The most common form of hereditary colorectal carcinoma is Lynch syndrome, a hereditary nonpolyposis colorectal cancer (HNPCC). Patients with Lynch syndrome also have an increased risk of urogenital tract cancer and other carcinomas. The inheritance of this disease is autosomal dominant. The cause of Lynch syndrome is a germline mutation in one of the genes responsible for repairing DNA replication errors (mismatch repair genes-MMR genes), resulting in the formation of tumors which are characteristic having a high degree of instability in short tandem repeats DNA (microsatellites). These tumors are therefore referred to as MSI-H (microsatellite instability-high). The guiding factor for finding mutated gene is immunohistochemical examination of the expression of MMR proteins hMLH1, hMSH2, hMSH6 and hPMS2. The gene with loss of expression is subsequently subjected to the examination by sequencing. For differential diagnostic of MSI-H there is also often used detection of the mutation BRAF (V600E). There is no such mutation in Lynch syndrome.
A subgroup of HNPCC is Muir–Torre syndrome (MTS) characteristic by benign and malignant sebaceous skin tumors in combination with internal malignancies. High microsatellite instability is present in approximately 70 % of these tumors. On the formation of MTS contribute mainly mutations in genes hMLH1 and hMLH2 wherein a germline mutation in the gene hMSH2 is predominant (90 %)
MMR proteins assemble into the functional complexes: hMSH2-hMSH6 heterodimer and hMLH1-hPMS2 heterodimer. The first of these dimers recognizes errors in DNA and indicates damage while the second one corrects these errors. At the same time it participates on cell cycle arrest and apoptosis induction in response to DNA damage.
hMLH1 gene is located on the chromosome 3 in the region p21.3 and has 19 exons. On the chromosome 2 in the region p21-22 there is located hMSH2 gene containing 16 exons and in the region p16 there is a gene hMSH6 consisting of 10 exons. The hPMS2 gene is located in the chromosomal region 7p22.
Microsatellite instability examination is performed by fragmentation analysis. We examine five mononucleotide (BAT-25, BAT-26, NR-21, NR-24, MONO-27) and three dinucleotide repetitions (D2S123, D17S250, D5S346), thus, a widespread Bethesda panel. We also detect mutations in coding regions of MMR genes hMLH1, hMSH2,hMSH6 and PMS2 Large deletions and duplications are examined via MLPA tests (MRC Holland). (Primers for PCR amplification are arranged in order to detect even an eventual splice site mutation. PCR products are analyzed by direct sequencing. Large deletions, which represent 12 - 15 % of all found mutations, and eventual methylation of MMR genes promoters are examined using MLPA kits. Examination can be performed from tissue (frozen and FFPE) and blood.
- analysis of the genes MLH1, MSH2, MSH6 - performed from gDNA which is isolated from anticoagulated blood collected into vacutainer with EDTA (usually purple cap), storage: in 4 - 8 °C
- analysis of the gene PMS2 - performed from RNA which is isolated from blood collected into so-called "PAX gene" vacutainer (includes agent that protects RNA from degradation), the tube will be delivered to you.
Blood collection into "PAXgene" vacutainer:
- before using, "PAX gene" vacutainer has to be stored in 18-25 °C
- fill "PAX gene" vacutainer as the last one
- "PAX gene" vacutainer contains a chemical additive, backflow can not occur
- turn the "PAX gene" vacutainer 8 - 10x upside down and back immediately after collection
- then let stand for 2h in 18-25 °C (upright)
- storage (upright): 4 - 8 °C, if the blood is picked up by our transport the same day, otherwise in -20 °C
According to the literature, MLH1 gene mutations were found in 50% of cases with Lynch syndrome (Peltomäki 2003). A combination of direct sequencing of gDNA (all exons and flanking intron sections) and MLPA-mP003 test, we are able to detect about 98% of known mutations in the gene MLH1 (see. HGMD database).
According to the literature, MSH2 gene mutations were found in 40% of cases with Lynch. syndrome (Peltomäki 2003). A combination of direct sequencing of gDNA (all exons and flanking intron sections) and MLPA-mP003 test, we are able to capture about 99% of known mutations in the gene MSH2 (see. HGMD database).
According to the literature, MSH6 gene mutations were found in 7 to 10% of cases with Lynch syndrome (Peltomäki 2003). A combination of direct sequencing of gDNA (all exons and flanking intron sections) and MLPA-mP072 test, we are able to capture about 98% of known mutations in the gene MSH6 (see. HGMD database).
According to the literature, PMS2 gene mutations were found in <5% of cases with Lynch. syndrome (Senter et al 2008). A combination of direct sequencing of cDNA and MLPA-mP008 test, we are able to capture about 87% of known mutations in the gene PMS2 (see. HGMD database). Mutational analysis of this gene is difficult due to the presence of pseudogenes in the genome.
The analytical sensitivity and specificity of sequencing: 99%
The analytical sensitivity of MLPA 90 % and specificity 98%.
Mutations deeply in intron and regulatory sequences are not captured. Rare polymorphisms in the annealing site of primers or probes could cause a diagnostic mistake.
Mutations will not be detected, in the case of somatic mutations analysis by sequencing, if the altered cell line will not represent at least 20%.