Eloisa S. Moreira

Limb-girdle muscular dystrophy type 2G is caused by mutations in the gene encoding the sarcomeric protein telethonin

Autosomal recessive limb-girdle muscular dystrophies (AR LGMDs) are a genetically heterogeneous group of disorders that affect mainly the proximal musculature. There are eight genetically distinct forms of AR LGMD, LGMD 2A–H (refs 2–10), and the genetic lesions underlying these forms, except for LGMD 2G and 2H, have been identified. LGMD 2A and LGMD 2B are caused by mutations in the genes encoding calpain 3 (ref. 11) and dysferlin, respectively, and are usually associated with a mild phenotype. Mutations in the genes encoding γ-(ref. 14), α-(ref. 5), β-(refs 6,7) and δ (ref. 15)-sarcoglycans are responsible for LGMD 2C to 2F, respectively. Sarcoglycans, together with sarcospan, dystroglycans, syntrophins and dystrobrevin, constitute the dystrophin-glycoprotein complex (DGC). Patients with LGMD 2C–F predominantly have a severe clinical course. The LGMD 2G locus maps to a 3-cM interval in 17q11–12 in two Brazilian families with a relatively mild form of AR LGMD (ref. 9). To positionally clone the LGMD 2G gene, we constructed a physical map of the 17q11–12 region and refined its localization to an interval of 1.2 Mb. The gene encoding telethonin, a sarcomeric protein, lies within this candidate region. We have found that mutations in the telethonin gene cause LGMD 2G, identifying a new molecular mechanism for AR LGMD.

The Seventh Form of Autosomal Recessive Limb-Girdle Muscular Dystrophy Is Mapped to 17q11-12

The group of autosomal recessive (AR) muscular dystrophies includes, among others, two main clinical entities, the limb-girdle muscular dystrophies (LGMDs) and the distal muscular dystrophies. The former are characterized mainly by muscle wasting of the upper and lower limbs, with a wide range of clinical severity. This clinical heterogeneity has been demonstrated at the molecular level, since the genes for six AR forms have been cloned and/or have been mapped to 15q15.1 (LGMD2A), 2p12-16 (LGMD2B), 13q12 (LGMD2C), 17q12-q21.33 (LGMD2D),4q12 (LGMD2E), and 5q33-34 (LGMD2F). The AR distal muscular dystrophies originally included two subgroups, Miyoshi myopathy, characterized mainly by extremely elevated serum creatine kinase (CK) activity and by a dystrophic muscle pattern, and Nonaka myopathy, which is distinct from the others because of the normal to slightly elevated serum CK levels and a myopathic muscle pattern with rimmed vacuoles. With regard to our unclassified AR LGMD families, analysis of the affected sibs from one of them (family LG61) revealed some clinical and laboratory findings (early involvement of the distal muscles, mildly elevated serum CK levels, and rimmed vacuoles in muscle biopsies) that usually are not observed in the analysis of patients with LGMD2A-LGMD2F. In the present investigation, through a genomewide search in family LG61, we demonstrated linkage of the allele causing this form of muscular dystrophy to a 3-cM region on 17q11-12. We suggest that this form, which, interestingly, clinically resembles AR Kugelberg-Welander disease, should be classified as LGMD2G. In addition, our results indicate the existence of still another locus causing severe LGMD.

Seven autosomal recessive limb-girdle muscular dystrophies in the Brazilian population: from LGMD2A to LGMD2G

The autosomal recessive limb-girdle muscular dystrophies (AR-LGMDs) are a heterogeneous group of disorders of progressive weakness of the pelvic and shoulder girdle musculature. The clinical course is characterized by great variability, ranging from severe forms with onset in the first decade and rapid progression resembling clinically Xp21 Duchenne muscular dystrophy (DMD) to milder forms with later onset and slower course. Eight genes are mapped for the AR-LGMDs; they are: LGMD2A (CAPN3) at 15q, LGMD2B (dysferlin) at 2p, LGMD2C (gamma-SG) at 13q, LGMD2D (alpha-SG) at 17q, LGMD2E (beta-SG) at 4q, LGMD2F (6-SG) at 5q, LGMD2G at 17q, and more recently LGMD2H at 9q. The LGMD2F (delta-SG) and LGMD2G genes were mapped in Brazilian AR-LGMD families. Linkage analysis in two unlinked families excluded the eight AR-LGMD genes, indicating that there is at least one more gene responsible for AR-LGMD. We have analyzed 140 patients (from 40 families) affected with one of seven autosomal recessive LGMD loci, that is, from LGMD2A to LGMD2G. The main observations were: 1) all LGMD2E and LGMD2F patients had a severe condition, but considerable inter- and intra-familial clinical variability was observed among patients from all other groups; 2) serum CK activities showed the highest values in LGMD2D (alpha-SG) patients among sarcoglycanopathies and LGMD2B (dysferlin) patients among nonsarcoglycanopathies; 3) comparison between LGMD2A (CAPN3) and LGMD2B (dysferlin) showed that the first have on average a more severe course and have calf hypertrophy more frequently (86% versus 13%); and 4) inability to walk on toes was observed in approximately 70% of LGMD2B patients.

Dysferlin protein analysis in limb-girdle muscular dystrophies

Dysferlin is the protein product of the DYSF gene mapped at 2p31, which mutations cause limb-girdle muscular dystrophy type 2B (LGMD2B) and Miyoshi myopathy. To date, nine autosomal recessive forms (AR-LGMD) have been identified: four genes, which code for the sarcoglycan glycoproteins, are associated with both mild and severe forms, the sarcoglycanopathies (LGMD2C, 2D, 2E and 2F). The other five forms, usually causing a milder phenotype are LGMD2A (calpain 3), LGMD2B (dysferlin), LGMD2G (telethonin), LGMD2H (9q31-11), and LGMD2I (19q13.3).We studied dysferlin expression in a total of 176 patients, from 166 LGMD families: 12 LGMD2B patients, 70 with other known forms of muscular dystrophies (LGMD2A, sarcoglycanopathies, LGMD2G), in an attempt to assess the effect of the primary gene-product deficiency on dysferlin. In addition, 94 still unclassified LGMD families were screened for dysferlin deficiency.In eight LGMD2B patients from five families, no dysferlin was observed in muscle biopsies, both through immunofluorescence (IF) and Western blot methodologies, while in two families, a very faint band was detected. Both patterns, negative or very faint bands, were concordant in patients belonging to the same families, suggesting that dysferlin deficiency is specific to LGMD2B.Myoferlin, the newly identified homologue of dysferlin was studied for the first time in LGMD2B patients. Since no difference was observed between patients mildly and severely affected, this protein do not seem to modify the phenotype in the present dysferlin-deficient patients.Dystrophin, sarcoglycans, and telethonin were normal in all LGMD2B patients, while patients with sarcoglycanopathies (2C, 2D, and 2E), LGMD2A, LGMD2G, and DMD showed the presence of a normal dysferlin band by Western blot and a positive pattern on IF. These data suggest that there is no interaction between dysferlin and these proteins. However, calpain analysis showed a weaker band in four patients from two families with intra-familial concordance. Therefore, this secondary deficiency of calpain in LGMD2B families, may indicate an interaction between dysferlin and calpain in muscle.Dysferlin was also present in cultured myotubes, in chorionic villus, and in the skin.Dysferlin deficiency was found in 24 out of a total of 166 Brazilian AR-LGMD families screened for muscle proteins (∼14%), thus representing the second most frequent known LGMD form, after calpainopathy, in our population.

Telethonin protein expression in neuromuscular disorders.

Telethonin is a 19-kDa sarcomeric protein, localized to the Z-disc of skeletal and cardiac muscles. Mutations in the telethonin gene cause limb-girdle muscular dystrophy type 2G (LGMD2G). We investigated the sarcomeric integrity of muscle fibers in LGMD2G patients, through double immunofluorescence analysis for telethonin with three sarcomeric proteins: titin, alpha-actinin-2, and myotilin and observed the typical cross striation pattern, suggesting that the Z-line of the sarcomere is apparently preserved, despite the absence of telethonin. Ultrastructural analysis confirmed the integrity of the sarcomeric architecture. The possible interaction of telethonin with other proteins responsible for several forms of neuromuscular disorders was also analyzed. Telethonin was clearly present in the rods in nemaline myopathy (NM) muscle fibers, confirming its localization to the Z-line of the sarcomere. Muscle from patients with absent telethonin showed normal expression for the proteins dystrophin, sarcoglycans, dysferlin, and calpain-3. Additionally, telethonin showed normal localization in muscle biopsies from patients with LGMD2A, LGMD2B, sarcoglycanopathies, and Duchenne muscular dystrophy (DMD). Therefore, the primary deficiency of calpain-3, dysferlin, sarcoglycans, and dystrophin do not seem to alter telethonin expression.

Does the P172H mutation at the TM4SF2 gene cause X-linked mental retardation?

The TM4SF2 gene mapped at Xp11.4 has recently been associated with non-syndromic X-linked mental retardation (MRX) by the analysis of the breakpoint of a t(X;2) balanced translocation in a female patient with mild mental retardation and minor autistic features [Zemni et al., 2000]. To date, three TM4SF2 mutations have been identified in three familial cases of MRX: the G218X (family L28), the P172H (family T15) [Zemni et al., 2000], and, more recently, a deletion of 2bp (564delGT) in exon 5 (familyMRX58) [Abidi et al., 2002]. Two of these changes create a premature stop codon and it has been predicted that theMRX in these cases results from the deficiency of the TM4SF2 product. The family T15, first described by Zemni et al. [2000], was recently reevaluated [Gomot et al., 2002]. Neuropsychological evaluation showed that five out of seven males and one of three women had mild to moderate mental retardation. The mutation was present in all mentally impaired individuals and also in one male reported to be normal. Further segregation analysis of markers surrounding the TM4SF2 gene suggested that in this family the disease gene should be localized between loci DXS556 (Xp11.4) and DXS441 (Xq13.2), thus excluding theTM4SF2 gene. The authors put forward two alternative explanations: (1) the TM4SF2 P172H mutation would be the cause of MR in association with intra-familial phenotypic variability or (2) this would be a rare mutation not detrimental to the functioning of theTM4SF2protein, and thereforenot causative of the phenotype [Gomot et al., 2002]. As part of an X-linked mental retardation (XLMR) candidate gene testing, we screened the seven exons of the TM4SF2 gene in 105males withmental retardation (25 familial and 80 isolated cases), whowere not carriers of the fragile X mutation. Blood samples were collected after informed consent. All the coding exons of the TM4SF2 gene were amplified using the primers and PCR conditions reported elsewhere [Zemni et al., 2000], and the amplification products were analyzed by SSCP (single strand conformation polimorfism) according to Splendore et al. [2000].One sample showedanabnormal migration pattern for the fragment corresponding to exon 5 (data not shown). Sequencing in both directions was performed using the BigDye Terminator Cycle Sequencing Kit (PE Biosystems, Foster City, CA) in an ABI Prism 377 sequencer (Applied Biosystems, Foster City, CA). A C!A transversion was found at nucleotide c.515 leading to the Pro!His substitution of amino acid 172 (P172H) (Fig. 1). This result was confirmed by the single-nucleotide primer extension assay (SNuPe, Amersham Biosciences, Piscataway, USA), a method that precisely localizes a SNP site. The patientwith the P172Hwas examined at 21 years of age and presented with mild to moderate mental retardation. He is able to write and read only simple texts. Developmental milestones had been slightly delayed, but speech acquisition was severely impaired, since he started talking at 31⁄2 years. Dysmorphic features included: a long narrow face with pronounced oral hypotonia; short forehead and filtrum; high palate; teeth malocclusion; abnormally folded ears; depressed sternum; hypoplastic nails; somewhat scarce hair with alopecia spots. Ophthalmological findings included nystagmus and myopia. His mother and sister, both withnormal intelligence,were found to be carriers of the P172H mutation, but not his mentally normal brother (Fig. 2). This mutation was not detected in the SNuPe assay screening of additional 320 unrelated X chromosomes from normal males. The finding of the mutation in a cohort of 105 individuals with mental retardation but not in 320 normalmenwith a similar ethnic background further supports the hypothesis that this might be a pathogenic mutation. A possibility of this being a common polymorphism has also been ruled out by Zemni et al. [2000] as they did not find it in 100 normal chromosomes. Gomot et al. [2002] advanced the possibility that thismutationwas not the cause ofmental retardation in family T15 because they found one mentally normal male carrier, an individual who has been previously Grant sponsor: FAPESP; Grant sponsor: PRONEX; Grant sponsor: CNPq.

Investigation of 15q11-q13, 16p11.2 and 22q13 CNVs in Autism Spectrum Disorder Brazilian Individuals with and without Epilepsy

Copy number variations (CNVs) are an important cause of ASD and those located at 15q11-q13, 16p11.2 and 22q13 have been reported as the most frequent. These CNVs exhibit variable clinical expressivity and those at 15q11-q13 and 16p11.2 also show incomplete penetrance. In the present work, through multiplex ligation-dependent probe amplification (MLPA) analysis of 531 ethnically admixed ASD-affected Brazilian individuals, we found that the combined prevalence of the 15q11-q13, 16p11.2 and 22q13 CNVs is 2.1% (11/531). Parental origin could be determined in 8 of the affected individuals, and revealed that 4 of the CNVs represent de novo events. Based on CNV prediction analysis from genome-wide SNP arrays, the size of those CNVs ranged from 206 kb to 2.27 Mb and those at 15q11-q13 were limited to the 15q13.3 region. In addition, this analysis also revealed 6 additional CNVs in 5 out of 11 affected individuals. Finally, we observed that the combined prevalence of CNVs at 15q13.3 and 22q13 in ASD-affected individuals with epilepsy (6.4%) was higher than that in ASD-affected individuals without epilepsy (1.3%; p<0.014). Therefore, our data show that the prevalence of CNVs at 15q13.3, 16p11.2 and 22q13 in Brazilian ASD-affected individuals is comparable to that estimated for ASD-affected individuals of pure or predominant European ancestry. Also, it suggests that the likelihood of a greater number of positive MLPA results might be found for the 15q13.3 and 22q13 regions by prioritizing ASD-affected individuals with epilepsy.