Prevalence of the 550delA mutation in calpainopathy (LGMD 2A) in Croatia

Mutations in the calpain 3 (CAPN3) gene are responsible for limb-girdle muscular dystrophy (LGMD) type 2A. We report five causal mutations: 550delA, DeltaFWSAL, R541W, Y357X and R49H found on 45/50 of alleles studied in 25 unrelated families from Croatia. The 550delA mutation was present on 76% of CAPN3 chromosomes that led us to screen general population for this mutation; 532 random blood samples from three different regions were analyzed using allele-specific PCR. Four healthy 550delA heterozygous were found suggesting a frequency of 1 in 133. All four carriers detected originated from an island and mountain region close to the Adriatic Sea. These findings combined with haplotype analysis confirm that our general population is rather "closed" with a probable founder effect in some parts of the country. In addition, the high frequency of 550delA mutation found in some neighboring European countries together with the easy detection of the 550delA mutation should streamline genetic analysis, especially bearing in mind the geographic and ethnic origin of the patients. Our results, combined with published haplotype studies suggest that 550delA originated in the Eastern Mediterranean from which it has probably spread widely across Europe. Extending this study to other areas would help to address this epidemiological question. Our data are relevant to accurate genetic counseling and patient testing since we lack sensitive and specific biopsy screening methods for detecting patients with calpainopathy. Thus, detection of patients relies on the direct detection of gene mutation and our findings may be helpful in establishing diagnostic screening strategy.     

Asymptomatic carriers for homozygous novel mutations in the FKRP gene: the other end of the spectrum

Autosomal recessive limb-girdle muscular dystrophy linked to 19q13.3 (LGMD2I) was recently related to mutations in the fukutin-related protein gene (FKRP) gene. Pathogenic changes in the same gene were detected in congenital muscular dystrophy patients (MDC1C), a severe disorder. We have screened 86 LGMD genealogies to assess the frequency and distribution of mutations in the FKRP gene in Brazilian LGMD patients. We found 13 Brazilian genealogies, including 20 individuals with mutations in the FKRP gene, and identified nine novel pathogenic changes. The commonest C826A European mutation was found in 30% (9/26) of the mutated LGMD2I alleles. One affected patient homozygous for the FKRP (C826A) mutation also carries a missense R125H change in one allele of the caveolin-3 gene (responsible for LGMD1C muscular dystrophy). Two of her normal sibs were found to be double heterozygotes. In two unrelated LGMD2I families, homozygous for novel missense mutations, we identified four asymptomatic carriers, all older than 20 years. Genotype-phenotype correlation studies in the present study as well as in patients from different populations suggests that the spectrum of variability associated with mutations in the FKRP gene seems to be wider than in other forms of LGMD. It also reinforces the observations that pathogenic mutations are not always determinant of an abnormal phenotype, suggesting the possibility of other mechanisms modulating the severity of the phenotype that opens new avenues for therapeutic approaches.     

Genomic screening for beta-sarcoglycan gene mutations: missense mutations may cause severe limb-girdle muscular dystrophy type 2E (LGMD 2E)

Autosomal recessive limb-girdle muscular dystrophies (LGMDs) are genetically heterogeneous. A subgroup of these disorders is caused by mutations in the dystrophin-associated sarcoglycan complex. Truncating mutations in the 43 kDa beta-sarcoglycan gene (LGMD 2E) were originally identified in a sporadic case of Duchenne-like muscular dystrophy, and a common missense mutation (T151R) was identified independently in Indiana Amish pedigrees with a milder form of LGMD. To facilitate mutational analysis of larger numbers of patients directly from genomic DNA, as opposed to reverse transcribed RNA from muscle biopsies, we have determined the genomic structure of the beta-sarcoglycan gene. The open reading frame of the beta-sarcoglycan coding region extends over six exons. Primers were designed for PCR amplification of single exons from genomic DNA and subsequent single strand conformation polymorphism (SSCP) analysis. We screened 15 patients from the Brazilian LGMD patient population, 13 of whom followed a severe course. Most of the patients had been assessed previously for deficiency of alpha- sarcoglycan immunofluorescence on muscle biopsy sections as a marker for disease of the sarcoglycan complex. Novel mutations in two familial and two sporadic cases of severe childhood-onset LGMD were identified. Only one of these patients carried a truncating mutation (homozygous 2 bp deletion, FS164TER), while the other three carried missense mutations (homozygous R91P, homozygous M100K, heterozygous recessive L108R; only one allele could be identified in this family). All three missense mutations occurred in exon 3, coding for the immediate extracellular domain. Complete absence for all three of the known sarcoglycans was noted by immunohistochemistry on muscle biopsy sections of the patients.      

Identical mutation in patients with limb girdle muscular dystrophy type 2B or Miyoshi myopathy suggests a role for modifier gene(s)

Limb girdle muscular dystrophy type 2B (LGMD2B) and Miyoshi myopathy (MM), a distal muscular dystrophy, are both caused by mutations in the recently cloned gene dysferlin, gene symbol DYSF. Two large pedigrees have been described which have both types of patient in the same families. Moreover, in both pedigrees LGMD2B and MM patients are homozygous for haplotypes of the critical region. This suggested that the same mutation in the same gene would lead to both LGMD2B or MM in these families and that additional factors were needed to explain the development of the different clinical phenotypes. In the present paper we show that in one of these families Pro791 of dysferlin is changed to an Arg residue. Both the LGMD2B and MM patients in this kindred are homozygous for this mutation, as are four additional patients from two previously unpublished families. Haplotype analyses suggest a common origin of the mutation in all the patients. On western blots of muscle, LGMD2B and MM patients show a similar abundance in dysferlin staining of 15 and 11%, respectively. Normal tissue sections show that dysferlin localizes to the sarcolemma while tissue sections from MM and LGMD patients show minimal staining which is indistinguishable between the two types. These findings emphasize the role for the dysferlin gene as being responsible for both LGMD2B and MM, but that the distinction between these two clinical phenotypes requires the identification of additional factor(s), such as modifier gene(s).     

A Chinese familial growth hormone deficiency with a deletion of 7.1 kb of DNA.

Using restriction endonuclease analysis and a human growth hormone cDNA probe, we have found a Chinese family with a human growth hormone gene deletion. Two affected sibs are homozygous for a deletion of approximately 7.1 kb of DNA, which contains the normal human growth hormone gene. The patients' parents and grandmothers are heterozygous for the deleted gene. Their grandfathers are normal and homozygous for the hGH-N gene. All of them have normal stature.     

Mutations in the fukutin-related protein gene (FKRP) identify limb girdle muscular dystrophy 2I as a milder allelic variant of congenital muscular dystrophy MDC1C.

The limb girdle and congenital muscular dystrophies (LGMD and CMD) are characterized by skeletal muscle weakness and dystrophic muscle changes. The onset of symptoms in CMD is within the first few months of life, whereas in LGMD they can occur in late childhood, adolescence or adult life. We have recently demonstrated that the fukutin-related protein gene (FKRP) is mutated in a severe form of CMD (MDC1C), characterized by the inability to walk, leg muscle hypertrophy and a secondary deficiency of laminin alpha2 and alpha-dystroglycan. Both MDC1C and LGMD2I map to an identical region on chromosome 19q13.3. To investigate whether these are allelic disorders, we undertook mutation analysis of FKRP in 25 potential LGMD2I families, including some with a severe and early onset phenotype. Mutations were identified in individuals from 17 families. A variable reduction of alpha-dystroglycan expression was observed in the skeletal muscle biopsy of all individuals studied. In addition, several cases showed a deficiency of laminin alpha2 either by immunocytochemistry or western blotting. Unexpectedly, affected individuals from 15 families had an identical C826A (Leu276Ileu) mutation, including five that were homozygous for this change. Linkage analysis identified at least two possible haplotypes in linkage disequilibrium with this mutation. Patients with the C826A change had the clinically less severe LGMD2I phenotype, suggesting that this is a less disruptive FKRP mutation than those found in MDC1C. The spectrum of LGMD2I phenotypes ranged from infants with an early presentation and a Duchenne-like disease course including cardiomyopathy, to milder phenotypes compatible with a favourable long-term outcome.     

Intragenic deletion of TRIM32 in compound heterozygotes with sarcotubular myopathy/LGMD2H

In 2005 the commonality of sarcotubular myopathy (STM) and limb girdle muscular dystrophy type 2H (LGMD2H) was demonstrated, as both are caused by the p D487N missense mutation in TRIM32 originally found in the Manitoba Hutterite population. Recently, three novel homozygous TRIM32 mutations have been described in LGMD patients. Here we describe a three generation Swedish family clinically presenting with limb girdle muscular weakness and histological features of a microvacuolar myopathy. The two index patients were compound heterozygotes for a frameshift mutation in TRIM32 (c.1560delC ) and a 30 kb intragenic deletion, encompassing parts of intron 1 and the entire exon 2 of TRIM32. In these patients, no full‐length or truncated TRIM32 could be detected. Interestingly, heterozygous family members carrying only one mutation showed mild clinical symptoms and vacuolar changes in muscle. In our family, the phenotype encompasses additionally a mild demyelinating polyneuropathic syndrome. Thus STM and LGMD2H are the result of loss of function mutations that can be either deletions or missense mutations. © 2009 Wiley‐Liss, Inc.     

Mutations of the cathepsin C gene are responsible for Papillon-Lefèvre syndrome

Papillon-Lefèvre syndrome (PLS) is an autosomal recessive disorder characterised by palmoplantar hyperkeratosis and severe early onset periodontitis that results in the premature loss of the primary and secondary dentitions. A major gene locus for PLS has been mapped to a 2.8 cM interval on chromosome 11q14. Correlation of physical and genetic maps of this interval indicate it includes at least 40 ESTs and six known genes including the lysosomal protease cathepsin C gene (CTSC). The CTSC message is expressed at high levels in a variety of immune cells including polymorphonuclear leucocytes, macrophages, and their precursors. By RT-PCR, we found CTSC is also expressed in epithelial regions commonly affected by PLS, including the palms, soles, knees, and oral keratinised gingiva. The 4.7 kb CTSC gene consists of two exons. Sequence analysis of CTSC from subjects affected with PLS from five consanguineous Turkish families identified four different mutations. An exon 1 nonsense mutation (856C-->T) introduces a premature stop codon at amino acid 286. Three exon 2 mutations were identified, including a single nucleotide deletion (2692delA) of codon 349 introducing a frameshift and premature termination codon, a 2 bp deletion (2673-2674delCT) that results in introduction of a stop codon at amino acid 343, and a G-->A substitution in codon 429 (2931G-->A) introducing a premature termination codon. All PLS patients were homozygous for cathepsin C mutations inherited from a common ancestor. Parents and sibs heterozygous for cathepsin C mutations do not show either the palmoplantar hyperkeratosis or severe early onset periodontitis characteristic of PLS. A more complete understanding of the functional physiology of cathepsin C carries significant implications for understanding normal and abnormal skin development and periodontal disease susceptibility.     

Mutations that impair interaction properties of TRIM32 associated with limb-girdle muscular dystrophy 2H

TRIM32 belongs to a large family of proteins characterized by a tripartite motif, possibly involved in the ubiquitination process, acting as an E3 ligase. In addition, TRIM32 has six NHL repeats with putative interaction properties. A homozygous mutation at the third NHL repeat (D487N) has been found in patients with limb girdle muscular dystrophy 2H (LGMD2H). This mutation was only identified in the inbred Manitoba Hutterite or their descendants. Interestingly, a mutation in the B-box domain of TRIM32 cosegregates with Bardet-Biedl syndrome type 11 (BBS11). The signs of BBS11 include obesity, pigmentary and retinal malformations, diabetes, polydactyly, and no muscular dystrophy, suggesting an alternative disease mechanism. We aim to ascertain whether D487N is the only pathological LGMD2H allele, limited to Hutterites. We studied the TRIM32 gene in 310 LGMD patients with no mutations at the other known loci. We identified four patients with novel mutated alleles. Two mutations were homozygous and missing in controls. These mutations also clustered at the NHL domain, suggesting that a specific (interaction) property might be abolished in LGMD2H patients. No mutations were found at the B-box region where the BBS11 mutation is found. We tested TRIM32 and its mutants by yeast-two-hybrid assay, developing an interaction test to validate mutations. All LGMD2H mutants, but not the BBS11, lost their ability to self-interact. The interaction of TRIM32 mutants with E2N, a protein involved in the ubiquitination process, was similarly impaired. In conclusion, the mutations here reported may cause muscular dystrophy by affecting the interaction properties of TRIM32.     

Mapping of the formin gene and exclusion as a candidate gene for the autosomal recessive form of limb-girdle muscular dystrophy.

Limb-Girdle Muscular Dystrophy (LGMD) is a myopathy with clinical and transmission heterogeneity. The recessive form, LGMD2, has been recently mapped by linkage analysis to 15q. As an attempt to identify the gene involved in this pathology, we tested as candidate gene the LD locus, called LD for limb deformity. This gene has recently been identified and mapped to chromosome 15q13-q14. It is homologous to the murine formin gene which is localized to mouse chromosome 2. Mutations in this murine gene have been shown to cause limb deformity and kidney defect. YAC clones containing the LD gene were isolated and utilised to confirm the cytogenetic localisation. Internal DNA polymorphisms of the LD locus were analyzed in LGMD2 and CEPH families. The LD gene was mapped between the alpha cardiac actin gene and the D15S24 locus. Crossovers between the LGMD2 and the LD loci excluded the LD gene as a candidate for LGMD2.     

A wide variety of mutations in the parkin gene are responsible for autosomal recessive parkinsonism in Europe. French Parkinson's Disease Genetics Study Group and the European Consortium on Genetic Susceptibility in Parkinson's Disease

Autosomal recessive juvenile parkinsonism (AR-JP, PARK2; OMIM 602544), one of the monogenic forms of Parkinson's disease (PD), was initially described in Japan. It is characterized by early onset (before age 40), marked response to levodopa treatment and levodopa-induced dyskinesias. The gene responsible for AR-JP was recently identified and designated parkin. We have analysed the 12 coding exons of the parkin gene in 35 mostly European families with early onset autosomal recessive parkinsonism. In one family, a homozygous deletion of exon 4 could be demonstrated. By direct sequencing of the exons in the index patients of the remaining 34 families, eight previously undescribed point mutations (homozygous or heterozygous) were detected in eight families that included 20 patients. The mutations segregated with the disease in the families and were not detected on 110-166 control chromosomes. Four mutations caused truncation of the parkin protein. Three were frameshifts (202-203delAG, 255delA and 321-322insGT) and one a nonsense mutation (Trp453Stop). The other four were missense mutations (Lys161Asn, Arg256Cys, Arg275Trp and Thr415Asn) that probably affect amino acids that are important for the function of the parkin protein, since they result in the same phenotype as truncating mutations or homozygous exon deletions. Mean age at onset was 38 +/- 12 years, but onset up to age 58 was observed. Mutations in the parkin gene are therefore not invariably associated with early onset parkinsonism. In many patients, the phenotype is indistinguishable from that of idiopathic PD. This study has shown that a wide variety of different mutations in the parkin gene are a common cause of autosomal recessive parkinsonism in Europe and that different types of point mutations seem to be more frequently responsible for the disease phenotype than are deletions.     

Detection of TRIM32 deletions in LGMD patients analyzed by a combined strategy of CGH array and massively parallel sequencing

Defects in TRIM32 were reported in limb-girdle muscular dystrophy type 2H (LGMD2H), sarcotubular myopathies (STM) and in Bardet-Biedl syndrome. Few cases have been described to date in LGMD2H/STM, but this gene is not systematically analysed because of the absence of specific signs and difficulties in protein analysis. By using high-throughput variants screening techniques, we identified variants in TRIM32 in two patients presenting nonspecific LGMD. We report the first case of total inactivation by homozygous deletion of the entire TRIM32 gene. Of interest, the deletion removes part of the ASTN2 gene, a large gene in which TRIM32 is nested. Despite the total TRIM32 gene inactivation, the patient does not present a more severe phenotype. However, he developed a mild progressive cognitive impairment that may be related to the loss of function of ASTN2 because association between ASTN2 heterozygous deletions and neurobehavioral disorders was previously reported. Regarding genomic characteristics at breakpoint of the deleted regions of TRIM32, we found a high density of repeated elements, suggesting a possible hotspot. These observations illustrate the importance of high-throughput technologies for identifying molecular defects in LGMD, confirm that total loss of function of TRIM32 is not associated with a specific phenotype and that TRIM32/ASTN2 inactivation could be associated with cognitive impairment.     

Loss of calpain-3 autocatalytic activity in LGMD2A patients with normal protein expression

The diagnosis of limb girdle muscular dystrophy (LGMD) type 2A (due to mutations in the gene encoding for calpain-3) is currently based on protein analysis, but mutant patients with normal protein expression have also been identified. In this study we investigated 150 LGMD patients with normal calpain-3 protein expression, identified gene mutations by an allele-specific polymerase chain reaction test, and analyzed the mutant calpain-3 catalytic activity. Four different mutations were found in eight patients (5.5%): a frame-shifting deletion (550 A del) and three missense (R490Q, R489Q, R490W). Patients with normal calpain-3 protein expression on Western blot are a considerable proportion (20%) of our total LGMD2A population. While in control muscle the calpain-3 Ca(++)-dependent autocatalytic activity was evident within 5 minutes and was prevented by ethylene diaminetetraacetic acid, in all mutant patient samples the protein was not degraded, indicating that the normal autocatalytic function had been lost. By this new functional test, we show that conventional protein diagnosis fails to detect some mutant proteins, and prove the pathogenetic role of R490Q, R489Q, R490W missense mutations. We suggest that these mutations impair protein activity by affecting interdomain protein interaction, or reduce autocatalytic activity by lowering the Ca(++) sensitivity.     

Unexpected genetic heterogeneity in a large consanguineous Brazilian pedigree presenting deafness

Nonsyndromic autosomal recessive deafness accounts for 80% of hereditary deafness. To date, 52 loci responsible for autosomal recessive deafness have been mapped and 24 genes identified. Here, we report a large inbred Brazilian pedigree with 26 subjects affected by prelingual deafness. Given the extensive consanguinity found in this pedigree, the most probable pattern of inheritance is autosomal recessive. However, our linkage and mutational analysis revealed, instead of an expected homozygous mutation in a single gene, two different mutant alleles and a possible third undetected mutant allele in the MYO15A gene (DFNB3 locus), as well as evidence for other causes for deafness in the same pedigree. Among the 26 affected subjects, 15 were homozygous for the novel c.10573delA mutation in the MYO15A gene, 5 were compound heterozygous for the mutation c.10573delA and the novel deletion c.9957_9960delTGAC and one inherited only a single c.10573delA mutant allele, while the other one could not be identified. Given the extensive consanguinity of the pedigree, there might be at least one more deafness locus segregating to explain the condition in some of the subjects whose deafness is not clearly associated with MYO15A mutations, although overlooked environmental causes could not be ruled out. Our findings illustrate a high level of etiological heterogeneity for deafness in the family and highlight some of the pitfalls of genetic analysis of large genes in extended pedigrees, when homozygosity for a single mutant allele is expected.     

Sarcoglycanopathies and the risk of undetected deletion alleles in diagnosis

We have designed Multiplex Amplifiable Probe Hybridization (MAPH) probes for 28 exons of the sarcoglycan genes SGCA, SGCB, SGCG, and SGCD. The set was used to screen DNA from limb-girdle muscular dystrophy (LGMD) patients for the presence of pathogenic deletion or duplication mutations. An unexpected heterozygous deletion of SGCG exon 7 was detected in a patient from a consanguineous family in which a known c.525delT mutation segregates. The exon 7 deletion was inherited from the father, who was part of the consanguineous c.525delT branch of the family but who did not carry the c.525delT mutation. A similar, homozygous deletion had been identified in two unrelated LGMD patients from southern Italy. The deletion breakpoints were mapped, isolated, and sequenced, and were identical in all cases. Haplotype analysis showed the same alleles segregating with the mutation in all three patients, suggesting a common ancestor. Exonic deletions in sarcoglycanopathies appear to be rare events. However, we recommend screening for exonic deletions/duplications in patients where a mutation has not been identified in both alleles, as well as in seemingly homozygous cases where segregation of the mutations can not be confirmed in the parents.     

A founder mutation in the gamma-sarcoglycan gene of gypsies possibly predating their migration out of India

We investigated the molecular basis of a severe form of early onset autosomal recessive muscular dystrophy with sarcoglycan (SG) deficiency in seven large Gypsy families living in different parts of Western Europe and apparently not closely related. They were linked to the LGMD2C locus (13q12) suggesting a primary defect in the gamma-SG gene coding for the 35 kDa dystrophin-associated glycoprotein. All of the 18 investigated patients were homozygous for the same G-->A transition in codon 283 producing the replacement of a conserved cysteine of the extra-cellular domain of the protein by a tyrosine. All affected chromosomes in homozygous and heterozygous relatives carried the same allele 5 of the intragenic marker D13S232. Flanking markers were studied to delineate a common ancestral haplotype, the size of which was used to compute the date of the founding mutation. We found evidence that the mutation occurred between 60 and 200 generations ago, therefore possibly predating the commonly accepted date of migration of the Gypsy ancestors out of India.      

Evidence of genetic heterogeneity in the autosomal recessive adult forms of limb-girdle muscular dystrophy following linkage analysis with 15q probes in Brazilian families.

The autosomal recessive limb-girdle muscular dystrophies (LGMD) represent a heterogeneous group of diseases which may be characterised by one or more autosomal loci. A gene at 15q has recently been found to be responsible for a mild form of LGMD in a group of families from the isolated island of Réunion, now classified as LGMD2. Based on results of eight out of 11 large Brazilian LGMD families of different racial background (which were informative for the closest available probe to the LGMD2 gene), we confirmed linkage to the LGMD2 gene at 15q in two of these families and exclusion in six others. These data provide the first evidence of genetic heterogeneity for the autosomal recessive limb-girdle muscular dystrophies.     

Restricted genetic defects underlie human complement C6 deficiency

Complement C6 homozygous deficiency (C6D) has been rarely observed in Caucasians but was reported at higher prevalence among African-Americans. We report on the molecular basis of C6D in seven unrelated black individuals of North or Central Africa descent who live in France. These patients have presented Neisseria meningitidis infection (four cases), focal and segmental glomerulosclerosis with hyalinosis (one case), systemic lupus erythematosus (one case) or Still's disease (one case). All patients exhibited undetectable antigenic C6 by using a sensitive ELISA assay. An additional four cases of complete C6 deficiency with no associated disease have been characterized after family studies. Exons 6, 7 and 12 have been described recently as the location of molecular defects on the C6 gene in randomly chosen black Americans. Genomic DNA from the seven patients were subjected to direct polymerase chain reaction amplification of these three exons. Nucleotide sequencing analysis of the amplified DNA fragments revealed a homozygous single-base deletion (1936delG) in exon 12 in three cases and four compound heterozygous deletions for a single base in exon 7 (1195delC) or in exon 6 (878delA) associated with the same deletion in exon 12 (1936delG). Our observations further establish the restricted pattern of genetic defects associated with homozygous C6 complement deficiency in individuals of African descent.     

Deoxyguanosine kinase mutations and combined deficiencies of the mitochondrial respiratory chain in patients with hepatic involvement

The activity of deoxyguanosine kinase (DGUOK), a mitochondrial enzyme involved in the anabolism of mitochondrial (mt) deoxyribonucleotides, governs the maintenance of the mtDNA. Deleterious mutations of the DGUOK gene are thus associated with mtDNA depletion and result in combined deficiencies of mtDNA-encoded respiratory chain enzymes. With the aim to estimate the prevalence of DGUOK mutations in a cohort of 30 patients with hepatocerebral disease and combined respiratory chain deficiencies, we studied the DGUOK gene and identified previously unreported mutations in five families. Two patients and their affected sibs, born to non-consanguineous parents, were homozygous for a missense mutation (M1T, and L250S, respectively). One patient presented a homozygous 4 pb insertion (796 insTGAT) and two other patients, and their affected sibs, were compound heterozygous (E165V/L266R and E211G/L266R, respectively). These findings allowed us to propose prenatal diagnosis in two families. In conclusion, we observed a high prevalence of DGUOK mutations (17%) in patients with hepatic involvement and combined respiratory chain deficiencies with hepatic involvement.