Abstract
Background: Some single-nucleotide polymorphisms are associated with higher risk of colorectal cancer development and are suggested to explain part of the genetic contribution to Lynch syndrome. Aim: To evaluate the mutL homolog 1 (MLH1) I219V polymorphism in 124 unrelated South American individuals suspected of having Lynch syndrome, based on frequency, association with pathogenic MLH1 and mutS homolog 2 (MSH2) mutation and clinical features. Materials and Methods: DNA was obtained from peripheral blood and polymerase chain reaction (PCR) was performed, followed by direct sequencing. Results: The Val allelic of the I219V polymorphism was found in 51.61% (64/124) of the individuals, with an allelic frequency of 0.3. MLH1 or MHS2 pathogenic mutations were found in 32.81% (21/64) and in 23.33% (14/60) of Val-carriers and non-carriers, respectively. Conclusion: The Val-carrying genotype was frequent in the studied population; however, it does not appear to exert any modifier effect on MLH1 or MSH2 pathogenic mutations and the development of colorectal cancer.
Lynch syndrome (LS) is the most common form of familial colorectal cancer (CRC) and is associated with germline mutations in the DNA mismatch repair (MMR) genes, i.e. mutL homolog 1 (MLH1) on 3p21.3, mutS homolog 2 (MSH2) on 2p22-p21, mutS homolog 6 (MSH6) on 2p16.3 and post-meiotic segregation increased 2 (PMS2) on 7p22.2. Mutations in the above mentioned genes have been reported to account for more than 85% of LS kindreds fulfilling the Amsterdam criteria and exhibiting microsatellite instability (MSI) (1). Recently, the term LS has been restricted to those families with germline mutations in one of the MMR genes. Familial aggregation of CRC with no evidence of MMR deficiency was shown to be clinically and molecularly distinct from classical LS tumors and, therefore, is designated as familial colorectal cancer type X (FCCTX). Given the above distinction, the term hereditary non-polyposis colorectal cancer (HNPCC), which was formerly used to refer to families clinically diagnosed with colorectal cancer that might or might not have mismatch repair deficiency, is replaced by one of the more informative names: LS and FCCTX (2).
To date, over 513 different DNA alterations have been reported in HNPCC, in which the majority are represented by single-nucleotide substitutions, deletions or insertions (3). In total, 308 different pathogenic mutations have been identified in MLH1, along with 45 polymorphisms (3-5). The characterization of the functional effects of MMR mutations is essential, not only for effective diagnosis of cancer predisposition, but also for selecting the appropriate screening and treatment (6).
The common definition of single-nucleotide polymorphisms (SNPs) requires that the relative frequency of the least frequent allele is greater than 0.01, except for two polymorphisms in MLH1: a TTC deletion in the 3’untranslated region and a CA repeat polymorphism assigned to locus D3S1611, whose allelic frequencies range from 0.4% to 50% (7).
One of the most common polymorphisms related to LS is I219V located on the MLH1 gene. The I219V polymorphism lies in exon 8 and is conserved throughout evolution, which can be seen as a sign of its importance for the good functioning of the protein. HNPCC databases, in silico programs and various functional studies described it as a benign polymorphism. Recently, a functional study based on a transient transfection of hMLH1 complementary DNA carrying the variant into a human embryonic kidney fibroblast cell line lacking hMLH1 expression, demonstrated in vivo that the I219V SNP does not affect the MMR capacity (8).
Several studies have shown a high frequency of the I1219 polymorphic variant in patients with LS, however, none have tested whether this variant exerts a modifier effect on pathogenic mutations. Therefore, the aim of our study was to evaluate the frequency of I219V polymorphism in 124 individuals and its association with MLH1/MSH2 pathogenic mutations and clinical characteristics, as part of a larger South American Collaborative Study evaluating MMR gene mutations in unrelated individuals with suspected LS (9).
Materials and Methods
Participating centers. One hundred and twenty-four unrelated individuals enrolled in this study were referred from the following South American hospitals and medical centers: 105 from the Department of Pelvic Surgery, A.C. Camargo Hospital, Sao Paulo, Brazil; 6 from the Colorectal Surgery Service, Uruguayan Collaborative Group, Montevideo, Uruguay; and 13 from the Department of Colorectal Surgery, Italian Hospital, Buenos Aires, Argentina. As part of a South American Collaborative Study, all cases were studied after signed informed consent was obtained.
Samples and DNA extraction. Genomic DNA from 124 patients fulfilling the Amsterdam I, II or Bethesda guidelines (10-13) was directly extracted from 3 ml of whole-blood using the Puregene Genomic DNA Isolation Kit (Gentra Systems, Minneapolis, MN, USA), according to the manufacturer's instructions.
PCR conditions. The MLH1 [GenBank: NM.000249] and MSH2 [GenBank: NM.000251.1] genes were divided into 20 fragments covering all 19 and 16 exons, respectively, and substantial parts of intronic regions. The PCR reactions contained 0.2 μM of each primer, 1.25 U AmpliTaq Gold (Applied Biosystems, SP, Brazil), 10 μM of each dNTP, 3 mM MgCl2 and 25 ng DNA in a final reaction volume of 25 μl. The following cycling conditions were used: initial denaturation at 95°C for 9 min, followed by 35 cycles of denaturation at 95°C for 45 s, annealing steps at 56-60°C for 45 s and extension at 72°C for 1 min, followed by 72°C for 7 min.
DNA sequencing and analysis. PCR products were purified with exonuclease I and alkaline phosphatase (EXO-SAP IT-USB/GE, SP, Brazil) and sequenced in both directions. Sequencing reactions were separated on an ABI Prism 3130 instrument (Applied Biosystems, SP, Brazil). All sequences were analyzed by the CLC software (CLCbio, Aarhus, Denmark).
Mutation nomenclature. Mutation nomenclature followed the Human Genome Variation Society (HGVS) guidelines (www.hgvs.org/mutnomen/). All identified mutations were compared with those previously reported in databases for MLH1 and MSH2 genes, maintained by the MMR Gene Unclassified Variants Database (www.mmruv.info), the Mismatch Repair Genes Variant Database (http://www.med.mun.ca/mmrvariants/) and the International Society for Gastrointestinal Hereditary Tumors (http://www.insight-group.org).
Statistical analysis. Statistical analyses were carried out to evaluate if the mutL homolog 1 (MLH1) I219V polymorphism has implications on age of tumor onset in patients harboring or not pathogenic mutations in MLH1/MSH2 genes. The comparison was performed by Wilcoxon and Kruskal-Wallis tests (with significance set at p<0.05), using the statistical software package IBM SPSS Statistics 20 (SPSS, Chicago, IL, USA).
Results
Genetic analyses of I219V polymorphism in individuals with suspected LS. The MLH1 I219V polymorphism frequencies are shown in Table I. Val-carriers were found in 64 (51.6%) out of 124 unrelated individuals with suspected LS, of whom 49 were from Brazil, two from Uruguay and 13 from Argentina. Of them, 54 (44%) and 10 (8%) were Ile/Val heterozygotes and Val/Val homozygotes, respectively. The allelic frequency of Ile and Val was 0.7 and 0.3, respectively.
Correlation of I219V polymorphism with MLH1 and/or MSH2 pathogenic mutations. The characterization of germline mutations of MLH1 and MSH2 in unrelated South American individuals with suspected LS were provided by the South American Collaborative Study (9). Twenty-one out of 64 (32.81%) of Val-carriers harbored pathogenic mutations in MLH1 and/or MHS2, and one case presented pathogenic mutations in both genes. In non-carriers (60/124), MLH1 and/or MHS2 pathogenic mutations were found in 23.33% (14/60) (Table II).
Clinical characteristics of I219V carriers associated with the presence of MLH1 and/or MSH2 pathogenic mutations. The Val polymorphism was found in 51.61% (64/124) of cases, out of which 51.5% (33/64) fulfilled the Bethesda guideline and 48.5% (31/64) the Amsterdam I or II criteria. The median age of diagnosis was 28-62 and 29-71 years in Val-carriers with and without MLH1/MSH2 pathogenic mutation (Table 3), respectively (Table III). Clinical criteria, type and number of tumors in these cases are presented in Table III.
In order to evaluate if this polymorphism exerts any influence on the age of tumor onset, we analyzed the mean age of carriers and non-carriers of the I219V polymorphism according to their mutation status (MLH1 and/or MSH2 mutation). However, no statistically significant association was found between these groups (Table IV).
Discussion
SNPs may play a small role in CRC risk. It is also possible that a combination of several variants in different genes might work synergistically to increase the risk of CRC (8). In this series, we investigated the I219V polymorphism in order to analyze its frequency, its correlation with MLH1/MSH2 pathogenic mutations and its influence in hereditary CRC risk (based on clinical characteristics).
Trojan et al. (14) postulated that the majority of MLH1 missense variants associated with LS, lead to structural changes within the amino- or carboxy-terminal regions containing the ATPase site and the domain of PMS2 interaction, respectively. However, according to Hudler et al. (15), the missense polymorphism located in the exon 8 leads to a substitution of conservative hydrophobic amino acid, namely isoleucine, by valine, which could affect the speed and fidelity of protein synthesis, due to the lower abundance of the tRNA or due to changes in the secondary structure of mRNA.
First studies regarding the I219V polymorphism reported an incidence of 31-80% in different populations (16-18). The allelic frequencies found in this series for Ile and Val were 0.7 and 0.3, respectively. Similar results were reported for German LS probands (0.69 and 0.31), Swedish families with LS (0.64 and 0.34) and Italian families with suspected LS (0.33 for Val) (16-19). This similarity might be explained by the African influence exerted through the slave population for three centuries, as well as the Native Indian and colonizing European populations in Brazil (20-22). It is known that ethnicity can impact molecular pathways of various types of human cancer and as a result, different clinical and pathological features can be found (23).
Regarding the association of the I219V polymorphism and MLH1/MSH2 pathogenic mutations, the results of this series showed no significant differences between Val-carriers with MLH1 and/or MSH2 pathogenic mutation (32.81%) and non-carriers with MLH1 and/or MSH2 pathogenic mutations (23.33%). We can deduce that the presence of I219V polymorphism is not linked to pathogenic mutation in MLH1 or MSH2 genes in LS.
Regarding clinical characteristics, 51.5% (33/64) of Val-carriers fulfilled the Bethesda criteria, whereas the MLH1/MSH2 pathogenic mutation was observed in 48.4% (15/31) of those fulfilling the Amsterdam criteria, confirming that the strict Amsterdam criteria are much better for selecting individuals suspected of having LS (9). However, no statistical difference was observed related to clinical criteria and tumor onset in carriers and non-carriers of Val in the I219V polymorphism.
Conclusion
To our knowledge, this is the first study regarding the evaluation of I219V according to MLH1 and/or MSH2 pathogenic mutations and clinical characteristics in unrelated South American LS individuals suspected of having LS. The Val-carrying variant was very frequent in this series and showed no association with MLH1/MSH2 pathogenic mutations, clinical criteria (Amsterdam or Bethesda), or age of tumor onset. According to the computational prediction methods, it is suggested that this polymorphism does not have a pathogenic consequence.
Acknowledgements
This work was supported by grants from the Brazilian National Institute of Science and Technology in Oncogenomics (Fundacao de Amparo à Pesquisa do Estado de Sao Paulo - FAPESP 05/05155-6 and 2008/57887-9) and CAPES.
Footnotes
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Conflicts of Interest
No Author has any conflict of interest with regard to this study.
- Received June 26, 2012.
- Revision received August 10, 2012.
- Accepted August 13, 2012.
- Copyright© 2012 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved