To determine whether cancer risks for carriers and noncarriers from families with a mismatch repair (MMR) gene mutation are increased above the risks of the general population.
We prospectively ...followed a cohort of 446 unaffected carriers of an MMR gene mutation (MLH1, n = 161; MSH2, n = 222; MSH6, n = 47; and PMS2, n = 16) and 1,029 their unaffected relatives who did not carry a mutation every 5 years at recruitment centers of the Colon Cancer Family Registry. For comparison of cancer risk with the general population, we estimated country-, age-, and sex-specific standardized incidence ratios (SIRs) of cancer for carriers and noncarriers.
Over a median follow-up of 5 years, mutation carriers had an increased risk of colorectal cancer (CRC; SIR, 20.48; 95% CI, 11.71 to 33.27; P < .001), endometrial cancer (SIR, 30.62; 95% CI, 11.24 to 66.64; P < .001), ovarian cancer (SIR, 18.81; 95% CI, 3.88 to 54.95; P < .001), renal cancer (SIR, 11.22; 95% CI, 2.31 to 32.79; P < .001), pancreatic cancer (SIR, 10.68; 95% CI, 2.68 to 47.70; P = .001), gastric cancer (SIR, 9.78; 95% CI, 1.18 to 35.30; P = .009), urinary bladder cancer (SIR, 9.51; 95% CI, 1.15 to 34.37; P = .009), and female breast cancer (SIR, 3.95; 95% CI, 1.59 to 8.13; P = .001). We found no evidence of their noncarrier relatives having an increased risk of any cancer, including CRC (SIR, 1.02; 95% CI, 0.33 to 2.39; P = .97).
We confirmed that carriers of an MMR gene mutation were at increased risk of a wide variety of cancers, including some cancers not previously recognized as being a result of MMR mutations, and found no evidence of an increased risk of cancer for their noncarrier relatives.
ABSTRACT
We studied 17,576 members of 166 MLH1 and 224 MSH2 mutation‐carrying families from the Colon Cancer Family Registry. Average cumulative risks of colorectal cancer (CRC), endometrial cancer ...(EC), and other cancers for carriers were estimated using modified segregation analysis conditioned on ascertainment criteria. Heterogeneity in risks was investigated using a polygenic risk modifier. Average CRC cumulative risks at the age of 70 years (95% confidence intervals) for MLH1 and MSH2 mutation carriers, respectively, were estimated to be 34% (25%–50%) and 47% (36%–60%) for male carriers and 36% (25%–51%) and 37% (27%–50%) for female carriers. Corresponding EC risks were 18% (9.1%–34%) and 30% (18%–45%). A high level of CRC risk heterogeneity was observed (P < 0.001), with cumulative risks at the age of 70 years estimated to follow U‐shaped distributions. For example, 17% of male MSH2 mutation carriers have estimated lifetime risks of 0%–10% and 18% have risks of 90%–100%. Therefore, average risks are similar for the two genes but there is so much individual variation about the average that large proportions of carriers have either very low or very high lifetime cancer risks. Our estimates of CRC and EC cumulative risks for MLH1 and MSH2 mutation carriers are the most precise currently available.
Our estimates of colorectal and endometrial cancer risks for MLH1 and MSH2 mutation carriers are the most precise currently available. Average risks are similar for the two genes but there is so much individual variation about the average that a sizeable proportion of carries are almost certain to develop cancer whereas another sizeable proportion only have population‐level risks.
Colorectal cancer (CRC) in densely affected families without Lynch Syndrome may be due to mutations in undiscovered genetic loci. Familial linkage analyses have yielded disparate results; the use of ...exome sequencing in coding regions may identify novel segregating variants.
We completed exome sequencing on 40 affected cases from 16 multicase pedigrees to identify novel loci. Variants shared among all sequenced cases within each family were identified and filtered to exclude common variants and single-nucleotide variants (SNV) predicted to be benign.
We identified 32 nonsense or splice-site SNVs, 375 missense SNVs, 1,394 synonymous or noncoding SNVs, and 50 indels in the 16 families. Of particular interest are two validated and replicated missense variants in CENPE and KIF23, which are both located within previously reported CRC linkage regions, on chromosomes 1 and 15, respectively.
Whole-exome sequencing identified DNA variants in multiple genes. Additional sequencing of these genes in additional samples will further elucidate the role of variants in these regions in CRC susceptibility.
Exome sequencing of familial CRC cases can identify novel rare variants that may influence disease risk.
Protein phosphatase I (PP1) is an essential eukaryotic serine/threonine phosphatase required for many cellular processes, including cell division, signaling, and metabolism. In mammalian cells there ...are three major isoforms of the PP1 catalytic subunit (PP1α, PP1β, and PP1γ) that are over 90% identical. Despite this high degree of identity, the PP1 catalytic subunits show distinct localization patterns in interphase cells; PP1α is primarily nuclear and largely excluded from nucleoli, whereas PP1γ and to a lesser extent PP1β concentrate in the nucleoli. The subcellular localization and the substrate specificity of PP1 catalytic subunits are determined by their interaction with targeting subunits, most of which bind PP1 through a so-called “RVXF” sequence. Although PP1 targeting subunits have been identified that direct PP1 to a number of subcellular locations and/or substrates, no targeting subunit has been identified that localizes PP1 to the nucleolus. Identification of nucleolar PP1 targeting subunit(s) is important because all three PP1 isoforms are included in the nucleolar proteome, enzymatically active PP1 is present in nucleoli, and PP1γ is highly concentrated in nucleoli of interphase cells. In this study, we identify NOM1 (nucleolar protein with MIF4G domain 1) as a PP1-interacting protein and further identify the NOM1 RVXF motif required for its binding to PP1. We also define the NOM1 nucleolar localization sequence. Finally, we demonstrate that NOM1 can target PP1 to the nucleolus and show that a specific NOM1 RVXF motif and the NOM1 nucleolar localization sequence are required for this targeting activity. We therefore conclude that NOM1 is a PP1 nucleolar targeting subunit, the first identified in eukaryotic cells.
Double heterozygotes for mutations in
APC
and a DNA mismatch repair gene are extremely rare. We report on an individual who had truncating mutations in
APC
and
MLH1
whose clinical presentation ...initially resembled Familial Adenomatous Polyposis but then emerged as a novel phenotype with multiple jejunal carcinomas. We have reviewed the relevant literature on double heterozygotes and based on what has been reported to date, this phenotype was not anticipated. It may be useful for clinicians to be aware of this observation as clinical screening guidelines are proposed for such individuals.
We studied 17,576 members of 166 MLH 1 and 224 MSH 2 mutation-carrying families from the Colon Cancer Family Registry. Average cumulative risks of colorectal cancer (CRC), endometrial cancer (EC), ...and other cancers for carriers were estimated using modified segregation analysis conditioned on ascertainment criteria. Heterogeneity in risks was investigated using a polygenic risk modifier. Average CRC cumulative risks at the age of 70 years (95% confidence intervals) for MLH 1 and MSH 2 mutation carriers, respectively, were estimated to be 34% (25%-50%) and 47% (36%-60%) for male carriers and 36% (25%-51%) and 37% (27%-50%) for female carriers. Corresponding EC risks were 18% (9.1%-34%) and 30% (18%-45%). A high level of CRC risk heterogeneity was observed (P < 0.001), with cumulative risks at the age of 70 years estimated to follow U-shaped distributions. For example, 17% of male MSH 2 mutation carriers have estimated lifetime risks of 0%-10% and 18% have risks of 90%-100%. Therefore, average risks are similar for the two genes but there is so much individual variation about the average that large proportions of carriers have either very low or very high lifetime cancer risks. Our estimates of CRC and EC cumulative risks for MLH 1 and MSH 2 mutation carriers are the most precise currently available. Our estimates of colorectal and endometrial cancer risks for MLH1 and MSH2 mutation carriers are the most precise currently available. Average risks are similar for the two genes but there is so much individual variation about the average that a sizeable proportion of carries are almost certain to develop cancer whereas another sizeable proportion only have population-level risks.
Abstract
Colorectal cancer (CRC) is estimated to be the third most common cancer in both men and women in the US in 2012, accounting for over 50,000 deaths. Familial CRC accounts for approximately ...30% of all CRC, yet only 5% of familial CRCs can be attributed to mutations in known hereditary colon cancer (HCC) genes. Many families with a strong family history of CRC do not have mutations in known CRC genes. Finding the genes underpinning CRC in these families may improve screening for disease and allow the development of more targeted therapeutics.
We completed targeted sequencing of over 2,000 genes and miRNAs in 1,500 cases of CRC from the Colon Cancer Family Registry (CCFR) in an effort to identify rare variants associated with CRC risk and to identify mutations in known CRC genes missed by conventional sequencing. Several categories of genes were included: 18 known HCC genes; 18 candidate HCC genes; 185 genes in previously reported CRC linkage peaks; 1,423 miRNAs; and 363 genes identified through whole exome sequencing of 40 affected cases in 16 families with hereditary CRC (n=2,007 total genes sequenced). Genomic DNA samples were indexed, then libraries were made using Agilent's SureSelect Targeted Enrichment kit. Samples were pooled (96 samples/pool), then each pool of samples was run on five lanes on an Illumina HiSeq2000 (average of 19.2 samples/lane). GenomeGPS, an in-house developed workflow, was used for read alignment, variant calling, and annotation.
In early analyses of the first 161 cases, the mean number of mapped reads exceeded 20 million per sample and over 90% of the target region exceeded 40x coverage. A total of 26,484 SNPs were identified and passed quality filtering. Sequencing identified known mutations in MMR genes in positive control cases as well as mutations in HCC genes in previously untested cases. Analysis is currently on-going on the remaining samples and we will report results on the full set of 1500 cases at the meeting.
Citation Format: Melissa S. DeRycke, Shanaka R. Gunawardena, Sumit Middha, Shannon K. McDonnell, Shaun M. Riska, Zachary C. Fogarty, Bruce W. Eckloff, Daniel J. Schaid, Ellen L. Goode, Stephen N. Thibodeau. Targeted sequencing to identify rare variants in colorectal cancer. abstract. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2548. doi:10.1158/1538-7445.AM2013-2548
Abstract
Several platforms for genome sequencing and methods for exon enrichment are now available and continue to evolve quickly. In this study, we compared the performance of whole exome sequencing ...using two instruments, Illumina's GAIIx and HiSeq 2000, comparing single versus duplicate runs. Eight runs of eight samples each were performed with genomic DNA derived from whole blood and sample libraries prepared with the Agilent SureSelect capture array. One run was conducted in duplicate on the GAIIx, and, for some analyses, the combined data were used. We used TREAT (Targeted RE-sequencing and Annotation Tool), developed in-house for data analysis, including sequence alignment (MAQ, BWA), local re-alignment (GATK), variant calling (utilizing MAQ), annotation (SIFT and Seattle Seq), and visualization.
Overall, duplicate runs on the GAIIx platform (compared to a single run) increased the number of total reads by approximately two-fold: from ∼66 M to ∼130 M. For both single and duplicate runs, ∼90% of reads mapped to the reference sequence, and ∼55% of reads mapped on-target. Of importance, the percent coverage of the target region increased substantially with duplicate runs; ∼72% of the target region was covered at 40-fold or more when run in duplicate compared to only ∼52% when run once. At 30x, 20x, and 10x, there was a 1.3- (62% v. 78%), 1.2- (72% v. 84%), and 1.1-fold (84% v. 91%) increase respectively, in the percent coverage of the target region for samples run twice. A similar number of filtered, on-target SNPs per sample (∼24 K) was found for both single and duplicate run analyses. However, a 1.3-fold increase in the number of on-target indels was seen in the duplicate run (∼1,200) compared to the single run (∼900).
When the combined data for samples run twice on a GAIIx were compared to the samples run using a HiSeq, the total number of reads were similar (∼130 M), although several of the samples had substantially more reads on the HiSeq platform. Samples run on a HiSeq had an increase in the percent coverage of the target region at 40x (72% v. 81%), 30x (78% v. 86%), 20x (84% v. 91%) and 10x (91% v. 95%) compared to the two-run approach on a GAIIx. A similar number of filtered on-target SNPs (∼24 K/sample) and indels (∼1200) was found with the HiSeq compared to two runs on a GAIIx.
In summary, our results demonstrate increases in the total number of reads and in overall coverage with the HiSeq 2000 instrument. The total number of SNPs and indel that mapped on target for the version 1 Agilent SureSelect capture array is ∼25 K and 1,200 per sample, respectively. As capture is of variable efficiency for individual runs, higher average coverage of the target region is necessary for sufficient coverage of poorly captured regions. Additional analyses comparing Agilent SureSelect versions 1 and 2 arrays are underway.
Citation Format: {Authors}. {Abstract title} abstract. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2742. doi:10.1158/1538-7445.AM2011-2742
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