Mutations in the fukutin-related protein (FKRP) have recently been demonstrated to cause limb girdle muscular dystrophy type 2I (LGMD2I), one of the most common forms of the autosomal recessive LGMDs in Europe. We performed a systematic clinical and muscle MRI assessment in 6 LGMD2I patients and compared these findings with those of 14 patients with genetically confirmed diagnosis of other forms of autosomal recessive LGMDs or dystrophinopathies. All LGMD2I patients had a characteristic clinical phenotype with predominant weakness of hip flexion and adduction, knee flexion and ankle dorsiflexion. These findings were also mirrored on MRI of the lower extremities which demonstrated marked signal changes in the adductor muscles, the posterior thigh and posterior calf muscles. This characteristic clinical and MRI phenotype was also seen in LGMD2A. However, in LGMD2A there was a selective involvement of the medial gastrocnemius and soleus muscle in the lower legs which was not seen in LGMD2I. The pattern in LGMD2A and LGMD2I were clearly different from the one seen in alpha-sarcoglycanopathy and dystrophinopathy type Becker which showed marked signal abnormalities in the anterior thigh muscles. Our results indicate that muscular MRI is a powerful tool for differentiating LGMD2I from other forms of autosomal recessive LGMDs and dystrophinopathies.

First described in 1974, FG syndrome (FGS) is an X-linked multiple congenital anomaly/mental retardation (MCA/MR) disorder, characterized by high clinical variability and genetic heterogeneity. Five loci (FGS1-5) have so far been linked to this phenotype on the X chromosome, but only one gene, MED12, has been identified to date. Mutations in this gene account for a restricted number of FGS patients with a more distinctive phenotype, referred to as the Opitz-Kaveggia phenotype. We report here that a p.R28L (c.83G-->T) missense mutation in CASK causes FGS phenotype in an Italian family previously mapped to Xp11.4-p11.3 (FGS4). The identified missense mutation cosegregates with the phenotype in this family and is absent in 1000 control X chromosomes of the same ethnic origin. An extensive analysis of CASK protein functions as well as structural and dynamic studies performed by molecular dynamics (MD) simulation did not reveal significant alterations induced by the p.R28L substitution. However, we observed a partial skipping of the exon 2 of CASK, presumably a consequence of improper recognition of exonic splicing enhancers (ESEs) induced by the c.83G-->T transversion. CASK is a multidomain scaffold protein highly expressed in the central nervous system (CNS) with specific localization to the synapses, where it forms large signaling complexes regulating neurotransmission. We suggest that the observed phenotype is most likely a consequence of an altered CASK expression profile during embryogenesis, brain development, and differentiation.

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.

Dysferlin is a 237-kDa transmembrane protein involved in calcium-mediated sarcolemma resealing. Dysferlin gene mutations cause limb-girdle muscular dystrophy (LGMD) 2B, Miyoshi myopathy (MM) and distal myopathy of the anterior tibialis. Considering that a secondary Dysferlin reduction has also been described in other myopathies, our original goal was to identify cases with a Dysferlin deficiency without dysferlin gene mutations. The dysferlin gene is huge, composed of 55 exons that span 233 140 bp of genomic DNA. We performed a thorough mutation analysis in 65 LGMD/MM patients with ≤20% Dysferlin. The screening was exhaustive, as we sequenced both genomic DNA and cDNA. When required, we used other methods, including real-time PCR, long PCR and array CGH. In all patients, we were able to recognize the primary involvement of the dysferlin gene. We identified 38 novel mutation types. Some of these, such as a dysferlin gene duplication, could have been missed by conventional screening strategies. Nonsense-mediated mRNA decay was evident in six cases, in three of which both alleles were only detectable in the genomic DNA but not in the mRNA. Among a wide spectrum of novel gene defects, we found the first example of a ‘nonstop' mutation causing a dysferlinopathy. This study presents the first direct and conclusive evidence that an amount of Dysferlin ≤20% is pathogenic and always caused by primary dysferlin gene mutations. This demonstrates the high specificity of a marked reduction of Dysferlin on western blot and the value of a comprehensive molecular approach for LGMD2B/MM diagnosis.