Carcinogenesis involves a series of events through acquisition of multiple genetic alterations which makes the milieu susceptible for further mutations and the precancerous cells genomically instable accelerating the process [5]. Majority of the CRC have CIN leading to changes in chromosome number or structure (aneuploidy) [6] due to gain, loss or translocation. This pathway involves hyperactivation of WNT signalling pathway mostly (80%) via inactivating mutation of APC gene, a negative regulator, (80%) or alterations in other components like β-catenin (5–10%) [7]. It may proceed with activation of KRAS oncogene, loss of 18q (DCC, SMAD2, SMAD4) and loss of TP53 expression. However, only few cases have all chromosomal anomalies [8].
In our case, we identified somatic mutations in APC (exon 16,5q22.2 NM_000038.6: c.3927_3931delAAAGA[1] p.Glu1309fs) and TP53 (exon 7,17p13.1 NM_000546.6:c.742C > T p.Arg248Trp). The former allelic variant reported with a frequency of 60% resulted from a frameshift insertion and deletion which might lead to truncated or absent APC protein. The latter allelic variant with a frequency of 51% has a missense mutation. Both the mutations have been established as pathogenic as per ClinVar database and data from numerous affected families in literature testify the same. Their coexistence support carcinogenesis along sporadic pathway.
Interestingly screening for hereditary cancer genes revealed presence of missense mutations in DNA mismatch repair genes- MSH6 (c.2039C > T) and PMS1 (c.321A > C), whose significance has not yet been established.MMR system functions to identify and correct errors during DNA replication- single base pair mismatch and insertion-deletion loops. HNPCC is a genetically acquired autosomal dominant disease resulting from germline defect in MMR genes which increases susceptibility to CRC as well as extra-colonic malignancies. It has incomplete penetrance and a variety of expressions depending upon the gene mutated. Mutations in MSH2 and MLH1 account for 80%, with MSH6 gene mutated in 7–20% cases, PMS2 in < 5% cases and other MMR genes are involved rarely [9].
MSH6 mutations are associated generally with endometrial cancer and also increase the likelihood of colorectal cancer by 8 fold [10]. Pathogenic variants of MSH6 produce colorectal cancer at an older age in contrast to probands with MSH2 or MLH1 mutations. MSH6 forms a heterodimer with MSH2 (hMutS Alpha), recognizes single pair mismatches and small InDeLs and corrects them [11]. Here a germline missense variant c.2039C > T was identified in coding exon 4 of MSH6 gene leading to alanine substitution by valine at codon 680 and alteration in a conserved residue in the protein. This variant has been reported in dbSNP database with identification number rs1558664035 and in Genome Aggregation Database (gnomAD) as a rare variant with frequency < 0.01%. It has not been shown to have clinical significance till date as per ClinVar database (VC000651717.4) in regard to hereditary cancer as per in silico prediction models. However, records of it have been submitted in ClinVar (accession: SCV0011748.2, SCV000947173.3, SCV001348668.2). Further studies are needed to establish its importance in disease causation.
PMS1, another MMR protein, forms complex with MLH1 (hMutL beta), hMutS complex and other proteins to facilitate excision of incorrect nucleotides and their repair [12]. Its mutation has been found to be associated with LS in very few cases [13, 14, 15]. In this case we report a novel heterozygous missense mutation in PMS1 gene, c.321A > C, p.Leu107Phe, in exon 4 on chromosome 2 which alters a conserved residue of protein. It has been found to be damaging by 2 out of 5 in silico missense prediction tools (FATHMM and Mutation Taster). It may point to an underlying susceptibility for LS. PMS1 is not classically implicated in LS and not routinely evaluated. Therefore, elaborate studies are required to establish its role in tumor development.
Microsatellite instability (MSI) testing is essentially important in phenotyping colorectal cancers and is indicated in all colorectal tumors. Instability results either due to defect in MMR gene or epigenetic silencing of promoter region mostly MLH1. Tumors with MSI have better prognosis compared to microsatellite stable (MSS) tumors. Although they don’t respond to 5-fluorouracil treatment, they are amenable to immunotherapy. It can be assessed by two ways: PCR and IHC. In the former nowadays instability at five quasi monomorphic microsatellite sites can be detected simultaneously: BAT-26, NR-21, BAT-25, MONO-27 and NR-24 [16]. In the present scenario, the patient was screened using the markers: BAT-25, BAT-26, NR21, NR22 and NR24 and was found to be MSS. A study by Schiemann Uet al. [17] proposed a protracted marker panel (BAT40, D10S197, D13S153, D18S58, MYCL1) for reassessment of MSS tumors and predicting presence of MSH6 mutations which might be actually MSI-L. All MSH6 mutations may not be detected either by PCR or IHC or both [18–20].