Audioprofiling identifies TECTA and GJB2-related deafness segregating in a single extended pedigree.

An audioprofile displays phenotypic data from several audiograms on a single graph that share a common genotype. In this report, we describe the application of audioprofiling to a large family in which a genome-wide screen failed to identify a deafness locus. Analysis of audiograms by audioprofiling suggested that two persons with hearing impairment had a different deafness genotype. On this basis, we reassigned affectation status and identified a p.Cys1837Arg autosomal dominant mutation in alpha-tectorin segregating in all family members except two persons, who segregated autosomal recessive deafness caused by p.Val37Ile and p.Leu90Pro mutations in Connexin 26. One nuclear family in the extended pedigree segregates both dominant and recessive non-syndromic hearing loss.

Single-nucleotide polymorphisms in the COL1A1 regulatory regions are associated with otosclerosis.

Otosclerosis (MIM 166800) has a prevalence of 0.2-1% among white adults, making it the single most common cause of hearing impairment in this ethnic group. Although measles virus, hormones, human leukocyte antigen alleles and genetic factors have been implicated in the development of otosclerosis, its etiology remains unknown. In a focused effort to identify genetic factors in otosclerosis, we have mapped four disease loci (MIM 166800/605727/608244/608787); however, cloning the disease-causing genes in these intervals has not been successful. Here, we used a case-control study design to investigate the association between collagen type I genes and otosclerosis. We identified susceptibility and protective haplotypes in COL1A1 that are significantly associated with otosclerosis in the Caucasian population. These haplotypes alter reporter gene activity in an osteoblast cell line by affecting binding of transcription factors to cis-acting elements. Our data suggest that increased amounts of collagen alpha1(I) homotrimers are causally related to the development of otosclerosis. Consistent with this hypothesis, mouse mutants homozygous for a Col1a2 frameshift mutation on a C57BL/6J background that deposit only homotrimeric type I collagen have hearing loss.

Mutation of COL11A2 causes autosomal recessive non-syndromic hearing loss at the DFNB53 locus.

BACKGROUND: Allele variants of COL11A2, encoding collagen type XI alpha2, cause autosomal dominant non-syndromic hearing loss (ARNSHL) at the DFNA13 locus (MIM 601868) and various syndromes that include a deafness phenotype. OBJECTIVE: To describe a genome-wide scan carried out on a consanguineous Iranian family segregating ARNSHL. RESULTS: Genotyping data identified a novel locus for ARNSHL on chromosome 6p21.3, which was designated DFNB53. Homozygosity for the P621T mutation of COL11A2 was present in all deaf persons in this family; this same variation was absent in 269 Iranian controls. Sequence comparison of collagen type XI alpha1 and alpha2 peptides across species shows that the replaced proline is an evolutionarily conserved amino acid. CONCLUSIONS: The P621T mutation of COL11A2 affects the Y position of the canonical -Gly-X-Y- repeat in collagens. It lies near the amino-terminus of the triple helical region and causes ARNSHL. This finding suggests that mutation type and location are critical determinants in defining the phenotype of COL11A2 associated diseases.

Genomic structures of SCN2A and SCN3A - candidate genes for deafness at the DFNA16 locus.

DFNA16 is a form of autosomal dominant non-syndromic hearing loss (ADNSHL) characterized by fluctuating progressive hearing impairment. Earlier, we mapped the deafness-causing gene to chromosome 2q23-24.3. In this paper, we describe fine mapping results using additional markers tightly linked to the DFNA16 candidate region. Critical recombinants at markers D2S354 and D2S124 define a 3.5-cM interval that contains the DFNA16 gene. Positional candidate genes include two members of the voltage-gated sodium channel family, the type 2 alpha subunit (SCN2A) and the type 3 alpha subunit (SCN3A). After showing that SCN2A is expressed in human fetal cochlea, we determined its genomic structure to facilitate mutation screening in our DFNA16 kindred. We also determined the genomic structure of SCN3A. These two genes are oriented head-to-head, with their 5' ends separated by approximately 40 kb; their homology is 82% at the nucleotide level, and 85% for identities and 90% for positives at the amino acid level. They share similar genomic structures and have alternative splice isoforms that are developmentally regulated and highly conserved between species. Although no DFNA16-causing mutations were found in either gene, haplotype analysis with polymorphic markers in SCN2A introns further narrowed the candidate gene interval to the region flanked by D2S354 and STS SHGC-82894.

A 1.1-Mb transcript map of the hereditary hemochromatosis locus.

In the process of positionally cloning a candidate gene responsible for hereditary hemochromatosis (HH), we constructed a 1.1-Mb transcript map of the region of human chromosome 6p that lies 4.5 Mb telomeric to HLA-A. A combination of three gene-finding techniques, direct cDNA selection, exon trapping, and sample sequencing, were used initially for a saturation screening of the 1.1-Mb region for expressed sequence fragments. As genetic analysis further narrowed the HH candidate locus, we sequenced completely 0.25 Mb of genomic DNA as a final measure to identify all genes. Besides the novel MHC class 1-like HH candidate gene HLA-H, we identified a family of five butyrophilin-related sequences, two genes with structural similarity to a type 1 sodium phosphate transporter, 12 novel histone genes, and a gene we named RoRet based on its strong similarity to the 52-kD Ro/SSA lupus and Sjogren's syndrome auto-antigen and the RET finger protein. Several members of the butyrophilin family and the RoRet gene share an exon of common evolutionary origin called B30-2. The B30-2 exon was originally isolated from the HLA class 1 region, yet has apparently "shuffled" into several genes along the chromosome telomeric to the MHC. The conservation of the B30-2 exon in several novel genes and the previously described amino acid homology of HLA-H to MHC class 1 molecules provide further support that this gene-rich region of 6p21.3 is related to the MHC. Finally, we performed an analysis of the four approaches for gene finding and conclude that direct selection provides the most effective probes for cDNA screening, and that as much as 30% of ESTs in this 1.1-Mb region may be derived from noncoding genomic DNA.

Clone-contig and STS maps of the hereditary hemochromatosis region on human chromosome 6p21.3-p22.

YAC-based and bacterial-clone based STS-content maps were constructed that served as the framework physical maps for the positional cloning of a candidate gene for hereditary hemochromatosis. The YAC-based map comprises 43 YACs and 86 STS and spans approximately 8 Mb of DNA between the class I region of the major histocompatibility complex on human chromosome 6p21.3 and D6S276 in 6p22. Comparison with published maps revealed a hole in the MIT/Whitehead and CEPH YAC maps that includes the immediate region around the hemochromatosis gene itself. Approximately 3 Mb of DNA was covered by a bacterial clone contig that consists of 38 BACs, 45 PACs, 26 PI clones and one lambda phage. The bacterial clone-based STS map comprises 153 STSs. A contiguous block of 8 STSs could be amplified from both human chromosome 6 and 5. Further characterization of selected STSs and bacterial clones by radiation hybrid mapping and fluorescence in situ hybridization, respectively, revealed the presence of a multicopy DNA segment, more than one bacterial clone length in size, which is duplicated near the chromosome-6 centromere and part of which is present in multiple copies on chromosome 5. Possible implications of the incomplete public YAC-contig map and of the multicopy segment for physical mapping and linkage disequilibrium studies of the hemochromatosis candidate region are discussed.

A novel MHC class I-like gene is mutated in patients with hereditary haemochromatosis.

Hereditary haemochromatosis (HH), which affects some 1 in 400 and has an estimated carrier frequency of 1 in 10 individuals of Northern European descent, results in multi-organ dysfunction caused by increased iron deposition, and is treatable if detected early. Using linkage-disequilibrium and full haplotype analysis, we have identified a 250-kilobase region more than 3 megabases telomeric of the major histocompatibility complex (MHC) that is identical-by-descent in 85% of patient chromosomes. Within this region, we have identified a gene related to the MHC class I family, termed HLA-H, containing two missense alterations. One of these is predicted to inactivate this class of proteins and was found homozygous in 83% of 178 patients. A role of this gene in haemochromatosis is supported by the frequency and nature of the major mutation and prior studies implicating MHC class I-like proteins in iron metabolism.

Purification of a maize dehydrin.

A maize dehydrin with an apparent molecular weight of 20 kDa was purified from whole kernels of maize inbred line G50. Kernels were ground in a seed mill, stirred overnight in extraction buffer, and centrifuged to extract soluble proteins. The sample was heated to 89 degrees C and centrifuged to remove heat-insoluble proteins. The remaining soluble proteins were fractionated in a three-step chromatographic process. Following cation exchange, hydrophobic interaction, and gel filtration chromatography, pure dehydrin samples were obtained.

Effect of thyroid hormones on the prolactin response to thyrotropin-releasing hormone in normal persons and euthyroid goitrous patients.

In nine euthyroid goitrous patients, increasing doses of T4 caused a significant decrease in the PRL response to TRH; the PRL response fell significantly at a dose of T4 of 100 micrograms/day for 1 month (P less than 0.02) and fell further with increasing doses so that at 300 micrograms T4/day, the PRL response was 40% of that in the untreated state. T4 treatment also blunted the PRL response to chlorpromazine (P less than 0.05) in a separate group of euthyroid goitrous patients. In contrast, there was only a small drop of the PRL response to TRH in normal subjects treated with T4 (n = 9) and none at all with T3 (n = 7). These data, together with previously published reports, suggest that thyroid hormone may affect PRL secretion in the presence of thyroid disease (hyperthyroidism, hypothyroidism, or euthyroid goiter), but that physiological amounts of thyroid hormone have little or no modulating effect on PRL secretion in normal persons.