Germline variants of the MMR/EPCAM genes in Russian patients with Lynch syndrome

Introduction

Lynch syndrome is a disease with an autosomal dominant type of inheritance characterized by the development of malignant tumors of various organs, the cause of which is a germline pathogenic variant in one of the genes of the DNA mismatch repair system (MMR system) or in the EPCAM gene. The frequency of Lynch syndrome in Europeans is approximately 1:300 people, which makes it the most common among all hereditary oncological syndromes [1]. Carriers of pathogenic variants can develop tumors of the colon, uterus, urinary system, stomach, etc. DNA diagnostics of blood relatives of patients with Lynch syndrome is extremely important, since lifelong clinical monitoring of carriers of the pathogenic variant significantly reduces both the incidence of malignant neoplasms and mortality caused by them [2].

Lynch syndrome causes only 3% of all cases of colorectal cancer [3], and the process of its diagnosis includes several stages. Initially, a sample of a colon tumor should be examined for microsatellite instability (MSI). This phenomenon occurs in approximately 12% of patients with sporadic colorectal cancer and in all Lynch-associated tumors [4]. Only if it is detected, a further search for a germline variant in the MMR system genes is carried out [2]. These genes were mapped in the 1990s in families of patients with predominantly hereditary colon cancer: MLH1 (OMIM:120436), MSH2 (OMIM:609309), MSH6 (OMIM:600678), PMS2 (OMIM:600259), PMS1 (OMIM:600258), etc. It is crucial to mention the EPCAM gene (OMIM:185535), the product of which is involved in the adhesion of epithelial cells and also takes part in proliferation. This gene is located immediately before the MSH2 gene, and large deletions of the 3' end of the EPCAM gene lead to epigenetic hypermethylation of the MSH2 gene promoter, causing the development of Lynch syndrome [5].

The Human Gene Mutation Database Professional (HGMD Pro) 2023.1 contains more than 3,500 described unique pathogenic variants in the MMR/EPCAM genes. At the same time, its data are constantly being updated, since previously undescribed variants are found in different patient samples from different populations.

This publication is dedicated to the study of germline variants in the MMR and EPCAM genes in Russian patients with Lynch syndrome.

Material and Methods

Patient selection

An observational single-center study aimed at identifying and studying patients with Lynch syndrome was conducted from 2012 through 2023 at the Ryzhikh National Medical Research Centre for Coloproctology (RNMRCC) of the Russian Federation Ministry of Healthcare (Moscow, Russia). The study material included data on 141 patients with a genetically confirmed diagnosis of Lynch syndrome from the local Registry of Hereditary Colorectal Cancer at RNMRCC. Among them, there were 78 men and 63 women aged 21 to 80 years. Informed consent was obtained from all patients included in the study. This study complied with the ethical principles of the Declaration of Helsinki and was approved by the Ethics Committee at RNMRCC.

The Amsterdam Criteria II or Bethesda Guidelines were employed to select patients with suspected Lynch syndrome from 2012 to 2014. Subsequently, from 2014 to 2023, the following more effective selection criteria developed at RNMRCC were used:

1. Colorectal cancer in a patient under 43 years of age (sensitivity: 88.9%; specificity: 82.9%);

2. In addition to colorectal cancer, two or more cases of Lynch syndrome-associated cancer in the patient or in his/her blood relatives in the same line, regardless of age (sensitivity: 100%; specificity: 64.7%).

Laboratory methods

The molecular genetic study, conducted from 2012 to 2018, included a primary search for MSI in colorectal cancer samples using fragment analysis by capillary electrophoresis on an ABI PRISM 3500 sequencer (Applied Biosystems, USA) using NR21, NR24, NR27, BAT25, BAT26 markers. When MSI was detected in the tumor, a search for pathogenic variants of the MLH1, MSH2 and MSH6 genes was carried out in DNA isolated from patients’ peripheral blood lymphocytes. For this purpose, polyacrylamide gel electrophoresis was performed on a Sequi-Gen GT Sequencing Cell (BIO RAD, USA). In case of differences in the electrophoretic pattern of the fragments of the patient’s genes under study from the control samples, they were sequenced using the Sanger method on an ABI PRISM 3500 device. In the absence of pathogenic variants in the MLH1/MSH2/MSH6 genes, a panel of genes, including the MMR genes, was sequenced using the Junior 454 instrument (Roche, Switzerland) in accordance with the manufacturer’s protocol. All identified hereditary variants were confirmed by the Sanger method [6].

The molecular genetic study, conducted from 2019 through 2023, included an initial search for MSI in the tumor using fragment analysis. When MSI was detected, germline pathogenic variants in the MLH1 and MSH2 genes were immediately searched for by Sanger sequencing on an ABI PRISM 3500 instrument. Large rearrangements were also searched by Multiplex Ligation-Dependent Probe Amplification (MLPA) in the MLH1, MSH2, MSH6, and EPCAM genes (SALSA MLPA Probemix P003 MLH1/MSH2; SALSA MLPA Probemix P072 MSH6) according to the manufacturer’s protocol. In the absence of pathogenic variants, whole-exome sequencing was performed; 100 ng of total genomic DNA was used to prepare paired-end enriched libraries using a tagmentation-based protocol and xGen Exome Research Panel v2 probes. Sequencing was performed on a NextSeq 550 platform (Illumina, USA) with a read length of 2*75 bp with an average coverage of 100x. The resulting reads were mapped to the reference GRCh38/hg38 genome using the BWA [7] and SAMtools [8] algorithms with recalibration. Obtained via the GATK [9] and Deepvariant [10] software, SNV variants were annotated using the databases (dpSNP v155, GnomAD v 2.1.1, ExAC v0.3.1). CNV analysis was conducted using the CODEX2 package [11]. Whole-exome sequencing was also performed for archival DNA samples in which pathogenic variants had not been previously identified. Variants identified by whole-exome sequencing were confirmed by the Sanger method. To establish the pathogenicity of previously undescribed variants, DNA diagnostics of patient relatives, routine MMR immunohistochemistry of tumor samples and pathogenicity assessment according to the ACMG and CanVIG criteria for MMR genes were performed [12, 13].

Results

The conducted molecular genetic study allowed diagnosing Lynch syndrome in 141 Russian patients. Previously undescribed germline variants were found in 17 of 141 probands (12.1%). All of them were analyzed in accordance with the most recent recommendations for the interpretation of variants detected in cancer predisposition genes (CanVIG) and a special supplement for the evaluation of variants in MMR genes [14].

Pathogenic and likely pathogenic variants in the MLH1 gene were revealed in 64 patients, including eight previously undescribed variants (Table 1).

Table 1. The spectrum of pathogenic and likely pathogenic variants in the MLH1 gene

In the MSH2 gene, 53 pathogenic/likely pathogenic variants were detected, including 8 undescribed before (Table 2).

Table 2. The spectrum of pathogenic and likely pathogenic variants in the MSH2 gene

It is necessary to consider in more detail the three most difficult to interpret germline variants in the MSH2 gene: c.1280_1378dup, c.1538T>C and c.1979A>G.

The first proband was found to have a 99-nucleotide insertion: c.1280_1378dup (p.Lys427_Asp459dup), which did not result in a frameshift. The patient’s family history was as follows: his father had rectal cancer at the age of 60 years, and his father’s mother had colon cancer at the age of 70 years. This proband was diagnosed with sebaceous carcinoma of the skin in the scapular region at the age of 36 years, and with synchronous/metachronous multiple primary malignancies of the sigmoid and cecum at the age of 48 years. MMR immunohistochemical examination of colon tumor biopsies revealed a dMMR phenotype with loss of MSH2/MSH6 proteins, and MSI was detected by PCR and fragment analysis. Variant c.1280_1378dup was classified by us as probably pathogenic based on the sum of the following criteria: PM2, PM4 and PP4 (moderate evidence).

Another proband with the hereditary variant c.1538T>C (p.Leu513Pro) was diagnosed with colon cancer at the age of 43 years. His grandfather died of esophageal cancer at the age of 65 years. The results of MMR immunohistochemical examination of the colon tumor revealed a dMMR phenotype with loss of MSH2/MSH6 proteins, and MSI was detected by PCR and fragment analysis. DNA diagnostics revealed the variant c.1538T>C in the MSH2 gene, leading to the replacement of nonpolar leucine with nonpolar proline. This variant is absent from population samples, it is present in a conserved region among all vertebrates (PhyloP100way 7.252), while functional studies (MAVE 2.43) and over 10 bioinformatic predictors (REVEL 0.943) indicate a high probability of pathogenic significance in this variant.

Consequently, the c.1538T>C variant was classified as likely pathogenic using the following criteria: PP3, PM2, PS3, PP4 (supporting), BP1.

The third patient was found to have the c.1979A>G variant, which was previously reported to have conflicting data regarding its association with Lynch syndrome. In this regard, we performed a functional study at the mRNA level, which confirmed the loss of nine amino acids (p.Asp660_Thr668del) (Figure 1). This allowed applying the PS3 criterion. In addition, a modified PP4 (moderate evidence) criterion was applied to this variant, since the patient’s pedigree showed an aggravated family history: the father’s mother died of colon cancer at the age of 65 years; the father was diagnosed with metachronous colorectal cancer at the age of 69 years, 70 years and 72 years; the father’s sister was diagnosed with colon cancer at the age of 40 years, and the patient per se was diagnosed with metachronous multiple primary cancer of the cecum at the age of 35 years, descending colon at the age of 46 years, and a sebaceous carcinoma of the nose skin at the age of 45 years. Hence, this variant was classified as likely pathogenic based on the sum of the criteria: PS3, PM2, PP5, PP4 (moderate), BP1.

Figure 1. Results of sequencing of the cDNA region of the MSH2 gene: A, control sample; B, patient with c.1979A>G variant (the arrow indicates the beginning of the deletion).

Eleven germline variants were revealed in the genes MSH6, PMS1, PMS2, including one new variant (Table 3).

Table 3. Pathogenic and likely pathogenic variants in the MSH6, PMS1, and PMS2 genes

MLPA method made it possible to find large deletions/duplications in 13 probands with Lynch syndrome (Table 4).

Table 4. Large rearrangements in the MMR/EPCAM genes

Discussion

Identification of patients with Lynch syndrome is an extremely difficult problem for two reasons. The first one is the issue of patient selection, and it lies in the fact that Lynch syndrome causes only 3% of colon cancer [3], which indicates the impossibility of conducting complete genetic diagnostics in all patients with colorectal cancer due to its high cost and time expenditures. At the same time, the lack of timely diagnosis of Lynch syndrome leads to incorrect treatment tactics and the exclusion of examination of the patient’s blood relatives. Also, the sensitivity of known patient selection guidelines, such as Amsterdam Criteria II and Bethesda Guidelines, is far from 100%, since they fail to identify 28% of patients with Lynch syndrome [15]. The second reason is genetic. There is a need to study a large number of genes to identify a pathogenic variant in patients with suspected Lynch syndrome: MLH1, MSH2, MSH6, PMS2, PMS1, EPCAM and some other. At the same time, various molecular genetic methods used in different laboratories allow reducing the cost and time of DNA diagnostics. Such methods include, in particular, electrophoresis (SSCP, conformation-sensitive, etc.) and HRM analysis (the sensitivity and specificity of which does not allow identifying all possible pathogenic variants). As a result of using the method of high throughput sequencing of a significant number of genes, a huge number of germline variants can be identified, often with previously unknown missense mutations and intron substitutions, the pathogenicity of which is not always possible to establish.

To solve the first problem (patient selection), after the first two years of clinical genetic testing, we developed our own criteria, the sensitivity of which significantly exceeded the sensitivity values of the revised Bethesda and Amsterdam II guidelines [16].

Solving the genetic problem took much longer. First of all, we found that the conformation-sensitive electrophoresis we used did not allow detecting single-nucleotide pathogenic variants in approximately 7% of patients, which led to a complete rejection of this method and its replacement with Sanger sequencing for all exons of the MLH1 and MSH2 genes. With the advent of next-generation sequencing, we initially used a gene panel to search for mutations, but later decided to use whole-exome sequencing, which allowed us to immediately find some of the large rearrangements. The MLPA method was used to identify the remaining large deletions/duplications. Finally, as one of the most important methods for determining the pathogenic significance of new variants (in addition to DNA diagnostics of relatives and the use of prognostic programs), we chose MMR immunohistochemical examination of tumor samples not only from the patient, but also from the patient’s relatives with oncological diseases. Thus, the period for developing the final diagnostic algorithm for Lynch syndrome was almost seven years (Figure 2).

Figure 2. Algorithm of diagnosing Lynch syndrome.

The results obtained using this algorithm allow stating with confidence that at the moment we can identify the most complete spectrum of pathogenic variants of the MMR/EPCAM genes in almost all of our patients suffering from Lynch syndrome. Moreover, as an example of its high efficiency, we would like to cite a unique instance of using the developed algorithm. As a result, in one of the examined patients we were able to genetically diagnose two severe hereditary syndromes at once: Lynch syndrome (gene MSH6: NM _000179.2:c.742C>T, p.Arg248Ter) and Diamond-Blackfan anemia (deletion of the chromosome 15 locus with the capture of the interval 82662932–84816747bp, including the complete sequence of the RPS17 gene). It should be noted that the estimated frequency of such patients was only 1 case per 480 million people, while such cases have not been previously described at all [17].

Unfortunately, not all laboratories of the world currently diagnose the genes EPCAM, PMS1, etc. Therefore, we can compare our own data with most other results only for four main genes: MLH1, MSH2, MSH6 and PMS2 (Figure 3).

Figure 3. The frequency of germline variants in the MMR genes in Lynch syndrome patients in different countries [18-22].

It is extremely important to emphasize that in Russia, among patients with Lynch syndrome, the most common hereditary mutations are found in the MLH1 gene (50.7%). However, such a high frequency of germline variants in one of the MMR genes has not been described in any other country (Figure 3). This fact indicates the advisability of initiating molecular genetic testing in Russian patients with the MLH1 gene, which will significantly reduce the time of DNA diagnostics of every second patient with Lynch syndrome. The combined frequency of pathogenic and likely pathogenic variants in the MLH1 and MSH2 genes in Russia is 91.9%, which is also the highest figure among all presented countries; the closest results to ours were obtained in Germany (87.6%) and Great Britain (81.3%) [19]. On the other hand, the minimum frequency of germline variants in Russia is described both in the MSH6 gene (7.4%) and in the PMS2 gene (0.7%), the combined frequency of which ranges from 12.4% in Germany to 51.8% in the Netherlands (Figure 2) [19, 22]. Nevertheless, the obtained results should be taken into account when conducting DNA diagnostics in Russian patients in order to make it as effective as possible.

Interestingly, among patients with colorectal cancer against the background of Lynch syndrome caused by a pathogenic variant in the MSH2 gene in Russia, male patients predominated (35:22). At the same time, in cases where Lynch syndrome was caused by pathogenic variants in other genes, the frequency of cancer patients in men and women did not differ: 36 men and 33 women had mutations in the MLH1 gene, 5 men and 5 women in MSH6; and only a total of 3 women and 2 men had mutations in PMS2, PMS1 and EPCAM genes. These data may indicate a higher risk of colon cancer in men carrying pathogenic variants in the MSH2 gene.

It is extremely important to emphasize that the current CanVIG recommendations [13, 14] do not take into account cases of sebaceous skin carcinomas in the family and personal anamnesis of carriers of germinal variants in the MSH2 gene, while their presence allows diagnosing Muir-Torre syndrome, which is a special variant of Lynch syndrome. We believe that it is appropriate to correct this situation in the updated version of these recommendations.

Conclusion

In conclusion, it should be noted that the analysis of data from Russian patients with Lynch syndrome allowed establishing that pathogenic and likely pathogenic variants are most often found in the MLH1 and MSH2 genes (91.9% of cases). Therefore, to conduct the most effective DNA diagnostics in patients suspected of having this syndrome, it is necessary to begin diagnostics with the study of these two genes. The frequency of major rearrangements in the MMR/EPCAM genes was 9.2%, which indicates the need to include the MLPA method in routine DNA diagnostics of patients. Germinal variants previously undescribed in the world population were found in 12.1% of probands, demonstrating the presence of population characteristics in Russian patients with Lynch syndrome.

Author contributions

AT: designed and conducted the study, analyzed data, prepared the draft manuscript. AB, VS and AL: performed genetic analysis and analyzed genetic data. DP: collected clinical data, identified patients, critically reviewed the draft manuscript, corrected the translated version of the manuscript. DS: critically reviewed the manuscript. YS and SA: supervised the project, reviewed the manuscript. All authors contributed to the study concept and design, as well as read and approved the final version of the manuscript.

Conflict of interest

The authors declare that no conflicts of interest pertaining to this study.

Ethical approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the Ethics Committee at Ryzhikh National Medical Research Centre for Coloproctology and with 1964 Declaration of Helsinki and its later amendments.