BackgroundCancer susceptibility germline variants generally require somatic alteration of the remaining allele to drive oncogenesis and, in some cases, tumor mutational profiles. Whether combined germline and somatic bi-allelic alterations are universally required for germline variation to influence tumor mutational profile is unclear. Here, we performed an exome-wide analysis of the frequency and functional effect of bi-allelic alterations in The Cancer Genome Atlas (TCGA).MethodsWe integrated germline varian… Show more
“…We investigated a set of 382 genes (referred to as cancer genes) involved in DNA repair, cancer susceptibility and progression (Additional file 1, Table S7). [32] Of those, 66 were altered by 82 variants, including 7 in the MLH1 gene of the LS patients, therefore confirming the panel sequencing results and validating the initial WES variant calling and filtering approach.…”
Section: Resultssupporting
confidence: 61%
“…To discover novel CPG, we focused on high-confidence (TLOD>12, FS<10, SEQQ>60, MQε60, STRANDQ>40, DP>10), rare (minor allele fraction was < 0.01 in ExAC of Eastern Asian population), and damaging (exonic/splicing, non-synonymous variants with >20 of CADD score) variants in 382 genes of 11 cancer-relevant pathways, [32] and they were defined as LoF germline variants. High-confidence of LoF variants were predicted by LOFTEE plugin for Ensembl VEP (v.99).…”
Section: Methodsmentioning
confidence: 99%
“…Event prevalence was set 0.1, and AI was defined when posterior probability of being in AI was over 90%. To assess loss of heterozygosity (LOH) of a given heterozygous germline variants in tumors as a secondary hit mechanism of inactivation of CPG, [32, 37] we evaluated germline and somatic bi-allelic alteration of corresponding tumors. When there was any one of somatic damaging mutation or AI event in candidate germline CPG, it was considered as bi-allelic alteration.…”
Section: Methodsmentioning
confidence: 99%
“…To compare the frequency of recurrent CPG in patients of eoMPCs to that of the cancer genome atlas (TCGA) cohort, germline data of East-Asian patients (≥80% of admixture) [38] with stomach cancer or colorectal cancer in TCGA cohort was used. [32, 39] Variants were filtered for rare and damaged variants similar to LoF germline variants in MPCs cohort. For recurrent variants and genes in eoMPCs cohort, the frequency was compared between eoMPCs cohort and selected TCGA cohort.…”
Background: Along with early onset cancers, multiple primary cancers (MPCs) are likely resulting from increased genetic susceptibility; however, the associated predisposition genes or prevalence of the pathogenic variants genes in MPC patients are often unknown. Methods: We screened 71 patients with MPC of the stomach, colorectal, and endometrium, sequencing 65 cancer predisposition genes. A subset of 19 patients with early onset MPC of stomach and colorectum were further evaluated for at DNA repair and cancer related genes using both normal (germline) and tumor (somatic) whole exome sequencing. Results: Among 71 patients with MPCs, variants predicted to be pathogenic were observed in 15 (21.1%) patients and affected Lynch Syndrome (LS) genes: MLH1 (n=10), MSH6 (n=2), PMS2 (n=2), and MSH2 (n=1). All carriers had tumors with high microsatellite instability and 13 of them (86.7%) were early-onset, consistent with LS. In 19 patients with early-onset MPCs, loss of function (LoF) variants in RECQL5, including a rare East-Asian specific variant, were more prevalent in non-LS MPC than in matched sporadic cancer patients (OR=31.6, p=0.001). Additional evaluation of bi-allelic alterations in the tumor correctly identified LS genes in LS patients and candidates genes in non-LS patients including high-confidence LoF variants in 2 patients, FANCG (c.307+1G>C) and CASP8 (p.R221Sfs*17) both accompanied by somatic loss of heterozygosity in a gastric and a colorectal tumor, respectively. Conclusions: The results suggest that genetic screening should be considered for synchronous cancers and metachronous MPCs of the LS tumor spectrum, particularly in early-onset patients. Susceptibility variants in non-LS genes for MPC patients may exist, but evidence for their role is more elusive than for LS patients.
“…We investigated a set of 382 genes (referred to as cancer genes) involved in DNA repair, cancer susceptibility and progression (Additional file 1, Table S7). [32] Of those, 66 were altered by 82 variants, including 7 in the MLH1 gene of the LS patients, therefore confirming the panel sequencing results and validating the initial WES variant calling and filtering approach.…”
Section: Resultssupporting
confidence: 61%
“…To discover novel CPG, we focused on high-confidence (TLOD>12, FS<10, SEQQ>60, MQε60, STRANDQ>40, DP>10), rare (minor allele fraction was < 0.01 in ExAC of Eastern Asian population), and damaging (exonic/splicing, non-synonymous variants with >20 of CADD score) variants in 382 genes of 11 cancer-relevant pathways, [32] and they were defined as LoF germline variants. High-confidence of LoF variants were predicted by LOFTEE plugin for Ensembl VEP (v.99).…”
Section: Methodsmentioning
confidence: 99%
“…Event prevalence was set 0.1, and AI was defined when posterior probability of being in AI was over 90%. To assess loss of heterozygosity (LOH) of a given heterozygous germline variants in tumors as a secondary hit mechanism of inactivation of CPG, [32, 37] we evaluated germline and somatic bi-allelic alteration of corresponding tumors. When there was any one of somatic damaging mutation or AI event in candidate germline CPG, it was considered as bi-allelic alteration.…”
Section: Methodsmentioning
confidence: 99%
“…To compare the frequency of recurrent CPG in patients of eoMPCs to that of the cancer genome atlas (TCGA) cohort, germline data of East-Asian patients (≥80% of admixture) [38] with stomach cancer or colorectal cancer in TCGA cohort was used. [32, 39] Variants were filtered for rare and damaged variants similar to LoF germline variants in MPCs cohort. For recurrent variants and genes in eoMPCs cohort, the frequency was compared between eoMPCs cohort and selected TCGA cohort.…”
Background: Along with early onset cancers, multiple primary cancers (MPCs) are likely resulting from increased genetic susceptibility; however, the associated predisposition genes or prevalence of the pathogenic variants genes in MPC patients are often unknown. Methods: We screened 71 patients with MPC of the stomach, colorectal, and endometrium, sequencing 65 cancer predisposition genes. A subset of 19 patients with early onset MPC of stomach and colorectum were further evaluated for at DNA repair and cancer related genes using both normal (germline) and tumor (somatic) whole exome sequencing. Results: Among 71 patients with MPCs, variants predicted to be pathogenic were observed in 15 (21.1%) patients and affected Lynch Syndrome (LS) genes: MLH1 (n=10), MSH6 (n=2), PMS2 (n=2), and MSH2 (n=1). All carriers had tumors with high microsatellite instability and 13 of them (86.7%) were early-onset, consistent with LS. In 19 patients with early-onset MPCs, loss of function (LoF) variants in RECQL5, including a rare East-Asian specific variant, were more prevalent in non-LS MPC than in matched sporadic cancer patients (OR=31.6, p=0.001). Additional evaluation of bi-allelic alterations in the tumor correctly identified LS genes in LS patients and candidates genes in non-LS patients including high-confidence LoF variants in 2 patients, FANCG (c.307+1G>C) and CASP8 (p.R221Sfs*17) both accompanied by somatic loss of heterozygosity in a gastric and a colorectal tumor, respectively. Conclusions: The results suggest that genetic screening should be considered for synchronous cancers and metachronous MPCs of the LS tumor spectrum, particularly in early-onset patients. Susceptibility variants in non-LS genes for MPC patients may exist, but evidence for their role is more elusive than for LS patients.
“…More specifically, silencing of SHPRH is associated with one of the characteristic cancer mutational signatures (mutational signature 6), which is common in uterine cancer. Interestingly, Signature 6 is also associated with mutations in DNA mismatch repair genes and is found in tumors showing elevated MSI [6]. Fig.…”
Background
Maintenance of genome integrity during DNA replication is crucial to the perpetuation of all organisms. In eukaryotes, the bypass of DNA lesions by the replication machinery prevents prolonged stalling of the replication fork, which could otherwise lead to greater damages such as gross chromosomal rearrangements. Bypassing DNA lesions and subsequent repair are accomplished by the activation of DNA damage tolerance pathways such as the template switching (TS) pathway. In yeast, the RAD5 (Radiation-sensitive 5) protein plays a crucial role in initiating the TS pathway by catalyzing the polyubiquitination of PCNA (Proliferation Cell Nuclear Antigen). Likewise, one of the mammalian RAD5-homologs, SHPRH (SNF2, histone linker, PHD, RING, helicase) mediates PCNA polyubiquitination. To date, the study of SHPRH enzymatic functions has been limited to this modification. It is therefore unclear how SHPRH carries out its function in DNA repair. Moreover, how this protein regulates gene transcription at the enzymatic level is also unknown.
Results
Given that SHPRH harbors domains found in chromatin remodeling proteins, we investigated its biochemical properties in the presence of nucleosomal substrates. We find that SHPRH binds equally well to double-stranded (ds) DNA and to nucleosome core particles, however, like ISWI and CHD-family remodelers, SHPRH shows a strong preference for nucleosomes presenting extranucleosomal DNA. Moreover, nucleosomes but not dsDNA strongly stimulate the ATPase activity of SHPRH. Intriguingly, unlike typically observed with SNF2-family enzymes, ATPase activity does not translate into conventional nucleosome remodeling, under standard assay conditions. To test whether SHPRH can act as a ubiquitin E3 ligase for nucleosomes, we performed a screen using 26 E2-conjugating enzymes. We uncover that SHPRH is a potent nucleosome E3-ubiquitin-ligase that can function with at least 7 different E2s. Mass spectrometry analyses of products generated in the presence of the UBE2D1-conjugating enzyme reveal that SHPRH can catalyze the formation of polyubiquitin linkages that are either branched or associated with the recruitment of DNA repair factors, as well as linkages involved in proteasomal degradation.
Conclusions
We propose that, in addition to polyubiquitinating PCNA, SHPRH promotes DNA repair or transcriptional regulation in part through chromatin ubiquitination. Our study sets a biochemical framework for studying other RAD5- and RAD16-related protein functions through the ubiquitination of nucleosomes.
Electronic supplementary material
The online version of this article (10.1186/s13072-019-0294-5) contains supplementary material, which is available to authorized users.
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