ABSTRACT
Age-related hearing impairment (ARHI), or presbycusis, is the most prevalent sensory impairment in the elderly. ARHI is a complex disease caused by an interaction between environmental and genetic factors. Here we describe the results of the first whole genome association study for ARHI. The study was performed using 846 cases and 846 controls selected from 3434 individuals collected by eight centers in six European countries. DNA pools for cases and controls were allelotyped on the Affymetrix 500K GeneChip for each center separately. The 252 top-ranked single nucleotide polymorphisms (SNPs) identified in a non-Finnish European sample group (1332 samples) and the 177 top-ranked SNPs from a Finnish sample group (360 samples) were confirmed using individual genotyping. Subsequently, the 23 most interesting SNPs were individually genotyped in an independent European replication group (138 samples). This resulted in the identification of a highly significant and replicated SNP located in GRM7, the gene encoding metabotropic glutamate receptor type 7. Also in the Finnish sample group, two GRM7 SNPs were significant, albeit in a different region of the gene. As the Finnish are genetically distinct from the rest of the European population, this may be due to allelic heterogeneity. We performed histochemical studies in human and mouse and showed that mGluR7 is expressed in hair cells and in spiral ganglion cells of the inner ear. Together these data indicate that common alleles of GRM7 contribute to an individual's risk of developing ARHI, possibly through a mechanism of altered susceptibility to glutamate excitotoxicity.
Subject(s)
Genetic Predisposition to Disease , Presbycusis/genetics , Receptors, Kainic Acid/genetics , Age Factors , Aged , Animals , Case-Control Studies , Ear, Inner/metabolism , Female , Genome-Wide Association Study , Humans , Male , Mice , Middle Aged , Polymorphism, Single Nucleotide , Presbycusis/metabolism , Receptors, Kainic Acid/metabolism , White People/genetics , GluK3 Kainate ReceptorABSTRACT
Mice homozygous for the hypomorphic allele Eya1 ( bor ) exhibit cochlear aplasia, with associated deafness, and renal hypoplasia, similar to Branchio-Oto-Renal syndrome (BOR) in humans. Although much is known about the genetics of the disease, little is known about the factors that modify its phenotypic expression. We have recently detailed two modifier loci (Mead1 and Mead2) in a C3HeB/FeJ-Eya1 ( bor/+ ) x C57BL/6 J intercross that suppress the ear-related phenotypes in our hypomorphic mutants. In this study we report corroborating evidence for our initial finding with the identification of two modifier loci mapping to the same region in CAST/EiJ and BALB/cJ. Furthermore, we describe an additional locus (Mead3) on chromosome 19 in CAST/EiJ, within which the previously cloned suppressor Nxf1 resides. The suppression effect on cochlear coiling was studied on congenic line(s) for each protective allele. The penetrance and suppressor strength of these alleles vary by strain and locus. Eya1 ( bor/bor ) hypomorphs, when homozygous for each of the three protective alleles (CAST/EiJ, C57BL/6 J, or BALB/cJ) at the Mead1 or Mead2 locus, exhibit completely penetrant suppression of cochlear agenesis. At the Mead1 locus, the C57BL/6 J and BALB/cJ alleles have comparable strengths. At the Mead2 locus, the C57BL/6 J and CAST/EiJ alleles have comparable strengths. In contrast, mice with genotype Eya1 ( bor/bor )Mead3(CAST/CAST) exhibit incomplete penetrance (50%) and a wide range of cochlear coiling (1/4-1(1/2) turns). The identification of these additional modifier alleles could provide crucial clues for evaluating the candidate genes.
Subject(s)
Cochlea/abnormalities , Deafness/genetics , Intracellular Signaling Peptides and Proteins/genetics , Nuclear Proteins/genetics , Protein Tyrosine Phosphatases/genetics , Alleles , Animals , Branchio-Oto-Renal Syndrome/genetics , Chromosome Mapping , Crosses, Genetic , Disease Models, Animal , Evoked Potentials, Auditory, Brain Stem , Female , Genes, Suppressor , Male , Mice , Mice, Congenic , Mice, Inbred BALB C , Mice, Mutant Strains , Mutation , PhenotypeABSTRACT
The Eya1(bor) mutant hypomorph contains an intracisternal A particle insertion in intron 7 of the Eya1 gene that results in a 50% reduction in wild-type mRNA levels. The homozygous mutants have middle and inner ear defects and variable kidney abnormalities. The severity of the disorder is affected by genetic background. In contrast to complete deafness and cochlear agenesis in the C3HeB/FeJ strain, F2 Eya1(bor/bor) mutants from an intercross between C3HeB/FeJ-Eya1(bor/+) and C57BL/6J showed variable auditory brain-stem responses and cochlear coiling. In this study, using these F2 Eya1(bor/bor) mutants, we have identified two major loci, Mead1 (modifier of Eya1-associated deafness 1) and Mead2, that are responsible for suppression of the original phenotypes. We have narrowed these two loci to 5.4 and 4.4 cM, respectively, in congenic lines. Quantitative PCR demonstrated that this modifying effect did not result from an increase in wild-type Eya1 mRNA, suggesting Mead1 and Mead2 are interacting directly or indirectly with Eya1 during inner ear development.
Subject(s)
Cochlea/embryology , Crosses, Genetic , Hearing Loss/genetics , Intracellular Signaling Peptides and Proteins/genetics , Nuclear Proteins/genetics , Protein Tyrosine Phosphatases/genetics , Quantitative Trait Loci/genetics , Animals , Cochlea/abnormalities , Disease Models, Animal , Genetic Linkage , Hearing Loss/pathology , Humans , Mice , Mice, Mutant StrainsABSTRACT
Cell fate specification during inner ear development is dependent upon regional gene expression within the otic vesicle. One of the earliest cell fate determination steps in this system is the specification of neural precursors, and regulators of this process include the Atonal-related basic helix-loop-helix genes, Ngn1 and NeuroD and the T-box gene, Tbx1. In this study we demonstrate that Eya1 signaling is critical to the normal expression patterns of Tbx1, Ngn1, and NeuroD in the developing mouse otocyst. We discuss a potential mechanism for the absence of neural precursors in the Eya1-/- inner ears and the primary and secondary mechanisms for the loss of cochleovestibular ganglion cells in the Eya1bor/bor hypomorphic mutant.
Subject(s)
Cochlea/embryology , Gene Expression Regulation, Developmental , Nerve Tissue Proteins/physiology , Neurotrophin 3/physiology , T-Box Domain Proteins/physiology , Trans-Activators/physiology , Animals , Basic Helix-Loop-Helix Transcription Factors , Brain-Derived Neurotrophic Factor/metabolism , Cell Death , Cell Lineage , Genotype , Immunohistochemistry , In Situ Hybridization , In Situ Nick-End Labeling , Intracellular Signaling Peptides and Proteins , Mice , Mice, Transgenic , Mutation , Nerve Tissue Proteins/biosynthesis , Neurons/metabolism , Neurotrophin 3/biosynthesis , Nuclear Proteins , Oligonucleotide Probes/chemistry , Phenotype , Protein Tyrosine Phosphatases , T-Box Domain Proteins/biosynthesis , Time Factors , Trans-Activators/biosynthesis , Transcription Factors/biosynthesis , Transcription Factors/physiologyABSTRACT
The p53 tumor suppressor protein is susceptible to oxidation, which prevents it from binding to its DNA response element. The goal of the current research was to determine the nature of the cysteine residue thiol oxidation that prevents p53 from binding its DNA target and its effect on p53 structure. Recombinant p53, purified in the presence of the reducing agent dithiothreitol (DTT), contains five free thiol groups on the surface of the protein. In the absence of DTT, p53 contains only four thiol groups, indicating that an average of one surface thiol group is readily susceptible to oxidation. Sulfite-mediated disulfide bond cleavage followed by reaction with 2-nitro-5-thiosulfobenzoate showed that oxidized p53 contains a single disulfide bond per monomer. By atomic force microscopy, we determined that reduced p53 binds to a double-stranded DNA containing the p53 promoter element of the MDM2 gene. The DNA-bound reduced p53 has an average cross-sectional diameter of 8.61 nm and a height of 4.12 nm. The amount of oxidized p53 that bound to the promoter element was ninefold lower, and it has an 18% larger average cross-sectional diameter. Electromobility shift assays showed that binding of oxidized p53 to DNA was enhanced upon addition of DTT, indicating that oxidation is reversible. The possibility that oxidized p53 contained significant amounts of sulfenic (-SOH), sulfinic (-SO2H), or sulfonic acid (-SO3H) was ruled out. Gel filtration chromatography indicated that oxidation increases the percentage of p53 monomers and high-molecular-weight oligomers (>1,000 kDa) relative to tetrameric p53. Protein modeling studies suggest that a mixed disulfide glutathione adduct on Cys182 could account for the observed stoichiometry of oxidized thiols and structural changes. The glutathione adduct may prevent proper helix-helix interaction within the DNA binding domain and contribute to tetramer dissociation.
