Introduction
Lynch syndrome (LS) accounts for 2–4% of all CRCs and is characterized by a high risk for developing malignancies, most notably colorectal cancer (CRC) and endometrial cancer (EC). The underlying cause is a germline mutation in one of the mismatch repair (MMR) genes:
MLH1, MSH2 (
EPCAM),
MSH6 or
PMS2. Mutations in all MMR genes are associated with a significantly increased cancer risk compared to the general population, although
MSH6 and
PMS2 carriers show lower penetrance compared to
MLH1 and
MSH2 carriers [
1‐
3]. Within and between family variability is commonly observed and a range of theories have been proposed to explain the phenomenon, such as genotype–phenotype correlations, parent-of-origin effects, lifestyle factors and the influence of common susceptibility loci. The latter, mainly single nucleotide polymorphisms (SNPs), were identified in genome wide association studies (GWAS) in large cohorts consisting of sporadic CRC cases [
4]. Among these candidate SNPs, previous studies have identified statistically significant effects of multiple SNPs in LS patients, and independent studies replicated the effect of SNPs rs3802842 (11q23.1) and rs16892766 (8q23.3) among
MLH1 carriers [
5,
6]. It should be noted, however, that others have failed to replicate these findings [
7,
8]. Although the latter studies analyzed cohorts of similar size to our own, few or no
PMS2 carriers were included [
7]. Due to a relatively low penetrance and high phenotypic variability, this specific subset of LS patients might be of particular interest [
9]. In a previous study among 377
PMS2 carriers, we found age at CRC diagnosis to vary widely (range 26–86 years) and mean age of index carriers and mutation-positive family members differed by 10 years [
3]. In the current study, we aim to determine whether these SNPs modify CRC risk in a large cohort of
PMS2 mutation carriers.
Discussion
PMS2 carriers currently represent a relatively small proportion of LS patients. However, the number of
PMS2-associated LS cases is expected to rise with the implementation of population-based screening protocols for all CRC below age 70. Identification of
PMS2 carriers has been challenging in the past due to difficulties in mutation analysis, a milder phenotype and many families not fulfilling clinical selection criteria [
3,
9,
15‐
17]. Obtaining a better understanding of the specific
PMS2-associated phenotype is particularly relevant, as it appears to differ markedly from phenotypes associated with other MMR mutations. Unfortunately, we were unable to confirm any risk modifying effects of rs3802842 (11q23.1) and rs16892766 (8q23.1), two SNPs previously shown to be associated with enhanced risk in
MLH1 mutation carriers [
5,
6]. Studies in
MLH1 mutation carriers reported that a higher number of risk alleles in a carrier is associated with a younger onset of disease (28 years younger for 3 compared to 0 risk alleles) [
6]. In our cohort, mean ages where 52.8 and 50.4 for 0 compared to more than 1 risk allele, respectively. As such, there seems to be no clinical utility of rs3802842 and rs16892766 in risk stratification for
PMS2 carriers.
Many studies on (genetic) modifiers in LS patients focus on
MLH1 and
MSH2, or
MSH6 carriers, while
PMS2 is seldom analyzed. The only study to include
PMS2 carriers (n = 40) found that carriers of the G-alleles of rs10795668 (10p14) and rs9929218 (16q22.1) were at lower risk of CRC, a notable finding in that this is the opposite effect compared to sporadic CRC [
7]. The authors conceded that their results should be confirmed in larger studies. As these findings have not been confirmed in our much larger cohort, we suggest that these previous findings may indeed have been false positives due to the small number of carriers included.
A relevant question is why our study did not confirm reported findings of previous studies of MMR carriers. One explanation might be that although patients have germline mutations in genes with similar functions, carriers are affected by genetic modifiers in different ways. Indeed, comparable studies in
BRCA1 or
BRCA2 mutation carriers have resulted in the identification of SNPs that clearly modify breast cancer risk. However,
BRCA2 carriers appear unaffected by SNPs that confer an increased breast cancer risk in
BRCA1 carriers, even though both genes play a role in homologous recombination [
18]. This could also hold for MMR mutation carriers, as illustrated by the observation that while rs3802842 and rs16892766 may increase risk in
MLH1 carriers, they do not appear to have an effect in
MSH2 or
PMS2 carriers [
5,
6]. Researchers should therefore concentrate on building cohorts large enough to analyze Lynch patients in a gene-stratified manner.
Gender stratification in our cohort led to the notable finding that male carriers of allele A at locus rs1321311 (6p21.31) show a per allele HR of 2.07 (95% CI 1.21–2.96), while the HR for females was 0.83 (95% CI 0.63–1.28). This SNP has been linked to the
CDKN1A gene that encodes the p21 protein. p21 is involved in several (p53-independent) pathways as a tumor suppressor, although it also has oncogenic characteristics [
19,
20]. Interestingly, down-regulation of p21 is inversely associated with MSI, the hallmark of Lynch-associated tumors. One study found that a larger proportion of Lynch-associated CRCs expressed p21 compared to sporadic CRCs (80 vs. 31%) [
21]. However, a recent study reported on expressive Quantitative Trait Loci (eQTL) in colonic tissue based on data from the GTEx project portal (
http://www.gtexportal.org/home/) and did not find a statistically significant effect of rs1321311 on CDKN1A expression in sigmoid and transverse colon tissue (p = 0.84 and p = 1.00 respectively) [
22,
23]. It is also unclear why this effect only appears to be present in male
PMS2 carriers. Although no gender difference was noted by the meta-analysis that identified the SNP [
19], it is possible that gender differences exist, as CRC risk in the general population and in Lynch patients is known to be higher in men compared to women [
24,
25]. Indeed, mutations are more often found in males than females when assessed for Lynch syndrome [
26]. Another possible explanation for this gender specific effect might lie in the effect of other risk modifiers. It is perceivable that other factors than SNPs have a stronger influence on (colorectal) cancer development in women, such as hormonal factors [
27,
28]. Unfortunately data on hormone levels or other factors previously shown to modify cancer risk in LS such as medication use (e.g. aspirin) or environmental factors were unavailable for analysis and as such we were unable to correct for this [
29‐
33]. Similarly, data on smoking and BMI were only available for a small proportion of carriers (n = 131, 26%). It should be emphasized that all results after gender stratification should be interpreted with caution because of small sample size and multiple testing. This could have led to false associations. Further studies are needed to validate these findings.
We also investigated the effect of the 24 SNPs on CRC risk in the PMS2 cohort by means of a polygenic risk score (PRS). While there did not appear to be a significant effect of the PRS based on ORs from sporadic CRC cohorts, there was a difference in the cumulative incidence of CRC for PMS2 carriers with a PRS2 (based on HRs in this study) in the highest quartile. Bootstrap validation however refuted this promising observation. Further studies are hence needed in other large cohorts.
There were some limitations to this study. Our study consisted exclusively of Dutch PMS2 carriers and thus had a relatively homogeneous genetic makeup, implying that differences between our results and previous studies might be due to population-specific effects.
A second limitation might be that we did not correct for the specific mutation present in each family, mainly because in the majority of families the segregating
PMS2 mutation is rare or even unique. A previous study by our group did not identify such a correlation with CRC risk in
PMS2 carriers (Supplemental Tables 2a + 2b Table: for more details) [
34].
Unfortunately, we were not able to validate our findings in an external cohort. To our best knowledge this is one of the largest
PMS2 cohorts currently collected, and bootstrap validation is a strong approach to assess discriminative ability of a prediction model [
35]. Stratifying our cohort into a discovery and validation cohort was not a viable option as this would have resulted in a substantial decrease in power. Our study might already have been underpowered to detect weak associations. However, while such associations are interesting from a scientific point of view and may be relevant to tumorigenesis, they are not necessarily useful in clinical practice when the effect is small. For the two SNPs previously found to increase risk in
MLH1 mutation carriers, we had 60–80% power to detect an HR of 1.5, which we would consider clinically relevant. The previously reported HR in
MLH1 carriers for rs3802842 was 2.7, an HR for which we have ample power to detect (Supplemental Fig. 1).
Families with a segregating PMS2 mutation show a high degree of phenotypic variability. We were not able to confirm the risk modifying effect of rs3802842 (11q23.1) and s16892766 (8q23.3), which were previously found to increase the risk in MLH1-associated LS. This, together with the established lower penetrance, raises the question of whether PMS2-associated LS should be considered a separate Lynch disease entity. Additional explanations for phenotypic variability that warrant greater exploration include gene-environment interactions and risk modification by other genetic variants.