, or MYH, gene is a member of the base-excision repair pathway involved in the detection and repair of oxidative DNA damage.30
Al-Tassan et al5
described 2 mutations in the MYH gene, Y165C and G382D, in a Welsh family with CRC and multiple adenomatous polyps. These 2 mutations appear to account for almost 90% of MYH mutations in Caucasian patients of Northern European ancestry. Additional mutations have been described in Caucasians13,31,32
and individuals of South Asian descent.7
The present study represents a large analysis of the association between germline MYH mutations and CRC risk. We characterized MYH mutations in 3811 population-derived CRC cases and 2802 controls from 3 sites of the C-CFR, in 3 countries: Canada, the United States, and Australia; and a fourth site including subjects from Newfoundland, Canada. As a population-derived series, the cases are more likely to represent the full spectrum of disease present in the general population and allow more thorough characterization of the phenotype associated with these mutations. More than 90% of cases and controls completed a detailed epidemiologic questionnaire that provided data for an analysis of potential modifiers of MYH mutations, although a definitive study of gene-environment interactions will require an even larger sample size. Unlike many previous population based studies15–19,22,33
that screened for only the 2 common mutations found in Caucasians, Y165C and G382D, we screened subjects for a larger panel of mutations. By using this 9-mutation panel, we identified an additional 10 heterozygous carriers and 1 additional compound heterozygous carrier who harbored confirmed pathogenic MYH mutations but did not carry either of the 2 common mutations.
Cases and controls at each study site were selected from population-based sampling frames providing comparable and representative groups for comparison. In addition, we observed an increased cancer risk for known CRC risk factors,34
suggesting that the study groups were representative of the general population.35
As with most case-control studies, there were several limitations. First, survival bias was possible because deceased cases were not recruited and response rates are lower for cases with later-stage disease. It is unlikely that this would affect our findings because MYH status does not appear to be associated with stage of disease. Response bias is possible when high response rates are not achieved.23,24
Although all 4 sites undertook population-based collection of CRC cases, the recruitment strategies of each site differed. The stratified recruitment method at the Ontario site24
may have lead to selection bias because MYH mutation carriers may not have a strong family history of CRC but may have certain pathologic characteristics that increased their likelihood of recruitment. However, adjustment for risk-strata and study site did not alter the findings of this study. Clinically obvious cases of florid polyposis were excluded from the C-CFR, which may have rendered some homozygous and compound heterozygous carriers ineligible for recruitment, leading to an underrepresentation of these mutations in this series.
We identified homozygous and compound heterozygous MYH mutations in 0.7% of the CRC cases in this series with a range of 0.4%–1% of cases from each site, indicating that these mutations likely contribute to a minority of CRC. Phenotypic characterization of CRC cases with homozygous and compound heterozygous MYH mutations showed that carriers had a younger age of diagnosis and a higher prevalence of polyps, right-sided and synchronous cancers. Although data on demographic and incident tumor characteristics were abstracted from original pathology reports, detailed polyp counts and colonoscopy reports were not available for all heterozygous and wild-type MYH cases; information for these subjects was based on synchronous polyps in the colectomy specimen or self-reported history of polyps or polypectomy. This combined with the exclusions of polyposis cases as well as the increased recruitment of multiple polyp CRC cases in Ontario limit the detailed interpretation of the phenotypic data for wild-type and heterozygous MYH carriers in this series. These findings do support the observation that the majority of carriers with mutations affecting both MYH alleles develop a mild polyposis syndrome as reported in clinic-based studies of MYH.6–14
However, as with other series15,18
we observed homozygous and compound heterozygous mutation carriers who developed CRC without multiple polyps. Homozygous and compound heterozygous carriers of the Y165C and G382D mutations developed CRC with and without polyps, indicating that neither the type nor location of the germline mutation affects the polyposis phenotype.
There is considerable debate whether an association exists between heterozygous MYH mutations and increased risk of CRC. Several studies have suggested that heterozygous MYH mutation may confer increased risk15,18,20–22
by showing a higher rate of heterozygous carriers among cases compared with controls, but each study alone lacked statistical power to definitively establish this relationship. Farrington et al18
reported an increased odds of CRC among heterozygous cases older than age 55, whereas Tenesa et al22
and Jenkins et al36
showed a statistically significant association by combining the results of several population-based series. Other studies19,33,37
have challenged this potential association by failing to find a relationship between heterozygous MYH mutations and CRC. The current study found a statistically significant association between heterozygous MYH mutation and CRC (AOR, 1.45; 95% CI, 1.01–2.10) and showed an increased risk for the G382D mutation (AOR, 1.6; 95% CI, 1.05–2.44). This risk associated with single MYH mutations is supported by the fact that mutations in other DNA repair pathways are inherited in a dominant fashion, loss of heterozygosity of 1p is a common somatic event in CRC,38,39
and higher rates of 1p loss of heterozygosity have been documented in heterozygous MYH carriers by our group15
Our ability to detect this association was aided by a greater sample size as well as the expanded mutation screening panel. If screening in this series had been limited to only Y165C and G382D, we would have detected heterozygous mutations in 78 cases and 43 controls, yielding an AOR of 1.45 (95% CI, 0.95–2.3). Furthermore, less common variants were detected in 15 cases (9 heterozygotes and 6 compound heterozygotes) and in only 1 control subject, suggesting that these rare variants may be more penetrant than the more common Y165C and G382D mutations. We have found that although most mutations abolish glycosylase and DNA binding activities of MYH, certain variants such as R260Q may retain reduced enzymatic function,26
raising the possibility that the functional characteristics of each MYH mutation may result in different risks or clinical manifestations.
We observed MSI-L tumors in 9% of MYH-wild-type cases, a frequency that is consistent with other large studies.40–42
MSI-L was detected in 18.8% and 23.5% of tumors from heterozygous and homozygous/compound heterozygous MYH-mutation carriers, respectively. We showed heterogeneity of the OR associated with heterozygous mutation status when stratified by MSI, indicating that the CRC risk associated with MYH variants differs by tumor MSI status. Although we did not show this finding for homozygous and compound heterozygous mutations owing to low numbers and instability of multivariate models, this effect modification also may hold for the risk associated with mutations affecting both alleles. Although the association between MYH and MSI was suggested by Kambara et al,37
it is difficult to draw comparisons with that study because the MYH V22M variant was considered pathogenic and comprised 60% of the variants detected in CRC cases in that series; in vitro data from our group26
have shown this variant is a polymorphism with no impact on glycosylase or DNA binding function. The nature and significance of MSI-L in CRC is unclear43
; however, there is an emerging consensus that although MSI-L cancers do not differ histologically from microsatellite stable cancers, they do have higher rates of mutations in K-ras and CpG-island methylation.44
Lipton et al45
showed the presence of G:C→T:A transversions in K-ras in 63% of MYH-associated cancers. We have shown that cancers associated with MYH mutations do not differ histologically from sporadic adenocarcinomas, which are associated commonly with chromosomal instability and are microsatellite stable.44,46
Furthermore, MYH has been shown to interact with the MSH2/MSH6 heterodimer and the function of MYH is enhanced by this interaction.47
The mechanism(s) by which germline MYH mutations would predispose to the development of MSI-L tumors is not clear; however, we offer a number of hypotheses. Because tumors with deficiencies in DNA repair may accumulate mutations in other DNA-damage signaling and repair pathways,48
cancers with disabled base-excision repair pathways caused by MYH mutations may develop somatic alterations in MMR genes resulting in a MSI-L phenotype. Our observation of intact mismatch repair proteins in MSI-L MYH mutation–positive cases would not appear to support this hypothesis. Alternatively, neoplastic progression along the MSI-L pathway may lead to mutations in MYH providing a second hit in heterozygous mutation carriers,49
thus accelerating tumorigenesis. Finally, because the MutSά complex is involved in both base excision and mismatch recognition and repair, it is possible that high levels of G:C→T:A transversions caused by deficiencies in MYH may overload the MutSά complex and lead to a MSI-L phenotype.45,47
In conclusion, we have shown homozygous and compound heterozygous mutations in a minority (<1%) of CRC cases and a single control (out of 2802) obtained from a large multisite population-derived registry. As with previous studies, not all biallelic carriers had an attenuated polyposis phenotype. Although biallelic carriers have an increased tendency to develop multiple polyps and right-sided CRC at a younger age than nonmutation carriers, none of these pathologic features are specific for homozygous and/or compound heterozygous MYH mutations. The absence of specific pathologic features and the lack of family history of CRC in these individuals indicates that identification of cases of MYH-associated CRC may be difficult. To date, a robust immunohistochemical assay to detect absent MYH protein in tumors from homozygous/compound heterozygous carriers is not available.46
The current study also conclusively shows an increased risk of CRC associated with heterozygous MYH mutations. Future studies are required to delineate the molecular pathways and genetic characteristics of MYH-associated CRC, to examine possible interaction between the base-excision repair and MMR pathways, and to determine optimal screening strategies for homozygous/compound heterozygous and heterozygous carriers.