Title: Recessive Mutations in the Putative Calcium-Activated Chloride Channel Anoctamin 5 Cause Proximal LGMD2L and Distal MMD3 Muscular Dystrophies
Abstract: The recently described human anion channel Anoctamin (ANO) protein family comprises at least ten members, many of which have been shown to correspond to calcium-activated chloride channels. To date, the only reported human mutations in this family of genes are dominant mutations in ANO5 (TMEM16E, GDD1) in the rare skeletal disorder gnathodiaphyseal dysplasia. We have identified recessive mutations in ANO5 that result in a proximal limb-girdle muscular dystrophy (LGMD2L) in three French Canadian families and in a distal non-dysferlin Miyoshi myopathy (MMD3) in Dutch and Finnish families. These mutations consist of a splice site, one base pair duplication shared by French Canadian and Dutch cases, and two missense mutations. The splice site and the duplication mutations introduce premature-termination codons and consequently trigger nonsense-mediated mRNA decay, suggesting an underlining loss-of-function mechanism. The LGMD2L phenotype is characterized by proximal weakness, with prominent asymmetrical quadriceps femoris and biceps brachii atrophy. The MMD3 phenotype is associated with distal weakness, of calf muscles in particular. With the use of electron microscopy, multifocal sarcolemmal lesions were observed in both phenotypes. The phenotypic heterogeneity associated with ANO5 mutations is reminiscent of that observed with Dysferlin (DYSF) mutations that can cause both LGMD2B and Miyoshi myopathy (MMD1). In one MMD3-affected individual, defective membrane repair was documented on fibroblasts by membrane-resealing ability assays, as observed in dysferlinopathies. Though the function of the ANO5 protein is still unknown, its putative calcium-activated chloride channel function may lead to important insights into the role of deficient skeletal muscle membrane repair in muscular dystrophies. The recently described human anion channel Anoctamin (ANO) protein family comprises at least ten members, many of which have been shown to correspond to calcium-activated chloride channels. To date, the only reported human mutations in this family of genes are dominant mutations in ANO5 (TMEM16E, GDD1) in the rare skeletal disorder gnathodiaphyseal dysplasia. We have identified recessive mutations in ANO5 that result in a proximal limb-girdle muscular dystrophy (LGMD2L) in three French Canadian families and in a distal non-dysferlin Miyoshi myopathy (MMD3) in Dutch and Finnish families. These mutations consist of a splice site, one base pair duplication shared by French Canadian and Dutch cases, and two missense mutations. The splice site and the duplication mutations introduce premature-termination codons and consequently trigger nonsense-mediated mRNA decay, suggesting an underlining loss-of-function mechanism. The LGMD2L phenotype is characterized by proximal weakness, with prominent asymmetrical quadriceps femoris and biceps brachii atrophy. The MMD3 phenotype is associated with distal weakness, of calf muscles in particular. With the use of electron microscopy, multifocal sarcolemmal lesions were observed in both phenotypes. The phenotypic heterogeneity associated with ANO5 mutations is reminiscent of that observed with Dysferlin (DYSF) mutations that can cause both LGMD2B and Miyoshi myopathy (MMD1). In one MMD3-affected individual, defective membrane repair was documented on fibroblasts by membrane-resealing ability assays, as observed in dysferlinopathies. Though the function of the ANO5 protein is still unknown, its putative calcium-activated chloride channel function may lead to important insights into the role of deficient skeletal muscle membrane repair in muscular dystrophies. Muscular dystrophies encompass a large and diverse group of inherited diseases defined by skeletal muscle weakness and atrophy. Among these, the limb-girdle muscular dystrophies (LGMD) represent a group of both dominant and recessive disorders, characterized by predominant proximal limb muscle weakness, with 18 known causal genes.1Guglieri M. Straub V. Bushby K. Lochmuller H. Limb-girdle muscular dystrophies.Curr. Opin. Neurol. 2008; 21: 576-584Crossref PubMed Scopus (91) Google Scholar, 2Norwood F. de Visser M. Eymard B. Lochmuller H. Bushby K. EFNS guideline on diagnosis and management of limb girdle muscular dystrophies.Eur. J. Neurol. 2007; 14: 1305-1312Crossref PubMed Scopus (92) Google Scholar The majority of the proteins involved in LGMD are important for maintaining the integrity of the sarcolemmal membrane, which is susceptible to injury because of the high mechanical stress imposed on muscle fibers during muscle contraction.1Guglieri M. Straub V. Bushby K. Lochmuller H. 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Dysferlinopathies.Neurol. India. 2008; 56: 289-297Crossref PubMed Scopus (50) Google Scholar The human Anoctamins (ANO) compose a family of at least ten proteins all exhibiting eight transmembrane domains and a DUF590 domain of unknown function.13Galindo B.E. Vacquier V.D. Phylogeny of the TMEM16 protein family: Some members are overexpressed in cancer.Int. J. Mol. Med. 2005; 16: 919-924PubMed Google Scholar, 14Hartzell H.C. Yu K. Xiao Q. Chien L.T. Qu Z. Anoctamin/TMEM16 family members are Ca2+-activated Cl- channels.J. Physiol. 2009; 587: 2127-2139Crossref PubMed Scopus (195) Google Scholar Recently, ANO1 (MIM 610108), ANO2 (MIM 610109), ANO6 (MIM 608663), ANO8 (MIM 610216), and ANO9 have been recognized to code for the elusive calcium-activated chloride channels (CaCC);15Caputo A. Caci E. Ferrera L. Pedemonte N. Barsanti C. Sondo E. Pfeffer U. Ravazzolo R. Zegarra-Moran O. Galietta L.J. 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Cygnar K.D. Reisert J. Zhao H. ANO2 is the cilial calcium-activated chloride channel that may mediate olfactory amplification.Proc. Natl. Acad. Sci. USA. 2009; 106: 11776-11781Crossref PubMed Scopus (243) Google Scholar, 20Almaca J. Tian Y. Aldehni F. Ousingsawat J. Kongsuphol P. Rock J.R. Harfe B.D. Schreiber R. Kunzelmann K. TMEM16 proteins produce volume regulated chloride currents that are reduced in mice lacking TMEM16A.J. Biol. Chem. 2009; 284: 28571-28578Crossref PubMed Scopus (134) Google Scholar however, the function of ANO5 (MIM 608662) is unknown. In this study we report that recessive mutations in Anoctamin 5 (ANO5) cause both a proximal muscular dystrophy, LGMD2L (MIM 611307), and the distal Miyoshi myopathy MMD3. A previous study mapped LGMD2L, a novel recessive form of LGMD associated with prominent asymmetrical quadriceps femoris and biceps brachii atrophy, to chromosome 11p12-p13 in a cohort of French Canadian (FC) families.21Jarry J. Rioux M.F. Bolduc V. Robitaille Y. Khoury V. Thiffault I. Tetreault M. Loisel L. Bouchard J.P. Brais B. A novel autosomal recessive limb-girdle muscular dystrophy with quadriceps atrophy maps to 11p13-p12.Brain. 2007; 130: 368-380Crossref PubMed Scopus (46) Google Scholar SNP genotyping via the Illumina HumanHap300 beadchip (317 503 SNPs; Illumina, San Diego, CA) was performed on the previously reported single large consanguineous FC Family IX21Jarry J. Rioux M.F. Bolduc V. Robitaille Y. Khoury V. Thiffault I. Tetreault M. Loisel L. Bouchard J.P. Brais B. A novel autosomal recessive limb-girdle muscular dystrophy with quadriceps atrophy maps to 11p13-p12.Brain. 2007; 130: 368-380Crossref PubMed Scopus (46) Google Scholar (Figure 1A) at the McGill University and Genome Québec Innovation Centre genotyping platform. Homozygosity analysis via AutoSNPa22Carr I.M. Flintoff K.J. Taylor G.R. Markham A.F. Bonthron D.T. Interactive visual analysis of SNP data for rapid autozygosity mapping in consanguineous families.Hum. Mutat. 2006; 27: 1041-1046Crossref PubMed Scopus (132) Google Scholar identified a 4.7 Mb region of homozygosity on chromosome 11p14.3-p15 (616 consecutive SNPs delimited by rs4073508 and rs10834273). This new region shifted telomerically by 11.9 Mb the previous published candidate interval (11p12-p13).21Jarry J. Rioux M.F. Bolduc V. Robitaille Y. Khoury V. Thiffault I. Tetreault M. Loisel L. Bouchard J.P. Brais B. A novel autosomal recessive limb-girdle muscular dystrophy with quadriceps atrophy maps to 11p13-p12.Brain. 2007; 130: 368-380Crossref PubMed Scopus (46) Google Scholar This region contained 11 annotated genes: NAV2 (MIM 607026), DBX1, HTATIP2 (MIM 605628), PRMT3 (MIM 603190), SLC6A5 (MIM 604159), NELL1 (MIM 602319), ANO5/TMEM16E, FANCF (MIM 603467), GAS2 (MIM 602835), SLC17A6 (MIM 607563), and SVIP. The exons and intron-exon junctions were sequenced for five of these genes: ANO5/TMEM16E, FANCF, GAS2, SLC17A6, and SVIP. Mutations were identified in ANO5/TMEM16E in family IX. Though the sequencing of ANO5 exons and intron-exon borders in the seven remaining families of the original cohort21Jarry J. Rioux M.F. Bolduc V. Robitaille Y. Khoury V. Thiffault I. Tetreault M. Loisel L. Bouchard J.P. Brais B. A novel autosomal recessive limb-girdle muscular dystrophy with quadriceps atrophy maps to 11p13-p12.Brain. 2007; 130: 368-380Crossref PubMed Scopus (46) Google Scholar did not identify mutations, the screening of two new FC LGMD2L families identified other mutations in ANO5. Informed consent approved by the Centre Hospitalier de l'Université de Montréal ethics committee was obtained for all participants. In Family IX we identified a homozygous nucleotide substitution (c.1295C>G) in genomic DNA, which creates a putative splice donor site within exon 13 as calculated by NetGene2 and Human Splicing Finder v2.4 (Figures 1A and 1B). Amplification and sequencing of patient muscle cDNA confirmed the aberrant splicing of exon 13 that results in the deletion of the last 38 nucleotides of this exon (Figures 1C and 1D), leading to a frameshift and a predicted premature truncation (p.Ala432GlyfsX49). Segregation of this variant in Family IX was confirmed by sequencing the genomic DNA of exon 13 in all siblings (Figure 1A). The same mutation was identified in homozygote state in the two cases belonging to the FC Family XXXI (Figure 1A). The two families are not known to be related and come from different regions, but share a six STR markers haplotype (covering 3.6 Mb), suggesting that it is the same historical mutation that segregates in these families (data not shown). The third LGMD2L FC Family XXIX was heterozygous for two other variants: c.191 dupA in exon 5 (p.Asn64LysfsX15) and c.692G>T in exon 8 (p.G231V) (Figure 1E). The G231 residue is evolutionary conserved (Figure 1F) and is predicted to be located in the putative intracellular N-terminal tail (Figure 2D). Sequencing of the entire 87 kb genomic region of ANO5 is underway to identify mutations in other FC LGMD families. Further evidence that ANO5 mutations cause muscular dystrophy came from an independent study of families presenting with a distal non-dysferlin Miyoshi myopathy. Informed consents approved by the Kainuu Central Hospital (Finland), the Academic Medical Centre (The Netherlands), and the University of Durham (United Kingdom) ethical committees were obtained for these families. Two Miyoshi myopathy families, in which mutations in DYSF and linkage to the 10p MMD2 locus had been excluded,23Linssen W.H. de Visser M. Notermans N.C. Vreyling J.P. Van Doorn P.A. Wokke J.H. Baas F. Bolhuis P.A. Genetic heterogeneity in Miyoshi-type distal muscular dystrophy.Neuromuscul. Disord. 1998; 8: 317-320Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar were genotyped via the Affymetrix GeneChip Human Mapping 50k (performed by AROS Applied Biotechnology, Aarhus Nord, Denmark). They were linked to a Miyoshi myopathy (MMD3) locus on chromosome 11p14.3-cen (rs722490 to rs509244, cumulative LOD score of >2.5) overlapping with the LGMD2L candidate region21Jarry J. Rioux M.F. Bolduc V. Robitaille Y. Khoury V. Thiffault I. Tetreault M. Loisel L. Bouchard J.P. Brais B. A novel autosomal recessive limb-girdle muscular dystrophy with quadriceps atrophy maps to 11p13-p12.Brain. 2007; 130: 368-380Crossref PubMed Scopus (46) Google Scholar (data not shown). Sequencing of the ANO5 gene identified mutations in both families. In the Finnish Family H,24Jaiswal J.K. Marlow G. Summerill G. Mahjneh I. Mueller S. Hill M. Miyake K. Haase H. Anderson L.V. Richard I. et al.Patients with a non-dysferlin Miyoshi myopathy have a novel membrane repair defect.Traffic. 2007; 8: 77-88Crossref PubMed Scopus (44) Google Scholar a homozygous nucleotide substitution (c.2272C>T) was identified in exon 20 that leads to the substitution of a conserved arginine to a cysteine residue (R758C; Figure 2A). The same heterozygote exon 5 mutation (c.191 dupA) observed in the FC Family XXIX (Figure 1E) was found in a homozygous state in the Dutch Family IV23Linssen W.H. de Visser M. Notermans N.C. Vreyling J.P. Van Doorn P.A. Wokke J.H. Baas F. Bolhuis P.A. Genetic heterogeneity in Miyoshi-type distal muscular dystrophy.Neuromuscul. Disord. 1998; 8: 317-320Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar (Figure 2B). Both variants were shown to segregate with the disease in the two families. None of the mutations have been reported as variants in the following databases: the National Center for Biotechnology Information (NCBI) database of genetic variation (dbSNP build 130), the Human Genome Diversity Project in collaboration with the Centre d'Étude du Polymorphisme Humain (HGDP-CEPH), and the International HapMap Project (Merged phases 1, 2, & 3). Considering that the five ANO5 families have diverse origins (three FC, one Dutch, and one Finnish), we tested their carrier frequencies in these three populations, as well as in panels of United Kingdom (UK) Caucasian controls (from the Health Protection Agency of United Kingdom) and CEPH controls. The c.1295C>G variant was absent in 210 FC and 162 CEPH control chromosomes, and the c.692G>T (G231V) variant was absent in 210 FC control chromosomes. The c.2272C>T (R758C) variant was not detected in 100 UK, nor in 208 FC, control chromosomes, but was detected in one out of 368 Finnish control chromosomes, indicating that this variant is present in the Finnish population at low frequency. The c.191 dupA mutation was absent in 210 FC and in 152 CEPH control chromosomes, but was identified in 1 out of 100 UK and in 2 out of 210 Dutch control chromosomes. The observed absence or very low population frequencies of the ANO5 mutations strongly support our conclusion that these cause LGMD2L and MMD3. ANO5 is predicted to produce several alternatively spliced isoforms, with the major isoform in muscle retaining all 22 exons.25Tsutsumi S. Inoue H. Sakamoto Y. Mizuta K. Kamata N. Itakura M. Molecular cloning and characterization of the murine gnathodiaphyseal dysplasia gene GDD1.Biochem. Biophys. Res. Commun. 2005; 331: 1099-1106Crossref PubMed Scopus (28) Google Scholar Based on the predicted full coding sequence (NCBI reference sequence NM_213599.2), it is expected that the mutations c.1295C>G (LGMD2L) and c.191 dupA (LGMD2L and MMD3) lead to a frameshift and premature truncation (p.Ala432GlyfsX49 and p.Asn64LysfsX15, respectively; Figure 2D). As is often the case with mRNAs containing premature termination codons, a translation-coupled nonsense-mediated RNA decay (NMD) mechanism may lead to their more rapid degradation.26Brogna S. Wen J. Nonsense-mediated mRNA decay (NMD) mechanisms.Nat. Struct. Mol. Biol. 2009; 16: 107-113Crossref PubMed Scopus (286) Google Scholar To test whether transcripts carrying the mutations c.1295C>G and c.191 dupA are subject to NMD, lymphoblastoid cell lines isolated from EBV-transformed lymphocytes were grown in IMDM medium (Invitrogen, Carlsbad, CA) supplemented with 10% Fetal Bovine serum (Invitrogen) and incubated for 9 hr with 0.1% DMSO (vehicle) or 100 μg/mL of cycloheximide (Sigma, St. Louis, MO), a translation inhibitor. Quantitative RT-PCR were performed in triplicate on extracted RNA, and expression of ANO5 was normalized to a set of three control genes (GAPDH [MIM 138400], PUM1 [MIM 607204], and RPL13A) via QBase, a modified ΔΔCt method,27Hellemans J. Mortier G. De Paepe A. Speleman F. Vandesompele J. qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data.Genome Biol. 2007; 8: R19Crossref PubMed Scopus (2653) Google Scholar at the Génome Québec and Université de Sherbrooke RNomics Centre. All controls and cases demonstrated an increase in the relative ANO5 expression as a consequence of cycloheximide treatment (Figure 3A); however, the differential ANO5 expression between cycloheximide-treated and untreated cells was significantly greater in patients homozygous for c.1295C>G (n = 2) versus controls (n = 3, p < 0.001, Student's t test; Figure 3B), suggesting that the mutated transcripts were protected from NMD when translation was inhibited. The approximate 5-fold increase observed for case XXIX-II-1 (Figure 3A), as compared to the 8- and 9-fold increases observed for the two c.1295C>G aberrant splicing mutation homozygote cases, is in agreement with her heterozygote state for a premature termination (c.191 dupA) and missense (c.692G>T, G231V) mutations. These results strongly suggest that the two variants c.1295C>G and c.191 dupA are associated with a loss of ANO5 function. Previously, dominant ANO5 mutations have been reported in two families with gnathodiaphyseal dysplasia (GDD [MIM 166260]), a rare skeletal syndrome characterized by bone fragility, cement-osseous lesions of the maxilla and mandible, and diaphyseal sclerosis of tubular bones.28Tsutsumi S. Kamata N. Vokes T.J. 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: 1255-1261Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar In these patients no other abnormalities in nonskeletal tissues have been reported.28Tsutsumi S. Kamata N. Vokes T.J. 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: 1255-1261Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar The missense mutations in GDD patients affect a conserved cysteine located in an extracellular loop of ANO5, similar to the R758C mutation found in MMD3 patients (Figure 2D). However, because GDD is a dominant disease, it was hypothesized that the mutations in these patients cause a gain-of-function effect predominantly in skeletal tissues.28Tsutsumi S. Kamata N. Vokes T.J. 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: 1255-1261Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar The only two available polyclonal antibodies generated against N- and C-terminal epitopes of mouse Ano529Mizuta K. Tsutsumi S. Inoue H. Sakamoto Y. Miyatake K. Miyawaki K. Noji S. Kamata N. Itakura M. Molecular characterization of GDD1/TMEM16E, the gene product responsible for autosomal dominant gnathodiaphyseal dysplasia.Biochem. Biophys. Res. Commun. 2007; 357: 126-132Crossref PubMed Scopus (79) Google Scholar failed to recognize the normal human protein on western blots generated from transfected HeLa and Cos-1 cell lysates. By immunofluorescence, nonspecific perimembranous and cytoplasmic staining was detected in skeletal muscle (data not shown), thereby precluding the testing of the impact of the mutations on the expression and localization of ANO5 in patient muscle. Table 1 summarizes the clinical findings for patients carrying ANO5 mutations that are also presented in greater details in Table S1 available online. LGMD2L patients are characterized by late-onset proximal scapular and pelvic girdle muscle weakness (mean age 34.4, 20–55), accompanied by asymmetrical atrophy of the quadriceps femoris and biceps brachii.21Jarry J. Rioux M.F. Bolduc V. Robitaille Y. Khoury V. Thiffault I. Tetreault M. Loisel L. Bouchard J.P. Brais B. A novel autosomal recessive limb-girdle muscular dystrophy with quadriceps atrophy maps to 11p13-p12.Brain. 2007; 130: 368-380Crossref PubMed Scopus (46) Google Scholar Though calf hypertrophy can be observed at presentation, they may develop later in the course asymmetrical mild calf atrophy usually not associated with weakness. No distal weakness in the upper limbs was observed. None of the LGMD2L patients lost walking, though the proximal weakness led to difficulties climbing stairs. The intrafamilial variability is striking with two patients reporting no symptoms at ages 68 (IX-II-3) and 61 (XXXI-II-3), though mild iliopsoas weakness was documented on examination and the creatine kinases (CK) levels were known to be elevated in the past years for both cases (Table 1). The phenotype is quite different for MMD3 patients. The Dutch patients (family IV) showed early calf weakness, along with difficulties of walking on tiptoes, without atrophy.23Linssen W.H. de Visser M. Notermans N.C. Vreyling J.P. Van Doorn P.A. Wokke J.H. Baas F. Bolhuis P.A. Genetic heterogeneity in Miyoshi-type distal muscular dystrophy.Neuromuscul. Disord. 1998; 8: 317-320Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 24Jaiswal J.K. Marlow G. Summerill G. Mahjneh I. Mueller S. Hill M. Miyake K. Haase H. Anderson L.V. Richard I. et al.Patients with a non-dysferlin Miyoshi myopathy have a novel membrane repair defect.Traffic. 2007; 8: 77-88Crossref PubMed Scopus (44) Google Scholar In the Finnish patients (family H), calf hypertrophy was seen in both cases during the early stages of their disease, but case H-II-6 later developed asymmetric calf atrophy. Despite the hypertrophy, calf weakness has always been an initial manifestation of all MMD3 cases. Asymmetric involvement of the proximal muscles of the lower and upper limb-girdles is a later manifestation with quadriceps atrophy being observed with time in two out of five MMD3 patients (Table 1 and Table S1). The FC case XXIX-II-1, which shares the exon 5 c.191 dupA mutation with the MMD3 Dutch Family IV, had a somewhat overlapping LGMD2L and MMD3 phenotype. At age 63, she had concomitant late-onset asymmetrical proximal upper limb (deltoid, biceps, triceps) and ilopsoas weakness and distal upper limb (wrist and finger extensors) and tibialis anterior weakness, while having asymmetrical normal strength gastrocnemius hypertrophy. CK were elevated in all patients (1032-15860IU, mean 5514IU). No cardiac abnormalities were detected on electrocardiomyogram (ECG), Holter ECG, and echocardiography for the FC and the Dutch patients. Electron microscopy performed on muscle from the LGMD2L patient IX-II-921Jarry J. Rioux M.F. Bolduc V. Robitaille Y. Khoury V. Thiffault I. Tetreault M. Loisel L. Bouchard J.P. Brais B. A novel autosomal recessive limb-girdle muscular dystrophy with quadriceps atrophy maps to 11p13-p12.Brain. 2007; 130: 368-380Crossref PubMed Scopus (46) Google Scholar and MMD3 patient H-II-6 (Figure 2C) both showed multifocal disruption of the sarcolemmal membrane but no subsarcolemmal vesicle accumulation, as has been reported in dysferlinopathies.30Selcen D. Stilling G. Engel A.G. The earliest pathologic alterations in dysferlinopathy.Neurology. 2001; 56: 1472-1481Crossref PubMed Scopus (123) Google Scholar, 31Cenacchi G. Fanin M. De Giorgi L.B. Angelini C. Ultrastructural changes in dysferlinopathy support defective membrane repair mechanism.J. Clin. Pathol. 2005; 58: 190-195Crossref PubMed Scopus (76) Google Scholar Furthermore, in the MMD3 Family H, membrane repair capability was found to be defective in patient fibroblasts by testing membrane resealing ability via either multiphoton laser irradiation or glass-bead-mediated wounding in the presence of fluorescent dyes, whereas lysosomal and enlargeosomal exocytosis was shown to occur normally, indicating that the conventional membrane repair pathways were not disrupted.24Jaiswal J.K. Marlow G. Summerill G. Mahjneh I. Mueller S. Hill M. Miyake K. Haase H. Anderson L.V. Richard I. et al.Patients with a non-dysferlin Miyoshi myopathy have a novel membrane repair defect.Traffic. 2007; 8: 77-88Crossref PubMed Scopus (44) Google ScholarTable 1Clinical Data of Patients Carrying ANO5 MutationsPhenotypeFam-IDaFamilies IX, H, and IV refer to original articles (see 21,23,24).Ethnic BckgSexANO5 Mutations (gDNA)Age (2009)Age of OnsetDistal Weakness ArmIliops. MRCQuad. AtrophyQuad. MRCCalf MusclesDistal Weakness LegLoss of WalkingMaximum CK (IU)LGMD2LIX-II-321Jarry J. Rioux M.F. Bolduc V. Robitaille Y. Khoury V. Thiffault I. Tetreault M. Loisel L. Bouchard J.P. Brais B. A novel autosomal recessive limb-girdle muscular dystrophy with quadriceps atrophy maps to 11p13-p12.Brain. 2007; 130: 368-380Crossref PubMed Scopus (46) Google ScholarFCFc.1295C>GbPatients homozygous for the mutation.68ASnone4/5+5/5nonenoneno1,649LGMD2LIX-II-421Jarry J. Rioux M.F. Bolduc V. Robitaille Y. Khoury V. Thiffault I. Tetreault M. Loisel L. Bouchard J.P. Brais B. A novel autosomal recessive limb-girdle muscular dystrophy with quadriceps atrophy maps to 11p13-p12.Brain. 2007; 130: 368-380Crossref PubMed Scopus (46) Google ScholarFCMc.1295C>GbPatients homozygous for the mutation.6740none4/5+4.5/5mild atrophynoneno1,032LGMD2LIX-II-521Jarry J. Rioux M.F. Bolduc V. Robitaille Y. Khoury V. Thiffault I. Tetreault M. Loisel L. Bouchard J.P. Brais B. A novel autosomal recessive limb-girdle muscular dystrophy with quadriceps atrophy maps to 11p13-p12.Brain. 2007; 130: 368-380Crossref PubMed Scopus (46) Google ScholarFCMc.1295C>GbPatients homozygous for the mutation.6537none4/5+4/5mild atrophynoneno1,156LGMD2LIX-II-921Jarry J. Rioux M.F. Bolduc V. Robitaille Y. Khoury V. Thiffault I. Tetreault M. Loisel L. Bouchard J.P. Brais B. A novel autosomal recessive limb-girdle muscular dystrophy with quadriceps atrophy maps to 11p13-p12.Brain. 2007; 130: 368-380Crossr