J Korean Med Sci.  2007 Jun;22(3):463-469. 10.3346/jkms.2007.22.3.463.

Mutations of CAPN3 in Korean Patients with Limb-Girdle Muscular Dystrophy

• Shin JH
• Kim HS
• Lee CH
• Kim CM
• Park KH
• Kim DS

• KMID: 1778355
• DOI: http://doi.org/10.3346/jkms.2007.22.3.463

Abstract

The limb-girdle muscular dystrophy type 2A (LGMD2A) is a recessively inherited disease caused by a mutation of the calpain 3 gene (CAPN3), and is considered one of the most prevalent subtypes of limb-girdle muscular dystrophy (LGMD). In this study, we aimed to identify CAPN3 mutations and to characterize the phenotype of Korean patients with LGMD2A. Among 35 patients with LGMD, four patients, who showed calpain 3 deficiency on western blot analysis, were analyzed in this study. Total RNA extracted from frozen muscle tissue was amplified by reverse transcriptase polymerase chain reaction (RT-PCR) using six primer pairs covering all coding sequences of CAPN3, and direct sequencing was performed. Clinical and pathological features of the patients were also reviewed. We found four different mutations in five alleles from three patients. Of the pathogenic mutations identified, two were novel (c.2125T>C and c.2355-2357delTTC), and the others had been reported elsewhere (c.440G>C, c.1076C>T). All patients showed a high CK level with predominant proximal leg weakness, and the onset was in their childhood except for one patient. Among two novel CAPN3 mutations, one was a missense mutation (c.2125T>C [p.709Ser>Pro]), and the other was a small in-frame deletion causing omission of a single amino acid (c.2355-2357delTTC [p.786delPhe]). The clinical features of our patients were generally compatible with the characteristics of LGMD2A patients described in the previous studies.

Keyword

Limb-Girdle Muscular Dystrophy; Calpain 3; Korean

MeSH Terms

Adolescent
Adult
Amino Acid Sequence
Base Sequence
Calpain/*genetics
DNA Primers/chemistry
Female
Humans
Korea
Male
Middle Aged
Molecular Sequence Data
Muscle Proteins/*genetics
Muscular Dystrophies, Limb-Girdle/*genetics
*Mutation
Sequence Homology, Amino Acid

Figure

• 

  Fig. 1 Western blot analysis of patient 1 (lane 5 of left column panels), patient 2 (lane 3 of middle column panels), and patient 3 and 4 (lane 2 and 4 of right column panels, respectively). Arrows indicate the presence of a band at the appropriate position, and asterisks (*) indicate the absence. The lanes other than described above are from the other samples that have been found to be negative for calpainopathy.

• 

  Fig. 2 Mutations identified in the study. (A) Heterozygous c.2125T>C in patient 1 (arrow). Original amino acid serine has been changed into proline by the mutation. (B) c.2355-2357delTTC in patient 4 (open arrow). Mutated allele misses the codon for phenylalanine. (C) Homozygous c.1076C>T in patient 2 (arrow) changes the codon for amino acid proline into leucine. (D) Heterozygous c.440G>C in patient 4 (arrow) changes codon for amino acid arginine into proline.

• 

  Fig. 3 Muscle biopsy findings of the patients. (A) In patient 1, markedly increased contents of connective tissue and clusters of regenerating muscle fibers are observed. Hematoxylin and eosin (H&E) stain. (B) Marked muscle fiber size variation with many hypertrophic fibers, and markedly increased contents of connective tissue are seen in muscle biopsy from patient 2. H&E stain. (C) In patient 2, type 1 fiber predominance is also observed. ATPase stain at pH 4.3. (D) In patient 3, marked muscle fiber size variation, markedly increased contents of connective tissue, and many fibers with rimmed vacuoles are seen. Modified Gomori-trichrome stain. Size of the bar=100 µm.

• 

  Fig. 4 Multiple alignment analysis of amino acid sequences of calpain 3 in different species. Both mutated amino acids serine (S, marked in panel A) and phenylalanine (F, marked in panel B) are highly conserved across several species. H. sapiens: human, M. mulatta: rhesus monkey, M. musculus: house mouse, R. norvegicus: Norway rat, G. gallus: chicken, C. familaris: dog.

Reference

1. Passos-Bueno MR, Moreira ES, Marie SK, Bashir R, Vasquez L, Love DR, Vainzof M, Iughetti P, Oliveira JR, Bakker E, Strachan T, Bushby K, Zatz M. Main clinical features of the three mapped autosomal recessive limb-girdle muscular dystrophies and estimated proportion of each form in 13 Brazilian families. J Med Genet. 1996. 33:97–102.

2. Dinçer P, Leturcq F, Richard I, Piccolo F, Yalnizoglu D, de Toma C, Äkçoren Z, Broux O, Deburgrave N, Brenguier L, Roudaut C, Urtizberea JA, Jung D, Tan E, Jeanpierre M, Campbell KP, Kaplan JC, Beckmann JS, Topaloglu H. A biochemical, genetic, and clinical survey of autosomal recessive limb girdle muscular dystrophies in Turkey. Ann Neurol. 1997. 42:222–229.

3. Richard I, Brenguier L, Dinçer P, Roudaut C, Bady B, Burgunder JM, Chemaly R, Garcia CA, Halaby G, Jackson CE, Kurnit DM, Lefranc G, Legum C, Loiselet J, Merlini L, Nivelon-Chevallier A, Ollagnon-Roman E, Restagno G, Topaloglu H, Beckmann JS. Multiple independent molecular etiology for limb-girdle muscular dystrophy type 2A patients from various geographical origins. Am J Hum Genet. 1997. 60:1128–1138.

4. Topaloglu H, Dincer P, Richard I, Akcoren Z, Alehan D, Ozme S, Caglar M, Karaduman A, Urtizberea JA, Beckmann JS. Calpain-3 deficiency causes a mild muscular dystrophy in childhood. Neuropediatrics. 1997. 28:212–216.

5. Kawai H, Akaike M, Kunishige M, Inui T, Adachi K, Kimura C, Kawajiri M, Nishida Y, Endo I, Kashiwagi S, Nishino H, Fujiwara T, Okuno S, Roudaut C, Richard I, Beckmann JS, Miyoshi K, Matsumoto T. Clinical, pathological, and genetic features of limb- girdle muscular dystrophy type 2A with new calpain 3 gene mutations in seven patients from three Japanese families. Muscle Nerve. 1998. 21:1493–1501.

6. Chae J, Minami N, Jin Y, Nakagawa M, Murayama K, Igarashi F, Nonaka I. Calpain 3 gene mutations: genetic and clinico-pathologic findings in limb-girdle muscular dystrophy. Neuromuscul Disord. 2001. 11:547–555.

7. de Paula F, Vainzof M, Passos-Bueno MR, de Cassia M Pavanello R, Matioli SR, V B Anderson L, Nigro V, Zatz M. Clinical variability in calpainopathy: what makes the difference? Eur J Hum Genet. 2002. 10:825–832.

8. Richard I, Broux O, Allamand V, Fougerousse F, Chiannilkulchai N, Bourg N, Brenguier L, Devaud C, Pasturaud P, Roudaut C, et al. Mutations in the proteolytic enzyme calpain 3 cause limb-girdle muscular dystrophy type 2A. Cell. 1995. 81:27–40.

9. Sorimachi H, Toyama-Sorimachi N, Saido TC, Kawasaki H, Sugita H, Miyasaka M, Arahata K, Ishiura S, Suzuki K. Muscle-specific calpain, p94, is degraded by autolysis immediately after translation, resulting in disappearance from muscle. J Biol Chem. 1993. 268:10593–10605.

10. Anderson LV, Davison K, Moss JA, Richard I, Fardeau M, Tome FM, Hubner C, Lasa A, Colomer J, Beckmann JS. Characterization of monoclonal antibodies to calpain 3 and protein expression in muscle from patients with limb-girdle muscular dystrophy type 2A. Am J Pathol. 1998. 153:1169–1179.

11. Anderson LV, Harrison RM, Pogue R, Vafiadaki E, Pollitt C, Davison K, Moss JA, Keers S, Pyle A, Shaw PJ, Mahjneh I, Argov Z, Greenberg CR, Wrogemann K, Bertorini T, Goebel HH, Beckmann JS, Bashir R, Bushby KM. Secondary reduction in calpain 3 expression in patients with limb girdle muscular dystrophy type 2B and Miyoshi myopathy (primary dysferlinopathies). Neuromuscul Disord. 2000. 10:553–559.

12. 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. 14:659–665.

13. Fokkema IF, den Dunnen JT, Taschner PE. LOVD: easy creation of a locus-specific sequence variation database using an "LSDB-in-a-box" approach. Hum Mutat. 2005. 26:63–68.

14. Ginjaar HB, Frankhuizen WS, de Mos M, Verschuuren JJ, Hoogendijk JE, van Doorn PA, van Engelen BG, Faber CG, Anderson LV, de Visser M, Baker E, van der Kooi AJ. Classification of limb girdle muscular dystrophy types 1C, 2A, and 2B based on protein and/or DNA studies. Neuromuscul Disord. 2002. 12:731.

15. Minami N, Nishino I, Kobayashi O, Ikezoe K, Goto Y, Nonaka I. Mutations of calpain 3 gene in patients with sporadic limb-girdle muscular dystrophy in Japan. J Neurol Sci. 1999. 171:31–37.

16. 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. 24:52–62.

17. 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, Paisan C, Goicoechea M, Blazguez L, Eymard B, Lochmuller H, Walter M, Bonnemann C, Figarella-Branger D, Kaplan JC, Urtizberea JA, Marti-Masso JF, Lopez de Munain A. LGMD2A: genotype-phenotype correlations based on a large mutational survey on the calpain 3 gene. Brain. 2005. 128:732–742.

18. Fardeau M, Hillaire D, Mignard C, Feingold N, Feingold J, Mignard D, de Ubeda B, Collin H, Tome FM, Richard I, Beckmann J. Juvenile limb-girdle muscular dystrophy. Clinical, histopathological and genetic data from a small community living in the Reunion Island. Brain. 1996. 119:295–308.

19. Beckmann JS, Bushby KM. Advances in the molecular genetics of the limb-girdle type of autosomal recessive progressive muscular dystrophy. Curr Opin Neurol. 1996. 9:389–393.

20. Urtasun M, Saenz A, Roudaut C, Poza JJ, Urtizberea JA, Cobo AM, Richard I, Garcia Bragado F, Leturcq F, Kaplan JC, Marti Masso JF, Beckmann JS, Lopez de Munain A. Limb-girdle muscular dystrophy in Guipuzcoa (Basque Country, Spain). Brain. 1998. 121:1735–1747.