Brain Advance Access originally published online on February 2, 2005
Brain 2005 128(4):732-742; doi:10.1093/brain/awh408
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
LGMD2A: genotype–phenotype correlations based on a large mutational survey on the calpain 3 gene
1 Unidad Experimental, 2 Unidad de Genética, 3 Servicio de Neurología and 4 Servicio de Epidemiología, Hospital Donostia, San Sebastián, Basque Country, 5 Servicio de Neurología, Hospital la Fe, Valencia and 6 Servicio de Neurología, Hospital Universitario 12 de Octubre, Madrid, Spain, 7 Laboratoire de Biochimie et Génétique Moléculaire, Hôpital Cochin, 8 Institut de Myologie, Groupe Hospitalier Pitié-Sapêtrière, París, 9 Service de Neurologie, Hôpital du Haut-Lévèque, Pessac and 10 Service d'Anatomie Pathologique et de Neuropathologie et Laboratoire de Biopathologie, Nerveuse et Musculaire La Timone-IPHM, Marseille, France, 11 Laboratorio di Patologia Neuromuscolare, Istituto Ortopedico Rizzoli, Bologna, Italy, 12 Friedrich-Baur-Institute, Department of Neurology, Ludwig-Maximilians-Universität, Munich and 13 Department of Pediatric Neurology, University Children's Hospital, Göttingen, Germany
Correspondence to: A. Sáenz, Unidad Experimental, Hospital Donostia, P° Dr Begiristain s/n, 20014 San Sebastián, Basque Country, Spain E-mail: uniexpe2{at}chdo.osakidetza.net
Received September 20, 2004. Revised November 16, 2004. Accepted December 21, 2004.
 |
Summary |
|---|
 Top Summary IntroductionPatients and methodsResultsDiscussionSupplementary materialReferences |
|---|
We present here the clinical, molecular and biochemical findings from 238 limb-girdle muscular dystrophy type 2A (LGMD2A) patients, representing
50% (238 out of 484) of the suspected calpainopathy cases referred for the molecular study of the calpain 3 (CAPN3) gene. The mean age at onset of LGMD2A patients was 14 years, and the first symptoms occurred between 6 and 18 years of age in 71% of patients. The mean age at which the patients became wheelchair bound was 32.2 years, with 84% requiring the use of a wheelchair between the age of 21 and 40 years. There was no correlation between the age at onset and the time at which the patient became wheelchair bound, nor between the sex of the patient and the risk of becoming wheelchair bound. Of the cases where the CAPN3 gene was not affected, 20% were diagnosed as LGMD2I muscular dystrophy, while facioscapulohumeral muscular dystrophy (FSHD) was uncommon in this sample. We identified 105 different mutations in the CAPN3 gene of which 50 have not been described previously. These were distributed throughout the coding region of the gene, although some exons remained free of mutations. The most frequent mutation was 2362AG
TCATCT (exon 22), which was present in 30.7% of the chromosomes analysed (146 chromosomes). Other recurrent mutations described were N50S, 550
A, G222R, IVS6-1GA, A483D, IVS17+1GT, 2069–2070AC, R748Q and R748X, each of which was found in >5 chromosomes. The type of mutation in the CAPN3 gene does not appear to be a risk factor for becoming dependent on a wheelchair at a determined age. However, in the cases with two null mutations, there were significantly fewer patients that were able to walk than in the group of patients with at least one missense mutation. Despite the fact that the results of phenotyping and western blot might be biased due to multiple referral centres, producing a diagnosis on the basis of the classical phenotype is neither sufficiently sensitive (86.7%) nor specific (69.3%), although western blot proved to be even less sensitive (52.5%) yet more specific (87.8%). In this case LGMD2I was a relevant cause of false-positive diagnoses. Considering both the clinical phenotype and the biochemical information together, the probability of correctly diagnosing a calpainopathy is very high (90.8%). However, if one of the analyses is lacking, the probability varies from 78.3 to 73.7% depending on the information available. When both tests are negative, the probability that the sample comes from a patient with LGMD2A was 12.2%.
Key Words: calpain 3; limb-girdle muscular dystrophy type 2A; western blot; clinical phenotype; Bayesian analysis; epidemiology
Abbreviations: FSHD = facioscapulohumeral muscular dystrophy; LGMD = limb-girdle muscular dystrophy; CK = creatine kinase
|
Introduction |
|---|
|
TopSummaryIntroductionPatients and methodsResultsDiscussionSupplementary materialReferences |
|---|
Limb-girdle muscular dystrophies (LGMDs) are a group of muscular dystrophies characterized by a predominant involvement of the scapula, pelvic girdle and trunk muscles without affecting the facial muscles. To date, >10 different autosomal recessive LGMDs (LGMD2) have been identified as distinct entities with a similar phenotype (Laval and Bushby, 2004
). Although these different entities show some distinctive phenotypic or chronopathological features, there is a clear clinical overlap that makes their differential diagnosis difficult. LGMD type 2A (LGMD2A) has been reported to be the most frequent recessive muscular dystrophy (Dinçer et al., 1997; Richard et al., 1997; Topaloglu et al., 1997; Vainzof et al., 1999; Chae et al., 2001; de Paula et al., 2002).
The gene responsible for LGMD2A coding for calpain 3 (CAPN3) was localized by linkage to the chromosomal region 15q15.1–15.3 and subsequently cloned (Richard et al., 1995). To date, >140 different mutations have been described, yet most of them are ‘private’ variants (Chou et al., 1999; Minami et al., 1999; Richard et al., 1999; Chae et al., 2001; Pollit et al., 2001; de Paula et al., 2002; Ginjaar et al., 2002; Meznaric-Petrusa et al., 2002; Chrobáková et al., 2004; Fanin et al., 2004; MGC Leiden database: www.dmd.nl).
LGMD2A was the first form of limb-girdle dystrophy identified that is caused by a deficiency of a non-structural protein, the enzyme calpain 3. Calpain 3 (p94) is a calcium-dependent protease that may be implicated in the contractile process since it can bind strongly to titin (Sorimachi et al., 1993). Its localization in the nucleus could suggest an important role in the regulation of transcription factors that indirectly control apoptosis in muscle fibres (Kaplan et al., 1996; Beckmann et al., 1998; Baghdiguian et al., 1999, 2001) although a recent study suggests that this could be a secondary consequence (Kramerova et al., 2004).
Because mutation screening of the CAPN3 gene is relatively labour intensive, expensive and time consuming, it is of great interest for clinicians and geneticists alike to have at their disposition a diagnostic algorithm that takes into account the predictive capacity of the clinical phenotype and of the muscle protein content.
We present here the clinical, molecular and biochemical findings of a large sample of LGMD2A patients from a cohort of LGMD2 patients that underwent molecular diagnosis of the CAPN3 gene at our laboratory.
|
Patients and methods |
|---|
|
TopSummaryIntroductionPatients and methodsResultsDiscussionSupplementary materialReferences |
|---|
Patients
Samples from 484 patients from different geographic origins were analysed by mutation screening of the CAPN3 gene. A neurologist (A.L.M.) revised the clinical information when available to determine if the patients indeed fulfilled the criteria of LGMD2A using the diagnostic criteria proposed at the European Neuromuscular Centre Workshop (Beckmann and Bushby, 1996
) and that previously published by Emery (1998). However, to specify a diagnosis of LGMD2A, we used the criteria based on the experience of the Reunion Island and Basque cohorts (Fardeau et al., 1996a, b; Urtasun et al., 1998; Table 1). Sufficient clinical information was available in 314 patients to verify whether patients presented LGMD2, and information was available in 263 patients to determine whether or not their phenotype was suggestive of LGMD2A.
|
CAPN3 western blot analysis
Muscle biopsies were taken from the quadriceps femoris, biceps brachii or deltoid muscles in each centre after the patients' informed consent was obtained. Western blots for calpain 3 were performed in the different laboratories; most of them in three laboratories (117 out of 146; 86 by F.L. in Paris, 12 by L.M. in Bologna and 19 by H.L. in Munich), and all using the Calp3d/2C4 (anti-exons 1 and 3) and Calp3c/12A2 antibodies (anti-exons 3 and 8; Novocastra) as described by Anderson et al. (1998)
. Under normal conditions, three calpain 3 bands of 94, 60 and 30 kDa were detected in western blots. In some cases, the amounts of each protein were diminished to below normal levels but they were clearly present. In others, only trace amounts of the proteins were detected and the bands were scarcely perceptible, or they were absent. We carried out two analyses to determine if the presence or absence of any particular combination of bands in the western blots of calpain 3 had greater diagnostic value. In the first, we considered as normal bands whose intensity was the equivalent of control levels, and abnormal those with a diminished intensity or bands that were absent. In a second more stringent approximation, we considered as abnormal only those at trace levels or that were absent. In both cases, we evaluated the sensitivity and the specificity of each possible combination. To ensure the accuracy of the results obtained, only cases with both characterized mutations in the CAPN3 gene were considered as LGMD2A to establish the predictive value of using the phenotypic criteria and western blotting as diagnostic tools.
Molecular study
DNA was extracted from peripheral blood samples obtained after informed consent according to standard procedures at the centre of origin.
When the geographic or ethnic origin of the patient suggested a higher probability of finding previously described mutations in the CAPN3 gene, allele-specific polymerase chain reaction (PCR) was performed with the appropriate primers for each of these mutations (Don et al., 1991; Urtasun et al., 1998). In cases in which the aforementioned mutations were ruled out, only one mutation was detected or the patient was of another geographic or ethnic origin, mutation screening of the CAPN3 gene was performed using the single strand conformation polymorphism (SSCP) technique according to Richard et al. (1995, 1999). All the electrophoretic variants were sequenced in an automated sequencer.
To confirm that the alterations found were mutations and not polymorphisms, a screening was carried out with 100 control Caucasian chromosomes. In order to assess the possible confusion of the clinical diagnosis with other muscular dystrophies, the FKRP gene related to LGMD2I and the facioscapulohumeral muscular dystrophy (FSHD)-linked 4q35 deletion were analysed in two randomly chosen subsets of LGMD2A-negative cases (79 and 42 patients, respectively) by procedures previously described (Lemmers et al., 1998; Brockington et al., 2001).
Genotype–phenotype correlations
In order to produce an adequate clinical description, we have taken into account the 238 cases in which mutations were found in the CAPN3 gene (one or two mutations; Fig. 1). To avoid possible bias when interpreting the correlation between genotype and phenotype, we have only considered the cases where two mutations were identified when calculating the prognostic value of a specific mutation, the age at the onset of the symptoms or the value of the information from western blots and/or the clinical criteria proposed as diagnostic tools. To evaluate the possible correlation between the age at onset and a more severe evolution, a homogeneous sample of patients (also with two mutations identified) was selected according to the period of evolution of the disease. Since the overall mean evolution of the disease until becoming dependent on a wheelchair is 19 years (SD ±6), we analysed a subset of patients in whom the disease had evolved for at least 25 years.
|
Statistical procedures
We estimated the sensitivity, specificity and predictive values of both the proposed clinical phenotype and the use of western blots as diagnostic tools using a 2 x 2 contingency table. Exact 95% confidence intervals were calculated using a binomial distribution. The sensitivity, specificity and predictive values for the combinations of results from these two tests were also calculated using a multivariate logistic model for those patients where the information from both tests was complete. A multivariate Bayesian model was used that incorporated the dependence between clinical phenotype and western blot in those cases where the information was incomplete (Spiegelhalter, 1985
). A correlation between the age of onset and the age at which the patient becomes wheelchair bound was made using a linear regression test. Correlations between the type of mutation and the age of onset or the age at which the patient becomes wheelchair bound were made using an ANOVA (analysis of variance) test with Bonferroni adjustment. All calculations were performed using SYSTAT 9.0. Statistical significance was established when P < 0.05. |
Results |
|---|
|
TopSummaryIntroductionPatients and methodsResultsDiscussionSupplementary materialReferences |
|---|
Out of the 484 patients in this study, clinical information was available for 314 patients. Of these, 282 presented a LGMD phenotype while 32 did not fulfil the clinical criteria for LGMD2. From the resulting cohort of 282 cases, 181 presented a phenotype strongly suggestive of a calpainopathy, while 82 did not fulfil the selected criteria. In 19 cases, the clinical information available was insufficient to be able to determine whether they may or may not be suffering from a calpainopathy (Fig. 1).
Clinico-epidemiological data
Total cohort
Of the 484 patients analysed, 243 were women and 236 were men (no information regarding sex was available in five cases). The mean age at the time of the study was 34.4 years (SD ±15.7; data from 338 cases). The age at onset was available in 290 patients and the mean was 16.1 years of age (SD ±11.3; range 1–67 years). Information regarding the current status of the disease was available in 194 patients (40.1%). Seventy-five of these patients were wheelchair bound at the time of the study, but information about the age at which they became wheelchair bound was only available in 64 cases. Of these, the mean age at which they became dependent on a wheelchair was 33 years (SD ±11.1), and 50 (78%) were between 21 and 40 years old at the time they became wheelchair bound. Finally, information about the evolution from the onset of symptoms until becoming wheelchair bound was available in 62 cases and occurred within an interval of 4–44 years, with a mean evolution of 19.2 years (SD ±8.3).
Clinical data of LGMD2A
From those 238 LGMD2A patients confirmed by molecular analysis, a LGMD2 phenotype was confirmed in 171 patients. In four cases, the phenotype did not coincide with generally described LGMD, all four of which were women. In three of these cases, the clinical symptoms were compatible with a metabolic myopathy with myalgia that produced no or only slight weakness. In the fourth, the clinical picture was compatible with a congenital myopathy with slow evolution, mainly involving the girdles, and with a dubious precedent of paternal late involvement. Information was not available for 63 patients. Out of the 171 cases considered as having a LGMD2 phenotype, the phenotype of 142 was suggestive of LGMD2A according to the criteria used, while 23 did not present a phenotype suggestive of a calpainopathy according to the clinical criteria used (Table 1). In six cases, insufficient information was available. The principal criteria for excluding these 23 cases was the age at onset in relation to established criteria (early or late) and the metabolic symptoms with myalgia or cramps after exercise but no significant limb weakness (Table 2).
|
Of the 238 LGMD2A patients, 121 were women (50.8%) and 117 were men (49.2%). The age at onset was known in 175 LGMD2A patients and a mean age of 13.8 years was calculated (SD ±8.1; range 2–49). The first symptoms occurred between 6 and 18 years of age in 125 patients (71%). Out of this LGMD2A cohort, information regarding the current status of the disease was available for 137 patients (57.6%). Fifty-five of these patients were wheelchair bound at the time of the study, but information about the age at which they became dependent on a wheelchair was only available in 50 cases. Of these 50 patients, the mean age at which they became wheelchair bound was 32.2 years (SD ±8.6) and 42 (84%) were between 21 and 40 years old at that time. Information regarding the evolution from the onset of symptoms until becoming wheelchair bound was available in 48 cases, and the mean evolution was 19 years (SD ± 6.1), with an interval of 7–36 years of disease evolution.
In the homogeneous subset of patients selected according to the evolution time of the disease (
25 years of evolution), no correlation was detected between the duration of the illness and the time until the patient became wheelchair bound (Student t test, P = 0.02). We also did not detect a correlation between the sex of the patient and a differential risk of being wheelchair bound (Pearson 
2 test, P = 0.247). In the sample of LGMD2A patients, the predictive value of the proposed clinical criteria as a diagnostic tool reached a sensitivity and a specificity of 86.7% (111 out of 128) and 69.3% (88 out of 127), respectively.
Molecular data
LGMD2A cases
The 238 LGMD2A patients belonged to 187 different families, yet in 55 of these patients (from 48 different families) we were only able to find one mutation. Out of the 282 cases with a LGMD phenotype, mutations in the CAPN3 gene were found in 171. In 132 instances, two mutations were characterized, while in 39 only one mutation was detected. Among the 32 patients that did not have a LGMD phenotype, mutations in the CAPN3 gene were detected in four cases. Of these four patients, mutations were detected in both alleles in two cases, whereas in the other two we were only able to detect one mutation.
We have identified 105 different mutations of which 55 were previously described mutations and 50 were new ones. Moreover, eight unclassified variants were also identified. (Table A in the Supplementary material). In 93 cases, homozygous mutations were identified, whereas 90 patients were compound heterozygotes and in 55 patients (23%) only one of the two mutations could be detected. The mutation found most frequently was 2362AGTCATCT (exon 22), which was present in 30.7% (146 chromosomes) of the chromosomes analysed, mainly in those from the Basque Country. Indeed, 51 patients were homozygous for the 2362AG TCATCT mutation, most of them from the Basque country, while 27 were heterozygous. Sixteen polymorphisms were also characterized, nine of which were new while seven had been described previously (Table B in the Supplementary material).
Other muscular dystrophies
Of the 79 cases that were analysed at the molecular level for the FKRP gene, in 20 a phenotype suggestive of LGMD2A was observed but not associated with mutations in the CAPN3 gene. Of these, mutations were detected in the FKRP gene in four cases: two patients from Germany and two from France. Mutations in this gene were detected in 19 of the FKRP cases analysed (24%) of which 12 were from Central Europe, five from Spain and two from the USA. The predominant L276I mutation was observed in 10 cases, 47.4% of the chromosomes analysed (18 out of 38).
In 42 patients without mutations in the CAPN3 gene, molecular analysis of the FSHD gene identified three incidences (7.1%), each from a different family, where a 28 kb fragment of the D4Z4 region was found in the 4q35 chromosome. Of the patients with a phenotype suggestive of LGMD2A, molecular studies of FSHD were performed in five cases, but mutations were not found in any of them.
Phenotype–genotype correlation
In patients with at least one missense mutation, the mean age of onset was later than in patients with two null mutations (15.67 ± 8.82 versus 11.36 ± 4.24; P = 0.001). The risk of patients that had at least one missense mutation (null–missense and missense–missense) becoming wheelchair bound at a determined age was not significantly different from that for those with two stop codon mutations (Table 3). Indeed, by analysing cases in which the disease had evolved for >25 years, we found that when two null mutations were present, more patients were reliant on a wheelchair than in the group of patients with at least one missense mutation (Pearson 2 test, P = 0.047; Fisher exact test, P = 0.085).
|
Western blot analysis
Information from western blots was available from 146 patients for different structural or enzymatic muscle proteins. To analyse the predictive value of the western blots, 27 cases were ignored due to the failure to detect two mutations, and a further eight were disregarded due to the lack of information regarding the quantity of any particular protein band (Table 4).
|
A total deficit of calpain 3 (absence of the 94, 60 and 30 kDa bands in the blot) was observed in 27 cases, and 21 of these were confirmed as LGMD2A cases in the molecular analysis due to the detection of both mutations. Normal protein levels for the three bands were observed in 15 cases, of which three had been confirmed as LGMD2A cases by the detection of both mutations in the CAPN3 gene (F397L/R748X, E553K/R572W, 1743
TG/S606L; Table C in the Supplementary material). This signified that as a diagnostic tool, western blotting has a sensitivity of 52.5% and a specificity of 87.8% (Table 4). If we considered the presence of protein to be the detection of any band in the western blot, the sensitivity was slightly reduced (42.8%) and the specificity was marginally improved (Table 4). An independent analysis of the sensitivity and specificity was also carried out where each of the bands in the western blots was considered independently (Table 4). In these analyses, the sensitivity and specificity varied depending on the way in which the results were treated. If we considered all bands whose intensity differed from that of the controls as an abnormal result, we obtained a sensitivity of >80% (and in some combinations of >90%), although the specificity was very low (<40%; Figure A in the Supplementary material). However, the specificity reached 70–80% if we considered only bands at trace levels or that were absent as abnormal, but at the cost of reducing the sensitivity to barely greater than 60% in the different combinations (Fig. B in the Supplementary material). In either case, the presence of two abnormal bands (whichever they were) indicated a positive result in this test, suggesting that we were dealing with a case of LGMD2A. Indeed, the absence of the 60 kDa band signified a sensitivity of 60% with a specificity of 85% (Figs A and B in the Supplementary material). The observed highest sensitivity and specificity corresponded to the absence of the 30 and 60 kDa bands, respectively (Table 4). In eight out of 25 patients (32%) where both mutations were characterized and normal or reduced protein levels were observed, at least one mutation was localized in the muscle-specific regions (NS, IS1 and IS2) of the CAPN3 gene. Conversely, in nine out of 11 patients with mutations detected in the muscle-specific regions, different levels of protein were detectable in western blots (Table C in the Supplementary material).
Information from western blots was available for 12 of the 19 LGMD2I cases characterized. In three cases, completely normal amounts of protein were observed, whereas in four others, all bands were diminished (except one for which no information was available regarding the 30 kDa band). In three cases, each band was found at trace levels except for one case in which the 94 kDa band was in trace amounts, the 60 kDa band was diminished and no 30 kDa band was detected. Finally, in two cases the 94 kDa band was absent, one where the 60 kDa band was diminished with traces of the 30 kDa band, and another where the 60 kDa was normal but the 30 kDa band was absent.
Bayesian analysis of the clinical phenotype and western blots as diagnostic tools
To assess the relevance of these two diagnostic tools, we determined the specificity and sensitivity for the patients upon whom a complete genetic screen had been performed and for those on whom both analyses had been performed. Only 77 patients fulfilled the latter criteria and, by logistic regression, we were not able to demonstrate the superiority of one test against the other.
To avoid the limitations of such a small series and given the absence of both types of information in all patients, we performed a Bayesian multiple analysis with dependence. If the phenotype and the western blot suggested a calpainopathy, the probability of this being true was very high (90.8%). However, if one of the analyses was missing, the probability varied from 78.3 to 73.7% depending on the information available (Table 5).
|
|
Discussion |
|---|
|
TopSummaryIntroductionPatients and methodsResultsDiscussionSupplementary materialReferences |
|---|
We present here a retrospective analysis of data derived from patients at multiple centres suspected of suffering from a LGMD due to a deficit in CAPN3, from whom samples were sent for molecular analysis. Thus, the nature of the study impedes us from reaching true epidemiological conclusions. However, despite these limitations, we estimate that 60% of the patients whose phenotype approximates to LGMD correspond to LGMD2A. If we exclude the Basque population to avoid the bias introduced due to a founder effect, the percentage is slightly greater than 50%, and it decreases to 37.7% if we consider only those cases in which the two mutations could be detected.
The fact that we were unable to find mutations in the CAPN3 gene for nearly half of the cases sent for molecular study could be interpreted in different ways. In the first place, the phenotype of >10% of cases was not consistent with a diagnosis of LGMD despite the fact that the patients were referred for a molecular study of the CAPN3 gene. Moreover, among those whose phenotype was in accordance with this diagnosis, some 45% (82 out of 181) presented characteristics clearly different from those considered for the classical type 2A phenotype, even though one-third of these (23 out of 82) actually had mutations in the CAPN3 gene. Conversely, we were unable to find mutations in the CAPN3 gene in nearly 25% of the cases with a phenotype suggestive of LGMD2A. The existence of phenotypes other than that classically considered as LGMD2A may account for up to 12% of the patients diagnosed with LGMD2A using molecular techniques (Pollit et al,., 2001). This was generally the case with patients that suffered a pseudometabolic myopathy with myalgia and exercise-related cramps, or a contractural phenotype. It should be emphasized that the clinical criteria used, based on the classic phenotype described by Erb (1884) and the studies carried out in the Reunion Island and the Basque Country, are fairly sensitive but not sufficiently specific. Indeed, this should be taken into account when only the clinical information is available prior to carrying out any molecular studies.
We should also highlight that due to the technical constraints, the number of mutations found may be an underestimate since the sensitivity of SSCP is between 80 and 90% (Hayashi, 1992). This is the most plausible reason to explain why only one mutation was detected in 22% of the patients here, or in up to 43% of cases in a recently published study (Fanin et al., 2004). These cases could present a real diagnostic problem, especially when no alterations are detected in western blots and if they occur in regions with a high prevalence of carriers. In such instances, only the complete sequencing of the gene, the study of the cDNA from muscle RNA (Chrobákova et al., 2004), of the protein in leukocytes (De Tullio et al., 2003), or eventually an assay of enzymatic activity (Ono et al., 2004) would enable us to improve the diagnosis.
According to our data, LGMD2I represents an important alternative diagnosis that must be taken into account. Here, LGMD2I represented 20% of the subgroup of patients with a LGMD2A phenotype but with no mutations in the CAPN3 gene, confirming the estimations made in the UK (Poppe et al., 2002) and Central European populations (Brockington et al., 2001). In contrast, the number of cases of FSHD in our cohort appears to be mainly anecdotic, although it should not be disregarded. In 25% of the cases of LGMD2I in our cohort, calpain 3 levels were totally normal whereas in 75% the levels were diminished or even highly diminished in the muscles of these patients, despite them not suffering from LGMD2A. However, in no case was a complete absence of the protein observed. This reduction in the quantity of protein has also been observed in other types of dystrophies in which the results of western blot for the CAPN3 protein may contrast with the results of molecular studies of the CAPN3 gene (Anderson et al., 1998; Minami et al., 1999; Chae et al., 2001; Fanin et al., 2001, 2003; Pogue et al., 2001; Pollit et al., 2001).
From the clinical point of view, we should emphasize that although 71% of patients begin to show symptoms between 6 and 18 years of age, the chronological limits have widened notably with respect to the age of the first appearance of the symptoms. Indeed, in 5.7% (10 out of 175) of our subjects and 13% of those in the cohort analysed by Fanin et al., onset was not observed until after reaching 30 years of age. The analysis of the subsequent evolution of the disease supports an earlier proposal that age of onset is not a predictive factor for the severity of the progression of the disease (Pollit et al., 2001), contradicting some other studies (Fanin et al., 2004).
This could be interpreted in accordance with the hypothesis that calpain 3-mediated cleavage produces an adaptive response of muscle cells to external and/or internal stimuli (Taveau et al., 2003). Thus, calpain 3 deficiency could be compensated for in the first few years by the development of alternative mechanisms, and the deficit will begin to be noted in a more or less gradual form in terms of function of the type of mutation present in the CAPN3 gene from the moment that these mechanisms fail (Spencer et al., 2002). In fact, in our set of patients, those with null–null mutations do indeed present a greater risk of being wheelchair bound after 25 years of disease evolution than patients with other more benign types of mutations. However, the precise moment of needing a wheelchair appears to be independent of the type of mutation. This implies the existence of additional factors, genetic and/or environmental, that might regulate the expression of the phenotype, even within the same family (Penisson-Besnier et al., 1998).
In general, our data offer fairly solid support for the notion that western blot analysis is less accurate than one might have thought (Chae et al., 2001; Pollit et al., 2001; Fanin et al., 2001, 2003, 2004; Pogue et al., 2001). This variability affects patients with the same mutations (Anderson et al., 1998; Minami et al., 1999), but could be due to technical artefacts introduced when processing the biopsy sample that might lead to protein degradation (Fanin et al., 2003). Nevertheless, the fact that we are dealing with a non-standard procedure in terms of the time of processing, and that this has been carried out in various different laboratories, could have introduced certain undetected errors in our study. Despite these possible technical artefacts or selection bias due to the contribution of different centres, other dystrophies whose deficient proteins are structurally or functionally related to CAPN3 may also provoke secondary deficits in CAPN3 in muscle, as shown previously (Anderson et al., 2000; Udd et al., 2000; Fanin et al., 2001; Haravuori et al., 2001; Pogue et al., 2001; Voit et al., 2001; Figarella-Branger et al., 2002; Hackman et al., 2002). In the six cases (22%) where the protein was absent in western blots but no mutation in the CAPN3 gene was detected, the final diagnosis remains to be resolved. In four of these cases, mutations in the FKRP gene and the deletion of FSHD have been ruled out.
In a similar manner to the clinical phenotype, false-negative results were also detected in western blots. While some authors have calculated these to represent some 20% (Fanin et al., 2004), in our study these reached nearly 48% when we consider the absence of all the bands as a pathological result. It is possible that the underestimate of Fanin et al. (2004) results from the less exhaustive screening for mutations than that applied here and because they considered a decrease in any of the bands as pathological. It appears that the mutations in these cases were situated preferentially in domain III of the protein, a region in which mutations can disrupt the interactions between domains II and III. These mutations may alter the autocatalytic activity of the protein and, as a consequence, justify the presence of the protein in western blots (Jia et al., 2001; Fanin et al., 2003).
In patients with a clinical and radiological phenotype of LGMD2A but with normal protein levels in the western blots, it appears to be most appropriate to commence such screens in the IS1, IS2 and flanking regions. Indeed, one of the mutations found in our cohort from a false-negative case in western blots, S606L, has also been isolated in another similar case (Talim et al., 2001). However, this approach should not be adopted in cases where the mutations are of the stop codon type, provoking the failure to produce protein. The combinations of the bands considered in the western blots should be made according to need, or alternatively to detect the maximum number of the cases or to optimize a molecular study. In both cases, the simultaneous absence of the 94 and 60 kDa bands, of the 60 and 30 kDa bands or the isolated absence of the 60 kDa band are the most reliable indicators in terms of specificity and sensitivity (Figs A and B in the Supplementary material).
From the molecular point of view, our study confirms that apart from the ethnic or geographic groupings, no single mutation predominates. Except for 2362AGTCATCT and a few other mutations, the mutations detected were new and non-recurrent ‘private’ mutations. The prevalence of carriers of the 2362AGTCATCT mutation in some areas of the Basque Country justifies a systematic screening of this mutation in the Basque population, as has been proposed for other recessive diseases with a very prevalent mutation such as cystic fibrosis (Dankert-Roelse and te Meerman, 1997; Vries et al., 1997; Gilbert, 2001; Bobadilla et al., 2002). While the 550A mutation previously was associated with patients from the East and South-East of Europe, and more recently Italy (Topaloglu et al., 1997; Pogoda et al., 2000; Canki-Klain et al., 2004; Fanin et al., 2004), it was found here in German and French patients, confirming a wider European distribution. On the other hand, in the CAPN3 gene, the mutations N50S, A483D and IVS12-1 GA have been found in the Maghreb, where a clear predominance of limb-girdle dystrophies due to mutations in the 
, 
-sarcoglycan and FKRP genes exists. We also identified the A483D mutation in a patient of Italian origin (A483D/R748X). Of the other frequently identified mutations, the G222R mutation has only been found in the Spanish population to date (all patients except one are Basque), whereas the R748Q mutation that is found in several Spanish patients had also previously been described in a Turkish population (Dinçer et al., 1997).
The distribution of mutations throughout the CAPN3 gene does not aid in identifying clear hot-spots, other than the relative accumulation in exons 10 and 11 encountered in other studies that might reflect the large number of CpG islands in this region (Fanin et al., 2004). Indeed, the CGTG or CGCA transitions could represent a mutational mechanism due to the spontaneous deamination of the 5-methylcytosine (Cooper and Youssoufian, 1998). While the absence of mutations in exons 12, 14, 18, 23 and 24 described in other studies is confirmed, there is insufficient evidence to suggest ignoring these exons in the analysis of suspicious cases. The clinical–molecular correlation is complicated when interpreting cases of double heterozygosity due to the different functional impact of each mutation on the activity of CAPN3. Hence other factors must also regulate the phenotype since variability is observed even between patients homozygous for a given mutation. Indeed, the differences in terms of the interval of onset (14–52 years; Penisson-Besnier et al., 1998) and the evolution in patients of a similar age (60 years), still walking as well as wheelchair bound, in patients with the R461C mutation is notable (Chae et al., 2001). Such variability is even seen between siblings, for example in two 2362AG TCATCT homozygous patients in our series the age of onset differed by 2 years and each had a very different evolution. One of the brothers became wheelchair bound 12 years after the onset of symptoms, whereas the other sibling was able to walk with support 17 years after the onset (Gardner–Medwin–Walton scale stage VI). Other siblings also showed a 10 year difference in becoming wheelchair bound.
This study also set out to obtain useful information to understand the functional role of CAPN3 in the genesis of this condition, to offer the clinician clues to help them reach a diagnosis in the easiest and most accurate manner, and to be able to better predict the evolution of the illness. In this sense, the Bayesian analysis demonstrated that the probability that a patient truly suffers LGMD2A is 90% when no protein can be detected in western blots and the phenotype coincides with the characteristic LGMD2A phenotype. The phenotype is a more accurate predictor than western blots when we compare patients from whom one of the two tests is indicative and the other is not.
|
Supplementary material |
|---|
|
TopSummaryIntroductionPatients and methodsResultsDiscussionSupplementary materialReferences |
|---|
The Supplementary material cited in this article is available at Brain on-line.
|
Acknowledgements |
|---|
We would like to acknowledge the efforts of all the clinicians involved in identifying the patients especially Drs Cartault, Colomer, Fardeau, Gámez, Grid, Hammouda, Illa, Olivé, Udd and Pou. We also wish to thank Drs Gallano and Lasa from Sant Pau Hospital in Barcelona for their collaboration, Nathalie Deburgrave for her technical assistance, the patients, families and organizations involved in FENEUME for their constant support, and Dr Sefton for editing of the manuscript. This work was supported by the Association Française contre les Myopathies (AFM 1995–1999), by the Spanish Ministry of Health (Fondo de Investigaciones Sanitarias FIS 98/0040/2 and FIS PI021426) and the Ministry of Science and Technology (MCYT, MAT2002-04265-C03-03). This study was also financed by the European Union through FEDER Funds, the Health Department of the Basque Government and Gipuzkoako Foru Aldundia (Berrikuntzarako eta Jakintzaren Gizarterako Departamentua). A.S. is a post-doctoral fellow supported by the Department of Education, University and Research of the Basque Government. A.M.C. is a research fellow from the Spanish Ministry of Health. D.O. is a pre-doctoral fellow from the Department of Education, University and Research of the Basque Government. P.C., M.G. and L.B. are fellows from the ‘Fundazioa Ilundain’. C.P. is a fellow from the CSIC (Instituto de Biomedicina de Valencia). H.L. and C.B. are members of the German network on muscular dystrophies (MD-NET, 01GM0302) funded by the German Ministry of Education and Research (BMBF, Bonn, Germany).
|
References |
|---|
|
TopSummaryIntroductionPatients and methodsResultsDiscussionSupplementary materialReferences |
|---|
Anderson LV, Davison K, Moss JA, Richard I, Fardeau M, Tome FM, et al. 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: 1169–79.
Anderson LV, Harrison RM, Pogue R, Vafiadaki E, Pollitt C, Davidson K, et al. Secondary reduction in calpain 3 expression in patients with limb girdle muscular dystrophy type 2B and Miyoshi myopathy (primary dysferlinopathies). Neuromuscul Disord 2000; 10: 553–9.[CrossRef][Web of Science][Medline]
Baghdiguian S, Martin M, Richard I, Pons F, Astier C, Bourg N, et al. Calpain 3 deficiency is associated with myonuclear apoptosis and profound perturbation of the IkappaB alpha/NF-kappaB pathway in limb-girdle muscular dystrophy type 2A. Nat Med 1999; 5: 503–11.[CrossRef][Web of Science][Medline]
Baghdiguian S, Richard I, Martin M, Coopman P, Beckmann JS, Mangeat P, et al. Pathophysiology of limb girdle muscular dystrophy type 2A: hypothesis and new insights into the IkappaB alpha/NF-kappaB survival pathway in skeletal muscle. J Mol Med 2001; 79: 254–61.[CrossRef][Web of Science][Medline]
Beckmann JS, Bushby KMD. Advances in the molecular genetics of the limb-girdle type of autosomal recessive progressive muscular dystrophy. Curr Opin Neurol 1996; 9: 389–93.[CrossRef][Web of Science][Medline]
Beckmann JS, Fardeau M. Limb-girdle muscular dystrophies. In: Emery AEH, editor. Neuromuscular disorders: clinical and molecular genetics. Chichester (UK): John Wiley and Sons; 1998. p 123–56.
Bobadilla JL, Macek M Jr, Fine JP, Farrell PM. Cystic fibrosis: a worldwide analysis of CFTR mutations—correlation with incidence data and application to screening. Hum Mutat 2002; 19: 575–606.[CrossRef][Web of Science][Medline]
Brockington M, Blake DJ, Prandini P, Brown SC, Torelli S, Benson MA, et al. 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: 1198–209.[CrossRef][Web of Science][Medline]
Canki-Klain N, Milic A, Kovac B, Trlaja A, Grgicevic D, Zurak N, et al. Prevalence of the 550delA mutation in calpainopathy (LGMD2A) in Croatia. Am J Med Genet 2004; 125A: 152–6.[CrossRef]
Chae J, Minami N, Jin Y, Nakagawa M, Murayama K, Igarashi F, et al. Calpain 3 mutations: genetic and clinico-pathologic findings in limb-girdle muscular dystrophy. Neuromuscul Disord 2001; 11: 547–55.[CrossRef][Web of Science][Medline]
Chrobáková T, Hermanová M, Kroupová I, Vondrácek P, Maríkova T, Mazanec R, et al. Mutation in Czech LGMD2A patients revealed by analysis of calpain3 mRNA and their phenotypic outcome. Neuromuscul Disord 2004; 14: 659–65.[CrossRef][Web of Science][Medline]
Chou F-L, Angelini C, Daentl D, Garcia C, Greco C, Hausmanowa-Petrusewicz I, et al. Calpain III mutation analysis of a heterogeneous limb-girdle muscular dystrophy population. Neurology 1999; 52: 1015.
Cooper DN, Youssoufian H. The CpG dinucleotide and human genetic disease. Hum Genet 1998; 78: 151–5.
Dankert-Roelse JE, te Meerman GJ. Screening for cystic fibrosis—time to change our position? N Engl J Med 1997; 337: 997–9.
de Paula F, Vainzof M, Passos-Bueno MR, Pavanello RCM, Matioli SR, Anderson LVB, et al. Clinical variability in calpainopathy: what makes the difference? Eur J Hum Genet 2002; 10: 825–32.[CrossRef][Web of Science][Medline]
De Tullio R, Stifanese R, Salamino F, Pontremoli S, Melloni E. Characterization of a new p94-like calpain form in human lymphocytes. Biochem J 2003; 375: 689–96.[CrossRef][Web of Science][Medline]
Dinçer P, Leturcq F, Richard I, Piccolo F, Yalnizoglu D, de Toma C, et al. A biochemical, genetic and clinical survey of autosomal recessive limb girdle muscular dystrophies in Turkey. Ann Neurol 1997; 42: 222–9.[CrossRef][Web of Science][Medline]
Don RH, Cox PT, Wainwright BJ, Baker K, Mattick JS. ‘Touchdown’ PCR to circumvent spurious priming during gene amplification. Nucleic Acids Res 1991; 19: 4008.
Emery AEH. Neuromuscular disorders: clinical and molecular genetics. Chichester (UK): John Wiley and Sons; 1998. p. 123–56.
Erb W. Ueber de ‘juvenile form’ der progressiven muskelatrophie ihre beziehungen zur sogennnanten pseudohypertrophie der muskeln. Dtsch Archiv Klin Med 1884; 34: 467.
Fanin M, Pegoraro E, Matsuda-Asada C, Brown RH Jr, Angelini C. Calpain-3 and dysferlin protein screening in patients with limb-girdle dystrophy and myopathy. Neurology 2001; 56: 660–5.
Fanin M, Nascimbeni AC, Fulizio L, Trevisan CP, Meznaric-Petrusa M, Angelini C. Loss of calpain-3 autocatalytic activity in LGMD2A patients with normal protein expression. Am J Pathol 2003; 163: 1929–36.
Fanin M, Fulizio L, Nascimbeni AC, Spinazzi M, Piluso G, Ventriglia VM, et al. Molecular diagnosis in LGMD2A: mutation analysis or protein testing? Hum Mutat 2004; 24: 52–62.[CrossRef][Web of Science][Medline]
Fardeau M, Hillaire D, Mignard C, Feingold N, Feingold J, Mignard D, et al. Juvenile limb-girdle muscular dystrophy. Clinical, histopathological and genetic data from a small community living in the Reunion Island. Brain 1996a; 119: 295–308.
Fardeau M, Eymard B, Mignard C, Tome FM, Richard I, Beckmann JS. Chromosome 15-linked limb-girdle muscular dystrophy: clinical phenotypes in Reunion Island and French metropolitan communities. Neuromuscul Disord 1996b; 6: 447–53.[CrossRef][Web of Science][Medline]
Figarella-Branger D, El-Dassouki M, Saenz A, Cobo AM, Malzac P, Tong S, et al. Myopathy with lobulated muscle fibers: evidence for heterogeneous etiology and clinical presentation. Neuromuscul Disord 2002; 12: 4–12.[CrossRef][Web of Science][Medline]
Gilbert F. Cystic fibrosis carrier screening: step in the development of a mutation panel. Genet Test 2001; 5: 223–7.[CrossRef][Web of Science][Medline]
Ginjaar HB, Frankhuizen WS, de Mos M, Verschuuren JJGM, Hoogendijk JE, van Doorn PA, et al. Classification of limb girdle muscular dystrophy types 1C, 2A and 2B based on protein and/or DNA studies. Neuromuscul Disord 2002; 12: 731.
Hackman P, Vihola A, Haravuori H, Marchand S, Sarparanta J, De Seze J, et al. Tibial muscular dystrophy is a titinopathy caused by mutations in TTN, the gene encoding the giant skeletal-muscle protein titin. Am J Hum Genet 2002; 71: 492–500.[CrossRef][Web of Science][Medline]
Haravuori H, Vihola A, Straub V, Auranen M, Richard I, Marchand S, et al. Secondary calpain3 deficiency in 2q-linked muscular dystrophy: titin is the candidate gene. Neurology 2001; 56: 869–77.
Hayashi K. PCR-SSCP: a method for detection of mutations. Genet Anal Tech Appl 1992; 9: 73–9.[Medline]
Jia Z, Petrounevitch V, Wong A, Moldoveanu T, Davies PL, Elce JS, et al. Mutations in calpain 3 associated with limb girdle muscular dystrophy: analysis by molecular modeling and by mutation in m-calpain. Biophys J 2001; 80: 2590–6.[Web of Science][Medline]
Kaplan JC, Jeanpierre M, Urtizberea JA, Beckmann JS. Bases moléculaires des dystrophies musculaires progressives à transmission autosomique récessive. Ann Inst Pasteur 1996; 7: 157–71.
Kawai H, Akaike M, Kunishige M, Inui T, Adachi K, Kimura C, et al. Clinical, pathological, and genetic features of limb-girdle muscular dystrophy type 2A with new calpain3 gene mutations in seven patients from three Japanese families. Muscle Nerve 1998; 21: 1493–501.[CrossRef][Web of Science][Medline]
Kramerova I, Kudryashova E, Tidball JG, Spencer MJ. Null mutation of calpain 3 (p94) in mice causes abnormal sarcomere formation in vivo and in vitro. Hum Mol Genet 2004; 13: 1373–88.
Laval SH, Bushby KM. Limb-girdle muscular dystrophies—from genetics to molecular pathology. Neuropathol Appl Neurobiol 2004; 30: 91–105.[CrossRef][Web of Science][Medline]
Lemmers RJ, van der Maarel S, van Deutekom JC, van der Wielen MJ, Deidda G, Dauwerse HG, et al. Inter- and intrachromosomal sub-telomeric rearrangements on 4q35: implications for facioscapulohumeral muscular dystrophy (FSHD) aetiology and diagnosis. Hum Mol Genet 1998; 7: 1207–14.
Meznaric-Petrusa MMP, Zidar J, Zupancic N, Fanin M, Angelini C, Piluso G, et al. Clinical, molecular and genetic features of calpainopathy in Slovenia. Neuromuscul Disord 2002; 12: 731.
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: 31–7.[CrossRef][Web of Science][Medline]
Ono Y, Kakinuma K, Tori F, Irie A, Nakagawa K, Labeit S, et al. Possible regulation of the conventional calpain system by skeletal muscle-specific calpain, p94/calpain 3. J Biol Chem 2004; 279: 2761–71.
Penisson-Besnier I, Richard I, Dubas F, Beckmann JS, Fardeau M. Pseudometabolic expression and phenotypic variability of calpain deficiency in two siblings. Muscle Nerve 1998; 21: 1078–80.[CrossRef][Web of Science][Medline]
Pogoda TV, Krakhmaleva IN, Lipatova NA, Shakhovskaya NI, Shishkin SS, Limborska SA. High incidence of 550del A of calpain3 in LGMD2A patients from Russia. Hum Mut 2000; 15: 295.[Medline]
Pogue R, Anderson LV, Pyle A, Sewry C, Pollitt C, Johnson MA, et al. Strategy for mutation analysis in the autosomal recessive limb-girdle muscular dystrophies. Neuromuscul Disord 2001; 11: 80–7.[CrossRef][Web of Science][Medline]
Politano LP, Nigro V, Aurino S, Belsito A, D'Amico F, Piluso G, et al. Multiple heterozygosity for different muscle genes is not rare among individuals with pauci-symptomatic hyperckemia. Neuromuscul Disord 2002; 12: 733.
Pollitt C, Anderson LV, Pogue R, Davison K, Pyle A, Bushby KM. The phenotype of calpainopathy: diagnosis based on a multidisciplinary approach. Neuromuscul Disord 2001; 11: 287–96.[CrossRef][Web of Science][Medline]
Poppe M, Eagle M, Bourke J, Bullock R, Cree L, Buddles M, et al. Respiratory and cardiac involvement are a primary part of limb-girdle muscular dystrophy 2I. Neuromuscul Disord 2002; 12: 732.
Richard I, Beckmann JS. How neutral are synonymous codon mutations? Nat Genet 1995; 10: 259.[CrossRef][Web of Science][Medline]
Richard I, Broux O, Allamand V, Fougerousse F, Chiannikulchai N, Bourg N, et al. Mutations in the proteolytic enzyme calpain 3 cause limb-girdle muscular dystrophy type 2A. Cell 1995; 81: 27–40.[CrossRef][Web of Science][Medline]
Richard I, Brenguier L, Dinçer P, Roudaut C, Bady B, Burgunder JM, et al. Multiple independent molecular etiology for limb-girdle muscular dystrophy type 2A patients from various geographical origins. Am J Hum Genet 1997; 60: 1128–38.[Web of Science][Medline]
Richard I, Roudaut C, Saenz A, Pogue R, Grimbergen JE, Anderson LV, et al. Calpainopathy—a survey of mutations and polymorphisms. Am J Hum Genet 1999; 64: 1524–40.[CrossRef][Web of Science][Medline]
Sorimachi H, Toyama-Sorimachi N, Saido TC, Kawasaki H, Sugita H, Miyasaka M, et al. Muscle-specific calpain, p94, is degraded by autolysis immediately after translation, resulting in disappearance from muscle. J Biol Chem 1993; 268: 10593–605.
Spencer MJ, Guyon JR, Sorimachi H, Potts A, Richard I, Herasse M, et al. Stable expression of calpain 3 from a muscle transgene in vivo: immature muscle in transgenic mice suggests a role for calpain 3 in muscle maturation. Proc Natl Acad Sci USA 2002; 99: 8874–9.
Spiegelhalter D. Statistical methodology for evaluating gastrointestinal symptoms. Clin Gastroenterol 1985; 14: 489–515.[Web of Science][Medline]
Talim B, Ognibene A, Mattioli E, Richard I, Anderson LV, Merlini L. Normal calpain expression in genetically confirmed limb-girdle muscular dystrophy type 2A. Neurology 2001; 56: 692–3.
Taveau M, Bourg N, Sillon G, Roudaut C, Bartoli M, Richard I. Calpain 3 is activated through autolysis within the active site and lyses sarcomeric and sarcolemmal components. Mol Cell Biol 2003; 23: 9127–35.
Topaloglu H, Dinçer P, Richard I, Akçören Z, Alehan D, Ozme S, et al. Calpain-3 deficiency causes a mild muscular dystrophy in childhood. Neuropediatrics 1997; 28: 212–6.[Web of Science][Medline]
Udd B, Vihola A, Haravuori H, Palotie L, Labeit S, Beckmann J. Calpain 3 involvement in the pathogenesis of tibial muscular dystrophy? Neuromuscul Disord 2000; 10: 352.
Urtasun M, Saenz A, Roudaut C, Poza JJ, Urtizberea JA, Cobo AM, et al. Limb-girdle muscular dystrophy in Guipuzcoa (Basque Country, Spain). Brain 1998; 121: 1735–47.
Vainzof M, Passos-Bueno MR, Pavanello RC, Marie SK, Oliveira AS, Mayana Z. Sarcoglycanopathies are responsible for 68% of severe autosomal recessive limb-girdle muscular dystrophy in the Brazilian population. J Neurol Sci 1999; 164: 3–6.[CrossRef][Web of Science][Medline]
Voit T, Neumann B, von Deimling F, Schaper J, Schöder JM, Herrmann R. Autosomal dominant distal myopathy with secondary reduction of calpain-3. Neuromuscul Disord 2001; 11: 628.
Vries HG, Collee JM, de Walle HE, van Veldhuizen MH, Smit Sibinga CT, Scheffer H, et al. Prevalence of delta F508 cystic fibrosis carriers in The Netherlands: logistic regression on sex, age, region of residence and number of offspring. Hum Genet 1997; 99: 74–9.[CrossRef][Web of Science][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  F. L. M. Norwood, C. Harling, P. F. Chinnery, M. Eagle, K. Bushby, and V. Straub Prevalence of genetic muscle disease in Northern England: in-depth analysis of a muscle clinic population Brain, November 1, 2009; 132(11): 3175 - 3186. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Kramerova, E. Kudryashova, B. Wu, S. Germain, K. Vandenborne, N. Romain, R. G. Haller, M. A. Verity, and M. J. Spencer Mitochondrial abnormalities, energy deficit and oxidative stress are features of calpain 3 deficiency in skeletal muscle Hum. Mol. Genet., September 1, 2009; 18(17): 3194 - 3205. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. A. Amato ADULTS WITH EOSINOPHILIC MYOSITIS AND CALPAIN-3 MUTATIONS Neurology, February 26, 2008; 70(9): 730 - 731. [Full Text] [PDF]
|
|
|
|
|
|
E. J. Groen, R. Charlton, R. Barresi, L. V. Anderson, M. Eagle, J. Hudson, M. S. Koref, V. Straub, and K. M. D. Bushby Analysis of the UK diagnostic strategy for limb girdle muscular dystrophy 2A Brain, December 1, 2007; 130(12): 3237 - 3249. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M Fanin, L Nardetto, A C Nascimbeni, E Tasca, M Spinazzi, R Padoan, and C Angelini Correlations between clinical severity, genotype and muscle pathology in limb girdle muscular dystrophy type 2A J. Med. Genet., October 1, 2007; 44(10): 609 - 614. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M Fanin, A C Nascimbeni, and C Angelini Screening of calpain-3 autolytic activity in LGMD muscle: a functional map of CAPN3 gene mutations J. Med. Genet., January 1, 2007; 44(1): 38 - 43. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||