Valerie A. Street

Mutations in a plasma membrane Ca2+-ATPase gene cause deafness in deafwaddler mice

Hearing loss is the most common sensory deficit in humans. Because the auditory systems of mice and humans are conserved, studies on mouse models have predicted several human deafness genes and identified new genes involved in hearing. The deafwaddler (dfw) mouse mutant is deaf and displays vestibular/motor imbalance. Here we report that the gene encoding a plasma membrane Ca2+-ATPase type 2 pump (Atp2b2 , also known as Pmca2) is mutated in dfw. An A→G nucleotide transition in dfw DNA causes a glycine-to-serine substitution at a highly conserved amino-acid position, whereas in a second allele, dfw 2J, a 2-base-pair deletion causes a frameshift that predicts a truncated protein. In the cochlea, the protein Atp2b2 is localized to stereocilia and the basolateral wall of hair cells in wild-type mice, but is not detected in dfw2J mice. This indicates that mutation of Atp2b2 may cause deafness and imbalance by affecting sensory transduction in stereocilia as well as neurotransmitter release from the basolateral membrane. These mutations affecting Atp2b2 in dfw and dfw2J are the first to be found in a mammalian plasma membrane calcium pump and define a new class of deafness genes that directly affect hair-cell physiology.

Mutation of a putative protein degradation gene LITAF/SIMPLE in Charcot-Marie-Tooth disease 1C

Background: Charcot-Marie-Tooth (CMT) neuropathy is a heterogeneous group of inherited disorders of the peripheral nervous system. The authors recently mapped an autosomal dominant demyelinating form of CMT type 1 (CMT1C) to chromosome 16p13.1-p12.3. Objective: To find the gene mutations underlying CMT1C. Methods: The authors used a combination of standard positional cloning and candidate gene approaches to identify the causal gene for CMT1C. Western blot analysis was used to determine relative protein levels in patient and control lymphocyte extracts. Northern blotting was used to characterize gene expression in 1) multiple tissues; 2) developing sciatic nerve; and 3) nerve-crush and nerve-transection experiments. Results: The authors identified missense mutations (G112S, T115N, W116G) in the LITAFgene (lipopolysaccharide-induced tumor necrosis factor-α factor) in three CMT1C pedigrees. LITAF, which is also referred to as SIMPLE, is a widely expressed gene encoding a 161-amino acid protein that may play a role in protein degradation pathways. The mutations associated with CMT1C were found to cluster, defining a domain of the LITAF protein having a critical role in peripheral nerve function. Western blot analysis suggested that the T115N and W116G mutations do not alter the level of LITAF protein in peripheral blood lymphocytes. The LITAF transcript is expressed in sciatic nerve, but its level of expression is not altered during development or in response to nerve injury. This finding is in stark contrast to that seen for other known genes that cause CMT1. Conclusions: Mutations in LITAF may account for a significant proportion of CMT1 patients with previously unknown molecular diagnosis and may define a new mechanism of peripheral nerve perturbation leading to demyelinating neuropathy.

The Type 1 Inositol 1,4,5-Trisphosphate Receptor Gene Is Altered in the opisthotonos Mouse

The opisthotonos (opt) mutation arose spontaneously in a C57BL/Ks-db2J colony and is the only known, naturally occurring allele of opt. This mutant mouse was first identified based on its ataxic and convulsive phenotype. Genetic and molecular data presented here demonstrate that the type 1 inositol 1,4,5-trisphosphate receptor (IP3R1) protein, which serves as an IP3-gated channel to release calcium from intracellular stores, is altered in the optmutant. A genomic deletion in the IP3R1 gene removes two exons from the IP3R1 mRNA but does not interrupt the translational reading frame. The altered protein is predicted to have lost several modulatory sites and is present at markedly reduced levels in opt homozygotes. Nonetheless, a strong calcium release from intracellular stores can be elicited in cerebellar Purkinje neurons treated with the metabotropic glutamate receptor (mGluR) agonist quisqualate (QA). QA activates Group I mGluRs linked to GTP-binding proteins that stimulate phospholipase C and subsequent production of the intracellular messenger IP3, leading to calcium mobilization via the IP3R1 protein. The calcium response in opt homozygotes shows less attenuation to repeated QA application than in control littermates. These data suggest that the convulsions and ataxia observed in opt mice may be caused by the physiological dysregulation of a functional IP3R1 protein.

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.

Mapping of Charcot-Marie-Tooth Disease Type 1C to Chromosome 16p Identifies a Novel Locus for Demyelinating Neuropathies

Charcot-Marie-Tooth (CMT) neuropathy represents a genetically heterogeneous group of diseases affecting the peripheral nervous system. We report genetic mapping of the disease to chromosome 16p13.1-p12.3, in two families with autosomal dominant CMT type 1C (CMT1C). Affected individuals in these families manifest characteristic CMT symptoms, including high-arched feet, distal muscle weakness and atrophy, depressed deep-tendon reflexes, sensory impairment, slow nerve conduction velocities, and nerve demyelination. A maximal combined LOD score of 14.25 was obtained with marker D16S500. The combined haplotype analysis in these two families localizes the CMT1C gene within a 9-cM interval flanked by markers D16S519 and D16S764. The disease-linked haplotypes in these two pedigrees are not conserved, suggesting that the gene mutation underlying the disease in each family arose independently. The epithelial membrane protein 2 gene (EMP2), which maps to chromosome 16p13.2, was evaluated as a candidate gene for CMT1C.

Charcot–Marie–Tooth neuropathy: clinical phenotypes of four novel mutations in the MPZ and Cx 32 genes

Charcot-Marie-Tooth Hereditary Neuropathy is a heterogeneous syndrome associated with mutations in several different genes including peripheral myelin protein 22, myelin P0, connexin 32, and early growth response 2. There is considerable variability in the phenotypic expression of this syndrome and the relationship of this variability to mutation genotypes requires extensive analysis. Here we describe the phenotypes and genotypes of four new mutations underlying the Charcot-Marie-Tooth syndrome and document segregation with disease. Four families with Charcot-Marie-Tooth were ascertained, examined, and evaluated electrophysiologically. Each family had peripheral blood DNA screened for mutations in myelin protein 22, myelin P0, and connexin 32. Two families were found with new mutations in the myelin P0 gene: S140T in the extracellular domain and K236del in the cytoplasmic domain. All families showed segregation of the mutations with the Charcot-Marie-Tooth phenotype as did a new family with the rare G163R mutation in the membrane domain. A 49-year-old man with the S140T mutation demonstrated conduction block on electrophysiological testing. A family with a novel S49P mutation in the connexin 32 gene had a neuropathy with very slow nerve conduction. These new mutations in the myelin P0 and connexin 32 genes help to clarify the pathophysiology of the clinical Charcot-Marie-Tooth syndrome. The S140T mutation in myelin P0 can be associated with conduction block and Charcot-Marie-Tooth should be part of the differential diagnosis of that phenomenon. Mutations in the cytoplasmic domain of myelin P0 can cause clinical neuropathy. The S49P mutation in the connexin 32 gene can produce aspects of a demyelinating type of X-linked hereditary neuropathy.

A novel DFNA9 mutation in the vWFA2 domain of COCH alters a conserved cysteine residue and intrachain disulfide bond formation resulting in progressive hearing loss and site-specific vestibular and central oculomotor dysfunction.

Mutations within the COCH gene (encoding the cochlin protein) lead to auditory and vestibular impairment in the DFNA9 disorder. In this study, we describe the genetic mapping of progressive autosomal dominant sensorineural hearing loss first affecting high‐frequency auditory thresholds within a human pedigree to the long arm of chromosome 14 in band q12. A maximal pairwise LOD score of 7.08 was obtained with marker D14S1021. We identified a c.1625G > T mutation in exon 12 of COCH that co‐segregates with auditory dysfunction in the pedigree. The mutation results in a predicted p.C542F substitution at an evolutionarily conserved cysteine residue in the C‐terminus of cochlin. The c.1625G > T transversion in COCH exon 12 represents the first reported mutation outside of the LCCL domain which is encoded by exons 4 and 5. The 542F mutant cochlin is translated and secreted by transfected mammalian cells. Western blot analysis under non‐reducing and reducing conditions suggests that the 542F mutation alters intramolecular cochlin disulfide bond formation. In the vestibular system, a progressive horizontal canal hypofunction and a probable saccular otolith challenge were detected in family members with the c.1625G > T COCH alteration. Abnormal central oculomotor test results in family members with the c.1625G > T COCH alteration imply a possible central nervous system change not previously noted in DFNA9 pedigrees harboring mutations within the LCCL domain.

A novel WFS1 mutation in a family with dominant low frequency sensorineural hearing loss with normal VEMP and EcochG findings.

BackgroundLow frequency sensorineural hearing loss (LFSNHL) is an uncommon clinical finding. Mutations within three different identified genes (DIAPH1, MYO7A, and WFS1) are known to cause LFSNHL. The majority of hereditary LFSNHL is associated with heterozygous mutations in the WFS1 gene (wolframin protein). The goal of this study was to use genetic analysis to determine if a small American familys hereditary LFSNHL is linked to a mutation in the WFS1 gene and to use VEMP and EcochG testing to further characterize the familys audiovestibular phenotype.MethodsThe clinical phenotype of the American family was characterized by audiologic testing, vestibular evoked myogenic potentials (VEMP), and electrocochleography (EcochG) evaluation. Genetic characterization was performed by microsatellite analysis and direct sequencing of WFS1 for mutation detection.ResultsSequence analysis of the WFS1 gene revealed a novel heterozygous mutation at c.2054G>C predicting a p.R685P amino acid substitution in wolframin. The c.2054G>C mutation segregates faithfully with hearing loss in the family and is absent in 230 control chromosomes. The p.R685 residue is located within the hydrophilic C-terminus of wolframin and is conserved across species. The VEMP and EcochG findings were normal in individuals segregating the WFS1 c.2054G>C mutation.ConclusionWe discovered a novel heterozygous missense mutation in exon 8 of WFS1 predicting a p.R685P amino acid substitution that is likely to underlie the LFSNHL phenotype in the American family. For the first time, we describe VEMP and EcochG findings for individuals segregating a heterozygous WFS1 mutation.

A DNA variant within the MYO7A promoter regulates YY1 transcription factor binding and gene expression serving as a potential dominant DFNA11 auditory genetic modifier

Mutations within MYO7A can lead to recessive and dominant forms of inherited hearing loss. We previously identified a large pedigree (referred to as the HL2 family) with hearing loss that first impacts the low and mid frequencies segregating a dominant MYO7A mutation in exon 17 at DNA residue G2164C. The MYO7AG2164C mutation predicts a nonconservative glycine-to-arginine (G722R) amino acid substitution at a highly conserved glycine residue. The degree of low and mid frequency hearing loss varies markedly in the family, suggesting the presence of a genetic modifier that either rescues or exacerbates the primary MYO7AG2164C mutation. Here we describe a single nucleotide polymorphism (SNP) T/C at position −4128 in the wild-type MYO7A promoter allele that sorts with the degree of hearing loss severity in the pedigree. Electrophoretic mobility shift assay analysis indicates that the SNP differentially regulates the binding of the YY1 transcription factor with the T−4128 allele creating an YY1 binding site. Immunocytochemistry demonstrates that Yy1 is expressed in hair cell nuclei within the cochlea. Given that Myo7a is also expressed in cochlear hair cells, Yy1 shows the appropriate localization to regulate Myo7a transcription within the inner ear. YY1 appears to be acting as a transcriptional repressor as the MYO7A promoter allele containing the T−4128 SNP drives 41 and 46% less reporter gene expression compared with the C−4128 SNP in the ARPE-19 and HeLa cell lines, respectively. The T−4128 SNP may be contributing to the severe hearing loss phenotype in the HL2 pedigree by reducing expression of the wild-type MYO7A allele.

In search of the DFNA11 myosin VIIA low- and mid-frequency auditory genetic modifier.

Objectives: To evaluate the auditory, vestibular, and retinal characteristics of a large American DFNA11 pedigree with autosomal dominant progressive sensorineural hearing loss that first impacts the low- and mid-frequency auditory range. The pedigree (referred to as the HL2 family) segregates a myosinVIIA (MYO7A) mutation in exon 17 at DNA residue G2164C (MYO7AG2164C) that seems to be influenced by a genetic modifier that either rescues or exacerbates the MYO7AG2164C alteration. DNA analysis to examine single-nucleotide polymorphisms in 2 candidate modifier genes (ATP2B2 and Wolfram syndrome 1 [WFS1]) is summarized in this report. Study Design: Family study. Results: The degree of low- and mid-frequency hearing loss in HL2 family members segregating the MYO7AG2164C mutation varies from mild to more severe, with approximately the same number of HL2 family members falling at each end of the severity spectrum. The extent of hearing loss in HL2 individuals can vary between family generations. Differences in the degree of hearing loss in MYO7AG2164C HL2 family members may be mirrored by vestibular function in at least 2 of these same individuals. The single-nucleotide polymorphisms examined within ATP2B2 and WFS1 did not segregate with the mild versus more severe auditory phenotype. Conclusion: The severity of the auditory and vestibular phenotypes in MYO7AG2164C HL2 family members may run in parallel, suggesting a common modifier gene within the inner ear. The putative MYO7AG2164C genetic modifier is likely to represent a common polymorphism that is not linked tightly to the MYO7A mutation on the MYO7A2164C allele.