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Background: The limb girdle muscular dystrophies (LGMD) are a heterogeneous group of Mendelian disorders highlighted by weakness of the pelvic and shoulder girdle muscles. Seventeen autosomal loci have been so far identified and genetic tests are mandatory to distinguish among the forms. Mutations at the calpain 3 locus (CAPN3) cause LGMD type 2A. Objective: To obtain unbiased information on the consequences of CAPN3 mutations. Patients: 530 subjects with different grades of symptoms and 300 controls. Methods: High throughput denaturing HPLC analysis of DNA pools. Results: 141 LGMD2A cases were identified, carrying 82 different CAPN3 mutations (45 novel), along with 18 novel polymorphisms/variants. Females had a more favourable course than males. In 94% of the more severely affected patient group, the defect was also discovered in the second allele. This proves the sensitivity of the approach. CAPN3 mutations were found in 35.1% of classical LGMD phenotypes. Mutations were also found in 18.4% of atypical patients and in 12.6% of subjects with high serum creatine kinase levels. Conclusions: A non-invasive and cost–effective strategy, based on the high throughput denaturing HPLC analysis of DNA pools, was used to obtain unbiased information on the consequences of CAPN3 mutations in the largest genetic study ever undertaken. This broadens the spectrum of LGMD2A phenotypes and sets the carrier frequency at 1:103.

Protein phosphatase 2A (PP2A) has been implicated to exert its tumor suppressive function via a small subset of regulatory subunits. In this study, we reported that the specific B regulatory subunits of PP2A B56γ1 and B56γ3 mediate dephosphorylation of p53 at Thr55. Ablation of the B56γ protein by RNAi, which abolishes the Thr55 dephosphorylation in response to DNA damage, reduces p53 stabilization, Bax expression and cell apoptosis. To investigate the molecular mechanisms, we have shown that the endogenous B56γ protein level and association with p53 increase after DNA damage. Finally, we demonstrate that Thr55 dephosphorylation is required for B56γ3‐mediated inhibition of cell proliferation and cell transformation. These results suggest a molecular mechanism for B56γ‐mediated tumor suppression and provide a potential route for regulation of B56γ‐specific PP2A complex function.

Limb girdle muscular dystrophy (LGMD) type 2A (LGMD2A) is caused by mutations in the CAPN3 gene encoding for calpain‐3, a muscle specific protease. While a large number of CAPN3 gene mutations have already been described in calpainopathy patients, the diagnosis has recently shifted from molecular genetics towards biochemical assay of defective protein. However, an estimate of sensitivity and specificity of protein analysis remains to be established. Thus, we first correlated protein and molecular data in our large LGMD2A patient population. By a preliminary immunoblot screening for calpain‐3 protein of 548 unclassified patients with various phenotypes (LGMD, myopathy, or elevated levels of serum creatine kinase [hyperCKemia]), we selected 208 cases for CAPN3 gene mutation analysis: 69 had protein deficiency and 139 had normal expression. Mutation search was conducted using SSCP, denaturing high performance liquid chromatography (DHPLC), amplification refractory mutation system (ARMS‐PCR), and direct sequencing methods. We identified 58 LGMD2A mutant patients: 46 (80%) had a variable degree of protein deficiency and 12 (20%) had normal amount of calpain‐3. We calculated that the probability of having LGMD2A is very high (84%) when patients show a complete calpain‐3 deficiency and progressively decreases with the amount of protein; this new data offers an important tool for genetic counseling when only protein data are available. A total of 37 different CAPN3 gene mutations were detected, 10 of which are novel. In our population, 87% of mutant alleles were concentrated in seven exons (exons 1, 4, 5, 8, 10, 11, and 21) and 61% correspond to only eight mutations, indicating the regions where future molecular analysis could be restricted. This study reports the largest collection of LGMD2A patients so far in which both protein and gene mutations were obtained to draw genotype–protein–phenotype correlations and provide insights into a critical protein domain. Hum Mutat 24:52–62, 2004. © 2004 Wiley‐Liss, Inc.

We have previously demonstrated that gene therapy can rescue the phenotype and extend lifespan in the delta-sarcoglycan deficient cardiomyopathic hamster. In patients with similar genetic defects, steroids have been largely used to slow down disease progression. Aim of our study was to evaluate the combined effects of steroid treatment and gene therapy on cardiac function. We injected the human delta-sarcoglycan cDNA by adeno-associated virus (AAV) 2/8 by a single intraperitoneal injection into BIO14.6 Syrian hamsters at ten days of age to rescue the phenotype. We then treated the hamsters with deflazacort. Treatment was administered to half of the hamsters that had received the AAV and the other hamsters without AAV, as well as to normal hamsters. Both horizontal and vertical activities were greatly enhanced by deflazacort in all groups. As in previous experiments, the AAV treatment alone was able to preserve the ejection fraction (70±7% EF). However, the EF value declined (52±14%) with a combination of AAV and deflazacort. This was similar with all the other groups of affected animals. We confirm that gene therapy improves cardiac function in the BIO14.6 hamsters. Our results suggest that deflazacort is ineffective and may also have a negative impact on the cardiomyopathy rescue, possibly by boosting motor activity. This is unexpected and may have significance in terms of the lifestyle recommendations for patients.

Mutations in the caveolin‐3 gene (CAV3) cause limb girdle muscular dystrophy (LGMD) type 1C (LGMD1C) and other muscle phenotypes. We screened 663 patients with various phenotypes of unknown etiology, for caveolin‐3 protein deficiency, and we identified eight unreported caveolin‐deficient patients (from seven families) in whom four CAV3 mutations had been detected (two are unreported). Following our wide screening, we estimated that caveolinopathies are 1% of both unclassified LGMD and other phenotypes, and demonstrated that caveolin‐3 protein deficiency is a highly sensitive and specific marker of primary caveolinopathy. This is the largest series of caveolinopathy families in whom the effect of gene mutations has been analyzed for protein level and phenotype. We showed that the same mutation could lead to heterogeneous clinical phenotypes and muscle histopathological changes. To study the role of the Golgi complex in the pathological pathway of misfolded caveolin‐3 oligomers, we performed a histopathological study on muscle biopsies from caveolinopathy patients. We documented normal caveolin‐3 immunolabeling at the plasmalemma in some regenerating fibers showing a proliferation of the Golgi complex. It is likely that caveolin‐3 overexpression occurring in regenerating fibers (compared with caveolin‐deficient adult fibers) may lead to an accumulation of misfolded oligomers in the Golgi and to its consequent proliferation. Hum Mutat 25:82–89, 2005. © 2004 Wiley‐Liss, Inc.

Neurofibromatosis type 1 (NF1) is a relatively common single-gene disorder, and is caused by heterozygous mutations in the NF1 gene that result in a loss of activity or in a nonfunctional neurofibromin protein. Despite the common association of NF1 with neurocutaneous features, its pathology can extend to numerous tissues not derived from the neural crest. Among the rare cerebrovascular abnormalities in NF1, more than 85% of cases are of purely occlusive or stenotic nature, with intracranial aneurysm being uncommon. Predominantly, the aneurysms are located in the internal carotid arteries (ICAs), being very rare bilateral aneurysms. This report describes a very unusual case of fusiform aneurysms of both ICAs in a Caucasian NF1 patient, with a new pathogenic intragenic heterozygous deletion of the NF1 gene, presenting at age 22 years with Tolosa-Hunt syndrome, because of partial thrombosis of the left giant intracavernous aneurysm. Medical treatment with anticoagulant therapy allowed a good outcome for the patient. In conclusion, early identification of cerebral arteriopathy in NF1 and close follow-up of its progression by neuroimaging may lead to early medical or surgical intervention and prevention of significant neurologic complications.

Background: Malignant hyperthermia (MH) is a fatal autosomal dominant pharmacogenetic disorder characterized by skeletal muscle hypertonicity that causes a sudden increase in body temperature after exposure to common anesthetic agents. The disease is genetically heterogeneous, with mutations in the gene encoding the skeletal muscle ryanodine receptor (RYR1) at 19q13.1 accounting for up to 80% of the cases. To date, at least 42 RYR1 mutations have been described that cause MH and/or central core disease. Because the RYR1 gene is huge, containing 106 exons, molecular tests have focused on the regions that are more frequently mutated. Thus the causative defect has been identified in only a fraction of families as linked to chromosome 19q, whereas in others it remains undetected. Methods: We used denaturing HPLC (DHPLC) to analyze the RYR1 gene. We set up conditions to scan the 27 exons to identify both known and unknown mutations in critical regions of the protein. For each exon, we analyzed members from 52 families with positive in vitro contracture test results, but without preliminary selection by linkage analysis. Results: We identified seven different mutations in 11 MH families. Among them, three were novel MH alleles: Arg44Cys, Arg533Cys, and Val2117Leu. Conclusion: Because of its sensitivity and speed, DHPLC could be the method of choice for the detection of unknown mutations in the RYR1 gene.

Co-immunoprecipitation experiments in cell extract from cultured cells or target tissues indicated that estrogen receptor was complexed with the retinoblastoma binding protein RIZ in a ligand-dependent manner. Mapping of interaction sites indicated that in both proteins the same regions and motifs responsible for the interaction of transcriptional co-activator and nuclear receptors were involved. In cultured cells, estradiol induced a redistribution of RIZ protein within the nucleus and in the cytoplasm. A similar effect was produced in vivo, in prepuberal rat endometrium, by administration of a physiological dose of estradiol. Therefore, RIZ protein could be a specific effector of estrogen action downstream of the hormone-receptor interaction, presumably involved in proliferation control.

Co-immunoprecipitation experiments in cell extract from cultured cells or target tissues indicated that estrogen receptor was complexed with the retinoblastoma binding protein RIZ in a ligand-dependent manner. Mapping of interaction sites indicated that in both proteins the same regions and motifs responsible for the interaction of transcriptional co- activator and nuclear receptors were involved. In cultured cells, estradiol induced a redistribution of RIZ protein within the nucleus and in the cytoplasm. A similar effect was produced in vivo, in prepuberal rat endometrium, by administration of a physiological dose of estradiol. Therefore, RIZ protein could be a specific effector of estrogen action downstream of the hormone-receptor interaction, presumably involved in proliferation control.