BACKGROUND: Autosomal recessive limb-girdle muscular dystrophies (LGMD2) include a number of disorders with heterogeneous etiology that cause predominantly weakness and wasting of the shoulder and pelvic girdle muscles. In this study, we determined the frequency of LGMD subtypes within a cohort of Czech LGMD2 patients using mutational analysis of the CAPN3, FKRP, SGCA, and ANO5 genes. METHODS: PCR-sequencing analysis; sequence capture and targeted resequencing. RESULTS: Mutations of the CAPN3 gene are the most common cause of LGMD2, and mutations in this gene were identified in 71 patients in a set of 218 Czech probands with a suspicion of LGMD2. Totally, we detected 37 different mutations of which 12 have been described only in Czech LGMD2A patients. The mutation c.550delA is the most frequent among our LGMD2A probands and was detected in 47.1% of CAPN3 mutant alleles. The frequency of particular forms of LGMD2 was 32.6% for LGMD2A (71 probands), 4.1% for LGMD2I (9 probands), 2.8% for LGMD2D (6 probands), and 1.4% for LGMD2L (3 probands).Further, we present the first results of a new approach established in the Czech Republic for diagnosis of neuromuscular diseases: sequence capture and targeted resequencing. Using this approach, we identified patients with mutations in the DYSF and SGCB genes. CONCLUSIONS: We characterised a cohort of Czech LGMD2 patients on the basis of mutation analysis of genes associated with the most common forms of LGMD2 in the European population and subsequently compared the occurrence of particular forms of LGMD2 among countries on the basis of our results and published studies.

Centre of Molecular Biology and Gene Therapy University Hospital Brno Černopolní 9 Brno 613 00 Czech Republic

Zobrazit více v PubMed

Kang PB, Feener CA, Estrella E, Thorne M, White AJ, Darras BT, Amato AA, Kunkel LM. LGMD2I in a North American population. BMC Musculoskelet Disord. 2007;8:115. doi: 10.1186/1471-2474-8-115. PubMed DOI PMC

Guglieri M, Magri F, D'Angelo MG, Prelle A, Morandi L, Rodolico C, Cagliani R, Mora M, Fortunato F, Bordoni A, Del Bo R, Ghezzi S, Pagliarani S, Lucchiari S, Salani S, Zecca C, Lamperti C, Ronchi D, Aguennouz M, Ciscato P, Di Blasi C, Ruggieri A, Moroni I, Turconi A, Toscano A, Moggio M, Bresolin N, Comi GP. Clinical, molecular, and protein correlations in a large sample of genetically diagnosed Italian limb girdle muscular dystrophy patients. Hum Mutat. 2008;29(2):258–266. doi: 10.1002/humu.20642. PubMed DOI

Lo HP, Cooper ST, Evesson FJ, Seto JT, Chiotis M, Tay V, Compton AG, Cairns AG, Corbett A, MacArthur DG, Yang N, Reardon K, North KN. Limb-girdle muscular dystrophy: diagnostic evaluation, frequency and clues to pathogenesis. Neuromuscul Disord. 2008;18(1):34–44. doi: 10.1016/j.nmd.2007.08.009. PubMed DOI

van der Kooi AJ, Frankhuizen WS, Barth PG, Howeler CJ, Padberg GW, Spaans F, Wintzen AR, Wokke JH, van Ommen GJ, de Visser M, Bakker E, Ginjaar HB. Limb-girdle muscular dystrophy in the Netherlands: gene defect identified in half the families. Neurology. 2007;68(24):2125–2128. doi: 10.1212/01.wnl.0000264853.40735.3b. PubMed DOI

Zatz M, Vainzof M, Passos-Bueno MR. Limb-girdle muscular dystrophy: one gene with different phenotypes, one phenotype with different genes. Curr Opin Neurol. 2000;13(5):511–517. doi: 10.1097/00019052-200010000-00002. PubMed DOI

Richard I, Broux O, Allamand V, Fougerousse F, Chiannilkulchai N, Bourg N, Brenguier L, Devaud C, Pasturaud P, Roudaut C, Hillaire D, Passos-Bueno MR, Zatz M, Tischfield JA, Fardeau M, Jackson CE, Cohen D, Beckmann JS. Mutations in the proteolytic enzyme calpain 3 cause limb-girdle muscular dystrophy type 2A. Cell. 1995;81(1):27–40. doi: 10.1016/0092-8674(95)90368-2. PubMed DOI

Sorimachi H, Kinbara K, Kimura S, Takahashi M, Ishiura S, Sasagawa N, Sorimachi N, Shimada H, Tagawa K, Maruyama K, Suzuki K. Muscle-specific calpain, p94, responsible for limb girdle muscular dystrophy type 2A, associates with connectin through IS2, a p94-specific sequence. J Biol Chem. 1995;270(52):31158–31162. doi: 10.1074/jbc.270.52.31158. PubMed DOI

Keira Y, Noguchi S, Minami N, Hayashi YK, Nishino I. Localization of calpain 3 in human skeletal muscle and its alteration in limb-girdle muscular dystrophy 2A muscle. J Biochem. 2003;133(5):659–664. doi: 10.1093/jb/mvg084. PubMed DOI

Fanin M, Fulizio L, Nascimbeni AC, Spinazzi M, Piluso G, Ventriglia VM, Ruzza G, Siciliano G, Trevisan CP, Politano L, Nigro V, Angelini C. Molecular diagnosis in LGMD2A: mutation analysis or protein testing? Hum Mutat. 2004;24(1):52–62. doi: 10.1002/humu.20058. PubMed DOI

Piluso G, Politano L, Aurino S, Fanin M, Ricci E, Ventriglia VM, Belsito A, Totaro A, Saccone V, Topaloglu H, Nascimbeni AC, Fulizio L, Broccolini A, Canki-Klain N, Comi LI, Nigro G, Angelini C, Nigro V. Extensive scanning of the calpain-3 gene broadens the spectrum of LGMD2A phenotypes. J Med Genet. 2005;42(9):686–693. doi: 10.1136/jmg.2004.028738. PubMed DOI PMC

Saenz A, Leturcq F, Cobo AM, Poza JJ, Ferrer X, Otaegui D, Camano P, Urtasun M, Vilchez J, Gutierrez-Rivas E, Emparanza J, Merlini L, Paisán C, Goicoechea M, Blázquez L, Eymard B, Lochmuller H, Walter M, Bonnemann C, Figarella-Branger D, Kaplan JC, Urtizberea JA, Martí-Massó JF, López De Munain A. LGMD2A: genotype-phenotype correlations based on a large mutational survey on the calpain 3 gene. Brain. 2005;128(Pt 4):732–742. doi: 10.1093/brain/awh408. PubMed DOI

Brockington M, Blake DJ, Prandini P, Brown SC, Torelli S, Benson MA, Ponting CP, Estournet B, Romero NB, Mercuri E, Voit T, Sewry CA, Guicheney P, Muntoni F. Mutations in the fukutin-related protein gene (FKRP) cause a form of congenital muscular dystrophy with secondary laminin alpha2 deficiency and abnormal glycosylation of alpha-dystroglycan. Am J Hum Genet. 2001;69(6):1198–1209. doi: 10.1086/324412. PubMed DOI PMC

Mercuri E, Brockington M, Straub V, Quijano-Roy S, Yuva Y, Herrmann R, Brown SC, Torelli S, Dubowitz V, Blake DJ, Romero NB, Estournet B, Sewry CA, Guicheney P, Voit T, Muntoni F. Phenotypic spectrum associated with mutations in the fukutin-related protein gene. Ann Neurol. 2003;53(4):537–542. doi: 10.1002/ana.10559. PubMed DOI

Walter MC, Petersen JA, Stucka R, Fischer D, Schroder R, Vorgerd M, Schroers A, Schreiber H, Hanemann CO, Knirsch U, Rosenbohm A, Huebner A, Barisic N, Horvath R, Komoly S, Reilich P, Müller-Felber W, Pongratz D, Müller JS, Auerswald EA, Lochmüller H. FKRP (826C > A) frequently causes limb-girdle muscular dystrophy in German patients. J Med Genet. 2004;41(4):e50. doi: 10.1136/jmg.2003.013953. PubMed DOI PMC

Frosk P, Greenberg CR, Tennese AA, Lamont R, Nylen E, Hirst C, Frappier D, Roslin NM, Zaik M, Bushby K, Straub V, Zatz M, de Paula F, Morgan K, Fujiwara TM, Wrogemann K. The most common mutation in FKRP causing limb girdle muscular dystrophy type 2I (LGMD2I) may have occurred only once and is present in Hutterites and other populations. Hum Mutat. 2005;25(1):38–44. doi: 10.1002/humu.20110. PubMed DOI

Angelini C, Fanin M, Freda MP, Duggan DJ, Siciliano G, Hoffman EP. The clinical spectrum of sarcoglycanopathies. Neurology. 1999;52(1):176–179. doi: 10.1212/WNL.52.1.176. PubMed DOI

Straub V, Bushby K. The childhood limb-girdle muscular dystrophies. Semin Pediatr Neurol. 2006;13(2):104–114. doi: 10.1016/j.spen.2006.06.006. PubMed DOI

Jarry J, Rioux MF, Bolduc V, Robitaille Y, Khoury V, Thiffault I, Tetreault M, Loisel L, Bouchard JP, Brais B. A novel autosomal recessive limb-girdle muscular dystrophy with quadriceps atrophy maps to 11p13-p12. Brain. 2007;130(Pt 2):368–380. doi: 10.1093/brain/awl270. PubMed DOI

Bolduc V, Marlow G, Boycott KM, Saleki K, Inoue H, Kroon J, Itakura M, Robitaille Y, Parent L, Baas F, Mizuta K, Kamata N, Richard I, Linssen WH, Mahjneh I, de Visser M, Bashir R, Brais B. Recessive mutations in the putative calcium-activated chloride channel Anoctamin 5 cause proximal LGMD2L and distal MMD3 muscular dystrophies. Am J Hum Genet. 2010;86(2):213–221. doi: 10.1016/j.ajhg.2009.12.013. PubMed DOI PMC

Tsutsumi S, Kamata N, Vokes TJ, Maruoka Y, Nakakuki K, Enomoto S, Omura K, Amagasa T, Nagayama M, Saito-Ohara F. et al.The novel gene encoding a putative transmembrane protein is mutated in gnathodiaphyseal dysplasia (GDD) Am J Hum Genet. 2004;74(6):1255–1261. doi: 10.1086/421527. PubMed DOI PMC

Schroeder BC, Cheng T, Jan YN, Jan LY. Expression cloning of TMEM16A as a calcium-activated chloride channel subunit. Cell. 2008;134(6):1019–1029. doi: 10.1016/j.cell.2008.09.003. PubMed DOI PMC

Yang YD, Cho H, Koo JY, Tak MH, Cho Y, Shim WS, Park SP, Lee J, Lee B, Kim BM, Raouf R, Shin YK, Oh U. TMEM16A confers receptor-activated calcium-dependent chloride conductance. Nature. 2008;455(7217):1210–1215. doi: 10.1038/nature07313. PubMed DOI

Stephan AB, Shum EY, Hirsh S, Cygnar KD, Reisert J, Zhao H. ANO2 is the cilial calcium-activated chloride channel that may mediate olfactory amplification. Proc Natl Acad Sci U S A. 2009;106(28):11776–11781. doi: 10.1073/pnas.0903304106. PubMed DOI PMC

Hicks D, Sarkozy A, Muelas N, Koehler K, Huebner A, Hudson G, Chinnery PF, Barresi R, Eagle M, Polvikoski T, Bailey G, Miller J, Radunovic A, Hughes PJ, Roberts R, Krause S, Walter MC, Laval SH, Straub V, Lochmüller H, Bushby K. A founder mutation in Anoctamin 5 is a major cause of limb-girdle muscular dystrophy. Brain. 2011;134(Pt 1):171–182. doi: 10.1093/brain/awq294. PubMed DOI PMC

Chrobakova T, Hermanova M, Kroupova I, Vondracek P, Marikova T, Mazanec R, Zamecnik J, Stanek J, Havlova M, Fajkusova L. Mutations in Czech LGMD2A patients revealed by analysis of calpain3 mRNA and their phenotypic outcome. Neuromuscul Disord. 2004;14(10):659–665. doi: 10.1016/j.nmd.2004.05.005. PubMed DOI

Hermanova M, Zapletalova E, Sedlackova J, Chrobakova T, Letocha O, Kroupova I, Zamecnik J, Vondracek P, Mazanec R, Marikova T, Vohánka S, Fajkusová L. Analysis of histopathologic and molecular pathologic findings in Czech LGMD2A patients. Muscle Nerve. 2006;33(3):424–432. doi: 10.1002/mus.20480. PubMed DOI

Stehlikova K, Zapletalova E, Sedlackova J, Hermanova M, Vondracek P, Marikova T, Mazanec R, Zamecnik J, Vohanka S, Fajkus J, Fajkusová L. Quantitative analysis of CAPN3 transcripts in LGMD2A patients: involvement of nonsense-mediated mRNA decay. Neuromuscul Disord. 2007;17(2):143–147. doi: 10.1016/j.nmd.2006.10.001. PubMed DOI

Magri F, Del Bo R, D’Angelo MG, Sciacco M, Gandossini S, Govoni A, Napoli L, Ciscato P, Fortunato F, Brighina E, Bonato S, Bordoni A, Lucchini V, Corti S, Moggio M, Bresolin N, Comi GP. Frequency and characterisation of anoctamin 5 mutations in a cohort of Italian limb-girdle muscular dystrophy patients. Neuromuscul Disord. 2012;22(11):934–943. doi: 10.1016/j.nmd.2012.05.001. PubMed DOI PMC

Sveen ML, Schwartz M, Vissing J. High prevalence and phenotype-genotype correlations of limb girdle muscular dystrophy type 2I in Denmark. Ann Neurol. 2006;59(5):808–815. doi: 10.1002/ana.20824. PubMed DOI

van der Kooi AJ, Ten Dam L, Frankhuizen WS, Straathof CS, van Doorn PA, de Visser M, Ginjaar IB. ANO5 mutations in the Dutch limb girdle muscular dystrophy population. Neuromuscul Disord. 2013;23(6):456–460. doi: 10.1016/j.nmd.2013.03.012. PubMed DOI

Sarkozy A, Hicks D, Hudson J, Laval SH, Barresi R, Hilton-Jones D, Deschauer M, Harris E, Rufibach L, Hwang E, Bashir R, Walter MC, Krause S, van den Bergh P, Illa I, Pénisson-Besnier I, De Waele L, Turnbull D, Guglieri M, Schrank B, Schoser B, Seeger J, Schreiber H, Gläser D, Eagle M, Bailey G, Walters R, Longman C, Norwood F, Winer J. et al.ANO5 gene analysis in a large cohort of patients with anoctaminopathy: confirmation of male prevalence and high occurrence of the common exon 5 gene mutation. Hum Mutat. 2013;34(8):1111–1118. doi: 10.1002/humu.22342. PubMed DOI

Fanin M, Nascimbeni AC, Aurino S, Tasca E, Pegoraro E, Nigro V, Angelini C. Frequency of LGMD gene mutations in Italian patients with distinct clinical phenotypes. Neurology. 2009;72(16):1432–1435. doi: 10.1212/WNL.0b013e3181a1885e. PubMed DOI

Fanin M, Nascimbeni AC, Tasca E, Angelini C. How to tackle the diagnosis of limb-girdle muscular dystrophy 2A. Eur J Hum Genet. 2009;17(5):598–603. doi: 10.1038/ejhg.2008.193. PubMed DOI PMC

Boito CA, Melacini P, Vianello A, Prandini P, Gavassini BF, Bagattin A, Siciliano G, Angelini C, Pegoraro E. Clinical and molecular characterization of patients with limb-girdle muscular dystrophy type 2I. Arch Neurol. 2005;62(12):1894–1899. doi: 10.1001/archneur.62.12.1894. PubMed DOI

Hanisch F, Grimm D, Zierz S, Deschauer M. Frequency of the FKRP mutation c.826C > A in isolated hyperCKemia and in limb girdle muscular dystrophy type 2 in German patients. J Neurol. 2010;257(2):300–301. doi: 10.1007/s00415-009-5349-2. PubMed DOI

Krahn M, Beroud C, Labelle V, Nguyen K, Bernard R, Bassez G, Figarella-Branger D, Fernandez C, Bouvenot J, Richard I, Ollagnon-Roman E, Bevilacqua JA, Salvo E, Attarian S, Chapon F, Pellissier JF, Pouget J, Hammouda El H, Laforêt P, Urtizberea JA, Eymard B, Leturcq F, Lévy N. Analysis of the DYSF mutational spectrum in a large cohort of patients. Hum Mutat. 2009;30(2):E345–375. doi: 10.1002/humu.20910. PubMed DOI

De Luna N, Freixas A, Gallano P, Caselles L, Rojas-Garcia R, Paradas C, Nogales G, Dominguez-Perles R, Gonzalez-Quereda L, Vilchez JJ, Márquez C, Bautista J, Guerrero A, Salazar JA, Pou A, Illa I, Gallardo E. Dysferlin expression in monocytes: a source of mRNA for mutation analysis. Neuromuscul Disord. 2007;17(1):69–76. doi: 10.1016/j.nmd.2006.09.006. PubMed DOI

Matsuda C, Hayashi YK, Ogawa M, Aoki M, Murayama K, Nishino I, Nonaka I, Arahata K, Brown RH Jr. The sarcolemmal proteins dysferlin and caveolin-3 interact in skeletal muscle. Hum Mol Genet. 2001;10(17):1761–1766. doi: 10.1093/hmg/10.17.1761. PubMed DOI

Tagawa K, Ogawa M, Kawabe K, Yamanaka G, Matsumura T, Goto K, Nonaka I, Nishino I, Hayashi YK. Protein and gene analyses of dysferlinopathy in a large group of Japanese muscular dystrophy patients. J Neurol Sci. 2003;211(1–2):23–28. doi: 10.1016/S0022-510X(03)00041-8. PubMed DOI

Rosales XQ, Gastier-Foster JM, Lewis S, Vinod M, Thrush DL, Astbury C, Pyatt R, Reshmi S, Sahenk Z, Mendell JR. Novel diagnostic features of dysferlinopathies. Muscle Nerve. 2010;42(1):14–21. doi: 10.1002/mus.21650. PubMed DOI PMC

Cagliani R, Magri F, Toscano A, Merlini L, Fortunato F, Lamperti C, Rodolico C, Prelle A, Sironi M, Aguennouz M, Ciscato P, Uncini A, Moggio M, Bresolin N, Comi GP. Mutation finding in patients with dysferlin deficiency and role of the dysferlin interacting proteins annexin A1 and A2 in muscular dystrophies. Hum Mutat. 2005;26(3):283. doi: 10.1002/humu.9364. PubMed DOI

Ueyama H, Kumamoto T, Nagao S, Masuda T, Horinouchi H, Fujimoto S, Tsuda T. A new dysferlin gene mutation in two Japanese families with limb-girdle muscular dystrophy 2B and Miyoshi myopathy. Neuromuscul Disord. 2001;11(2):139–145. doi: 10.1016/S0960-8966(00)00168-1. PubMed DOI

A founder COL4A4 pathogenic variant resulting in autosomal recessive Alport syndrome accounts for most genetic kidney failure in Romani people

Two Novel Mutations in the JAG1 Gene in Pediatric Patients with Alagille Syndrome: The First Case Series in Czech Republic

Bending of DNA duplexes with mutation motifs