BRIEF COMMUNICATION

Association Between Biallelic and Monoallelic Germline MYH Gene Mutations and Colorectal Cancer Risk

Marina E. Croitoru, Sean P. Cleary, Nando Di Nicola, Michael Manno, Teresa Selander, Melyssa Aronson, Mark Redston, Michelle Cotterchio, Julia Knight, Robert Gryfe, Steven Gallinger

Affiliations of authors: Samuel Lunenfeld Research Institute (MEC, SPC, NDN, MM, JK, RG, SG) and Department of Surgery (RG, SG), Mount Sinai Hospital, Toronto, ON, Canada; Ontario Familial Colorectal Cancer Registry, Cancer Care Ontario, Toronto (MM, TS, MA, MC, JK, SG); Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA (MR)

Correspondence to: Steven Gallinger, MD, MSc, FRCS, Rm. 1225, Mount Sinai Hospital, 600 University Ave., Toronto, ON, Canada M5G 1X5 (e-mail: sgallinger{at}mtsinai.on.ca)


    ABSTRACT
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The MutY human homologue (MYH) gene encodes a member of the base excision repair pathway that is involved in repairing oxidative damage to DNA. Two germline MYH gene mutations that result in Myh proteins containing amino acid substitutions Y165C and G382D (hereafter called the Y165C and G382D mutations) are associated with adenomatous poly-posis and colorectal cancer among patients from several European poly-posis registries. We used a population-based series of 1238 colorectal cancer patients and 1255 healthy control subjects from Ontario, Canada, to examine the risk of colorectal cancer among biallelic and monoallelic germline MYH Y165C and G382D mutation carriers. The entire MYH gene coding region was screened in all MYH Y165C and G382D mutation carriers. Compared with noncarriers, biallelic and monoallelic germline MYH gene mutation carriers had an increased risk of colorectal cancer and were more likely to have first-or second-degree relatives with colorectal cancer (relative risk = 1.54, 95% confidence interval = 1.10 to 2.16). The increased risk of colorectal cancer in biallelic and monoallelic MYH gene mutation carriers was not consistently associated with the development of multiple adenomatous polyps. Loss of heterozygosity in at least one of four loci in MYH was detected in eight (47%) of 17 colorectal tumors from monoallelic MYH gene mutation carriers but in only two (20%) of 10 colorectal tumors from biallelic MYH gene mutation carriers. These two MYH gene mutations may account for a substantial fraction of hereditary colorectal cancer.


Colorectal cancer is the third most common malignancy and the second most common cause of cancer-related deaths in North America (1). As many as 20% of colorectal cancer patients have a family history of the disease (24); however, the highly penetrant autosomal dominant familial syndromes—familial adenomatous polyposis and hereditary nonpolyposis colorectal cancer—account for less than 3% of all colorectal cancer cases (5). It is therefore likely that most familial colorectal cancer cases are associated with mutations in unidentified genes or in genes that display incomplete penetrance.

One gene potentially involved in familial colorectal cancer, MutY human homologue (MYH), is located on chromosome 1p and encodes a member of the base excision repair pathway that is involved in repairing oxidative damage to DNA (6). Al-Tassan et al. (7) described a British family in which three siblings with colorectal cancer and adenomatous polyposis were compound heterozygotes for two germline mutations in the MYH gene that result in Myh proteins containing amino acid substitutions Y165C and G382D (hereafter called the Y165C and G382D mutations). Al-Tassan et al. further showed that proteins containing these substitutions in Escherichia coli lead to severe impairment of base excision repair (7). Other investigators subsequently confirmed the association between these and other biallelic germline MYH gene mutations and colorectal polyposis and colorectal cancer among patients known not to have germline mutations in the adenomatous polyposis coli gene. In these reports, biallelic MYH gene mutations were identified in approximately 20%–30% of patients who had 15–100 adenomas (811). In addition, the Y165C and G382D mutations have been found to be the two most common changes in the MYH gene, accounting for more than 80% of all MYH variants reported to date in white populations (710,12,13).

Although the increased risk of colorectal cancer and polyposis among biallelic MYH gene mutation carriers has been confirmed by several groups, the risk among monoallelic MYH gene mutation carriers is unclear (7,9,10,13,14). Heterozygous MYH mutation carriers were detected in the aforementioned studies (7,10,13); however, they did not appear to have increased risks for colorectal cancer or adenomatous polyps. Vertical transmission of the polyposis phenotype was not evident in these studies (7,8,10,11) because the small numbers of monoallelic mutation carrier relatives were reported as unaffected with colorectal cancer at the time of analysis. It was therefore concluded that biallelic MYH gene mutations are required for colorectal polyposis on the basis of an autosomal recessive inheritance model (710). However, relatively small numbers of primarily clinic-based subjects were tested in those studies, and the case patients were selected on the basis of having the polyposis phenotype.

We conducted the first population-based, case–control study in North America to examine the nature of the association between monoallelic and biallelic germline MYH gene mutations and colorectal cancer risk. Our primary objective was to examine the prevalence of germline MYH gene mutations and the association between those mutations and colorectal cancer risk in the population of Ontario, Canada. As a secondary objective, we examined the hypothesis that monoallelic germline MYH gene mutations may display a weakly penetrant autosomal dominant inheritance pattern.

Our study was performed using the resources of the Ontario Familial Colorectal Cancer Registry (OFCCR) (15), a member of the (U.S.) National Cancer Institute's Cooperative Family Registries for Colorectal Cancer Studies. The OFCCR identified individuals who were diagnosed with colorectal cancer from July 1, 1997, through June 30, 2000, and were aged 20–74 years at diagnosis. Cases of familial adenomatous polyposis were excluded from the registry. Age- and sex-matched control subjects with no personal history of colorectal cancer were recruited by telephone from a list of randomly selected residential telephone numbers for Ontario and from population-based Tax Assessment Rolls of the Ontario Ministry of Finance. All study participants provided informed written consent. This study was approved by the research ethics boards of Mount Sinai Hospital and Samuel Lunenfeld Research Institute.

We used Ficoll-Paque to isolate peripheral lymphocytes from venous blood obtained from each subject, according to the manufacturer's protocol (Amersham Biosciences, Baie d'Urfé, Quebec, PQ, Canada); phenol–chloroform was used to extract genomic DNA from the lymphocytes. We used genomic DNA from 1238 case patients and 1255 control subjects to screen for MYH Y165C and G382D mutations by using denaturing high-performance liquid chromatography (Transgenomic Wave 3500HT System; Transgenomic, Omaha, NE). Each genomic DNA sample was subjected to polymerase chain reaction (PCR) amplification using primers (see Supplemental Table available at: http://jncicancerspectrum.oupjournals.org/jnci/content/vol96/issue21) designed by the Transgenomics Mutation Discovery program (www.mutationdiscovery.com). The GenBank accession number is MYH: NM_012222. Online Mendelian Inheritance in Man (OMIM) information is available at http://www.ncbi.nlm.nih.gov/Omim/. To ensure detection of Y165C and G382D homozygous mutations, the resulting test PCR products were mixed with PCR products amplified from a wild-type sample (25% of the test sample amount). The mixed samples were heated to 95 °C for 5 minutes and then cooled for 50 minutes at a rate of –1.5 °C per cycle for 46 cycles to enhance heteroduplex formation prior to analysis (8). Samples were passed through a SepHT Cartridge (Transgenomic) under partially denatured conditions; temperatures were calculated using Wavemaker version 4.1 software for each amplicon (see Supplemental Table). Samples showing variant mobility patterns were sequenced. We then used denaturing high-performance liquid chromatography to screen the entire MYH gene (16 exons) of all monoallelic and biallelic MYH Y165C and/or G382D mutation carriers for additional mutations. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated from proportions of mutation carriers in case patients and control subjects using logistic regression. Continuous variables were compared using Student's t test. Family history data for mutation carriers and noncarriers were compared by Poisson regression with an offset to correct for family size. All statistical tests were two-sided.

Pathogenic biallelic MYH mutations [Y165C, G382D, the truncating Y90X mutation, and the predicted 891+3A>C splice site mutation (16)] were detected in 12 (1.0%) of the 1238 case patients (Table 1). None (0%) of the 1255 control subjects carried these biallelic mutations. Twenty-nine (2.34%) case patients and 21 (1.67%) control subjects were heterozygous for the Y165C mutation or the G382D mutation and had no other MYH gene mutations. The frequency of MYH Y165C heterozygotes was 0.65% among case patients and 0.32% among control subjects; the frequency of MYH G382D heterozygotes was 1.70% among case patients and 1.35% among control subjects. We detected two previously identified MYH gene polymorphisms, V22M and Q324H (7), in two case patients and in 14 subjects (nine case patients and five control subjects), respectively.


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Table 1. Frequency of germline MYH gene mutations in colorectal cancer case patients and control subjects and odds ratio (OR) estimates*

 
In our study population, carrying a germline MYH Y165C and/or G382D mutation was associated with a doubling of the risk of colorectal cancer (OR = 2.0, 95% CI = 1.2 to 3.4) compared with that among subjects who had no such mutations. Carriers of either single mutation had a combined estimated OR of 1.4 (95% CI = 0.8 to 2.5). More specifically, Y165C mutation carriers had an OR of 2.1 (95% CI = 0.6 to 6.8), and G382D mutation carriers had an OR of 1.3 (95% CI = 0.7 to 2.4).

We then characterized the clinicopathologic features of the MYH gene mutation carriers in our study. Previous reports (8,10,11) did not focus on age at diagnosis of colorectal cancer; instead, they emphasized the polyposis phenotype that was associated with biallelic MYH gene mutation carrier status. The mean age at diagnosis of colorectal cancer for patients with germline MYH Y165C and/or G382D mutations was statistically significantly younger than the mean age at diagnosis of colorectal cancer for patients who carried neither of these mutations (56.3 years versus 59.8 years; P = .01, Student's t test). In addition, nine of the 29 case patients who carried the heterozygous Y165C or G382D mutation had more than one synchronous polyp (range = 2–12 polyps) in their colectomy specimen, which suggests that carrying a heterozygous MYH gene mutation may be associated with an increased risk of colorectal polyps (Table 2). Conversely, only seven of the 12 case patients who carried both Y165C and G382D or Y165C or G382D and another pathogenic mutation had more than one synchronous polyp (range = 4 to at least 48, some reported as "polyposis"), suggesting that multiple adenomatous polyposis may not be an obligatory phenotype of MYH gene mutations.


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Table 2. Demographic and phenotypic features of case patients with germline MYH Y165C and G382D mutations*

 
We used information obtained from the OFCCR to examine the association between MYH gene mutations and a family history of colorectal cancer by using a Poisson regression with offset to correct for family size. Case patients who carried MYH Y165C and/or G382D mutations were more likely than case patients who did not carry either mutation to have a first- or second-degree relative affected with colorectal cancer (relative risk [RR] = 1.54, 95% CI = 1.10 to 2.16). Furthermore, case patients who were monoallelic carriers of either the MYH Y165C or G382D mutation were more likely than noncarrier case patients to have a first- or second-degree relative affected with colorectal cancer (RR = 1.57, 95% CI = 1.05 to 2.36). These findings provide further evidence that heterozygous MYH Y165C and G382D mutations are associated with an autosomal dominant transmission pattern of increased colorectal cancer risk.

Both the MYH Y165C and G382D mutations were in Hardy–Weinberg equilibrium among control subjects. We observed more homozygous carriers of the Y165C and G382D mutations among case patients than would be predicted by the Hardy–Weinberg model. In addition, the observed frequency of MYH gene mutations and the associated odds ratios predict that MYH gene mutation carrier status is associated with a population-attributable risk of 1.7% (17).

To provide additional evidence for an association between heterozygous MYH gene mutations and colorectal cancer, we tested 17 colorectal tumor specimens from 16 monoallelic MYH gene mutation carriers and 10 colorectal tumor specimens from 10 biallelic MYH gene mutation carriers for loss of heterozygosity of the MYH gene by using four closely linked microsatellite loci (D1S2677, D1S1182, D1S451, and D1S211), as previously described (18). We detected loss of heterozygosity in at least one of these loci in eight (47%) of the 17 tumors from monoallelic MYH gene mutation carriers but in only two (20%) of the 10 tumors from biallelic MYH gene mutation carriers (data not shown). Allelic loss of chromosome 1p is an early event in colorectal tumorigenesis (19,20); therefore, loss of heterozygosity in some monoallelic MYH gene mutation carriers may provide an explanation for the increased risk of colorectal cancer in these subjects.

By using a population-based case–control series of colorectal cancer patients and healthy individuals, we found an association between MYH Y165C and G382D mutations and colorectal cancer risk. Our finding—that colorectal cancer patients who carry monoallelic or biallelic MYH gene mutations have more first- and second-degree relatives with colorectal cancer than do colorectal cancer patients that lack MYH mutations—suggests that even a monoallelic mutant MYH genotype may confer a potentially important low-penetrant risk of colorectal cancer. We detected a higher frequency of heterozygous MYH Y165C or G382D mutation carriers among population-based colorectal cancer patients than among control subjects. Although these differences did not achieve statistical significance, they provide suggestive evidence for an association between monoallelic MYH Y165C or G382D variants and colorectal cancer risk. We combined our results with those from previous studies (714,2123), generating totals of 29 of 4452 case patients and nine of 2871 control subjects heterozygous for Y165C, and 46 of 4452 case patients and 21 of 2871 control subjects heterozygous for G382D. These combined data produced estimated ORs of 2.1 (95% CI = 1.0 to 4.4) for Y165C carriers and 1.4 (95% CI = 0.8 to 2.4) for G382D carriers. Clearly, larger studies with sufficient statistical power are necessary to accurately estimate the magnitude of the risk associated with these mutations. Nevertheless, this association should be considered in current genetic testing practices and in screening colonoscopy recommendations for individuals at high risk of colorectal cancer.


    NOTES
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M. E. Croitoru and S. P. Cleary contributed equally to this manuscript.

Supported by Public Health Service grant RFA CA-95-011 (to S. Gallinger) from the National Cancer Institute, National Institutes of Health, Department of Health and Human Services, grant U01 CA074783 from the Ontario Registry for Studies of Familial Colorectal Cancer, and through cooperative agreements with members of the Colon Cancer Family Registry (CFRs), and grant 13304 (to S. Gallinger) from the National Cancer Institute of Canada. M. E. Croitoru is supported by an Interdisciplinary Health Research Team Scholarship through the Canadian Institutes of Health Research. S. P. Cleary is a Research Fellow of the National Cancer Institute of Canada and is supported by funds from the Canadian Cancer Society. R. Gryfe is a Charles H. Hollenberg Senior Fellow/Clinician Scientist and is supported by funds from Eli-Lilly Canada, Cancer Care Ontario, and the Canadian Institutes of Health Research.

We thank Deborah Hogarth for technical assistance.


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Manuscript received June 9, 2004; revised August 12, 2004; accepted August 19, 2004.


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