Radim Mazanec

Hot-spot residue in small heat-shock protein 22 causes distal motor neuropathy

Distal hereditary motor neuropathies are pure motor disorders of the peripheral nervous system resulting in severe atrophy and wasting of distal limb muscles. In two pedigrees with distal hereditary motor neuropathy type II linked to chromosome 12q24.3, we identified the same mutation (K141N) in small heat-shock 22-kDa protein 8 (encoded by HSPB8; also called HSP22). We found a second mutation (K141E) in two smaller families. Both mutations target the same amino acid, which is essential to the structural and functional integrity of the small heat-shock protein αA-crystallin. This positively charged residue, when mutated in other small heat-shock proteins, results in various human disorders. Coimmunoprecipitation experiments showed greater binding of both HSPB8 mutants to the interacting partner HSPB1. Expression of mutant HSPB8 in cultured cells promoted formation of intracellular aggregates. Our findings provide further evidence that mutations in heat-shock proteins have an important role in neurodegenerative disorders.

Relative contribution of mutations in genes for autosomal dominant distal hereditary motor neuropathies: a genotype–phenotype correlation study

Distal hereditary motor neuropathy (HMN) is a clinically and genetically heterogeneous group of disorders affecting spinal alpha-motor neurons. Since 2001, mutations in six different genes have been identified for autosomal dominant distal HMN; glycyl-tRNA synthetase (GARS), dynactin 1 (DCTN1), small heat shock 27 kDa protein 1 (HSPB1), small heat shock 22 kDa protein 8 (HSPB8), Berardinelli-Seip congenital lipodystrophy (BSCL2) and senataxin (SETX). In addition a mutation in the (VAMP)-associated protein B and C (VAPB) was found in several Brazilian families with complex and atypical forms of autosomal dominantly inherited motor neuron disease. We have investigated the distribution of mutations in these seven genes in a cohort of 112 familial and isolated patients with a diagnosis of distal motor neuropathy and found nine different disease-causing mutations in HSPB8, HSPB1, BSCL2 and SETX in 17 patients of whom 10 have been previously reported. No mutations were found in GARS, DCTN1 and VAPB. The phenotypic features of patients with mutations in HSPB8, HSPB1, BSCL2 and SETX fit within the distal HMN classification, with only one exception; a C-terminal HSPB1-mutation was associated with upper motor neuron signs. Furthermore, we provide evidence for a genetic mosaicism in transmitting an HSPB1 mutation. This study, performed in a large cohort of familial and isolated distal HMN patients, clearly confirms the genetic and phenotypic heterogeneity of distal HMN and provides a basis for the development of algorithms for diagnostic mutation screening in this group of disorders.

Highly unstable sequence interruptions of the CTG repeat in the myotonic dystrophy gene.

Myotonic dystrophy type 1 is caused by the expansion of a CTG repeat in the 3′ UTR of the DMPK gene. A length exceeding 50 CTG triplets is pathogenic. Intermediate alleles with 35–49 triplets are not disease‐causing but show instability in intergenerational transmissions. We report on the identification of multiple patients with different patterns of CCG and CTC interruptions in the DMPK CTG repeat tract that display unique intergenerational instability. In patients bearing interrupted expanded alleles, the location of the interruptions changed dramatically between generations and the repeats tended to contract. The phenotype for these patients corresponded to the classical form of the disease, but in some cases without muscular dystrophy and possibly with a later onset than expected. Symptomatic patients bearing interrupted intermediate length repeat tracts were also identified, although the role of the interruptions in their phenotype remains unclear. The identification of interruptions in the DMPK repeat has important consequences for molecular genetic testing where they can lead to false negative conclusions.

SIMPLE mutation in demyelinating neuropathy and distribution in sciatic nerve

Charcot–Marie–Tooth neuropathy type 1C (CMT1C) is an autosomal dominant demyelinating peripheral neuropathy caused by missense mutations in the small integral membrane protein of lysosome/late endosome (SIMPLE) gene. To investigate the prevalence of SIMPLE mutations, we screened a cohort of 152 probands with various types of demyelinating or axonal and pure motor or sensory inherited neuropathies. SIMPLE mutations were found only in CMT1 patients, including one G112S and one W116G missense mutations. A novel I74I polymorphism was identified, yet no splicing defect of SIMPLE is likely. Haplotype analysis of STR markers and intragenic SNPs linked to the gene demonstrated that families with the same mutation are unlikely to be related. The clustering of the G112S, T115N, and W116G mutations within five amino acids suggests this domain may be critical to peripheral nerve myelination. Electrophysiological studies showed that CMT1C patients from six pedigrees (n = 38) had reduced nerve conduction velocities ranging from 7.5 to 27.0m/sec (peroneal). Two patients had temporal dispersion of nerve conduction and irregularity of conduction slowing, which is unusual for CMT1 patients. We report the expression of SIMPLE in various cell types of the sciatic nerve, including Schwann cells, the affected cell type in CMT1C.

Mutations in Czech LGMD2A patients revealed by analysis of calpain3 mRNA and their phenotypic outcome

Calpain3 (CAPN3, p94) is a muscle-specific nonlysosomal cysteine proteinase. Loss of proteolytic function or change of other properties of this enzyme (such as stability or ability to interact with other muscular proteins) is manifested as limb girdle muscular dystrophy type 2A (LGMD2A, calpainopathy). These pathological changes in properties of calpain3 are caused by mutations in the calpain3 gene. The fact that the human gene for calpain3 is quite long led us to analyse its coding sequence by reverse transcription-PCR followed by sequence analysis. This study reports nine mutations that we found by analysing mRNA of seven unrelated LGMD patients in the Czech Republic. Three of these mutations were novel, not described on the Leiden muscular dystrophy pages so far. Further, we observed a reduction of dysferlin in muscle membrane in five of our seven LGMD2A patients by immunohistochemical analysis of muscle sections.

Analysis of histopathologic and molecular pathologic findings in Czech LGMD2A patients

Limb‐girdle muscular dystrophy type 2A (LGMD2A) is an autosomal‐recessive disorder characterized by selective atrophy and progressive weakness of proximal girdle muscles. LGMD2A, the most prevalent form of LGMD, is caused by mutations in the CAPN3 gene that encodes the skeletal muscle–specific member of the calpain family, calpain‐3 (p94). We examined the histopathologic and molecular pathologic findings in 14 Czech LGMD2A patients. Analysis of the CAPN3 gene was performed at the mRNA level, using reverse transcription–polymerase chain reaction (RT‐PCR) and sequencing, and/or DNA level, using PCR and denaturing high‐performance liquid chromatography (DHPLC). Our results confirm that mutation 550delA is the most frequent CAPN3 defect in Czech LGMD2A patients (9 alleles of 28). Furthermore, we established that, in a patient with the 550delA/R490W genotype, mRNA carrying frameshift mutation 550delA was not detected, probably due to its degradation by nonsense‐mediated mRNA decay. In muscle biopsies of two LGMD2A patients, a neurogenic pattern simulating a neurogenic lesion was observed. Immunoblot analysis revealed the deficiency of p94 in all genetically confirmed cases of LGMD2A, and secondary dysferlin deficiency was demonstrated on muscle membranes in 6 patients using immunofluorescence. Thus, we find a combination of DNA and mRNA mutational analysis to be useful in the diagnosis of LGMD2A. Moreover, our study expands the spectrum of calpainopathies to cases that simulate a neurogenic lesion in muscle biopsies, and the knowledge of possible secondary deficiencies of muscular proteins also contributes to a diagnosis of LGMD2A. Muscle Nerve, 2006

Spinal Deformities in Hereditary Motor and Sensory Neuropathy A Retrospective Qualitative, Quantitative, Genotypical, and Familial Analysis of 175 Patients

Study Design. Retrospective study of 175 patients with hereditary motor and sensory neuropathy (HMSN), i.e., Charcot-Marie-Tooth (CMT) disease. Objective. To investigate the frequency, age of onset, character, familial, and genotypical incidence of spinal deformities among HMSN patients. Summary of Background Data. Prior studies addressing HMSN discuss the associated spinal deformities. However, these data vary significantly while inconsistently including genotypes within the classification framework. Methods. Plain-film radiographic spine studies of 175 HMSN patients were performed to determine the incidence, character, and severity of spinal deformity. The degree of the spinal deformity was evaluated measuring Cobb’s angle of the main curve. The results of the entire cohort were initially assessed before being classified by genotype. Results. The incidence of spinal deformity for the entire group was 26%. Of these, 58% demonstrated scoliosis, 31% had kyphoscoliosis, and 11% had thoracic hyperkyphosis; 73% of patients with spinal deformity were classified as HMSN Type I with confirmed duplication of the PMP 22 (peripheral myelin protein) gene on chromosome 17. The incidence of spinal deformity by genotype was: duplication of the PMP 22 gene: 29% (25 of 87); deletion of the PMP 22 gene: 0% (0 of 15); Cx32 (connexin 32) gene mutation: 24% (8 of 34); and MPZ (myelin protein zero) gene mutation: 100% (6 of 6). Familial incidence of spinal deformity was found in “MPZ gene mutation” and “duplication of PMP 22 gene” subgroups. Conclusion. This study demonstrates a 26% incidence of spinal deformity among HMSN patients. Spinal deformity was most frequently observed in patients with the MPZ gene mutation, where the most common familial incidence was also found.

High frequency of SH3TC2 mutations in Czech HMSN I patients.

Laššuthová P, Mazanec R, Vondráček P, Šišková D, Haberlová J, Sabová J, Seeman P. High frequency of SH3TC2 mutations in Czech HMSN I patients.

Novel EGR2 mutation R359Q is associated with CMT type 1 and progressive scoliosis

Mutations in the early growth response 2 gene (EGR2) cause demyelinating neuropathies differing in severity and age of onset. We tested 46 unrelated Czech patients with dominant or sporadic demyelinating CMT neuropathy for mutations in the EGR2 gene. One novel de-novo mutation (Arg359Gln, R359Q) was identified in heterozygous state in a patient with a typical CMT1 phenotype, progressive moderate thoracolumbar scoliosis and without clinical signs of cranial nerve dysfunction. This patient is presently less affected compared to previously described Dejerine-Sottas neuropathy (DSN) patients carrying another substitution at codon 359 (Arg359Trp, R359W). This report shows that EGR2 mutations are rare in Czech patients with demyelinating type of CMT and suggests that different substitutions at codon 359 of EGR2 can cause significantly different phenotypes.

Loss of function mutations in HARS cause a spectrum of inherited peripheral neuropathies

Inherited peripheral neuropathies are a genetically heterogeneous group of disorders characterized by distal muscle weakness and sensory loss. Mutations in genes encoding aminoacyl-tRNA synthetases have been implicated in peripheral neuropathies, suggesting that these tRNA charging enzymes are uniquely important for the peripheral nerve. Recently, a mutation in histidyl-tRNA synthetase (HARS) was identified in a single patient with a late-onset, sensory-predominant peripheral neuropathy; however, the genetic evidence was lacking, making the significance of the finding unclear. Here, we present clinical, genetic, and functional data that implicate HARS mutations in inherited peripheral neuropathies. The associated phenotypic spectrum is broad and encompasses axonal and demyelinating motor and sensory neuropathies, including four young patients presenting with pure motor axonal neuropathy. Genome-wide linkage studies in combination with whole-exome and conventional sequencing revealed four distinct and previously unreported heterozygous HARS mutations segregating with autosomal dominant peripheral neuropathy in four unrelated families (p.Thr132Ile, p.Pro134His, p.Asp175Glu and p.Asp364Tyr). All mutations cause a loss of function in yeast complementation assays, and p.Asp364Tyr is dominantly neurotoxic in a Caenorhabditis elegans model. This study demonstrates the role of HARS mutations in peripheral neuropathy and expands the genetic and clinical spectrum of aminoacyl-tRNA synthetase-related human disease.