Familial Cancer (2012) 11:13–17 DOI 10.1007/s10689-011-9478-2
Two novel mutations in hMLH1 gene in Iranian hereditary non-polyposis colorectal cancer patients Somayeh Shahmoradi • Ali Bidmeshkipour • Ahmad Salamian • Mohammad Hasan Emami Zahra Kazemi • Mansoor Salehi
•
Published online: 7 September 2011 Ó Springer Science+Business Media B.V. 2011
Abstract Hereditary non-polyposis colorectal cancer (HNPCC) is one of the most common forms of hereditary colorectal cancer. It is an autosomal dominant disorder resulting from germline mutations in DNA mismatch repair genes. In this study, we screened hMLH1 gene in a group of Iranian HNPCC patients using polymerase chain reaction-single strand conformational polymorphism and direct sequencing methods. Here we report two novel frameshift mutations in this gene in our studied population. One of them results from a deletion of ‘‘T’’ at codon 36, exon 1 which causes premature stop codon and a truncated protein. The other results from a deletion of ‘‘T’’ at codon 753, exon 19 causing a delayed stop codon. There are a variety of the reported novel mutations in hMLH1 gene studies. Identification of these mutations is necessary in different populations and can help the management of colorectal cancer in these populations by screening, by prevention
S. Shahmoradi A. Bidmeshkipour Department of Biology, Faculty of Sciences, Razi University, Bagh Abrisham, Kermanshah, Iran A. Salamian M. Salehi (&) Department of Genetics, Medical School, Isfahan University of Medical Sciences, Isfahan, Iran e-mail:
[email protected] M. H. Emami Z. Kazemi Poursina Hakim Research Center, Bozorgmehr Ave., Isfahan, Iran M. H. Emami Department of Internal Medicine, Medical School, Isfahan University of Medical Sciences, Isfahan, Iran M. Salehi Medical Genetics Center of Genome, No 208, Shariati St. (West), Isfahan, Iran
strategies, and by following up the suspected HNPCC families. Keywords Colorectal cancer Frameshift mutation Germline mutation Hereditary non-polyposis colorectal cancer hMLH1 Mismatch repair genes Abbreviations APS Ammonium persulphate CRC Colorectal cancer HNPCC Hereditary non-polyposis colorectal cancer MMR Mismatch repair PCR Polymerase chain reaction SSCP Single strand conformational polymorphism TBE Tris-borate-EDTA TEMED N,N,N0 ,N0 -tetramethylethylenediamine
Introduction Colorectal cancer (CRC) is one of the most common cancers worldwide especially in Western and European countries [1–4]. It can be divided into three patterns of sporadic, inherited and familial [5]. Hereditary non-polyposis colorectal cancer (HNPCC) or Lynch syndrome is the most common form of hereditary colorectal cancer and is responsible for 1–5% of all CRC cases [1, 6–8]. This syndrome is inherited in an autosomal dominant manner [3, 9, 10]. In 1990, an International collaborative group on HNPCC developed some criteria called ‘‘Amsterdam Criteria I’’ [11, 12]. These indicate that there should be at least 3 relatives with CRC and, in addition, all of the following
123
14
criteria should be present: (1) one of them should be a firstdegree relative to the other two, (2) at least two sequential generations should be affected, (3) at least one CRC should be diagnosed before the age of 50 in one of the patients [12]. Since these criteria were too limited and stringent, the ‘‘Bethesda Guidelines’’ and ‘‘Amsterdam Criteria II’’ were developed later [13, 14]. HNPCC is characterized by CRC onset at younger age and also is associated with right-sided colon cancer. Extracolonic cancers, including cancers of the ovary, stomach, endometrium, small bowel, uroepithelial tract, ureter, renal pelvis, bile ducts and brain are also observed in this cancer [3, 7, 11]. HNPCC results from germline mutations in mismatch repair (MMR) genes. So far, ten MMR genes have been reported in the human genome including hMSH2, hMLH1, hMSH6, hPMS1, hPMS2, hMLH3, EXO1, hMSH3, hMSH4, hMSH5 [5, 9, 15, 16]. Among them, germline mutations in hMLH1 located on chromosome 3p21 and hMSH2 on 2p16 are the main molecular causes for HNPCC (in about 90% of cases with identified mutations). Germline mutations in hPMS2 on chromosome 7p22 and hMSH6 on 2p16 cause the other 10% [1, 7, 17, 18]. In this cancer, hMLH1, which is the human homologue of bacterial MutL, is the major gene. Until 2004 approximately 250 different germline mutations were detected in hMLH1 which were 50% of all HNPCC-related mutations [16, 19]. In this study, the hMLH1 gene was screened in a subset of Iranian HNPCC suspected families in Isfahan. Here we report two novel mutations in the hMLH1 gene in our studied population, both of which are expected to alter the protein product.
Materials and methods Specimens Colorectal cancer patients were collected in a 2-year period from 2007 to 2009. After genetic counseling, 20 patients that fulfilled Amsterdam criteria were selected. 5 ml of peripheral blood was obtained from each of them and was transferred to the genetic laboratory of Isfahan University of Medical Sciences for molecular studies. Genomic DNA was extracted from blood samples by DNGTMplus DNA extraction kit, produced by Cinnagen Company, Iran.
S. Shahmoradi et al.
were performed in 50 ll volumes of component mixture using the following cycling program: initial denaturation at 95°C for 5 min followed by 35 cycles of denaturation at 95°C for 30 s, annealing at specific annealing temperature for each exon for 30 s, extension at 72°C for 30 s; and a final extension at 72°C for 10 min. The PCR products were analysed using agarose gel electrophoresis and SSCP analysis. SSCP analysis and direct sequencing For each gel, 20 ml non-denaturating polyacrylamide gel (10%) was prepared containing: 5 ml acrylamide-bisacrylamide 40% (ratio of acrylamide-bisacrylamide was 38:2), 2 ml TBE (109), 13 ml distilled water, 200 ll APS (0.1%) and 20 ll TEMED. After polymerization of polyacrylamid gel for about 30 min, the gel was pre-run at 130 V for 10 min. Samples were prepared through mixing 10 ll PCR product with 3 ll loading dye, heated at 95°C for 5 min and then chilled on ice-bath and immediately loaded in wells. Electrophoresis was performed at 70 V for 8 h at room temperature. DNA bands on gel were visualized by silver staining using standard methods [21]. The PCR products, which showed an abnormal mobility on SSCP gel, were confirmed by direct sequencing (Takapou Zist Company; Tehran; Iran).
Results We analyzed PCR products of all exons of the hMLH1 gene by SSCP method to search for germline mutations in 20 Iranian HNPCC families resident in Isfahan. Seven germline mutations were identified in the hMLH1 gene of 12 samples. Of all the detected mutations, five have been previously reported; these are summarized in Table 1. The other two mutations, in samples 9Z and 14S, were novel. Figures 1a, b and 2a, b represent the SSCP gel and results of direct sequencing of samples 9Z and 14S respectively. One frameshift mutation that was in sample 9Z resulted from a deletion of ‘‘T’’ at nucleotide 108 of exon 1 and the one in sample 14S resulted from a deletion of ‘‘T’’ at nucleotide 2,259 of exon 19. These results are summarized in Table 2.
PCR amplification
Discussion
Polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) technique was applied for analyzing the hMLH1 gene. Primer sequences were taken from an article by Nicholas et al. [20]. PCR amplifications
In this study, we screened the hMLH1 gene in a group of HNPCC patients selected using Amsterdam criteria from the Isfahan region. In this studied population, we detected two novel mutations in the hMLH1 gene. The novelties of
123
Novel mutations in hMLH1 gene in HNPCC patients
15
Table 1 Detected mutations in the hMLH1 gene in HNPCC families that were reported in previous studies (InSiGHT database at www.insight-group.org) Affected patients 3
Exon 3
Altered codon 89
Nucleotide alteration
Mutation
Consequence
c.265G[T
Nonsense
p.Glu89X
3
19
714
c.2142G[A
Nonsense
p.Trp714X
2
15
565
c.1693A[T
Missense
p.Ile565Phe
1
8
217
c.649C[T
Missense
p.Arg217Cys
1
2
62
c.184C[T
Nonsense
p.Gln62X
Fig. 1 Representative of one of the gels of PCR-SSCP of the hMLH1 gene exon 1 a, and result of direct sequencing of template strand b. Arrows show the location of abnormality
Fig. 2 Representative of one of the gels of PCR-SSCP of the hMLH1 gene exon 19 a, and result of direct sequencing of template strand b. Arrows show the location of abnormality
the detected mutations are based on InSiGHT database (www.insight-group.org). These two novel mutations result from a deletion of ‘‘T’’ at codon 36, exon 1 and a deletion of ‘‘T’’ at codon 753, exon 19. Both of them change the reading frame causing alteration in the protein product. HNPCC (Lynch syndrome) is caused by germline mutations in mismatch repair genes. Many studies have been performed to screen mutations in these genes in patients in many different populations. Among 10 MMR genes which have been identified until now, hMLH1 and hMSH2 are the most frequently altered genes [5, 7, 17]. In many studies hMLH1 has been reported as the major gene. For example, in Portuguese and Brazilian populations
mutations in hMLH1 were very frequent [22, 23] and in a survey on Spanish families 64.3% of mutations were detected in hMLH1 and 35.7% in hMSH2 [24]. However, in some reports, such as a study by Irmejs et al. [25] in Latvia, more mutations were reported in the hMSH2 gene. There are also some other studies that have found more mutations in the hMLH1 gene. For example, studies on the Colombian and Korean HNPCC families demonstrated the greater number of germline mutations in hMLH1 [26, 27]. In a study in the USA, it is reported that point mutations in the hMLH1 gene are much more frequent than in other MMR genes [28]. In a recent HNPCC study that has been performed on Iranian families, the majority of the detected mutations were in the hMLH1 gene [29].
123
16
S. Shahmoradi et al.
Table 2 Hereditary non-polyposis colorectal cancer (HNPCC) novel mutations in the hMLH1 gene in HNPCC families Sample
Exon
Altered codon
Nucleotide alteration
Mutation
Consequence
9Z
1
36
c.108del T
Frameshift
Premature stop at codon 40
14S
19
753
c.2259del T
Frameshift
Alteration in coding frame and delayed stop codon
Until now, various types of mutations have been reported in the hMLH1 gene in different populations. In some studies novel mutations were reported as well as known mutations. In a study on 20 Portuguese families, Fidalgo et al. [22] detected 6 mutations in the hMLH1 gene, 4 of which were novel. In a similar study by Rossi et al. [23] on 25 Brazilian families 7 different mutations were detected in the hMLH1 gene, 5 of which were novel. In some research, such as the Long Cui et al. [30] study on Chinese CRC patients, all of the detected mutations in the hMLH1 gene were novel. Most of the reported mutations in the hMLH1 gene were nonsense, frameshift and splice-site mutations which change protein specifications. Missense mutations were also reported in many cases, but their effects in pathogenesis remain unclear and need confirmation by functional experiments. There have been a variety of reports of detected novel mutations in the hMLH1 gene in different studies. In some populations, such as Portuguese and Brazilian, most of the novel mutations were missense [22, 23]. However, most of the novel mutations that were reported by Cui et al. [30] were frameshift. In another study, Rey et al. reported 6 novel mutations in MMR genes, 2 of which were in hMLH1. Both of these mutations were frameshift, resulting in premature stop codon [31]. Similar to this, both novel mutations in our study were frameshift, which were caused from a single-base deletion. These mutations lead to abnormal proteins which result from alteration in coding frame. Determining mutation spectrum in the hMLH1 gene in each region can help to decrease mortality and morbidity of CRC. Finding novel mutations as well as known mutations is important in all populations for screening, for prevention strategies and for following up the suspected HNPCC families. Acknowledgments This work was funded by the Deputy for Research, Isfahan University of Medical Sciences. We also would like to thank Poursina Hakim Research Center for their help and support.
References 1. Abdel-Rahman WM, Mecklin JP, Peltoma¨ki P (2006) The genetics of HNPCC: application to diagnosis and screening. Crit Rev Oncol Hematol 58(3):208–220
123
2. Zlobec I, Lugli A (2008) Prognostic and predictive factors in colorectal cancer. J Clin Pathol 61(5):561–569 3. Martinez SR, Young SE, Hoedema RE et al (2006) Colorectal cancer screening and surveillance: current standards and future trends. Ann Surg Oncol 13(6):768–775 4. Rustgi AK (2007) The genetics of hereditary colon cancer. Genes Dev 21(20):2525–2538 5. Calvert PM, Frucht H (2002) The genetics of colorectal cancer. Ann Intern Med 137(7):603–612 6. Vasen HFA, Morreau H (2002) Familial and hereditary colorectal cancer with emphasis on the hereditary non-polyposis colorectal cancer syndrome. Curr Diagn Pathol 8(4):241–248 7. Kemp Z, Thirlwell C, Sieber O et al (2004) An update on the genetics of colorectal cancer. Hum Mol Genet 13(2):177–185 8. Mitchell RJ, Farrington SM, Dunlop MG et al (2002) Mismatch repair genes hMLH1 and hMSH2 and colorectal cancer: a HuGE review. Am J Epidemiol 156(10):885–902 9. Plotz G, Zeuzem S, Raedle J (2006) DNA mismatch repair and Lynch syndrome. J Mol Histol 37(5–7):271–283 10. Aarnio M, Sankila R, Pukkala E et al (1999) Cancer risk in mutation carriers of DNA-mismatch-repair genes. Int J Cancer 81(2):214–218 11. Vasen HF, Mo¨slein G, Alonso A et al (2007) Guidelines for the clinical management of Lynch syndrome (hereditary non-polyposis cancer). J Med Genet 44(6):353–362 12. Vasen HF, Mecklin JP, Khan PM et al (1991) The international collaborative group on hereditary nonpolyposis colorectal cancer (ICG-HNPCC). Dis Colon Rectum 34(5):424–425 13. Vasen HF, Watson P, Mecklin JP et al (1999) New clinical criteria for hereditary nonpolyposis colorectal cancer (HNPCC, Lynch syndrome) proposed by the International Collaborative group on HNPCC. Gastroenterology 116(6):1453–1456 14. Rodriguez-Bigas MA, Boland CR, Hamilton SR et al (1997) A National Cancer Institute Workshop on Hereditary Nonpolyposis Colorectal Cancer Syndrome: meeting highlights and Bethesda guidelines. J Natl Cancer Inst 89(23):1758–1762 15. Narayan S, Roy D (2003) Role of APC and DNA mismatch repair genes in the development of colorectal cancers. Mol Cancer 2: 41–48 16. Peltoma¨ki P (2005) Lynch syndrome genes. Fam Cancer 4(3): 227–232 17. Nicolaides NC, Papadopoulos N, Liu B et al (1994) Mutations of two PMS homologues in hereditary nonpolyposis colon cancer. Nature 371(6492):75–80 18. Zhang J, Lindroos A, Ollila S et al (2006) Gene conversion is a frequent mechanism of inactivation of the wild-type allele in cancers from MLH1/MSH2 deletion carriers. Cancer Res 66(2): 659–664 19. Peltoma¨ki P, Vasen H (2004) Mutations associated with HNPCC predisposition—update of ICG-HNPCC/INSiGHT mutation database. Dis Markers 20(4–5):269–276 20. Beck NE, Tomlinson IP, Homfray T et al (1997) Use of SSCP analysis to identify germline mutations in HNPCC families fulfilling the Amsterdam criteria. Hum Genet 99(2):219–224 21. Sanguinetti CJ, Dias Neto E, Simpson AJ (1994) Rapid silver staining and recovery of PCR products separated on polyacrylamide gels. Biotechniques 17(5):914–921
Novel mutations in hMLH1 gene in HNPCC patients 22. Fidalgo P, Almeida MR, West S et al (2000) Detection of mutations in mismatch repair genes in Portuguese families with hereditary non-polyposis colorectal cancer (HNPCC) by a multimethod approach. Eur J Hum Genet 8(1):49–53 23. Rossi BM, Lopes A, Oliveira Ferreira F et al (2002) hMLH1 and hMSH2 gene mutation in Brazilian families with suspected hereditary nonpolyposis colorectal cancer. Ann Surg Oncol 9(6):555–561 24. Caldes T, Godino J, de la Hoya M et al (2002) Prevalence of germline mutations of MLH1 and MSH2 in hereditary nonpolyposis colorectal cancer families from Spain. Int J Cancer 98(5):774–779 25. Irmejs A, Borosenko V, Melbarde-Gorkusa I et al (2007) Nationwide study of clinical and molecular features of hereditary non-polyposis colorectal cancer (HNPCC) in Latvia. Anticancer Res 27(1B):653–658 26. Giraldo A, Go´mez A, Salguero G et al (2005) MLH1 and MSH2 mutations in Colombian families with hereditary nonpolyposis colorectal cancer (Lynch syndrome)—description of four novel mutations. Fam Cancer 4(4):285–290
17 27. Shin YK, Heo SC, Shin JH et al (2004) Germline mutations in MLH1, MSH2 and MSH6 in Korean hereditary non-polyposis colorectal cancer families. Hum Mutat 24(4):351–358 28. Wagner A, Barrows A, Wijnen JT et al (2003) Molecular analysis of hereditary nonpolyposis colorectal cancer in the United States: high mutation detection rate among clinically selected families and characterization of an American founder genomic deletion of the MSH2 gene. Am J Hum Genet 72(5):1088–1100 29. Salehi M, Amani S, Javan S et al (2009) Evaluation of MLH1 and MSH2 gene mutations in a subset of Iranian families with hereditary nonpolyposis colorectal cancer (HNPCC). J Sci I R Iran 20(1):7–12 30. Cui L, Jin HY, Cheng HY et al (2004) Genetic detection of Chinese hereditary nonpolyposis colorectal cancer. World J Gastroenterol 10(2):209–213 31. Rey JM, Noruzinia M, Brouillet JP et al (2004) Six novel heterozygous MLH1, MSH2, and MSH6 and one homozygous MLH1 germline mutations in hereditary nonpolyposis colorectal cancer. Cancer Genet Cytogenet 155(2):149–151
123
