Classification of Genetic Variants

  • Maurizio Genuardi
  • Elke Holinski-Feder
  • Andreas Laner
  • Alexandra Martins
Chapter

Abstract

Widespread resequencing for research and diagnostic purposes has disclosed a huge amount of genetic variability in the human genome, including the genes associated with inherited predisposition to colorectal cancer. The functional and clinical consequences of the gene variants identified are often difficult to predict. Therefore, it has becoming increasingly evident that standardized approaches for the clinical interpretation of gene variants are needed in order to maximize the clinical utility of molecular testing. In this chapter, we discuss strategies for variant classification, with special reference to hereditary colorectal cancer genes and to the functional and clinical points of evidence that are available for their interpretation.

Keywords

mRNA functional studies Alternative splicing Nonsense- mediated mRNA decay In vitro protein assays In silico prediction tools Multifactorial Bayesian analysis Variants of uncertain significance (VUS) 

References

  1. 1.
    Gonzaga-Juaregui C, Lupski JR, Gibbs RA. Human genome sequencing in health and disease. Annu Rev Med. 2012;63:35–61.  https://doi.org/10.1146/annurev-med-051010-162644.CrossRefGoogle Scholar
  2. 2.
    Goldgar DE, Easton DF, Deffenbaugh AM, Monteiro AN, Tavtigian SV, Couch FJ, Breast Cancer Information Core (BIC) Steering Committee. Integrated evaluation of DNA sequence variants of unknown clinical significance: application to BRCA1 and BRCA2. Am J Hum Genet. 2004;75:535–44.CrossRefGoogle Scholar
  3. 3.
    Møller P, Seppälä T, Bernstein I, Holinski-Feder E, Sala P, Evans DG, et al. Cancer incidence and survival in lynch syndrome patients receiving colonoscopic and gynaecological surveillance: first report from the prospective lynch syndrome database. Gut. 2017;66:464–72.  https://doi.org/10.1136/gutjnl-2015-309675.CrossRefPubMedGoogle Scholar
  4. 4.
    Møller P, Seppälä T, Bernstein I, Holinski-Feder E, Sala P, Evans DG, et al. Incidence of and survival after subsequent cancers in carriers of pathogenic MMR variants with previous cancer: a report from the prospective lynch syndrome database. Gut. 2017;66:1657–64.  https://doi.org/10.1136/gutjnl-2016-311403.CrossRefPubMedGoogle Scholar
  5. 5.
    Wimmer K, Kratz CP, Vasen HF, Caron O, Colas C, Entz-Werle N, et al. Diagnostic criteria for constitutional mismatch repair deficiency syndrome: suggestions of the European consortium ‘care for CMMRD’ (C4CMMRD). J Med Genet. 2014;51:355–65.  https://doi.org/10.1136/jmedgenet-2014-102284.CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Thompson BA, Spurdle AB, Plazzer JP, Greenblatt MS, Akagi K, Al-Mulla F, et al. Application of a five-tiered scheme for standardized classification of 2,360 unique mismatch repair gene variants lodged on the InSiGHT locus-specific database. Nat Genet. 2014;46:107–15.  https://doi.org/10.1038/ng.2854.CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Richards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, et al. ACMG Laboratory Quality Assurance Committee. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med. 2015;17:405–24.CrossRefGoogle Scholar
  8. 8.
    Genome Aggregation Database (gnomAD). 2016. http://gnomad.broadinstitute.org/. Accessed 17 Mar 2018.
  9. 9.
    Stevens KN, Vachon CM, Couch FJ. Genetic susceptibility to triple-negative breast cancer. Cancer Res. 2013;73:2025–30.  https://doi.org/10.1158/0008-5472.CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Zugazagoitia J, Pérez-Segura P, Manzano A, Blanco I, Vega A, Custodio A, et al. Limited family structure and triple-negative breast cancer (TNBC) subtype as predictors of BRCA mutations in a genetic counseling cohort of early-onset sporadic breast cancers. Breast Cancer Res Treat. 2014;148:415–21.  https://doi.org/10.1007/s10549-014-3167-4.CrossRefPubMedGoogle Scholar
  11. 11.
    Lucci-Cordisco E, Risio M, Venesio T, Genuardi M. The growing complexity of the intestinal polyposis syndromes. Am J Med Genet A. 2013;161A:2777–87.  https://doi.org/10.1002/ajmg.a.36253.CrossRefPubMedGoogle Scholar
  12. 12.
    Lucci-Cordisco E, Boccuto L, Neri G, Genuardi M. The use of microsatellite instability, immunohistochemistry and other variables in determining the clinical significance of MLH1 and MSH2 unclassified variants in lynch syndrome. Cancer Biomark. 2006;2:11–27.CrossRefGoogle Scholar
  13. 13.
    Van Puijenbroek M, Nielsen M, Tops CM, Halfwerk H, Vasen HF, Weiss MM, et al. Identification of patients with (atypical) MUTYH-associated polyposis by KRAS2 c.34G > T prescreening followed by MUTYH hotspot analysis in formalin-fixed paraffin-embedded tissue. Clin Cancer Res. 2008;14:139–42.  https://doi.org/10.1158/1078-0432.CCR-07-1705.CrossRefPubMedGoogle Scholar
  14. 14.
    Weren RD, Ligtenberg MJ, Kets CM, de Voer RM, Verwiel ET, Spruijt L, et al. A germline homozygous mutation in the base-excision repair gene NTHL1 causes adenomatous polyposis and colorectal cancer. Nat Genet. 2015;47:668–71.  https://doi.org/10.1038/ng.3287.CrossRefGoogle Scholar
  15. 15.
    Briggs S, Tomlinson I. Germline and somatic polymerase ε and δ mutations define a new class of hypermutated colorectal and endometrial cancers. J Pathol. 2013;230:148–53.  https://doi.org/10.1002/path.4185.CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Jansen AM, van Wezel T, van den Akker BE, Ventayol Garcia M, Ruano D, Tops CM, et al. Combined mismatch repair and POLE/POLD1 defects explain unresolved suspected lynch syndrome cancers. Eur J Hum Genet. 2016;24:1089–92.  https://doi.org/10.1038/ejhg.2015.252.CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Howitt BE, Shukla SA, Sholl LM, Ritterhouse LL, Watkins JC, Rodig S, Stover E, Strickland KC, D’Andrea AD, Wu CJ, Matulonis UA, Konstantinopoulos PA. Association of olymerase e-mutated and microsatellite-instable endometrial cancers with neoantigen load, number of tumor-infiltrating lymphocytes, and expression of PD-1 and PD-L1. JAMA Oncol. 2015;1:1319–23.  https://doi.org/10.1001/jamaoncol.2015.2151.CrossRefPubMedGoogle Scholar
  18. 18.
    Tricarico R, Kasela M, Mareni C, Thompson BA, Drouet A, Staderini L, et al. Assessment of the InSiGHT interpretation criteria for the clinical classification of 24 MLH1 and MSH2 gene variants. Hum Mutat. 2017;38:64–77.  https://doi.org/10.1002/humu.23117.CrossRefPubMedGoogle Scholar
  19. 19.
    Baralle D, Lucassen A, Buratti E. Missed threads. The impact of pre-mRNA splicing defects on clinical practice. EMBO Rep. 2009;10:810–6.  https://doi.org/10.1038/embor.2009.170.CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Baralle D, Buratti E. RNA splicing in human disease and in the clinic. Clin Sci (Lond). 2017;131:355–68.  https://doi.org/10.1042/CS20160211.CrossRefGoogle Scholar
  21. 21.
    Cartegni L, Chew SL, Krainer AR. Listening to silence and understanding nonsense: exonic mutations that affect splicing. Nat Rev Genet. 2002;3:285–98.CrossRefGoogle Scholar
  22. 22.
    Ward AJ, Cooper TA. The pathobiology of splicing. J Pathol. 2010;220:152–63.  https://doi.org/10.1002/path.2649.CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Klamt B, Koziell A, Poulat F, Wieacker P, Scambler P, Berta P, Gessler M. Frasier syndrome is caused by defective alternative splicing of WT1 leading to an altered ratio of WT1 +/-KTS splice isoforms. Hum Mol Genet. 1998;7:709–14.CrossRefGoogle Scholar
  24. 24.
    Park SA, Ahn SI, Gallo J-M. Tau mis-splicing in the pathogenesis of neurodegenerative disorders. BMB Rep. 2016;49:405–13.CrossRefGoogle Scholar
  25. 25.
    Spurdle AB, Couch FJ, Hogervorst FBL, Radice P, Sinilnikova OM, IARC Unclassified Genetic Variants Working Group. Prediction and assessment of splicing alterations: implications for clinical testing. Hum Mutat. 2008;29:1304–13.  https://doi.org/10.1002/humu.20901.CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Houdayer C, Caux-Moncoutier V, Krieger S, Barrois M, Bonnet F, Bourdon V, et al. Guidelines for splicing analysis in molecular diagnosis derived from a set of 327 combined in silico/in vitro studies on BRCA1 and BRCA2 variants. Hum Mutat. 2012;33:1228–38.  https://doi.org/10.1002/humu.22101.CrossRefPubMedGoogle Scholar
  27. 27.
    Di Giacomo D, Gaildrat P, Abuli A, Abdat J, Frébourg T, Tosi M, Martins A. Functional analysis of a large set of BRCA2 exon 7 variants highlights the predictive value of hexamer scores in detecting alterations of exonic splicing regulatory elements. Hum Mutat. 2013;34:1547–57.  https://doi.org/10.1002/humu.22428.CrossRefPubMedGoogle Scholar
  28. 28.
    Erkelenz S, Theiss S, Otte M, Widera M, Peter JO, Schaal H. Genomic HEXploring allows landscaping of novel potential splicing regulatory elements. Nucleic Acids Res. 2014;42:10681–97.  https://doi.org/10.1093/nar/gku736.CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Xiong HY, Alipanahi B, Lee LJ, Bretschneider H, Merico D, Yuen RK, et al. RNA splicing. The human splicing code reveals new insights into the genetic determinants of disease. Science. 2015;347:1254806.  https://doi.org/10.1126/science.1254806.CrossRefPubMedGoogle Scholar
  30. 30.
    Soukarieh O, Gaildrat P, Hamieh M, Drouet A, Baert-Desurmont S, Frébourg T, et al. Exonic splicing mutations are more prevalent than currently estimated and can be predicted by using in Silico tools. PLoS Genet. 2016;12:e1005756.  https://doi.org/10.1371/journal.pgen.1005756.CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Teraoka SN, Telatar M, Becker-Catania S, Liang T, Onengüt S, Tolun A, et al. Splicing defects in the ataxia-telangiectasia gene, ATM: underlying mutations and consequences. Am J Hum Genet. 1999;64:1617–31.CrossRefGoogle Scholar
  32. 32.
    Ars E, Serra E, García J, Kruyer H, Gaona A, Lázaro C, Estivill X. Mutations affecting mRNA splicing are the most common molecular defects in patients with neurofibromatosis type 1. Hum Mol Genet. 2000;9:237–47.CrossRefGoogle Scholar
  33. 33.
    Lim KH, Ferraris L, Filloux ME, Raphael BJ, Fairbrother WG. Using positional distribution to identify splicing elements and predict pre-mRNA processing defects in human genes. Proc Natl Acad Sci U S A. 2011;108:11093–8.  https://doi.org/10.1073/pnas.1101135108.CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Aretz S, Uhlhaas S, Sun Y, Pagenstecher C, Mangold E, Caspari R, et al. Familial adenomatous polyposis: aberrant splicing due to missense or silent mutations in the APC gene. Hum Mutat. 2004;24:370–80.CrossRefGoogle Scholar
  35. 35.
    Auclair J, Busine MP, Navarro C, Ruano E, Montmaigne G, Desseigne F, et al. Systematic mRNA analysis for the effect of MLH1 and MSH2 missense and silent mutations on aberrant splicing. Hum Mutat. 2006;27:145–54.CrossRefGoogle Scholar
  36. 36.
    Adam R, Spier I, Zhao B, Kloth M, Marquez J, Hinrichsen I, et al. Exome sequencing identifies Biallelic MSH3 Germline mutations as a recessive subtype of colorectal adenomatous polyposis. Am J Hum Genet. 2016;99:337–51.  https://doi.org/10.1016/j.ajhg.2016.06.015.CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Plaschke J, Linnebacher M, Kloor M, Gebert J, Cremer FW, Tinschert S, et al. Compound heterozygosity for two MSH6 mutations in a patient with early onset of HNPCC-associated cancers, but without hematological malignancy and brain tumor. Eur J Hum Genet. 2006;14:561–6.CrossRefGoogle Scholar
  38. 38.
    Dallosso AR, Dolwani S, Jones N, Jones S, Colley J, Maynard J, et al. Inherited predisposition to colorectal adenomas caused by multiple rare alleles of MUTYH but not OGG1, NUDT1, NTH1 or NEIL 1, 2 or 3. Gut. 2008;57:1252–5.  https://doi.org/10.1136/gut.2007.145748.CrossRefGoogle Scholar
  39. 39.
    Pin E, Pastrello C, Tricarico R, Papi L, Quaia M, Fornasarig M, et al. MUTYH c.933+3A>C, associated with a severely impaired gene expression, is the first Italian founder mutation in MUTYH-associated polyposis. Int J Cancer. 2013;132:1060–9.  https://doi.org/10.1002/ijc.27761.CrossRefPubMedGoogle Scholar
  40. 40.
    Borràs E, Pineda M, Cadiñanos J, Del Valle J, Brieger A, Hinrichsen I, et al. Refining the role of PMS2 in lynch syndrome: germline mutational analysis improved by comprehensive assessment of variants. J Med Genet. 2013;50:552–63.  https://doi.org/10.1136/jmedgenet-2012-101511.CrossRefPubMedGoogle Scholar
  41. 41.
    Pachlopnik Schmid J, Lemoine R, Nehme N, Cormier-Daire V, Revy P, Debeurme F, et al. Polymerase ε1 mutation in a human syndrome with facial dysmorphism, immunodeficiency, livedo, and short stature (“FILS syndrome”). J Exp Med. 2013;209:2323–30.  https://doi.org/10.1084/jem.20121303.CrossRefGoogle Scholar
  42. 42.
    Agrawal S, Pilarski R, Eng C. Different splicing defects lead to differential effects downstream of the lipid and protein phosphatase activities of PTEN. Hum Mol Genet. 2005;14:2459–68.CrossRefGoogle Scholar
  43. 43.
    Suphapeetiporn K, Kongkam P, Tantivatana J, Sinthuwiwat T, Tongkobpetch S, Shotelersuk V. PTEN c.511C>T nonsense mutation in a BRRS family disrupts a potential exonic splicing enhancer and causes exon skipping. Jpn J Clin Oncol. 2006;36:814–21.CrossRefGoogle Scholar
  44. 44.
    Papp J, Kovacs ME, Solyom S, Kasler M, Børresen-Dale A-L, Olah E. High prevalence of germline STK11 mutations in Hungarian Peutz-Jeghers syndrome patients. BMC Med Genet. 2010;11:169.  https://doi.org/10.1186/1471-2350-11-169.CrossRefPubMedPubMedCentralGoogle Scholar
  45. 45.
    van der Klift HM, Jansen AML, van der Steenstraten N, Bik EC, Tops CMJ, Devilee P, Wijnen JT. Splicing analysis for exonic and intronic mismatch repair gene variants associated with lynch syndrome confirms high concordance between minigene assays and patient RNA analyses. Mol Genet Genomic Med. 2015;3:327–45.  https://doi.org/10.1002/mgg3.145.CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    Trojan J, Plotz G, Brieger A, Raedle J, Meltzer SJ, Wolter M, Zeuzem S. Activation of a cryptic splice site of PTEN and loss of heterozygosity in benign skin lesions in Cowden disease. J Invest Dermatol. 2001;117:1650–3.CrossRefGoogle Scholar
  47. 47.
    Reifenberger J, Rauch L, Beckmann MW, Megahed M, Ruzicka T, Reifenberger G. Cowden’s disease: clinical and molecular genetic findings in a patient with a novel PTEN germline mutation. Br J Dermatol. 2003;148:1040–6.CrossRefGoogle Scholar
  48. 48.
    Kaufmann A, Vogt S, Uhlhaas S, Stienen D, Kurth I, Hameister H, et al. Analysis of rare APC variants at the mRNA level: six pathogenic mutations and literature review. J Mol Diagn. 2009;11:131–9.  https://doi.org/10.2353/jmoldx.2009.080129.CrossRefPubMedPubMedCentralGoogle Scholar
  49. 49.
    Sharp A, Pichert G, Lucassen A, Eccles D. RNA analysis reveals splicing mutations and loss of expression defects in MLH1 and BRCA1. Hum Mutat. 2004;24:272.CrossRefGoogle Scholar
  50. 50.
    Yan H, Papadopoulos N, Marra G, Perrera C, Jiricny J, Boland CR, et al. Conversion of diploidy to haploidy. Nature. 2000;403:723–4.CrossRefGoogle Scholar
  51. 51.
    Thompson BA, Goldgar DE, Paterson C, Clendenning M, Walters R, Arnold S, et al. A multifactorial likelihood model for MMR gene variant classification incorporating probabilities based on sequence bioinformatics and tumor characteristics: a report from the Colon Cancer family registry. Hum Mutat. 2013;34:200–9.  https://doi.org/10.1002/humu.22213.CrossRefPubMedGoogle Scholar
  52. 52.
    Celebi JT, Wanner M, Ping XL, Zhang H, Peacocke M. Association of splicing defects in PTEN leading to exon skipping or partial intron retention in Cowden syndrome. Hum Genet. 2000;107:234–8.CrossRefGoogle Scholar
  53. 53.
    Tao H, Shinmura K, Hanaoka T, Natsukawa S, Shaura K, Koizumi Y, et al. A novel splice-site variant of the base excision repair gene MYH is associated with production of an aberrant mRNA transcript encoding a truncated MYH protein not localized in the nucleus. Carcinogenesis. 2004;25:1859–66.CrossRefGoogle Scholar
  54. 54.
    Abed AA, Günther K, Kraus C, Hohenberger W, Ballhausen WG. Mutation screening at the RNA level of the STK11/LKB1 gene in Peutz-Jeghers syndrome reveals complex splicing abnormalities and a novel mRNA isoform (STK11 c.597(insertion mark)598insIVS4). Hum Mutat. 2001;18:397–410.CrossRefGoogle Scholar
  55. 55.
    Spier I, Horpaopan S, Vogt S, Ulhaas S, Morak M, Stienen D, et al. Deep intronic APC mutations explain a substantial proportion of patients with familial or early-onset adenomatous polyposis. Hum Mutat. 2012;33:1045–50.  https://doi.org/10.1002/humu.22082.CrossRefPubMedPubMedCentralGoogle Scholar
  56. 56.
    Nieminen TT, Pavicic W, Porkka N, Kankainen M, Järvinen HJ, Lepistö A, Peltomäki P. Pseudoexons provide a mechanism for allele-specific expression of APC in familial adenomatous polyposis. Oncotarget. 2016;7:70685–98.  https://doi.org/10.18632/oncotarget.12206.CrossRefPubMedPubMedCentralGoogle Scholar
  57. 57.
    Clendenning M, Buchanan DD, Walsh MD, Nagler B, Rosty C, Thompson B, et al. Mutation deep within an intron of MSH2 causes lynch syndrome. Familial Cancer. 2011;10:297–301.  https://doi.org/10.1007/s10689-011-9427-0.CrossRefPubMedPubMedCentralGoogle Scholar
  58. 58.
    Sjursen W, Bjørnevoll I, Engebretsen LF, Fjelland K, Halvorsen T, Myrvold HE. A homozygote splice site PMS2 mutation as cause of Turcot syndrome gives rise to two different abnormal transcripts. Familial Cancer. 2009;8:179–86.  https://doi.org/10.1007/s10689-008-9225-5.CrossRefPubMedGoogle Scholar
  59. 59.
    Liu Q, Thompson BA, Ward RL, Hesson LB, Sloane MA. Understanding the pathogenicity of noncoding mismatch gepair gene promoter variants in lynch syndrome. Hum Mutat. 2016;37:417–26.  https://doi.org/10.1002/humu.22971.CrossRefGoogle Scholar
  60. 60.
    Yan H, Jin H, Xue G, Mei Q, Ding F, Hao L, Sun SH. Germline hMSH2 promoter mutation in a Chinese HNPCC kindred: evidence for dual role of LOH. Clin Genet. 2007;72:556–61.CrossRefGoogle Scholar
  61. 61.
    Kadiyska TK, Todorov TP, Bichev SN, Vazharova RV, Nossikoff AV, Savov AS, et al. APC promoter 1B deletion in familial polyposis--implications for mutation-negative families. Clin Genet. 2014;85:452–7.  https://doi.org/10.1111/cge.12210.CrossRefPubMedGoogle Scholar
  62. 62.
    Lin Y, Lin S, Baxter MD, Lin L, Kennedy SM, Zhang Z, et al. Novel APC promoter and exon 1B deletion and allelic silencing in three mutation-negative classic familial adenomatous polyposis families. Genome Med. 2015;7:42.  https://doi.org/10.1186/s13073-015-0148.CrossRefPubMedPubMedCentralGoogle Scholar
  63. 63.
    Tutlewska K, Lubinski J, Kurzawski G. Germline deletions in the EPCAM gene as a cause of lynch syndrome - literature review. Hered Cancer Clin Pract. 2013;11:9.  https://doi.org/10.1186/1897-4287-11-9.CrossRefPubMedPubMedCentralGoogle Scholar
  64. 64.
    Diederichs S, Bartsch L, Berkmann JC, Fröse K, Heitmann J, Hoppe C, et al. The dark matter of the cancer genome: aberrations in regulatory elements, untranslated regions, splice sites, non-coding RNA and synonymous mutations. EMBO Mol Med. 2016;8:442–57.  https://doi.org/10.15252/emmm.201506055.CrossRefPubMedPubMedCentralGoogle Scholar
  65. 65.
  66. 66.
    Thompson BA, Martins A, Spurdle AB. A review of mismatch repair gene transcripts: issues for interpretation of mRNA splicing assays. Clin Genet. 2015;87:100–8.  https://doi.org/10.1111/cge.12450.CrossRefPubMedGoogle Scholar
  67. 67.
    Tournier I, Vezain M, Martins A, Charbonnier F, Baert-Desurmont S, Olschwang S, et al. A large fraction of unclassified variants of the mismatch repair genes MLH1 and MSH2 is associated with splicing defects. Hum Mutat. 2008;29:1412–24.  https://doi.org/10.1002/humu.20796.CrossRefPubMedGoogle Scholar
  68. 68.
    Conesa A, Madrigal P, Tarazona S, Gomez-Cabrero D, Cervera A, McPherson A, et al. A survey of best practices for RNA-seq data analysis. Genome Biol. 2016;17:13.  https://doi.org/10.1186/s13059-016-0881-8.CrossRefPubMedPubMedCentralGoogle Scholar
  69. 69.
    Saliba A-E, Westermann AJ, Gorski SA, Vogel J. Single-cell RNA-seq: advances and future challenges. Nucleic Acids Res. 2014;42:8845–60.  https://doi.org/10.1093/nar/gku555.CrossRefPubMedPubMedCentralGoogle Scholar
  70. 70.
    Liu Q, Hesson LB, Nunez AC, Packham D, Williams R, Ward RL, Sloane MA. A cryptic paracentric inversion of MSH2 exons 2-6 causes lynch syndrome. Carcinogenesis. 2016;37:10–7.  https://doi.org/10.1093/carcin/bgv154.CrossRefGoogle Scholar
  71. 71.
    De Lellis L, Aceto GM, Curia MC, et al. Integrative analysis of hereditary nonpolyposis colorectal cancer: the contribution of allele-specific expression and other assays to diagnostic algorithms. PLoS One. 2013;8:e81194.  https://doi.org/10.1371/journal.pone.0081194.CrossRefPubMedPubMedCentralGoogle Scholar
  72. 72.
    Hesson LB, Packham D, Kwok C-T, Nunez AC, Ng B, Schmidt C, et al. Lynch syndrome associated with two MLH1 promoter variants and allelic imbalance of MLH1 expression. Hum Mutat. 2015;36:622–30.  https://doi.org/10.1002/humu.22785.CrossRefPubMedPubMedCentralGoogle Scholar
  73. 73.
    Lastella P, Surdo NC, Resta N, Guanti G, Stella A. In silico and in vivo splicing analysis of MLH1 and MSH2 missense mutations shows exon- and tissue-specific effects. BMC Genomics. 2006;7:243.CrossRefGoogle Scholar
  74. 74.
    Kwok C-T, Ward RL, Hawkins NJ, Hitchins MP. Detection of allelic imbalance in MLH1 expression by pyrosequencing serves as a tool for the identification of germline defects in lynch syndrome. Familial Cancer. 2010;9:345–56.  https://doi.org/10.1007/s10689-009-9314-0.CrossRefPubMedGoogle Scholar
  75. 75.
    Santibanez Koref M, Wilson V, Cartwright N, Cunnington MS, Mathers JC, et al. MLH1 differential allelic expression in mutation carriers and controls. Ann Hum Genet. 2010;74:479–88.  https://doi.org/10.1111/j.1469-1809.2010.00603.x.CrossRefPubMedGoogle Scholar
  76. 76.
    Etzler J, Peyrl A, Zatkova A, Schildhaus H-U, Ficek A, Merkelbach-Bruse S, et al. RNA-based mutation analysis identifies an unusual MSH6 splicing defect and circumvents PMS2 pseudogene interference. Hum Mutat. 2008;29:299–305.CrossRefGoogle Scholar
  77. 77.
    van der Klift HM, Mensenkamp AR, Drost M, Bik EC, Vos YJ, et al. Comprehensive mutation analysis of PMS2 in a large cohort of probands suspected of lynch syndrome or constitutional mismatch repair deficiency syndrome. Hum Mutat. 2016;37:1162–79.  https://doi.org/10.1002/humu.23052.CrossRefGoogle Scholar
  78. 78.
    Nasif S, Contu L, Mühlemann O. Beyond quality control: the role of nonsense-mediated mRNA decay (NMD) in regulating gene expression. Semin Cell Dev Biol. 2018;75:78–87.  https://doi.org/10.1016/j.semcdb.2017.08.053.CrossRefPubMedGoogle Scholar
  79. 79.
    Bonnet C, Krieger S, Vezain M, Rousselin A, Tournier I, Martins A, et al. Screening BRCA1 and BRCA2 unclassified variants for splicing mutations using reverse transcription PCR on patient RNA and an ex vivo assay based on a splicing reporter minigene. J Med Genet. 2008;45:438–46.  https://doi.org/10.1136/jmg.2007.056895.CrossRefPubMedGoogle Scholar
  80. 80.
    Ward RL, Dobbins T, Lindor NM, Rapkins RW, Hitchins MP. Identification of constitutional MLH1 epimutations and promoter variants in colorectal cancer patients from the Colon Cancer family registry. Genet Med. 2013;15:25–35.  https://doi.org/10.1038/gim.2012.91.CrossRefPubMedGoogle Scholar
  81. 81.
    Waidmann MS, Bleichrodt FS, Laslo T, Riedel CU. Bacterial luciferase reporters: the Swiss army knife of molecular biology. Bioeng Bugs. 2011;2:8–16.  https://doi.org/10.4161/bbug.2.1.13566.CrossRefPubMedGoogle Scholar
  82. 82.
    Gaildrat P, Krieger S, Théry JC, Killian A, Rousselin A, Berthet P, et al. The BRCA1 c.5434C->G (p.Pro1812Ala) variant induces a deleterious exon 23 skipping by affecting exonic splicing regulatory elements. J Med Genet. 2010;47:398–403.  https://doi.org/10.1136/jmg.2009.074047.CrossRefPubMedGoogle Scholar
  83. 83.
    Shimodaira H, Filosi N, Shibata H, Suzuki T, Radice P, Kanamaru R, et al. Functional analysis of human MLH1 mutations in Saccharomyces cerevisiae. Nat Genet. 1998;19:384–9.CrossRefGoogle Scholar
  84. 84.
    Nyström-Lahti M, Perrera C, Räschle M, Panyushkina-Seiler E, Marra G, Curci A, et al. Functional analysis of MLH1 mutations linked to hereditary nonpolyposis colon cancer. Genes Chromosomes Cancer. 2002;33:160–7.CrossRefGoogle Scholar
  85. 85.
    Trojan J, Zeuzem S, Randolph A, Hemmerle C, Brieger A, Raedle J, Plotz G, Jiricny J, Marra G. Functional analysis of hMLH1 variants and HNPCC-related mutations using a human expression system. Gastroenterology. 2002;122:211–9.CrossRefGoogle Scholar
  86. 86.
    Kondo E, Suzuki H, Horii A, Fukushige S. A yeast two-hybrid assay provides a simple way to evaluate the vast majority of hMLH1 germ-line mutations. Cancer Res. 2003;63:3302–8.PubMedGoogle Scholar
  87. 87.
    Plotz G, Welsch C, Giron-Monzon L, Friedhoff P, Albrecht M, Piiper A, et al. Mutations in the MutSalpha interaction interface of MLH1 can abolish DNA mismatch repair. Nucleic Acids Res. 2006;34:6574–86.CrossRefGoogle Scholar
  88. 88.
    Ollila S, Dermadi Bebek D, Jiricny J, Nystrom M. Mechanisms of pathogenicity in human MSH2 missense mutants. Hum Mutat. 2008;29:1355–63.  https://doi.org/10.1002/humu.20893.CrossRefPubMedGoogle Scholar
  89. 89.
    Drost M, Zonneveld JB, van Hees S, Rasmussen LJ, Hofstra RM, de Wind N. A rapid and cell-free assay to test the activity of lynch syndrome-associated MSH2 and MSH6 missense variants. Hum Mutat. 2012;33:488–94.  https://doi.org/10.1002/humu.22000.CrossRefPubMedGoogle Scholar
  90. 90.
    Wielders EA, Dekker RJ, Holt I, Morris GE, te Riele H. Characterization of MSH2 variants by endogenous gene modification in mouse embryonic stem cells. Hum Mutat. 2011;32:389–96.  https://doi.org/10.1002/humu.21448.CrossRefPubMedGoogle Scholar
  91. 91.
    Marra G, D’Atri S, Yan H, Perrera C, Cannavo E, Vogelstein B, Jiricny J. Phenotypic analysis of hMSH2 mutations in mouse cells carrying human chromosomes. Cancer Res. 2001;61:7719–21.Google Scholar
  92. 92.
    Blasi MF, Ventura I, Aquilina G, Degan P, Bertario L, Bassi C, et al. A human cell-based assay to evaluate the effects of alterations in the MLH1 mismatch repair gene. Cancer Res. 2006;66:9036–44.CrossRefGoogle Scholar
  93. 93.
    Mastrocola AS, Heinen CD. Lynch syndrome-associated mutations in MSH2 alter DNA repair and checkpoint response functions in vivo. Hum Mutat. 2010;31:E1699–708.  https://doi.org/10.1002/humu.21333.CrossRefPubMedPubMedCentralGoogle Scholar
  94. 94.
    Takahashi M, Shimodaira H, Andreutti-Zaugg C, Iggo R, Kolodner RD, Ishioka C. Functional analysis of human MLH1 variants using yeast and in vitro mismatch repair assays. Cancer Res. 2007;67:4595–604.CrossRefGoogle Scholar
  95. 95.
    Belvederesi L, Bianchi F, Galizia E, Loretelli C, Bracci R, Catalani R, Amati M, Cellerino R. MSH2 missense mutations and HNPCC syndrome: pathogenicity assessment in a human expression system. Hum Mutat. 2008;29:E296–309.  https://doi.org/10.1002/humu.2087.CrossRefPubMedGoogle Scholar
  96. 96.
    Perera S, Bapat B. The MLH1 variants p.Arg265Cys and p.Lys618Ala affect protein stability while p.Leu749Gln affects heterodimer formation. Hum Mutat. 2008;29:332.  https://doi.org/10.1002/humu.9523.CrossRefPubMedGoogle Scholar
  97. 97.
    Borràs E, Pineda M, Blanco I, Jewett EM, Wang F, Teulé A, et al. MLH1 founder mutations with moderate penetrance in Spanish lynch syndrome families. Cancer Res. 2010;70:7379–91.  https://doi.org/10.1158/0008-5472.CrossRefPubMedGoogle Scholar
  98. 98.
    Hardt K, Heick SB, Betz B, Goecke T, Yazdanparast H, Küppers R, et al. Missense variants in hMLH1 identified in patients from the German HNPCC consortium and functional studies. Familial Cancer. 2011;10:273–84.  https://doi.org/10.1007/s10689-011-9431-4.CrossRefPubMedGoogle Scholar
  99. 99.
    Hinrichsen I, Brieger A, Trojan J, Zeuzem S, Nilbert M, Plotz G. Expression defect size among unclassified MLH1 variants determines pathogenicity in lynch syndrome diagnosis. Clin Cancer Res. 2013;19:2432–41.  https://doi.org/10.1158/1078-0432.CCR-12-3299.CrossRefPubMedGoogle Scholar
  100. 100.
    Borràs E, Pineda M, Brieger A, Hinrichsen I, Gómez C, Navarro M, et al. Comprehensive functional assessment of MLH1 variants of unknown significance. Hum Mutat. 2012;33:1576–88.  https://doi.org/10.1002/humu.22142.CrossRefPubMedGoogle Scholar
  101. 101.
    Raevaara TE, Korhonen MK, Lohi H, Hampel H, Lynch E, Lönnqvist KE, et al. Functional significance and clinical phenotype of nontruncating mismatch repair variants of MLH1. Gastroenterology. 2005;129:537–49.PubMedGoogle Scholar
  102. 102.
    Gassman NR, Clodfelter JE, McCauley AK, Bonin K, Salsbury FR Jr, Scarpinato KD. Cooperative nuclear localization sequences lend a novel role to the N-terminal region of MSH6. PLoS One. 2011;6:e17907.  https://doi.org/10.1371/journal.pone.0017907.CrossRefPubMedPubMedCentralGoogle Scholar
  103. 103.
    Belvederesi L, Bianchi F, Loretelli C, Bracci R, Cascinu S, Cellerino R. Sub-cellular localization analysis of MSH6 missense mutations does not reveal an overt MSH6 nuclear transport impairment. Familial Cancer. 2012;11:675–80.  https://doi.org/10.1007/s10689-012-9558-y.CrossRefPubMedGoogle Scholar
  104. 104.
    Guerrette S, Wilson T, Gradia S, Fishel R. Interactions of human hMSH2 with hMSH3 and hMSH2 with hMSH6: examination of mutations found in hereditary nonpolyposis colorectal cancer. Mol Cell Biol. 1998;18:6616–23.CrossRefGoogle Scholar
  105. 105.
    Guerrette S, Acharya S, Fishel R. The interaction of the human MutL homologues in hereditary nonpolyposis colon cancer. J Biol Chem. 1999;274:6336–41.CrossRefGoogle Scholar
  106. 106.
    Yuan ZQ, Gottlieb B, Beitel LK, Wong N, Gordon PH, Wang Q, et al. Polymorphisms and HNPCC: PMS2-MLH1 protein interactions diminished by single nucleotide polymorphisms. Hum Mutat. 2002;19:108–13.CrossRefGoogle Scholar
  107. 107.
    Clark AB, Cook ME, Tran HT, Gordenin DA, Resnick MA, Kunkel TA. Functional analysis of human MutS alpha and MutS beta complexes in yeast. Nucleic Acids Res. 1999;27:736–42.CrossRefGoogle Scholar
  108. 108.
    Drotschmann K, Clark AB, Tran HT, Resnick MA, Gordenin DA, Kunkel TA. Mutator phenotypes of yeast strains heterozygous for mutations in the MSH2 gene. Proc Natl Acad Sci U S A. 1999;96:2970–5.CrossRefGoogle Scholar
  109. 109.
    Iaccarino I, Marra G, Dufner P, Jiricny J. Mutation in the magnesium binding site of hMSH6 disables the hMutSalpha sliding clamp from translocating along DNA. J Biol Chem. 2000;275:2080–6.CrossRefGoogle Scholar
  110. 110.
    Heinen CD, Wilson T, Mazurek A, Berardini M, Butz C, Fishel R. HNPCC mutations in hMSH2 result in reduced hMSH2-hMSH6 molecular switch functions. Cancer Cell. 2002;1:469–78.CrossRefGoogle Scholar
  111. 111.
    Molatore S, Russo MT, D’Agostino VG, Barone F, Matsumoto Y, Albertini AM, et al. MUTYH mutations associated with familial adenomatous polyposis: functional characterization by a mammalian cell-based assay. Hum Mutat. 2010;31:159–66.  https://doi.org/10.1002/humu.21158.CrossRefPubMedGoogle Scholar
  112. 112.
    Komine K, Shimodaira H, Takao M, Soeda H, Zhang X, Takahashi M, Ishioka C. Functional complementation assay for 47 MUTYH variants in a MutY-disrupted Escherichia coli strain. Hum Mutat. 2015;36:704–11.  https://doi.org/10.1002/humu.22794.CrossRefPubMedPubMedCentralGoogle Scholar
  113. 113.
    Shinmura K, Kato H, Goto M, Yamada H, Tao H, Nakamura S, Sugimura H. Functional evaluation of nine missense-type variants of the human DNA Glycosylase enzyme MUTYH in the Japanese population. Hum Mutat. 2016;37:350–3.  https://doi.org/10.1002/humu.22949104.CrossRefGoogle Scholar
  114. 114.
    Ali M, Kim H, Cleary S, Cupples C, Gallinger S, Bristow R. Characterization of mutant MUTYH proteins associated with familial colorectal cancer. Gastroenterology. 2008;135:499–507.  https://doi.org/10.1053/j.gastro.2008.04.035.CrossRefPubMedPubMedCentralGoogle Scholar
  115. 115.
    D’Agostino VG, Minoprio A, Torreri P, Marioni I, Bossa C, Petrucci TC, et al. Functional analysis of MUTYH mutated proteins associated with familial adenomatous polyposis. DNA Repair. 2010;9:700–7.  https://doi.org/10.1016/j.dnarep.2010.03.008.CrossRefPubMedGoogle Scholar
  116. 116.
    Ou J, Niessen RC, Lützen A, Sijmons RH, Kleibeuker JH, de Wind N, et al. Functional analysis helps to clarify the clinical importance of unclassified variants in DNA mismatch repair genes. Hum Mutat. 2007;28:1047–54.CrossRefGoogle Scholar
  117. 117.
    Rasmussen LJ, Heinen CD, Royer-Pokora B, Drost M, Tavtigian S, Hofstra RM, de Wind N. Pathological assessment of mismatch repair gene variants in lynch syndrome: past, present, and future. Hum Mutat. 2012;33:1617–25.  https://doi.org/10.1002/humu.22168.CrossRefPubMedGoogle Scholar
  118. 118.
    Thusberg J, Olatubosun A, Vihinen M. Performance of mutation pathogenicity prediction methods on missense variants. Hum Mutat. 2011;32:358–68.  https://doi.org/10.1002/humu.21445.CrossRefPubMedGoogle Scholar
  119. 119.
    Choi Y, Sims GE, Murphy S, Miller JR, Chan AP. Predicting the functional effect of amino acid substitutions and indels. PLoS One. 2012;7:e46688.  https://doi.org/10.1371/journal.pone.0046688.CrossRefPubMedPubMedCentralGoogle Scholar
  120. 120.
    Chan PA, Duraisamy S, Miller PJ, Newell JA, McBride C, Bond JP, et al. Interpreting missense variants: comparing computational methods in human disease genes CDKN2A, MLH1, MSH2, MECP2, and tyrosinase (TYR). Hum Mutat. 2007;28:683–93.CrossRefGoogle Scholar
  121. 121.
    Stone EA, Sidow A. Physicochemical constraint violation by missense substitutions mediates impairment of protein function and disease severity. Genome Res. 2005;15:978–86.CrossRefGoogle Scholar
  122. 122.
    Adzhubei I, Jordan DM, Sunyaev SR. Predicting functional effect of human missense mutations using PolyPhen-2. Curr Protoc Hum Genet. 2013.  https://doi.org/10.1002/0471142905.hg0720s76.
  123. 123.
    Plon SE, Eccles DM, Easton D, Foulkes WD, Genuardi M, Greenblatt MS, et al. Sequence variant classification and reporting: recommendations for improving the interpretation of cancer susceptibility genetic test results. Hum Mutat. 2008;29:1282–91.  https://doi.org/10.1002/humu.20880.CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer International Publishing AG, part of Springer Nature 2018

Authors and Affiliations

  • Maurizio Genuardi
    • 1
    • 2
  • Elke Holinski-Feder
    • 3
    • 4
  • Andreas Laner
    • 3
  • Alexandra Martins
    • 5
  1. 1.Institute of Genomic Medicine, Catholic University of the Sacred HeartRomeItaly
  2. 2.Fondazione Policlinico Universitario “A. Gemelli”RomeItaly
  3. 3.Medizinische Klinik und Poliklinik IV, Campus Innenstadt, Klinikum der Universität MünchenMunichGermany
  4. 4.MGZ – Medizinisch Genetisches ZentrumMunichGermany
  5. 5.Inserm-U1245-IRIB, Normandy Centre for Genomic and Personalized Medicine, University of RouenRouenFrance

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