As most disease pedigrees are small, it is difficult to obtain a sufficient number of samples from affected and informative unaffected individuals. Moreover, LS deleterious variants are not completely penetrant. For these reasons, it is rarely possible to categorize variants as deleterious based on segregation alone. The co-occurrence of another known deleterious variant reduces the likelihood that an UV is truly deleterious, especially when both variants are located in trans [3]. To our knowledge, co-occurrence of a deleterious variant in one of the LS genes with the p.Lys618Ala variant has been observed in only two families. Liu et al [14] described an index subject from a LS family with two heterozygous variants (c.546-2A>G and c.1852_1853AA>GC); only the former segregated with LS in the family. Similarly, Steinke et al [15], described the co-occurrence of the p.Lys618Ala (c.1852_1853AA>GC) variant with the MSH6 p.Arg1068X (c.3202C>T) deleterious variant.

Herein, we describe the coexistence of the p.Lys618Ala variant with deleterious variants in another two unrelated LS families. In one family, the allele distribution of the pathogenic and unclassified variant was in trans, in the other family the pathogenic variant was detected in the MSH6 gene and only the deleterious variant co-segregated with the disease in both families. This evidence indicates that the p.Lys618Ala variant is not deleterious.

The molecular hallmark of LS tumours is an MSI phenotype, a functional consequence of MMR deficiency. It is expected that the putative germ-line mutation responsible for LS would confer the MSI phenotype. We tested the MSI status of 17 tumours from p.Lys618Ala carriers and detected only two cases of MSI (11.8%). Taking into consideration the bias caused by the over-representation of Bethesda Criteria-positive tumours in this subset of cases (8/17), the MSI frequency was not significantly different from that in the unselected CRC group [7]. This is further proof that the presence of this variant is irrelevant to the functional inactivation of MLH1 in CRC patients.

Nonetheless, we cannot exclude the possibility that this variant may result in a small increase in susceptibility to CRC or adenomas, as was suggested by Fearnhead et al [16]. Further studies with appropriate sample sizes are required to address the low penetrance effect of this variant in CRC.

Finally, we hypothesize that the clinical significance of a genetic variant may differ according to genetic background. Gene functionality may be the net result of the effects of allelic structures and their interactions with environmental factors. It is possible that low-penetrance variants behave differently in different populations, making it difficult to make predictions in terms of conferred risk.

