Auditory neuropathy is a rare form of deafness characterized by an absent or abnormal auditory brainstem response with preservation of outer hair cell function. We have identified Diaphanous homolog 3 (DIAPH3) as the gene responsible for autosomal dominant nonsyndromic auditory neuropathy (AUNA1), which we previously mapped to chromosome 13q21-q24. Genotyping of additional family members narrowed the interval to an 11-Mb, 3.28-cM gene-poor region containing only four genes, including DIAPH3. DNA sequencing of DIAPH3 revealed a c.-172G > A, g. 48G > A mutation in a highly conserved region of the 5′ UTR. The c.-172G > A mutation occurs within a GC box sequence element and was not found in 379 controls. Using genome-wide expression arrays and quantitative RT-PCR, we demonstrate a 2-to 3-fold overexpression of DIAPH3 mRNA in lymphoblastoid cell lines from affected individuals. Likewise, a significant increase (≈1.5-fold) in DIAPH3 protein was found by quantitative immunoblotting of lysates from lymphoblastoid cell lines derived from affected individuals in comparison with controls. In addition, the c.-172G > A mutation is sufficient to drive overexpression of a luciferase reporter. Finally, the expression of a constitutively active form of diaphanous protein in the auditory organ of Drosophila melanogaster recapitulates the phenotype of impaired response to sound. To date, only two genes, the otoferlin gene OTOF and the pejvakin gene PJVK, are known to underlie nonsyndromic auditory neuropathy. Genetic testing for DIAPH3 may be useful for individuals with recessive as well as dominant inheritance of nonsyndromic auditory neuropathy.

Objective To identify the genetic etiology in a family with autosomal dominant progressive sensorineural hearing loss. Design Prospective molecular genetic research study. Setting Academic genetic research laboratory. Participants Seventeen members of a family with dominant progressive nonsyndromic sensorineural hearing loss: 9 affected, 6 unaffected, and 2 spouses. Interventions Clinical data from questionnaires, interviews, serial audiograms, and medical records; genetic data from genome-wide linkage analysis and candidate gene mutation analysis. Main Outcome Measures Symptoms, age at onset, serial audiometric data, and the presence or absence of a deafness-associated mutation. Results Affected individuals in this family presented with autosomal dominant nonsyndromic high-frequency progressive sensorineural hearing loss, with age at onset ranging from 1 to 21 years. Genome-wide linkage analysis of single-nucleotide polymorphisms yielded evidence of linkage to an 18.9-Mb region on chromosome 1p34–p36, with a multipoint logarithm of odds score of 3.6. This interval contains a known deafness gene, KCNQ4, which underlies DNFA2 deafness. Sequencing of the 14 coding exons and intron-exon junctions of KCNQ4 revealed a novel heterozygous missense mutation, c.859G>C, p.Gly287Arg. The mutation disrupts the highly conserved GYG motif (glycine-tyrosine-glycine) of the phosphate- binding loop, hypothesized to be critical in maintaining pore structure and function. All 274 controls were negative for the mutation. Conclusions Autosomal dominant high-frequency hearing loss is genetically heterogeneous, and linkage analysis is an efficient means of identifying the etiology in larger families. Deafness in this family is caused by a novel mutation in KCNQ4.

The pelvic support structures of rats and mice are anatomically and histologically similar to those of humans. We propose that both mice and rats are appropriate, cost-effective models for directed studies in POP research.

The genome size and chromosome conformation of Treponema pallidum subsp. pallidum, Nichols strain, were determined by contour-clamped homogeneous electric field electrophoresis, a pulsed-field gel electrophoresis technique. Digestion of T. pallidum subsp. pallidum DNA with the restriction endonucleases NotI and SpeI produced 12 and 26 fragments, respectively. Summation of the physical lengths of the fragments produced by NotI and SpeI cleavage yielded average sizes of 900 and 913 kbp, respectively, for the genome of T. pallidum subsp. pallidum. Contour-clamped homogeneous electric field electrophoresis of T. pallidum subsp. pallidum DNA exposed to 4 krads of gamma irradiation resolved a single band of 800 to 1,000 kbp; treatment of the DNA with 16 krads of gamma irradiation resulted in the production of smaller fragments, whereas untreated DNA did not migrate into the gels. The gamma irradiation results indicate that T. pallidum subsp. pallidum has a single, circular chromosome that was linearized at a dosage of 4 krads of gamma irradiation. The size estimate provided by restriction endonuclease digestion with NotI and SpeI shows that the genome of T. pallidum subsp. pallidum, at approximately 900 kbp, is considerably smaller than the 13,700-kbp genome size calculated from renaturation kinetics.

Low-speed centrifugation-mediated adsorption was evaluated as an enhancement of infectivity of clinical and laboratory strains of cytomegalovirus (CMV) occurring with cells grown in conventional culture tubes. The time required for reporting of primary isolates of CMV from urine specimens adsorbed onto monolayers of WI-38 cells in culture tubes was calculated. Of 668 specimens adsorbed by the stationary phase (SP) method, 98 were positive by cytopathic effect (CPE) that required an average of 16.8 days for recovery in culture. However, the appearance of CPE required a shorter average time of 11.9 days for 70 CMV strains isolated from 283 specimens adsorbed in tube cultures by the spin-amplified (SA) method. In another phase of clinical CMV recovery, urine specimens were adsorbed by the SA method onto cell cultures grown in both shell vials and test tubes. Of 594 specimens inoculated, a total of 74 were positive by either CPE in test tubes or immunostaininglocalized early antigen in shell vials. Approximately one-third of these CMV isolates were recovered only by CPE from specimens adsorbed by the SA method in test-tube cultures. In a related study to further evaluate differences between adsorption methods, the AD-169 laboratory strain of CMV was adsorbed by SP and SA methods onto MRC-5 cells grown in both culture vessels. Early antigen detection by immunomicroscopy was found in the infected cells at least 2 to 4 days prior to the appearance of CPE, regardless of adsorption procedure. In both vessels, the replication of AD-169 virus in cultures adsorbed by the SA method consistently exceeded that of virus adsorbed by the SP procedure. CPE occurred 24 to 48 h earlier and progressed two to four times more extensively; early antigen was expressed twoto fourfold greater within 24 to 48 h postinfection; and foci of infected cells containing late antigen were two to four times greater in number at 1, 2, and 5 days postinfection. Overall, the replication and enhancement of infectivity of laboratory and clinical strains of CMV as determined by CPE and early and late antigen expression occurred most efficiently with specimens adsorbed by the SA method onto cultures grown in conventional tubes or shell vials. * Corresponding author. t This report is dedicated to the memory of Sue Hwa Loo for her inspiration, scientific contributions, and dedication.