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Familial Cancer

, Volume 9, Issue 3, pp 365–376 | Cite as

Analysis of mismatch repair gene mutations in Turkish HNPCC patients

  • Berrin TuncaEmail author
  • Monica Pedroni
  • Gulsah Cecener
  • Unal Egeli
  • Enrica Borsi
  • Abdullah Zorluoglu
  • Carmela Di Gregorio
  • Tuncay Yilmazlar
  • Omer Yerci
  • Maurizio Ponz de Leon
Article

Abstract

Hereditary non-polyposis colorectal cancer (HNPCC or Lynch syndrome) is caused by the inheritance of a mutant allele of a DNA mismatch repair gene. We aimed to investigate types and frequencies of mismatch repair (MMR) gene mutations in Turkish patients with HNPCC and to identify specific biomarkers for early diagnosis of their non-symptomatic kindred’s. The molecular characteristics of 28 Turkish colorectal cancer patients at high-risk for HNPCC were investigated by analysis of microsatellite instability (MSI), immunohistochemistry and methylation-specific PCR in order to select tumors for mutation analysis. Ten cases (35.7%) were classified as MSI (+). Lack of expression of the main MMR proteins was observed in MSI (+) tumors. Hypermethylation of the MLH1 promoter region was observed in one tumor. Nine Lynch syndrome cases showed novel germ-line alterations of the MMR gene: two frame-shifts (MLH1 c.1843dupC and MLH1 c.1743delG) and three missense mutations (MLH1 c.293G>C, MLH1 c.954_955delinsTA and MSH2 c.2210G>A). Unclassified variants were evaluated as likely to be pathogenic by using the in-silico analyses. In addition, the MSH2 c.2210G>A alteration could be considered as a founder mutation for the Turkish population due to its identification in five different Lynch syndrome families and absence in control group. The present study adds new information about MMR gene mutation types and their role in Lynch syndrome. This is the first detailed research on Turkish Lynch syndrome families.

Keywords

HNPCC Lynch syndrome MMR genes IHC MSI Methylation Mutation analysis In-silico analysis of the unclassified variants 

Abbreviations

CRC

Colorectal cancer

HNPCC

Hereditary non-polyposis colorectal cancer

MMR

Mismatch repair

MSI

Microsatellite instability

IHC

Immunohistochemistry

NCI

National Cancer Institute

PCR

Polymerase chain reaction

SSCP

Single-strand conformational polymorphism

MLPA

Multiplex ligation-dependent probe amplification

HSF

Human splicing finder

ESE

Exonic splicing enhancer

ESSs

Exonic splicing silencers

ISEs

Intronic splicing enhancers

ISSs

Intronic splicing silencers

BDGP

Berkely drosophila genome project

NG2

NetGene 2

SSF

SpliceSiteFinder

ASSA

Automated splice-site analysis

MES

MaxEntScan

UMD

Universal mutation database

EIE

Exon-identity element

Notes

Acknowledgments

We would like to thank Prizma, Elips, Teknomed and Beckman Coulter Ltds for their support in supplying the experimental equipment. This manuscript was supported by the Society of Investigation and Prevention of Genetic Diseases. Programs: Human Splicing Finder (HSF): http://www.umd.be/HSF/, SIFT: http://blocks.fhcrc.org/sift/SIFT.html, SNPs3D: http://www.snps3d.org, PhD-SNP: http://gpcr.biocomp.unibo.it, A-GVGD: http://agvgd.iarc.fr, human gene mutation database: www.hgmd.org, Ensembl human genome database: http://www.ensembl.org.

References

  1. 1.
    Turkish Ministry of Health (1999) http://www.saglik.gov.tr
  2. 2.
    Lynch HT, Smyrk T (1996) Hereditary non-polyposis colorectal cancer (Lynch syndrome). An updated review. Cancer 78:1149–1167CrossRefPubMedGoogle Scholar
  3. 3.
    Lynch HT, de la Chapelle A (1999) Genetic susceptibility to non-poyposis colorectal cancer. J Med Genet 36:801–818PubMedGoogle Scholar
  4. 4.
    Vasen HF (2005) Clinical description of the Lynch Syndrome [Hereditary Nonpolyposis Colorectal Cancer (HNPCC)]. Fam Cancer 4:219–225CrossRefPubMedGoogle Scholar
  5. 5.
    Ricciardiello L, Boland CR (2005) Lynch syndrome (Hereditary Nonpolyposis Colorectal Cancer): current concepts and approaches to management. Curr Gastroenterol Rep 7:412–420CrossRefPubMedGoogle Scholar
  6. 6.
    Lynch HT, Lynch JF, Lynch PM et al (2007) Toward a consensus in molecular diagnosis of hereditary nonpolyposis colorectal cancer (Lynch syndrome). J Natl Cancer Inst 99:261–263CrossRefPubMedGoogle Scholar
  7. 7.
    Brian DH, Sue JR (2000) DNA mismatch repair and genetic instability. Annu Rev Genet 34:359–399CrossRefGoogle Scholar
  8. 8.
    Kurzawski G, Safranow K, Suchy J et al (2002) Mutation analysis of MLH1 and MSH2 genes performed by denaturing high-performance liquid chromatography. J Biochem Biophys Methods 51:89–100CrossRefPubMedGoogle Scholar
  9. 9.
    Wang Q, Lasset C, Desseigne F et al (1999) Prevalence of germline mutations of hMLH1, hMSH2, hPMS1, hPMS2 and hMSH6 genes in 75 French kindreds with nonpolyposis colorectal cancer. Hum Genet 105:79–85CrossRefPubMedGoogle Scholar
  10. 10.
    Umar A, Boland CR, Terdiman JP et al (2004) Revised Bethesda guidelines for hereditary nonpolyposis colorectal cancer (Lynch Syndrome) and microsatellite instability. J Nat Cancer Inst 96:261–268CrossRefPubMedGoogle Scholar
  11. 11.
    Yan HL, Hao LQ, Jin HY et al (2008) Clinical features and mismatch repair genes analyses of Chinese suspected hereditary non-polyposis colorectal cancer: a cost-effective screening strategy proposal. Cancer Sci 99:770–780CrossRefPubMedGoogle Scholar
  12. 12.
    Tunca B, Bekar A, Cecener G et al (2006) Impact of novel PTEN mutations in Turkish patients with glioblastoma multiforme. J Neurooncol 82:263–269CrossRefPubMedGoogle Scholar
  13. 13.
    Xicola RM, Llor X, Pons E et al (2007) Gastrointestinal oncology group of the Spanish gastroenterological association. Performance of different microsatellite marker panels for detection of mismatch repair-deficient colorectal tumors. J Natl Cancer Inst 99:244–252CrossRefPubMedGoogle Scholar
  14. 14.
    Buhard O, Cattaneo F, Wong YF et al (2006) Multipopulation analysis of polymorphisms in five mononucleotide repeats used to determine the microsatellite instability status of human tumors. J Clin Oncol 24:241–251CrossRefPubMedGoogle Scholar
  15. 15.
    Pedroni M, Roncari B, Maffei S et al (2007) A mononucleotide markers panel to identify hMLH1/hMSH2 germline mutations. Dis Markers 23:179–187PubMedGoogle Scholar
  16. 16.
    Menigatti M, Di Gregorio C, Borghi F et al (2001) Methylation pattern of different regions of the MLH1 promoter and silencing of gene expression in hereditary and sporadic colorectal cancer. Genes Chromosomes Cancer 31:357–361CrossRefPubMedGoogle Scholar
  17. 17.
    Deng G, Chen A, Hong J et al (1999) Methylation of CpG in a small region of the hMLH1 promoter in variably correlates with the absence of gene expression. Cancer Res 59:2029–2033PubMedGoogle Scholar
  18. 18.
    Losi L, Di Gregorio C, Pedroni M et al (2005) Molecular genetic alterations and clinical features in early-onset colorectal carcinomas and their role for the recognition of hereditary cancer syndromes. Am J Gastroenterol 100(10):2280–2287CrossRefPubMedGoogle Scholar
  19. 19.
    Ng PC, Henikoff S (2003) SIFT: predicting amino acid changes that affect protein function. Nucleic Acids Res 31(13):3812–3814CrossRefPubMedGoogle Scholar
  20. 20.
    Yue P, Melamud E, Moult J (2006) 1SNPs3D: candidate gene and SNP selection for association studies. BMC Bioinform 7:1–15CrossRefGoogle Scholar
  21. 21.
    Capriotti E, Calabrese R, Casadio R (2006) Predicting the insurgence of human genetic diseases associated to single point protein mutations with support vector machines and evolutionary information. Bioinformatics 22:2729–2734CrossRefPubMedGoogle Scholar
  22. 22.
    Tavtigian SV, Deffenbaugh AM, Yin L et al (2006) Comprehensive statistical study of 452 BRCA1missense substitutions with classification of eightrecurrent substitutions as neutral. J Med Genet 43:295–305CrossRefPubMedGoogle Scholar
  23. 23.
    Desmet FO, Hamroun D, Lalande M et al. (2009) Human Splicing Finder: an online bioinformatics tool to predict splicing signals. Nucleic Acids Res 1–14 doi: 10.1093/nar/gkp215
  24. 24.
    Dobrovic A, Kristensen LS (2009) DNA methylation, epimutations and cancer predisposition. Intl J Biochem Cell Biol 41:34–39CrossRefGoogle Scholar
  25. 25.
    Hitchins MP, Ward RL (2009) Constitutional (germline) MLH1 epimutation as an aetiological mechanism for hereditary non-polyposis colorectal cancer. J Med Genet. doi: 10.1136/jmg.2009.068122
  26. 26.
    Gazzoli I, Loda M, Garber J et al (2002) A hereditary nonpolyposis colorectal carcinoma case associated with hypermethylation of the MLH1 gene in normal tissue and loss of heterozygosity of the unmethylated allele in the resulting microsatellite instability-high tumor. Cancer Res 62:3925–3928PubMedGoogle Scholar
  27. 27.
    Miyakura Y, Sugano K, Akasu T et al (2004) Extensive but hemiallelic methylation of the hMLH1 promoter region in early-onset sporadic colon cancers with microsatellite instability. Clin Gastroenterol Hepatol 2:147–156CrossRefPubMedGoogle Scholar
  28. 28.
    Suter CM, Martin DI, Ward RL (2004) Germline epimutation of MLH1 in individuals with multiple cancers. Nat Genet 36:497–501CrossRefPubMedGoogle Scholar
  29. 29.
    Hitchins MP, Wong JJ, Suthers G et al (2007) Inheritance of a cancer-associated MLH1 germ-line epimutation. N Engl J Med 356:697–705CrossRefPubMedGoogle Scholar
  30. 30.
    Valle L, Carbonell P, Fernandez V (2007) MLH1 germline epimutations in selected patients with early-onset non-polyposis colorectal cancer. Clin Genet 71:232–237CrossRefPubMedGoogle Scholar
  31. 31.
    Morak M, Schackert HK, Rahner N et al (2008) Further evidence for heritability of an epimutation in one of 12 cases with MLH1 promoter methylation in blood cells clinically displaying HNPCC. Eur J Hum Genet 16:804–811CrossRefPubMedGoogle Scholar
  32. 32.
    Niessen RC, Hofstra RMW, Westers H et al (2009) Germline hypermethylation of MLH1 and EPCAM deletions are a frequent cause of Lynch syndrome. Genes Chromosomes Cancer 48:737–744CrossRefPubMedGoogle Scholar
  33. 33.
    Flicek P, Aken BL, Beal K et al (2008) Ensembl 2008. Nucleic Acids Res 36:707–714CrossRefGoogle Scholar
  34. 34.
    Wang Q, Desseigne F, Lasset C et al (1997) Germline hMSH2 and hMLH1 gene mutations in incomplete HNPCC families. Int J Cancer 73:831–836CrossRefPubMedGoogle Scholar
  35. 35.
    Güran S, Ozet A, Dede M et al (2005) Hereditary breast cancer syndromes in a Turkish population. Results of molecular germline analysis. Cancer Genet Cytogenet 160:164–168CrossRefPubMedGoogle Scholar
  36. 36.
    Chan PA, Duraisamy S, Miller PJ et al (2007) Interpreting missense variants: comparing computational methods in human disease genes CDKN2A, MLH1, MSH2, MECP2, and tyrosinase (TYR). Hum Mutat 28:683–693CrossRefPubMedGoogle Scholar
  37. 37.
    Thusberg J, Vihinen M (2009) Pathogenic or not? And ıf so, then how? Studying the effects of missense mutations using bioinformatics methods. Hum Mutat 30:703–714CrossRefPubMedGoogle Scholar
  38. 38.
    Hastings ML, Krainer AR (2001) Pre-mRNA splicing in the new millennium. Curr Opin Cell Biol 13:302–309CrossRefPubMedGoogle Scholar
  39. 39.
    Jacob M, Gallinaro H (1989) The 50 splice site: phylogenetic evolution and variable geometry of association with U1RNA. Nucleic Acids Res 17:2159–2180CrossRefPubMedGoogle Scholar
  40. 40.
    Krawczak M, Thomas NST, Hundrieser B et al (2007) Single base-pair substitutions in exon–intron junctions of human genes: nature, distribution, and consequences for mRNA splicing. Hum Mutat 28:150–158CrossRefPubMedGoogle Scholar
  41. 41.
    Faustino NA, Cooper TA (2003) Pre-mRNA splicing and human disease. Genes Dev 17:419–437CrossRefPubMedGoogle Scholar
  42. 42.
    Baralle D, Baralle M (2005) Splicing in action: assessing disease causing sequence changes. J Med Genet 42:737–748CrossRefPubMedGoogle Scholar
  43. 43.
    Blencowe BJ (2000) Exonic splicing enhancers: mechanism of action, diversity and role in human genetic diseases. Trends Biochem Sci 25:106–110CrossRefPubMedGoogle Scholar
  44. 44.
    Zhu J, Mayeda A, Krainer AR (2001) Exon identity established through differential antagonism between exonic splicing silencer-bound hnRNP A1 and enhancer-bound SR proteins. Mol Cell 8:1351–1361CrossRefPubMedGoogle Scholar
  45. 45.
    Vreeswijk MPG, Kraan JN, Klift HM et al (2009) Intronic variants in BRCA1 and BRCA2 that affect RNA splicing can be reliably selected by splice-site prediction programs. Hum Mutat 30:107–114CrossRefPubMedGoogle Scholar
  46. 46.
    Reese MG, Eeckman FH, Kulp D, Haussler D (1997) Improved splice site detection in Genie. J Comput Biol 4:311–323CrossRefPubMedGoogle Scholar
  47. 47.
    Hebsgaard SM, Korning PG, Tolstrup N et al (1996) Splice site prediction in Arabidopsis thaliana pre-mRNA by combining local and global sequence information. Nucleic Acids Res 24:3439–3452CrossRefPubMedGoogle Scholar
  48. 48.
    Shapiro MB, Senapathy P (1987) RNA splice junctions of different classes of eukaryotes: sequence statistics and functional implications in gene expression. Nucleic Acids Res 15:7155–7174CrossRefPubMedGoogle Scholar
  49. 49.
    Nalla VK, Rogan PK (2005) Automated splicing mutation analysis by information theory. Hum Mutat 25:334–342CrossRefPubMedGoogle Scholar
  50. 50.
    Yeo G, Burge CB (2004) Maximum entropy modeling of short sequence motifs with applications to RNA splicing signals. J Comput Biol 11:377–394CrossRefPubMedGoogle Scholar
  51. 51.
    Beroud C, Hamroun D, Collod-Beroud G et al (2005) UMD (Universal Mutation Database): 2005 update. Hum Mutat 26:184–191CrossRefPubMedGoogle Scholar
  52. 52.
    Fairbrother WG, Yeo GW, Yeh R et al (2004) RESCUE-ESE identifies candidate exonic splicing enhancers in vertebrate exons. Nucleic Acids Res 32:187–190CrossRefGoogle Scholar
  53. 53.
    Cartegni L, Wang J, Zhu Z (2003) ESEfinder: a web resource to identify exonic splicing enhancers. Nucleic Acids Res 31:3568–3571CrossRefPubMedGoogle Scholar
  54. 54.
    Zhang XH, Chasin LA (2004) Computational definition of sequence motifs governing constitutive exon splicing. Genes Dev 18:1241–1250CrossRefPubMedGoogle Scholar
  55. 55.
    Sironi M, Menozzi G, Riva L et al (2004) Silencer elements as possible inhibitors of pseudoexon splicing. Nucleic Acids Res 32:1783–1791CrossRefPubMedGoogle Scholar
  56. 56.
    Zhang C, Li WH, Krainer AR, Zhang MQ (2008) RNA landscape of evolution for optimal exon and intron discrimination. Proc Natl Acad Sci USA 105:5797–5802CrossRefPubMedGoogle Scholar
  57. 57.
    Salovaara R, Loukola A, Kristo P et al (2000) Population-based molecular detection of hereditary nonpolyposis colorectal cancer. J Clin Oncol 18:2193–2200PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  • Berrin Tunca
    • 1
    Email author
  • Monica Pedroni
    • 2
  • Gulsah Cecener
    • 1
  • Unal Egeli
    • 1
  • Enrica Borsi
    • 2
  • Abdullah Zorluoglu
    • 3
  • Carmela Di Gregorio
    • 4
  • Tuncay Yilmazlar
    • 5
  • Omer Yerci
    • 6
  • Maurizio Ponz de Leon
    • 2
  1. 1.Department of Medical Biology, Medical FacultyUludag UniversityBursaTurkey
  2. 2.Department of Internal Medicine, Medical FacultyModena and Reggio Emilia UniversityModenaItaly
  3. 3.Department of General SurgeryAcıbadem HospitalBursaTurkey
  4. 4.Service of PathologyHospital of CarpiCarpiItaly
  5. 5.Department of General Surgery, Medical FacultyUludag UniversityBursaTurkey
  6. 6.Department of Pathology, Medical FacultyUludag UniversityBursaTurkey

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