MUTYH Variants Research Articles

A human MUTYH variant linking colonic polyposis to redox degradation of the [4Fe4S]2+ cluster.

The human DNA repair enzyme MUTYH excises mispaired adenine residues in oxidized DNA. Homozygous MUTYH mutations underlie the autosomal, recessive cancer syndrome MUTYH-associated polyposis. We report a MUTYH variant, p.C306W (c.918C>G), with a tryptophan residue in place of native cysteine, that ligates the [4Fe4S] cluster in a patient with colonic polyposis and family history of early age colon cancer. In bacterial MutY, the [4Fe4S] cluster is redox active, allowing rapid localization to target lesions by long-range, DNA-mediated signalling. In the current study, using DNA electrochemistry, we determine that wild-type MUTYH is similarly redox-active, but MUTYH C306W undergoes rapid oxidative degradation of its cluster to [3Fe4S]+, with loss of redox signalling. In MUTYH C306W, oxidative cluster degradation leads to decreased DNA binding and enzyme function. This study confirms redox activity in eukaryotic DNA repair proteins and establishes MUTYH C306W as a pathogenic variant, highlighting the essential role of redox signalling by the [4Fe4S] cluster.

Direct Observation of MUTYH and the Cancer-Associated Y150C Variant Binding to OG: A Mismatches at the Single Molecule Level

Of the four DNA bases, guanine is the most likely to be damaged by reactive oxygen species. Oxidized guanine (specifically, 8-oxoguanine) mispairs with adenine (instead of cytosine) during DNA replication (an OG:A mismatch), ultimately resulting in genomic G -> T transversion mutations. MUTYH is a mammalian DNA repair glycosylase that locates and removes the adenine in these OG:A mismatches. Y150C is the mouse homolog of a human MUTYH variant that is associated with colorectal adenomas and carcinomas. Structural information indicates that tyrosine 150 is analogous to the critical “wedge residue” present in many other DNA glycosylases, which is essential for lesion recognition. Here, we directly observe, at the single molecule level in vitro, fluorescently tagged wildtype (WT) MUTYH and the Y150C variant while they scan suspended DNA “tightropes” comprised of concatenated, linearized plasmids that contain specifically positioned OG:A mismatches every 2700 base pairs. In bulk assays, Y150C demonstrates reduced catalytic activity. Although we find that both the WT and Y150C diffuse along undamaged DNA with the same diffusion constant (1x105nm2/s) as they scan for damage, Y150C remains bound to and scans the undamaged DNA for a much shorter period of time than WT (3.9s and 32.7s, respectively). Both the WT and Y150C recognize and pause at OG:A mismatches, suggesting that Y150C lesion recognition is not impaired. However, once recognized Y150C pauses at a mismatch for only a short time (median 1.9s), while the WT enzyme remains bound at these mismatches for at least the duration of our 5-minute recordings. Taken together, our data show that mutation of the tyrosine “wedge residue” does not impair MUTYH's ability recognize and pause at OG:A mismatches, although the variant is unable to form a long-lived complex.

Somatic APC mosaicism and oligogenic inheritance in genetically unsolved colorectal adenomatous polyposis patients

Germline variants in the APC gene cause familial adenomatous polyposis. Inherited variants in MutYH, POLE, POLD1, NTHL1, and MSH3 genes and somatic APC mosaicism have been reported as alternative causes of polyposis. However, ~30–50% of cases of polyposis remain genetically unsolved. Thus, the aim of this study was to investigate the genetic causes of unexplained adenomatous polyposis. Eight sporadic cases with >20 adenomatous polyps by 35 years of age or >50 adenomatous polyps by 55 years of age, and no causative germline variants in APC and/or MutYH, were enrolled from a cohort of 56 subjects with adenomatous colorectal polyposis. APC gene mosaicism was investigated on DNA from colonic adenomas by Sanger sequencing or Whole Exome Sequencing (WES). Mosaicism extension to other tissues (peripheral blood, saliva, hair follicles) was evaluated using Sanger sequencing and/or digital PCR. APC second hit was investigated in adenomas from mosaic patients. WES was performed on DNA from peripheral blood to identify additional polyposis candidate variants. We identified APC mosaicism in 50% of patients. In three cases mosaicism was restricted to the colon, while in one it also extended to the duodenum and saliva. One patient without APC mosaicism, carrying an APC in-frame deletion of uncertain significance, was found to harbor rare germline variants in OGG1, POLQ, and EXO1 genes. In conclusion, our restrictive selection criteria improved the detection of mosaic APC patients. In addition, we showed for the first time that an oligogenic inheritance of rare variants might have a cooperative role in sporadic colorectal polyposis onset.

Characteristics of MUTYH variants in Japanese colorectal polyposis patients.

The base excision repair gene MUTYH is the causative gene of colorectal polyposis syndrome, which is an autosomal recessive disorder associated with a high risk of colorectal cancer. Since few studies have investigated the genotype-phenotype association in Japanese patients with MUTYH variants, the aim of this study was to clarify the clinicopathological findings in Japanese patients with MUTYH gene variants who were detected by screening causative genes associated with hereditary colorectal polyposis. After obtaining informed consent, genetic testing was performed using target enrichment sequencing of 26 genes, including MUTYH. Of the 31 Japanese patients with suspected hereditary colorectal polyposis, eight MUTYH variants were detected in five patients. MUTYH hotspot variants known for Caucasians, namely p.G396D and p.Y179D, were not among the detected variants.Of five patients, two with biallelic MUTYH variants were diagnosed with MUTYH-associated polyposis, while two others had monoallelic MUTYH variants. One patient had the p.P18L and p.G25D variants on the same allele; however, supportive data for considering these two variants 'pathogenic' were lacking. Two patients with biallelic MUTYH variants and two others with monoallelic MUTYH variants were identified among Japanese colorectal polyposis patients. Hotspot variants of the MUTYH gene for Caucasians were not hotspots for Japanese patients.

Fe-S Clusters and MutY Base Excision Repair Glycosylases: Purification, Kinetics, and DNA Affinity Measurements.

A growing number of iron-sulfur (Fe-S) cluster cofactors have been identified in DNA repair proteins. MutY and its homologs are base excision repair (BER) glycosylases that prevent mutations associated with the common oxidation product of guanine (G), 8-oxo-7,8-dihydroguanine (OG) by catalyzing adenine (A) base excision from inappropriately formed OG:A mispairs. The finding of an [4Fe-4S]2+ cluster cofactor in MutY, Endonuclease III, and structurally similar BER enzymes was surprising and initially thought to represent an example of a purely structural role for the cofactor. However, in the two decades subsequent to the initial discovery, purification and in vitro analysis of bacterial MutYs and mammalian homologs, such as human MUTYH and mouse Mutyh, have demonstrated that proper Fe-S cluster coordination is required for OG:A substrate recognition and adenine excision. In addition, the Fe-S cluster in MutY has been shown to be capable of redox chemistry in the presence of DNA. The work in our laboratory aimed at addressing the importance of the MutY Fe-S cluster has involved a battery of approaches, with the overarching hypothesis that understanding the role(s) of the Fe-S cluster is intimately associated with understanding the biological and chemical properties of MutY and its unique damaged DNA substrate as a whole. In this chapter, we focus on methods of enzyme expression and purification, detailed enzyme kinetics, and DNA affinity assays. The methods described herein have not only been leveraged to provide insight into the roles of the MutY Fe-S cluster but have also been provided crucial information needed to delineate the impact of inherited variants of the human homolog MUTYH associated with a colorectal cancer syndrome known as MUTYH-associated polyposis or MAP. Notably, many MAP-associated variants have been found adjacent to the Fe-S cluster further underscoring the intimate relationship between the cofactor, MUTYH-mediated DNA repair, and disease.

NTHL1 and MUTYH polyposis syndromes: two sides of the same coin?

It is now well established that germline genomic aberrations can underlie high-penetrant familial polyposis and colorectal cancer syndromes, but a genetic cause has not yet been found for the major proportion of patients with polyposis. Since next-generation sequencing has become widely accessible, several novel, but rare, high-penetrant risk factors for adenomatous polyposis have been identified, all operating in pathways responsible for genomic maintenance and DNA repair. One of these is the base excision repair pathway. In addition to the well-established role of the DNA glycosylase gene MUTYH, biallelic mutations in which predispose to MUTYH-associated polyposis, a second DNA glycosylase gene, NTHL1, has recently been associated with adenomatous polyposis and a high colorectal cancer risk. Both recessive polyposis syndromes are associated with increased risks for several other cancer types as well, but the spectrum of benign and malignant tumours in individuals with biallelic NTHL1 mutations was shown to be broader; hence the name NTHL1-associated tumour syndrome. Colorectal tumours encountered in patients with these syndromes show unique, clearly distinct mutational signatures that may facilitate the identification of these syndromes. On the basis of the prevalence of pathogenic MUTYH and NTHL1 variants in the normal population, we estimate that the frequency of the novel NTHL1-associated tumour syndrome is five times lower than that of MUTYH-associated polyposis. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Clinical and genetic characterization of hereditary breast cancer in a Chinese population

BackgroundBreast cancer develops as a result of multiple gene mutations in combination with environmental risk factors. Causative variants in genes such as BRCA1 and/or BRCA2 have been shown to account for hereditary nature of certain breast cancers. However,other genes, such as ATM, PALB2, BRIP1, CHEK, BARD1, while lower in frequency, may also increase breast cancer risk. There are few studies examining the role of these causative variants. Our study aimed to examine the clinical and genetic characterization of hereditary breast cancer in a Chinese population.MethodsWe tested a panel of 27 genes implicated in breast cancer risk in 240 participants using Next-Generation Sequencing. The prevalence of genetic causative variants was determined and the association between causative variants and clinico-pathological characteristics was analyzed.ResultsCausative variant rate was 19.2% in the breast cancer (case) group and 12.5% in the high-risk group. In the case group 2.5% of patients carried BRCA1 causative variant, 7.5% BRCA2 variants, 1.7% patients had MUTYH, CHEK or PALB2 variants, and 0.8% patients carried ATM, BARD1, NBN, RAD51C or TP53 variants. In the high-risk group 5.8% women carried MUTYH causative variants, 2.5% had causative variants in ATM, 1.7% patients had variants in BRCA2 and 0.8% in BARD1, BRIP1 or CDH1. There was no significant difference in the presence of causative variants among clinical stages of breast cancer, tumor size and lymph nodes status. However, eight of the 12 BRCA1/2 causative variants were found in the TNBC group.ConclusionsWe found increased genetic causative variants in the familial breast cancer group and in high-risk women with a family history of breast cancer. However, the variant MUTYH c.892-2A > G may not be directly associated with hereditary breast carcinoma.

Elucidating the molecular basis of MSH2-deficient tumors by combined germline and somatic analysis.

In a proportion of patients presenting mismatch repair (MMR)-deficient tumors, no germline MMR mutations are identified, the so-called Lynch-like syndrome (LLS). Recently, MMR-deficient tumors have been associated with germline mutations in POLE and MUTYH or double somatic MMR events. Our aim was to elucidate the molecular basis of MSH2-deficient LS-suspected cases using a comprehensive analysis of colorectal cancer (CRC)-associated genes at germline and somatic level. Fifty-eight probands harboring MSH2-deficient tumors were included. Germline mutational analysis of MSH2 (including EPCAM deletions) and MSH6 was performed. Pathogenicity of MSH2 variants was assessed by RNA analysis and multifactorial likelihood calculations. MSH2 cDNA and methylation of MSH2 and MSH6 promoters were studied. Matched blood and tumor DNA were analyzed using a customized next generation sequencing panel. Thirty-five individuals were carriers of pathogenic or probably pathogenic variants in MSH2 and EPCAM. Five patients harbored 4 different MSH2 variants of unknown significance (VUS) and one had 2 novel MSH6 promoter VUS. Pathogenicity assessment allowed the reclassification of the 4 MSH2 VUS and 6 probably pathogenic variants as pathogenic mutations, enabling a total of 40 LS diagnostics. Predicted pathogenic germline variants in BUB1, SETD2, FAN1 and MUTYH were identified in 5 cases. Three patients had double somatic hits in MSH2 or MSH6, and another 2 had somatic alterations in other MMR genes and/or proofreading polymerases. In conclusion, our comprehensive strategy combining germline and somatic mutational status of CRC-associated genes by means of a subexome panel allows the elucidation of up to 86% of MSH2-deficient suspected LS tumors.

Type and frequency of MUTYH variants in Italian patients with suspected MAP: a retrospective multicenter study.

To determine prevalence, spectrum and genotype-phenotype correlations of MUTYH variants in Italian patients with suspected MAP (MUTYH-associated polyposis), a retrospective analysis was conducted to identify patients who had undergone MUTYH genetic testing from September 2002 to February 2014. Results of genetic testing and patient clinical characteristics were collected (gender, number of polyps, age at polyp diagnosis, presence of colorectal cancer (CRC) and/or other cancers, family data). The presence of large rearrangements of the MUTYH gene was evaluated by Multiplex Ligation-dependent Probe Amplification analysis. In all, 299 patients with colorectal neoplasia were evaluated: 61.2% were males, the median age at polyps or cancer diagnosis was 50 years (16-80 years), 65.2% had <100 polyps and 51.8% had CRC. A total of 36 different MUTYH variants were identified: 13 (36.1%) were classified as pathogenetic, whereas 23 (63.9%) were variants of unknown significance (VUS). Two pathogenetic variants were observed in 78 patients (26.1%). A large homozygous deletion of exon 15 was found in one patient (<1.0%). MAP patients were younger than those with negative MUTYH testing at polyps diagnosis (P<0.0001) and at first cancer diagnosis (P=0.007). MAP patients carrying the p.Glu480del variant presented with a younger age at polyp diagnosis as compared to patients carrying p.Gly396Asp and p.Tyr179Cys variants. A high heterogeneity of MUTYH variants and a high rate of VUS were identified in a cohort of Italian patients with suspected MAP. Genotype-phenotype analysis suggests that the p.Glu480del variant is associated with a severe phenotype.

Abstract LB-372: Robust targeted sequencing strategy identifies highly penetrant mutations in hereditary colorectal cancer cases from Colon Cancer Family Registry

Abstract Highly penetrant mutations responsible for Mendelian cancer syndromes have not been systematically identified in large populations. Historically, highly penetrant cancer mutations have been identified in large families with multiple cancer cases using linkage analysis. Next-generation sequencing (NGS) allows for efficient genomic screening of large numbers of familial cancer cases. Highly penetrant mutations such as those found in the APC or mismatch repair (MMR) genes for colorectal cancer (CRC) or BRCA1/2 genes for breast cancer, are rare and often population-specific. Therefore, to identify these rare, highly penetrant cancer-predisposing mutations using NGS requires a large sample size that can make this approach prohibitively expensive. Here, we present a new, robust NGS pooling strategy for identification of rare, highly penetrant mutations. Our strategy is at least five times less expensive than traditional germline NGS of cancer patients. We screened 1,046 CRC cases and 1,006 unaffected controls from Colon Cancer Family Registry (Colon CFR), to identify highly penetrant CRC mutations in 40 known or candidate CRC genes. Cases were patients with familial CRC and controls were spouses or unrelated healthy individuals. DNA from 1,046 cases and 1,006 controls were combined into 480 pools (3 cases/pool and 8 controls/pool) for targeted sequencing of the 40 selected genes with mean depth of &amp;gt;1000X in cases and &amp;gt;700X in controls using Qiagen GeneRead Customized Targeted Panel. Following quality control, alignment of reads, and calculation of allele frequencies, we selected likely functional variants (nonsynonymous and nonsense) in cases that were not found in controls and absent or very rare (minor allele frequency &amp;lt;0. 001) in other populations. We found 264 rare variants in 273 cases: six stopgain and 258 nonsynonymous variants. While, &amp;gt;40% (n = 106) of the variants have never been reported, almost all the variants identified in the MMR genes (MLH1, MSH2, MSH6, and PMS2) were reported in dbSNP. However, only 11 of 39 MMR variants have sufficient evidence to be considered pathogenic in ClinVar. In addition to known mutations, we found novel, deleterious variants in ABCC1, APC (including stopgain), AXIN2, BMPR1A, CHEK2, CTNNB1, ENG, MUTYH, POLD1, POLE, PTCH1, SH2B3, TGFBR2, and TP53 among other genes. Previously observed variants of unknown significance (VUS) were found in BMPR1A, EPCAM, and SMAD4. Deleterious variants were selected for Sanger sequencing to identify individual genotypes and validate NGS results. Our study identified a large proportion of previously unreported, likely deleterious variants in known or candidate CRC susceptibility genes. The targeted NGS pooling strategy used in this study proved to be a robust and cost-effective alternative for screening a large cohort of familial CRC cases and controls for mutations in CRC susceptibility genes, making it applicable for screening other cancer types and complex diseases with dominant mode of inheritance. Citation Format: Leon Raskin, Yan Guo, Stephen B. Gruber, Daniel D. Buchanan. Robust targeted sequencing strategy identifies highly penetrant mutations in hereditary colorectal cancer cases from Colon Cancer Family Registry. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-372.

Abstract 5226: Genomic analysis of pancreatic ductal adenocarcinoma in a patient with MUTYH-associated polyposis

Abstract Biallelic pathogenic germline variants in the DNA repair glycosylase, MUTYH, cause MUTYH-associated polyposis, characterised by an increased susceptibility to colorectal adenomas and carcinomas secondary to defective base excision repair. We report a patient diagnosed with Stage IIB distal pancreatic ductal adenocarcinoma (PDAC) at the age of 45 years. Prior colonoscopy and gastroscopy noted three colonic tubular adenomas and a gastric fundic gland polyp. The patient was consented to whole genome and transcriptome sequencing of the PDAC and matched normal blood DNA through the British Columbia Personalized Onco-Genomics (POG) program. Analysis of germline and somatic variants including single nucleotide variants, copy number determination, loss of heterozygosity detection and mutational signatures was undertaken. Expression fold-changes were calculated against Illumina BodyMap pancreatic tissue averages and compared against The Cancer Genome Atlas PDAC cases. Germline analysis revealed biallelic mutations in the MUTYH gene. In light of this patient's personal and family history of adenomatous colon polyps, clinic-initiated panel testing of 14 cancer susceptibility genes, including MUTYH, via Illumina sequencing with reflex Sanger confirmation revealed the same biallelic MUTYH changes. Analysis of the patient's PDAC revealed a base excision repair pathway signature, demonstrated by an increased frequency of C:G&amp;gt;A:T transversions, consistent with deficient MUTYH activity. This is the first association of germline MUTYH biallelic pathogenic variants with PDAC and provides evidence of the contribution of aberrant MUTYH function to the genomic landscape of a PDAC. Detection of the base excision repair mutational signature may be a sensitive way to screen tumors for aberrant MUTYH function that can reveal potential germline MUTYH-related cancer susceptibility, and allow inference of pathogenicity of detected MUTYH variants, which may have cancer prevention and therapeutic implications. Citation Format: Kasmintan A. Schrader, Carolyn Chu’ng, Eric Zhao, Hui-li Wong, Yaoqing Shen, Martin Jones, Tom Thomson, Howard Lim, Sean Young, Carol Cremin, Robert Holt, Peter Eirew, Joanna Karasinska, Jacquie Schein, Yongjun Zhao, Andy Mungall, Richard Moore, Yussanne Ma, Alexandra Fok, Robyn Roscoe, Stephen Yip, Gillian Mitchell, Aly Karsan, Steven Jones, David Schaeffer, Janessa Laskin, Marco Marra, Daniel Renouf. Genomic analysis of pancreatic ductal adenocarcinoma in a patient with MUTYH-associated polyposis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5226.

Functional Evaluation of Nine Missense-Type Variants of the Human DNA Glycosylase Enzyme MUTYH in the Japanese Population.

Biallelic germline mutations of MUTYH, the gene encoding DNA glycosylase, cause MUTYH-associated polyposis (MAP), characterized by multiple colorectal adenomas and carcinoma(s). However, a considerable number of MUTYH variants are still functionally uncharacterized. Herein, we report the results of functional evaluation of nine missense-type MUTYH variant proteins in the Japanese population. The DNA glycosylase activity and ability to suppress mutations caused by 8-hydroxyguanine, an oxidized form of guanine, were examined for the nine variants of type 2 MUTYH, a nuclear form of the enzyme, by DNA cleavage activity assay and supF forward mutation assay, respectively. Both activities were severely defective in the p.N210S MUTYH type 2 variant corresponding to p.N238S in the reference MUTYH form and partially defective in p.R219G variant corresponding to p.R247G, but nearly fully retained in seven other variants examined. Our results suggest that p.N238S and p.R247G are likely to be pathogenic alleles for MAP.

MG-107 Multiple pathogenic variants identified by next-generation sequencing hereditary cancer panel testing – a case report

Background The utilisation of next-generation sequencing technology to interrogate multiple genes simultaneously is being utilised more frequently in hereditary cancer testing. While this has benefits of reducing cost and allowing clinicians to cast a wide net in the elucidation of their patient’s cancer, panel testing has the potential to reveal unexpected information. We report on a proband with pathogenic variants in two different hereditary colon cancer syndromes. Case report A 39 year-old male with history of colon cancer diagnosed at 38, with normal IHC, and >20 colon polyps presented for genetic counselling. Family history was significant for a paternal aunt and paternal uncle with colon cancer in their early 50s. Both parents reportedly had colon polyps requiring frequent colonoscopy; his mother had a TAH-BSO at 40 for unknown reasons. Testing with a 7-gene high-risk hereditary colon cancer panel identified a homozygous pathogenic variant, c.1187G >A (p. Gly396Asp), in MUTYH and a likely pathogenic duplication of exon 7 in MSH2. Due to this finding, his parents were referred for genetic counselling and testing; his mother, who was diagnosed with colon cancer in the interim, was found to carry the MSH2 duplication. Both parents were obligate carriers of the MUTYH variant. Conclusion This case example demonstrates the impact of identifying multiple hereditary syndromes. Being aware of all conditions has significant impact on at-risk family members particularly those who test negative for a known familial pathogenic variant, yet could still be at risk for cancer due to a second familial pathogenic variant.

Distinct functional consequences of MUTYH variants associated with colorectal cancer: Damaged DNA affinity, glycosylase activity and interaction with PCNA and Hus1.

MUTYH is a base excision repair (BER) enzyme that prevents mutations in DNA associated with 8-oxoguanine (OG) by catalyzing the removal of adenine from inappropriately formed OG:A base-pairs. Germline mutations in the MUTYH gene are linked to colorectal polyposis and a high risk of colorectal cancer, a syndrome referred to as MUTYH-associated polyposis (MAP). There are over 300 different MUTYH mutations associated with MAP and a large fraction of these gene changes code for missense MUTYH variants. Herein, the adenine glycosylase activity, mismatch recognition properties, and interaction with relevant protein partners of human MUTYH and five MAP variants (R295C, P281L, Q324H, P502L, and R520Q) were examined. P281L MUTYH was found to be severely compromised both in DNA binding and base excision activity, consistent with the location of this variation in the iron-sulfur cluster (FCL) DNA binding motif of MUTYH. Both R295C and R520Q MUTYH were found to have low fractions of active enzyme, compromised affinity for damaged DNA, and reduced rates for adenine excision. In contrast, both Q324H and P502L MUTYH function relatively similarly to WT MUTYH in both binding and glycosylase assays. However, P502L and R520Q exhibited reduced affinity for PCNA (proliferation cell nuclear antigen), consistent with their location in the PCNA-binding motif of MUTYH. Whereas, only Q324H, and not R295C, was found to have reduced affinity for Hus1 of the Rad9-Hus1-Rad1 complex, despite both being localized to the same region implicated for interaction with Hus1. These results underscore the diversity of functional consequences due to MUTYH variants that may impact the progression of MAP.

Functional Complementation Assay for 47 MUTYH Variants in a MutY-Disrupted Escherichia coli Strain.

MUTYH-associated polyposis (MAP) is an adenomatous polyposis transmitted in an autosomal-recessive pattern, involving biallelic inactivation of the MUTYH gene. Loss of a functional MUTYH protein will result in the accumulation of G:T mismatched DNA caused by oxidative damage. Although p.Y179C and p.G396D are the two most prevalent MUTYH variants, more than 200 missense variants have been detected. It is difficult to determine whether these variants are disease-causing mutations or single-nucleotide polymorphisms. To understand the functional consequences of these variants, we generated 47 MUTYH gene variants via site-directed mutagenesis, expressed the encoded proteins in MutY-disrupted Escherichia coli, and assessed their abilities to complement the functional deficiency in the E. coli by monitoring spontaneous mutation rates. Although the majority of variants exhibited intermediate complementation relative to the wild type, some variants severely interfered with this complementation. However, some variants retained functioning similar to the wild type. In silico predictions of functional effects demonstrated a good correlation. Structural prediction of MUTYH based on the MutY protein structure allowed us to interpret effects on the protein stability or catalytic activity. These data will be useful for evaluating the functional consequences of missense MUTYH variants detected in patients with suspected MAP.

Abstract P4-12-04: Clinical evaluation of multigene testing for hereditary breast and ovarian cancer

Abstract Background: Advances in DNA sequencing technology have fueled the development of multigene panels for hereditary cancer testing. While such assays are potentially both practical and affordable for routine clinical genetic testing, there remains uncertainty regarding their proper application and interpretation. Among the unanswered questions are which patients are appropriate candidates for such expanded testing; what is the likelihood that finding deleterious variants will alter risk assessment and management, and what is the prevalence of variants of unknown significance (VUS) that may create uncertainty for providers and anxiety for patients. Methods: 821 patients who met NCCN guidelines for BRCA1/2 testing for hereditary breast/ovarian cancer (HBOC) were prospectively recruited at two major academic medical centers. These patients received traditional BRCA1/2 tests as part of their clinical care and also were later tested for a panel of 29 known cancer risk genes. This panel also re-tested BRCA1/2. Both sequence changes and deletions/duplications were reported, and the panel-testing laboratory was blind to the earlier results. Panel testing results were validated by comparison with the earlier data or by independent testing using established technologies. Family history information was collected directly by genetic counselors. Results: 13.8% of the patients carried BRCA1 or BRCA2 mutations, with &amp;gt;99% concordance between the traditional and panel results for these two genes. 53 (7.6%) of the BRCA1/2-negative patients carried mutations in other cancer risk genes. Some of these findings confer a high risk for breast/ovarian cancer (e.g. TP53), while others (i.e. CHEK2, BRIP1, PALB2, RAD51C, CDKN2A, ATM, and NBN) confer a moderate increase in risk. 10 of these 55 patients were positive for genes involved in Lynch syndrome (MLH1, MSH2, MSH6, and PMS2), although breast and ovarian cancer are not uniformly accepted as part of this syndrome. 40% of these patients were heterozygous carriers of pathogenic variants in MUTYH, which have a less clear impact on breast/ovarian cancer, although this rate (3%) is higher than the expected carrier frequency in this population. About 60% of patients had one or more VUS in the 29 genes. The rate of VUS was highly gene dependent, with ATM, APC, and PTCH1 providing the largest number. A small but significant subset of patients did not have personal or family histories consistent with the classical presentation of their identified mutation. Nevertheless, a majority of the non-BRCA1/2 findings would have prompted consideration of a management change for the tested patient even considering the personal and family cancer history. Conclusions: Multigene panel testing for hereditary cancer risk assessment increased the yield of findings with potential clinical impact for almost 8% of patients. This is consistent with prior data from our laboratory and others. This study, carried out in a uniform clinical practice setting and with data collected directly by health care providers, provides a representative view of the benefit from such testing in an unselected patient population. Citation Format: Leif Ellisen, Allison Kurian, Stephen Lincoln, Andrea Desmond, Meredith Mills, Kristen Shannon, Michelle Gabree, Michael Anderson, Yuya Kobayashi, Federico Monzon, James Ford. Clinical evaluation of multigene testing for hereditary breast and ovarian cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-12-04.

Increased risk for colorectal adenomas and cancer in mono-allelic MUTYH mutation carriers: results from a cohort of North-African Jews.

Bi-allelic MUTYH gene mutations are associated with a clinical phenotype of multiple colorectal adenomas and an increased risk for colorectal cancer (CRC). It is unclear whether mono-allelic MUTYH gene carriers (heterozygotes) are also at increased risk for even few adenomas or cancer. In order to clarify an association between MUTYH heterozygotes and adenomas, we evaluated the frequency and types of MUTYH mutations and variants in 72 North-African Jews having few (≥3) colorectal adenomas with or without early onset (<50 years) CRC compared to 29 healthy controls. Germ-line DNA was analyzed for a panel of 6 MUTYH mutations and variants, and Sanger sequencing of the entire MUTYH gene was performed for mono-allelic MUTYH mutation carriers. APC gene mutations and Lynch syndrome were excluded in the relevant cases according to accepted clinical criteria. Twenty-two of the 72 adenoma subjects (30.5%) had MUTYH mutations or variants. Nine were homozygotes or compound heterozygotes: all had >10 adenomas and one had CRC. Thirteen others were mono-allelic carriers (heterozygotes) of a single MUTYH mutation: six had more than ten adenomas and seven had less than ten adenomas; of these 13 mono-allelic carriers, six had a neoplasm: three CRCs and three extra-intestinal tumors. Eleven of the thirteen mono-allelic carriers with adenomas had a family history of cancer in first or second degree relatives. A multivariable model showed positive correlation between G396D, Y179C and 1186 ins GG mutations and number of adenomas (OR 8.6, 10.2 and 14.4, respectively). The Q324H variant was negatively associated with the number of adenomatous polyps (OR -5.23). In conclusion, MUTYH mutations are prevalent among Jews of North-African origin with colorectal adenomas with or without early onset CRC. Mono-allelic MUTYH carriers with a family history of cancer had a clinical phenotype that varied from having only few adenomas to multiple (>10) adenomas. These findings support MUTYH testing in patients with even few adenomas and suggest the consideration of increased surveillance in mono-allelic carriers with a family history of cancer.

Common colorectal cancer risk alleles contribute to the multiple colorectal adenoma phenotype, but do not influence colonic polyposis in FAP

The presence of multiple (5–100) colorectal adenomas suggests an inherited predisposition, but the genetic aetiology of this phenotype is undetermined if patients test negative for Mendelian polyposis syndromes such as familial adenomatous polyposis (FAP) and MUTYH-associated polyposis (MAP). We investigated whether 18 common colorectal cancer (CRC) predisposition single-nucleotide polymorphisms (SNPs) could help to explain some cases with multiple adenomas who phenocopied FAP or MAP, but had no pathogenic APC or MUTYH variant. No multiple adenoma case had an outlying number of CRC SNP risk alleles, but multiple adenoma patients did have a significantly higher number of risk alleles than population controls (P=5.7 × 10−7). The association was stronger in those with ≥10 adenomas. The CRC SNPs accounted for 4.3% of the variation in multiple adenoma risk, with three SNPs (rs6983267, rs10795668, rs3802842) explaining 3.0% of the variation. In FAP patients, the CRC risk score did not differ significantly from the controls, as we expected given the overwhelming effect of pathogenic germline APC variants on the phenotype of these cases. More unexpectedly, we found no evidence that the CRC SNPs act as modifier genes for the number of colorectal adenomas in FAP patients. In conclusion, common colorectal tumour risk alleles contribute to the development of multiple adenomas in patients without pathogenic germline APC or MUTYH variants. This phenotype may have ‘polygenic' or monogenic origins. The risk of CRC in relatives of multiple adenoma cases is probably much lower for cases with polygenic disease, and this should be taken into account when counselling such patients.

Tu1893 MUTYH Mutations and Variants in Jews of North-African Origin With or Without Minimal Colorectal Adenomatous Polyposis

Background: Israeli-Jews of North-African origin are ~ 1/6 of the Israeli population, and have been considered as a low-risk population for colorectal cancer (CRC). In recent years CRC risk has been increased in this population. However, the contributing genetic susceptibility is currently unknown. Mutations in the MUTYH gene, which cause multiple colorectal adenomas and early onset colon cancer, might play a role in their increased CRC risk. Objectives: Evaluate frequency and types of MUTYH mutations and variants in a population of Jews fromNorth-African origin having colorectal adenomas and early onset CRC. Methods: The study population included North-African Jews having colonoscopy between 2007-12, having ≥3 colorectal adenomas and/or CRC, and were matched to controls having normal colonoscopy. Germ-line DNA was tested for a panel of 6 MUTYH mutations or variants and Sanger sequencing of the entire MUTYH gene for heterozygotes. Familial Adenomatous Polyposis (FAP) and Lynch Syndrome were excluded in the relevant cases. Results: 99 subjects, 69 with adenomas had MUTYH analysis; 19 (27.5%) had mutations or variants identified. In adenoma patients there were eight homozygotes or compound heterozygotes to either Y179C or G396D mutations: all had >10 adenomas and 5 had familial neoplasia. Six others were heterozygotes to only one mutation: 4 with 10 adenomas; 5 had familial neoplasia and 3 a neoplasm. Four subjects found as S512F heterozygotes: two had 10 adenomas. Two heterozygotes had the Q324H variant. In 23 non-adenoma controls: 1 had the S512F another the L417M variant, another the R509C variant and 16 the Q324H variant. Conclusions: MUTYH mutations are prevalent among Jews of North-African origin having colonic adenomas and/or early onset CRC. The clinical phenotype can be associated with only few adenoma and with family and personal history of cancer similar to sporadic CRC. The S512F mutation appears to be pathogenic, the Q324H a frequent variant that was not found to be associated with adenomas. The findings suggest MUTYH evaluation in these patients with even few adenomas, and need to follow-up heterozygotes carriers for CRC.

Genetic instability in lymphoblastoid cell lines expressing biallelic and monoallelic variants in the human MUTYH gene

The MUTYH DNA glycosylase counteracts mutagenesis by removing adenine misincorporated opposite DNA 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG). Biallelic germline mutations in MUTYH cause the autosomal recessive MUTYH-associated polyposis (MAP). The impact on genetic instability of the p.Tyr179Cys and p.Arg245His MUTYH variants was evaluated in lymphoblastoid cell lines (LCLs) derived from MAP patients and their relatives in comparison to wild-type LCLs. No difference in MUTYH expression was identified between wild type and LCLs with the p.Tyr179Cys, while the p.Arg245His mutation was associated with an unstable MUTYH protein. LCLs homozygous for the p.Tyr179Cys or the p.Arg245His variant contained increased DNA 8-oxodG levels and exhibited a mutator phenotype at the PIG-A gene. The extent of the increased spontaneous mutation frequency was 3-fold (range 1.6- to 4.6-fold) in four independent LCLs carrying the p.Tyr179Cys variant, while a larger increase (6-fold) was observed in two p.Arg245His LCLs. A similar hypermutability and S-phase delay following treatment with KBrO3 was observed in LCLs homozygous for either variant. When genetic instability was investigated in monoallelic p.Arg245His carriers, mutant frequencies showed an increase which is intermediate between wild-type and homozygous cells, whereas the mutator effect in heterozygous p.Tyr179Cys LCLs was similar to that in homozygotes. These findings indicate that the type of MUTYH mutation can affect the extent of genome instability associated with MUTYH inactivation. In addition, the mild spontaneous mutator phenotype observed in monoallelic carriers highlights the biological importance of this gene in the protection of the genome against endogenous DNA damage.