R. Pavanello

Asymptomatic carriers and gender differences in facioscapulohumeral muscular dystrophy (FSHD)

Facioscapulohumeral muscular dystrophy is an autosomal dominant muscle disorder, mapped to 4q35. It is characterized by remarkable inter- and intrafamilial clinical variability ranging from severe phenotype to asymptomatic carriers. The aim of the present study was to assess the size of the Eco RI fragment in a large sample of asymptomatic or minimally affected carriers as well as symptomatic patients, comparing both sexes, in order to verify if asymptomatic carriers are randomly distributed or concentrated in some particular families and if there is preferential parental transmission (maternal or paternal) resulting in non-penetrant carriers. We have analysed a total of 506 individuals from 106 unrelated families with at least one affected facioscapulohumeral muscular dystrophy proband. In all patients the molecular diagnosis was confirmed following double digestion (Eco RI/Bln I fragment <35 kb). About 20% among probands relatives who were found to carry the small fragment were asymptomatic or minimally affected, without preferential parental transmission, but with a significantly higher proportion of females (n=37) than males (n=14). Although asymptomatic carriers were found in about 30% of the families, some genealogies seem to concentrate more non-penetrant cases. A significant correlation between the size of the Eco RI fragment and severity of the phenotype was observed in the total sample but surprisingly this correlation is significant only among affected females. The gender difference in clinical manifestation as well as the observation that asymptomatic carriers are not rare should be taken into consideration in genetic counseling of affected patients or at-risk relatives.

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.

Maternal MTHFR interacts with the offspring's BCL3 genotypes, but not with TGFA, in increasing risk to nonsyndromic cleft lip with or without cleft palate

The 677 C → T polymorphism in the 5-10 methylenetetrahydrofolate reductase (MTHFR) gene has been associated with nonsyndromic cleft lip with or without cleft palate (CL/P) in some populations, but not others. Previous studies (ie, case–control and transmission disequilibrium tests (TDT)) in Brazilian families with CL/P have been unable to replicate this putative association. However, our group observed a lower proportion of CT heterozygotes among the mothers of CL/P probands, suggesting that the maternal genotype for this polymorphism might influence predisposition to CL/P. In order to further examine this issue, we performed a case–control study of the 677 C → T/MTHFR polymorphism in families with CL/P ascertained in two regions of Brazil: 172 from São Paulo (SP) and 252 from Ceará (CE). The control samples included 243 individuals from SP and 401 from CE. TDT was carried out in 102 patients with CL/P and their parents. No evidence of an association was observed between the 677 C → T/MTHFR polymorphism and CL/P using the case–control design, while borderline significance was obtained with the TDT (P=0.055). We have also looked for an interaction between maternal MTHFR genotypes and the propositi offsprings genotypes at two candidate susceptibility loci for CL/P, TGFA and BCL3. Interestingly, we observed an interaction between the maternal MTHFR and offsprings BCL3 genotypes (OR: 2.3; 95% CI: 1.1–4.8; P=0.03) but not with the offsprings TGFA genotypes. Therefore, our results reinforce the idea that the maternal MTHFR genotype plays a significant role in susceptibility to CL/P, but its teratogenic effect depends on the genotype of the offspring.

Facioscapulohumeral (FSHD1) and other forms of muscular dystrophy in the same family: is there more in muscular dystrophy than meets the eye?

We report on two unrelated Brazilian families with members affected by two different forms of muscular dystrophy. In the first one, the 35-year-old male proband has limb-girdle muscular dystrophy with proximal weakness, elevated creatine kinase and a myopathic muscle biopsy. All the proteins known to be associated with limb-girdle muscular dystrophy were normal. Two of his sisters also complained of muscle weakness. The oldest sister showed clinical signs consistent with facioscapulohumeral muscular dystrophy, confirmed through molecular analysis. She presented a 30 kb EcoRI/BlnI fragment which was found in another six relatives, but surprisingly not in the affected proband or the other sister. In the second family, a 57-year-old male with a typical facioscapulohumeral muscular dystrophy phenotype has a 17 kb EcoRI/BlnI fragment, which was also present in other affected relatives. However in a 14-year-old severely affected male cousin, confined to a wheelchair since age 12, but without facial weakness, the small fragment was absent. These families illustrate the importance of testing all affected individuals in a family.

P.4.13 Central core disease (CCD): Improving the screening for mutations in RYR1 gene

Central core disease (CCD) is a congenital myopathy, characterized by the presence of central core-like areas in muscle fibers. Patients with RYR-related CCD usually have mild or moderate axial and proximal weakness, hypotonia and motor developmental delay. CCD is associated with susceptibility to malignant hyperthermia (MH), and both conditions have been linked to mutations in human RYR1 gene, which encodes a calcium release channel known as ryanodine receptor (RyR1). RYR1 mutational spectrum linked with CCD includes more than 200 described mutations mostly heterozygous dominant missense mutations and minor deletions or duplications. Definite diagnosis of CCD is done by clinical history and evaluation, histopathological analyses of muscle biopsy and, recently, genetic testing of gene’s “hot spots”, because RYR1 is a large gene and direct sequencing of each exon is laborious. Here, we tested Multiplex Ligation-dependent Probe Amplification (MLPA) as an alternative and simple method for screening patients with histological diagnosis or family history of CCD. This technique uses high-performance PCR to quantify up to 45 transcripts in small human DNA samples. It is based on annealing of two hemi-probes into its complementary sequence and was designed to detect the 33 most common mutations in RYR1 gene. From 41 patients diagnosed with CCD in three different institutions, 24 (58,5%) had mutations found in direct sequencing and 11 (26,8%) in MLPA. Seventeen patients (41,5%) had consistent results: 6 (14,6%) had their mutation diagnosed with sequencing and MLPA and neither method was able to find mutations in 11 patients (26,8%). Mutations in exon 101 were the most prevalent in sequencing (11 patients) and MLPA (4 patients). MLPA had shown to be a relevant tool to help diagnosing CCD; moreover, it is especially useful in diagnosing family members of patients with known mutations detectable by MLPA. Financial support: FAPESP-CEPID, CNPQ-INCT, FINEP.

P.4.12 Association of two mutations in the RYR1 gene in central core disease: Recessive or modifying effect?

Central core disease (CCD) is a congenital myopathy, characterized by the presence of central core-like areas in muscle fibers. Patients usually have mild or moderate axial and proximal weakness, hypotonia and motor developmental delay. CCD is associated with mutations in human RYR1 gene, which encodes a calcium release channel known as ryanodine receptor (RyR1). Here we present a family diagnosed with CCD with distinct phenotypes. A 48-year old female was referred to our service with clinical features suggesting a congenital myopathy. Histological analysis confirmed the diagnosis of CCD. Nine exons from the C-terminal region of RYR1 were screened for mutations in the index case. Two different missense mutations were identified: c.14256 Axa0>xa0C in exon 98 and c.14693 Txa0>xa0C in exon 102. Trans position of the 2 mutations was confirmed because patient’s daughter, mother and sister carried the exon 98 mutation, but not the one in exon 102. The mutation in exon 102 have already been described as pathogenic, and is compatible with both severe and mild phenotypes, observed as intra and inter family variability. On the other hand, the c.14256 Axa0>xa0C mutation in exon 98 is a single nucleotide variation and its clinical significance is still untested. Considering the fact that patient’s relatives showed no CCD phenotype, it could be inferred that this polymorphism alone is not responsible for the disease. Moreover, the effect of association of this polymorphism to exon 102’s pathogenic mutation is not clear. Further studies about patients with both exon 98 polymorphism and exon 102 mutation could help establishing a better relation between these two findings. The case described above illustrates the present reality where new methods for wide genome screening are becoming more accessible and able to identify a great variety of mutations and polymorphisms in patients and their families. FAPESP-CEPID, CNPq-INCT, FINEP.