Pavel Seeman

GJB2 Mutations and Degree of Hearing Loss: A Multicenter Study

Hearing impairment (HI) affects 1 in 650 newborns, which makes it the most common congenital sensory impairment. Despite extraordinary genetic heterogeneity, mutations in one gene, GJB2, which encodes the connexin 26 protein and is involved in inner ear homeostasis, are found in up to 50% of patients with autosomal recessive nonsyndromic hearing loss. Because of the high frequency of GJB2 mutations, mutation analysis of this gene is widely available as a diagnostic test. In this study, we assessed the association between genotype and degree of hearing loss in persons with HI and biallelic GJB2 mutations. We performed cross-sectional analyses of GJB2 genotype and audiometric data from 1,531 persons, from 16 different countries, with autosomal recessive, mild-to-profound nonsyndromic HI. The median age of all participants was 8 years; 90% of persons were within the age range of 0-26 years. Of the 83 different mutations identified, 47 were classified as nontruncating, and 36 as truncating. A total of 153 different genotypes were found, of which 56 were homozygous truncating (T/T), 30 were homozygous nontruncating (NT/NT), and 67 were compound heterozygous truncating/nontruncating (T/NT). The degree of HI associated with biallelic truncating mutations was significantly more severe than the HI associated with biallelic nontruncating mutations (P<.0001). The HI of 48 different genotypes was less severe than that of 35delG homozygotes. Several common mutations (M34T, V37I, and L90P) were associated with mild-to-moderate HI (median 25-40 dB). Two genotypes--35delG/R143W (median 105 dB) and 35delG/dela(GJB6-D13S1830) (median 108 dB)--had significantly more-severe HI than that of 35delG homozygotes.

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.

Mutations in the RNA exosome component gene EXOSC3 cause pontocerebellar hypoplasia and spinal motor neuron degeneration

RNA exosomes are multi-subunit complexes conserved throughout evolution and are emerging as the major cellular machinery for processing, surveillance and turnover of a diverse spectrum of coding and noncoding RNA substrates essential for viability. By exome sequencing, we discovered recessive mutations in EXOSC3 (encoding exosome component 3) in four siblings with infantile spinal motor neuron disease, cerebellar atrophy, progressive microcephaly and profound global developmental delay, consistent with pontocerebellar hypoplasia type 1 (PCH1; MIM 607596). We identified mutations in EXOSC3 in an additional 8 of 12 families with PCH1. Morpholino knockdown of exosc3 in zebrafish embryos caused embryonic maldevelopment, resulting in small brain size and poor motility, reminiscent of human clinical features, and these defects were largely rescued by co-injection with wild-type but not mutant exosc3 mRNA. These findings represent the first example of an RNA exosome core component gene that is responsible for a human disease and further implicate dysregulation of RNA processing in cerebellar and spinal motor neuron maldevelopment and degeneration.

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.

Mutations in the SPTLC2 Subunit of Serine Palmitoyltransferase Cause Hereditary Sensory and Autonomic Neuropathy Type I

Hereditary sensory and autonomic neuropathy type I (HSAN-I) is an axonal peripheral neuropathy associated with progressive distal sensory loss and severe ulcerations. Mutations in the first subunit of the enzyme serine palmitoyltransferase (SPT) have been associated with HSAN-I. The SPT enzyme catalyzes the first and rate-limiting step in the de novo sphingolipid synthesis pathway. However, different studies suggest the implication of other genes in the pathology of HSAN-I. Therefore, we screened the two other known subunits of SPT, SPTLC2 and SPTLC3, in a cohort of 78 HSAN patients. No mutations were found in SPTLC3, but we identified three heterozygous missense mutations in the SPTLC2 subunit of SPT in four families presenting with a typical HSAN-I phenotype. We demonstrate that these mutations result in a partial to complete loss of SPT activity in vitro and in vivo. Moreover, they cause the accumulation of the atypical and neurotoxic sphingoid metabolite 1-deoxy-sphinganine. Our findings extend the genetic heterogeneity in HSAN-I and enlarge the group of HSAN neuropathies associated with SPT defects. We further show that HSAN-I is consistently associated with an increased formation of the neurotoxic 1-deoxysphinganine, suggesting a common pathomechanism for HSAN-I.

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.

Pontocerebellar hypoplasia type 1 Clinical spectrum and relevance of EXOSC3 mutations

Objectives: Pontocerebellar hypoplasia with spinal muscular atrophy, also known as PCH1, is a group of autosomal recessive disorders characterized by generalized muscle weakness and global developmental delay commonly resulting in early death. Gene defects had been discovered only in single patients until the recent identification of EXOSC3 mutations in several families with relatively mild course of PCH1. We aim to genetically stratify subjects in a large and well-defined cohort to define the clinical spectrum and genotype–phenotype correlation. Methods: We documented clinical, neuroimaging, and morphologic data of 37 subjects from 27 families with PCH1. EXOSC3 gene sequencing was performed in 27 unrelated index patients of mixed ethnicity. Results: Biallelic mutations in EXOSC3 were detected in 10 of 27 families (37%). The most common mutation among all ethnic groups was c.395A>C, p.D132A, responsible for 11 (55%) of the 20 mutated alleles and ancestral in origin. The mutation-positive subjects typically presented with normal pregnancy, normal birth measurements, and relative preservation of brainstem and cortical structures. Psychomotor retardation was profound in all patients but lifespan was variable, with 3 subjects surviving beyond the late teens. Abnormal oculomotor function was commonly observed in patients surviving beyond the first year. Major clinical features previously reported in PCH1, including intrauterine abnormalities, postnatal hypoventilation and feeding difficulties, joint contractures, and neonatal death, were rarely observed in mutation-positive infants but were typical among the mutation-negative subjects. Conclusion: EXOSC3 mutations account for 30%–40% of patients with PCH1 with variability in survival and clinical severity that is correlated with the genotype.

Spectrum and frequencies of mutations in the GJB2 (Cx26) gene among 156 Czech patients with pre-lingual deafness.

Mutations in the gene gap junction beta 2 (GJB2), the gene for the connexin 26, are the most common cause of pre‐lingual deafness worldwide. The mutation 35delG within GJB2 is prevalent in Europe. To date, there are no data about GJB2 mutation spectrum and frequencies from the Czech population. We investigated and report here the spectrum and frequencies of mutations in the GJB2 gene among 156 unrelated, congenital deafness Czech patients. Allele‐specific polymerase chain reaction, together with fluorescent fragment analysis, were used for the detection of the 35delG mutation. The entire coding region of the GJB2 was directly sequenced in all patients who were not homozygous for the 35delG. No pathogenic mutation was detected in 51.9% of patients. At least one pathogenic mutation was found in 48.1% of patients, and both pathogenic mutations were detected in 37.8% of patients. Single mutations in a heterozygous state were detected in 10.3% of patients. The mutation 35delG accounts for 82.8% of detected disease mutations, Trp24stop accounts for 9.7% of pathogenic alleles and was found in patients with gypsy heritage. Mutation 313del14 accounts for 3.7% of pathogenic alleles. The frequency of 35delG heterozygotes in the Czech Republic is 1 : 29.6. Testing for only the three most common mutations would detect over 96% of all pathogenic alleles in the Czech Republic.

Phenotypic variability of patients homozygous for the GJB2 mutation 35delG cannot be explained by the influence of one major modifier gene

Hereditary hearing loss (HL) is a very heterogeneous trait, with 46 gene identifications for non-syndromic HL. Mutations in GJB2 cause up to half of all cases of severe-to-profound congenital autosomal recessive non-syndromic HL, with 35delG being the most frequent mutation in Caucasians. Although a genotype–phenotype correlation has been established for most GJB2 genotypes, the HL of 35delG homozygous patients is mild to profound. We hypothesise that this phenotypic variability is at least partly caused by the influence of modifier genes. By performing a whole-genome association (WGA) study on 35delG homozygotes, we sought to identify modifier genes. The association study was performed by comparing the genotypes of mild/moderate cases and profound cases. The first analysis included a pooling-based WGA study of a first set of 255 samples by using both the Illumina 550K and Affymetrix 500K chips. This analysis resulted in a ranking of all analysed single-nucleotide polymorphisms (SNPs) according to their P-values. The top 250 most significantly associated SNPs were genotyped individually in the same sample set. All 192 SNPs that still had significant P-values were genotyped in a second independent set of 297 samples for replication. The significant P-values were replicated in nine SNPs, with combined P-values between 3 × 10−3 and 1 × 10−4. This study suggests that the phenotypic variability in 35delG homozygous patients cannot be explained by the effect of one major modifier gene. Significantly associated SNPs may reflect a small modifying effect on the phenotype. Increasing the power of the study will be of greatest importance to confirm these results.

High prevalence of the IVS 1 + 1 G to A/GJB2 mutation among Czech hearing impaired patients with monoallelic mutation in the coding region of GJB2

Biallelic pathogenic GJB2 gene mutations cause pre‐lingual genetic hearing loss in up to 50% of individuals with bilateral sensorineural hearing loss worldwide. Sequencing of the entire GJB2 gene‐coding region in Czech patients with pre‐lingual bilateral hearing loss revealed that 10.3% of Czech patients carry only one monoallelic pathogenic mutation in the coding region of the GJB2 gene, which is significantly more than the population frequency of 3.4%. The 309‐kb GJB6 deletion, frequent in Spain and France, is very rare in the Czech population. In order to evaluate the impact of the IVS1 + 1 G to A splice site mutation in the non‐coding part of the GJB2 gene among Czech patients, we tested all available patients with pre‐lingual hearing loss with only one monoallelic mutation in the coding part of GJB2. By sequencing of the exon 1 region of the GJB2 gene and HphI restriction analysis in 20 Czech patients we identified nine patients carrying IVS1 + 1 G to A. Testing for this mutation explained deafness in 45% of Czech GJB2 monoallelic patients. This mutation represents now 4% of GJB2 pathogenic mutations in Czech patients and is the third most common GJB2 mutation found in our cohort of 242 unrelated Czech patients with prelingual hearing loss. A similar frequency may also be expected in other Central European or Slavic populations.