A multi-exonic SPG4 duplication underlies sex-dependent penetrance of hereditary spastic paraplegia in a large Brazilian pedigree.

SPG4 mutations are the most frequent cause of autosomal-dominant hereditary spastic paraplegia (HSP). SPG4 HSP is characterized by large inter- and intrafamilial variability in age at onset (AAO) and disease severity. The broad spectrum of SPG4 mutations has recently been further extended by the finding of large genomic deletions in SPG4-linked pedigrees negative for 'small' mutations. We had previously reported a very large pedigree, linked to the SPG4 locus with many affected members, which showed gender difference in clinical manifestation. Screening for copy number aberrations revealed the first case of a multi-exonic duplication (exon10_12dup) in the SPG4 gene. The mutation leads to a premature stop codon, suggesting that the protein product is not functional. The analysis of 30 individuals who carry the mutation showed that males have on average an earlier AAO and are more severely affected. The present family suggests that this HSP pathogenesis may be modulated by factors related to individual background and gender as observed for other autosomal dominant conditions, such as facio-scapulohumeral muscular dystrophy or amyloidosis. Understanding why some individuals, particularly women, are 'partially protected' from the effects of this and other pathogenic mutations is of utmost importance.

Spastic paraplegia, optic atrophy, and neuropathy is linked to chromosome 11q13.

We report an autosomal recessive neurodegenerative disorder in 25 white members from a large inbred Brazilian family, 22 of whom were evaluated clinically. This condition is characterized by (1) subnormal vision secondary to apparently nonprogressive congenital optic atrophy; (2) onset of progressive spastic paraplegia in infancy; (3) onset of progressive motor and sensory axonal neuropathy in late childhood/early adolescence; (4) dysarthria starting in the third decade of life; (5) exacerbated acoustic startle response; and (6) progressive joint contractures and spine deformities. Motor handicap was severe, and all patients were wheelchair bound after 15 years old. We performed a genome-wide screen including 25 affected individuals and 49 of their unaffected relatives. Linkage was detected at 11q13 region with a maximum logarithm of odds score of +14.43, obtained with marker D11S1883. The candidate region, which lies between D11S1908 and D11S1889, encompasses approximately 4.8Mb and has more than 100 genes and expressed sequences. We propose the acronym SPOAN (spastic paraplegia, optic atrophy, and neuropathy) for this complex syndrome.

A new form of autosomal dominant limb-girdle muscular dystrophy (LGMD1G) with progressive fingers and toes flexion limitation maps to chromosome 4p21.

Limb-girdle muscular dystrophy (LGMD) is a genetic disorder characterized by progressive weakness of pelvic and scapular girdles and great clinical variability. It is a highly heterogeneous disease with 16 identified loci: six of them autosomal dominant (AD) (LGMD1) and 10 autosomal recessive (AR) (LGMD2). The responsible genes are known for three of the AD-LGMD and for all 10 AR-LGMD. Linkage analysis excluded these 16 loci in a Brazilian-Caucasian family with 12 patients affected by AD late-onset LGMD associated with progressive fingers and toes flexion limitation. Biceps muscle biopsy from one of the patients showed a predominantly myopathic histopathological pattern, associated with rimmed vacuoles. A genomewide scan was performed which mapped a new locus for this disorder at 4p21 with a maximum two-point lod score of 6.62 for marker D4S2964. Flanking markers place this locus between D4S2947 and D4S2409, within an interval of 9 cM. We propose to classify this AD form of LGMD as LGMD1G.

Calpainopathy: how broad is the spectrum of clinical variability?

Five affected siblings were referred with a probable diagnosis of proximal adult-type spinal muscular atrophy (SMA) based on lower motor neuron signs (muscle weakness and atrophy, hypotony, hypoactive or absent reflexes, and fasciculations), normal or borderline serum creatine kinase levels, and a neurogenic pattern on electromyography, compatible with motor neuron disease, in one patient. No exon 7-8 deletion in the survival motor neuron (SMN) gene was found. Linkage analysis excluded the SMN and all known autosomal recessive limb girdle muscular dystrophy loci, with the exception of LGMD-2A. A homozygous R769Q mutation in the calpain-3 gene and absence of muscle calpain-3 protein confirmed a calpainopathy. This family suggests that the clinical spectrum of calpainopathy might be broader and that this diagnosis might be considered in patients with an atypical motor neuron disease.

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.

The 10 autosomal recessive limb-girdle muscular dystrophies.

Fifteen forms of limb-girdle muscular dystrophies (5 autosomal dominant and 10 autosomal recessive) have already been found. The 10 genes responsible for the autosomal recessive forms, which account for more than 90% of the cases, had their product identified. This review will focus on the most recent data on autosomal recessive-limb-girdle muscular dystrophy and on our own experience of more than 300 patients studied from 120 families who were classified (based on DNA, linkage and muscle protein analysis) in eight different forms of autosomal recessive-limb-girdle muscular dystrophy. Genotype-phenotype correlations in this highly heterogeneous group confirm that patients with mutations in different genes may be clinically indistinguishable. On the other hand, for most forms of autosomal recessive-limb-girdle muscular dystrophy a discordant phenotype, ranging from a relatively severe course to mildly affected or asymptomatic carriers may be seen in patients carrying the same mutation even within the same family. A gender difference in the severity of the phenotype might exist for some forms of autosomal recessive-limb-girdle muscular dystrophy, such as calpainopathy and telethoninopathy but not for others, such as dysferlinopathies or sarcoglycanopathies. Understanding similarities in patients affected by mutations in different genes, differences in patients carrying the same mutations or why some muscles are affected while others are spared remains a major challenge. It will depend on future knowledge of gene expression, gene and protein interactions and on identifying modifying genes and other factors underlying clinical variability.

Evaluation of alternative reporter genes for the yeast two-hybrid system.

The yeast two-hybrid system is a powerful tool for screening protein-protein interactions and has also been used for large-scale studies. We evaluated two protein-coding sequences as reporter genes for the yeast two-hybrid system, to determine if it was suitable as an alternative screening strategy. Aspergillus awamori glucoamylase activity results in clear haloes around colonies producing this enzyme after growth on starch plates and staining with iodine vapors. However, transcription activation by Gal4 on Gal-regulated promoters was insufficient for this type of phenotypic visualization. A modified gene of Aequoria victoria enhanced green fluorescent protein (EGFP) was tested to determine its suitability for interaction screenings with flow cytometry. When the EGFP reporter gene system was incorporated into the cells, Gal4 transcriptional activation produced sufficient fluorescence for detection with the flow cytometer, especially when there were strong interactions.