Marci M. Lesperance

Impairment of SLC17A8 Encoding Vesicular Glutamate Transporter-3, VGLUT3, Underlies Nonsyndromic Deafness DFNA25 and Inner Hair Cell Dysfunction in Null Mice

Autosomal-dominant sensorineural hearing loss is genetically heterogeneous, with a phenotype closely resembling presbycusis, the most common sensory defect associated with aging in humans. We have identified SLC17A8, which encodes the vesicular glutamate transporter-3 (VGLUT3), as the gene responsible for DFNA25, an autosomal-dominant form of progressive, high-frequency nonsyndromic deafness. In two unrelated families, a heterozygous missense mutation, c.632C-->T (p.A211V), was found to segregate with DFNA25 deafness and was not present in 267 controls. Linkage-disequilibrium analysis suggested that the families have a distant common ancestor. The A211 residue is conserved in VGLUT3 across species and in all human VGLUT subtypes (VGLUT1-3), suggesting an important functional role. In the cochlea, VGLUT3 accumulates glutamate in the synaptic vesicles of the sensory inner hair cells (IHCs) before releasing it onto receptors of auditory-nerve terminals. Null mice with a targeted deletion of Slc17a8 exon 2 lacked auditory-nerve responses to acoustic stimuli, although auditory brainstem responses could be elicited by electrical stimuli, and robust otoacoustic emissions were recorded. Ca(2+)-triggered synaptic-vesicle turnover was normal in IHCs of Slc17a8 null mice when probed by membrane capacitance measurements at 2 weeks of age. Later, the number of afferent synapses, spiral ganglion neurons, and lateral efferent endings below sensory IHCs declined. Ribbon synapses remaining by 3 months of age had a normal ultrastructural appearance. We conclude that deafness in Slc17a8-deficient mice is due to a specific defect of vesicular glutamate uptake and release and that VGLUT3 is essential for auditory coding at the IHC synapse.

Autosomal dominant stapes ankylosis with broad thumbs and toes, hyperopia, and skeletal anomalies is caused by heterozygous nonsense and frameshift mutations in NOG, the gene encoding noggin.

Although fixation of the stapes is usually progressive and secondary to otosclerosis, it may present congenitally, with other skeletal manifestations, as an autosomal dominant syndrome-such as proximal symphalangism (SYM1) or multiple-synostoses syndrome (SYNS1), both of which are caused by mutations in NOG, the gene encoding noggin. We describe a family that was ascertained to have nonsyndromic otosclerosis but was subsequently found to have a congenital stapes ankylosis syndrome that included hyperopia, a hemicylindrical nose, broad thumbs and great toes, and other minor skeletal anomalies but lacked symphalangism. A heterozygous nonsense NOG mutation-c.328C-->T (Q110X), predicted to truncate the latter half of the protein-was identified, and a heterozygous insertion in NOG-c.252-253insC, in which the frameshift is predicted to result in 96 novel amino acids before premature truncation-was identified in a previously described second family with a similar phenotype. In contrast to most NOG mutations that have been reported in kindreds with SYM1 and SYNS1, the mutations observed in these families with stapes ankylosis without symphalangism are predicted to disrupt the cysteine-rich C-terminal domain. These clinical and molecular findings suggest that (1) a broader range of conductive hearing-loss phenotypes are associated with NOG mutations than had previously been recognized, (2) patients with sporadic or familial nonsyndromic otosclerosis should be evaluated for mild features of this syndrome, and (3) NOG alterations should be considered in conductive hearing loss with subtle clinical and skeletal features, even in the absence of symphalangism.

Squamous cell carcinoma arising in inverted papilloma

A retrospective review of all cases of inverted papilloma at the University of Michigan from 1975 to 1992 revealed 51 cases of inverted papilloma. Of these, 14 (27%) had an associated squamous cell carcinoma (SCC). Eight (16%) were metachronous and 6 (11%) were synchronous. At a mean follow‐up of 53 months, the disease‐free survival of patients with carcinoma limited to the nasal cavity and paranasal sinuses was 57% (4/7) compared to 14% (1/7) of those patients presenting with disease extending beyond the nasal cavity and paranasal sinuses. The data also support the lateral rhinotomy approach with medial maxillectomy and ethmoidectomy as a minimum procedure followed by postoperative radiation therapy. The mean interval between the diagnosis of inverted papilloma and development of SCC was 63 months (range, 6 months to 13 years). Therefore, long‐term follow‐up with clinical examination and computed tomography (CT) scan is indicated for all patients with inverted papilloma.

A Dominantly Inherited Progressive Deafness Affecting Distal Auditory Nerve and Hair Cells

We have studied 72 members belonging to a large kindred with a hearing disorder inherited in an autosomal dominant pattern. We used audiological, physiological, and psychoacoustic measures to characterize the hearing disorders. The initial phenotypic features of the hearing loss are of an auditory neuropathy (AN) with abnormal auditory nerve and brainstem responses (ABRs) and normal outer hair cell functions [otoacoustic emissions (OAEs) and cochlear microphonics (CMs)]. Psychoacoustic studies revealed profound abnormalities of auditory temporal processes (gap detection, amplitude modulation detection, speech discrimination) and frequency processes (difference limens) beyond that seen in hearing impairment accompanying cochlear sensory disorders. The hearing loss progresses over 10–20 years to also involve outer hair cells, producing a profound sensorineural hearing loss with absent ABRs and OAEs. Affected family members do not have evidence of other cranial or peripheral neuropathies. There was a marked improvement of auditory functions in three affected family members studied after cochlear implantation with return of electrically evoked auditory brainstem responses (EABRs), auditory temporal processes, and speech recognition. These findings are compatible with a distal auditory nerve disorder affecting one or all of the components in the auditory periphery including terminal auditory nerve dendrites, inner hair cells, and the synapses between inner hair cells and auditory nerve. There is relative sparing of auditory ganglion cells and their axons.

Increased activity of Diaphanous homolog 3 (DIAPH3)/diaphanous causes hearing defects in humans with auditory neuropathy and in Drosophila

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.

Presence of human papillomavirus predicts recurrence of inverted papilloma

Recent evidence suggests that human papillomavirus may play a role in the pathogenesis of inverted papilloma, a benign but locally aggressive neoplasm with a high recurrence rate and an association with squamous cell carcinoma. Histologic features of inverted papilloma have not been useful in discriminating lesions at high risk for recurrence. We studied archival pathology specimens from 32 patients with inverted papilloma treated at the University of Michigan between 1980 and 1994 with polymerase chain reaction techniques and human papillomavirus E6 and L1 consensus primers. Twenty (63%) specimens tested positive for human papillomavirus. The clinical status of the remaining 25 patients was reviewed after seven patients with recent diagnosis or who were lost to follow-up were excluded. A significant association was identified between the presence of human papillomavirus DNA in inverted papilloma and recurrence after surgical excision. Thirteen of 15 patients whose tumors tested positive for HPV recurred, whereas none of the 10 patients whose tumors were human papillomavirus negative recurred (p < 0.00002). This strongly suggests that the presence of human papillomavirus predicts recurrence of inverted papilloma.

Human papillomavirus types important in progression of inverted papilloma.

Recent evidence suggests that human papillomavirus may play a role in the pathogenesis of inverted papilloma, a benign but locally aggressive neoplasm with a high recurrence rate and an association with squamous cell carcinoma. Histologic features of inverted papilloma have not been useful in discriminating lesions at high risk for malignant transformation. We studied archival pathology specimens from 39 patients with inverted papilloma treated at the University of Michigan between 1980 and 1994 using polymerase chain reaction techniques and human papillomavirus L1 and E6 consensus primers. Previously we reported that 63 of these specimens tested positive for human papillomavirus sequences and that presence of human papillomavirus predicted recurrence of inverted papilloma. We used type-specific primer pairs and polymerase chain reaction techniques as well as hybridization with type-specific oligonucleotide probes to determine human papillomavirus type. A significant correlation was observed between the severity of the lesion (dysplasia or carcinoma) and high risk human papillomavirus type (p < 0.01). All 12 benign inverted papilloma specimens that contained human papillomavirus tested positive for human papillomavirus 6 or 11. Of seven inverted papilloma specimens that exhibited dysplasia, five were human papillomavirus positive, three contained human papillomavirus 6, one contained human papillomavirus 11, and one contained human papillomavirus 18. In each of the three specimens that contained inverted papilloma in association with squamous cell carcinoma, the inverted papilloma portion of the specimen tested positive for a single human papillomavirus type: human papillomavirus 6, 11, or 16. Of the four human papillomavirus-positive specimens with squamous cell carcinoma alone (patients who had an inverted papilloma previously resected at the same site), three tested positive for human papillomavirus 16, and 1 was untyped. (OTOLARYNGOL HEAD NECK SURG 1995;113:558-63.)

A gene responsible for autosomal dominant auditory neuropathy (AUNA1) maps to 13q14–21

Hearing loss is most commonly defined as either conductive, affecting the sound conduction mechanism comprised of the external auditory canal, tympanic membrane, and middle ear ossicles, or sensorineural (SNHL), affecting the cochlea, the auditory nerve, or the central auditory pathway. However, the recent discovery that outer hair cells (OHC) generate otoacoustic emissions (OAEs) has allowed differentiation of sensory hearing loss (in which OAEs are absent) from neural hearing loss, which is caused by a lesion of inner hair cells and/or the auditory nerve. The hallmark of auditory neuropathy (AN), a neural type of hearing loss, is preservation of OAEs and abnormal or absent auditory brainstem responses.1 Most patients with SNHL are found to have a sensory type of hearing loss, and numerous genes for both syndromic and non-syndromic forms have been identified (Hereditary Hearing Loss Homepage, http://www.uia.ac.be/dnalab/hhh/). However, none of the approximately 50 dominant (DFNA) loci are known to represent an auditory neuropathy phenotype. AN may accompany peripheral neuropathy in a variety of dominant syndromes such as Charcot-Marie-Tooth disease2 and Freidreich’s ataxia.3 AN unassociated with peripheral neuropathy most commonly occurs as a sporadic or recessive trait,4–6 but X linked recessive6 and autosomal dominant7 forms have also been described. We have mapped a gene responsible for autosomal dominant auditory neuropathy in a multigenerational family from the United States to a novel locus, AUNA1 (auditory neuropathy, dominant, 1) on 13q14–21. The family is of European descent and was ascertained through two different probands by both the University of Michigan and the University of California at Irvine. The Institutional Review Boards of the University of Michigan Medical School, Louisiana State University Health Sciences Center, and the University of California at Irvine approved the study, and informed consent was obtained from all subjects. Four generations were available …

Identification of p.A684V Missense Mutation in the WFS1 Gene as a Frequent Cause of Autosomal Dominant Optic Atrophy and Hearing Impairment

Optic atrophy (OA) and sensorineural hearing loss (SNHL) are key abnormalities in several syndromes, including the recessively inherited Wolfram syndrome, caused by mutations in WFS1. In contrast, the association of autosomal dominant OA and SNHL without other phenotypic abnormalities is rare, and almost exclusively attributed to mutations in the Optic Atrophy‐1 gene (OPA1), most commonly the p.R445H mutation. We present eight probands and their families from the US, Sweden, and UK with OA and SNHL, whom we analyzed for mutations in OPA1 and WFS1. Among these families, we found three heterozygous missense mutations in WFS1 segregating with OA and SNHL: p.A684V (six families), and two novel mutations, p.G780S and p.D797Y, all involving evolutionarily conserved amino acids and absent from 298 control chromosomes. Importantly, none of these families harbored the OPA1 p.R445H mutation. No mitochondrial DNA deletions were detected in muscle from one p.A684V patient analyzed. Finally, wolframin p.A684V mutant ectopically expressed in HEK cells showed reduced protein levels compared to wild‐type wolframin, strongly indicating that the mutation is disease‐causing. Our data support OA and SNHL as a phenotype caused by dominant mutations in WFS1 in these additional eight families. Importantly, our data provide the first evidence that a single, recurrent mutation in WFS1, p.A684V, may be a common cause of ADOA and SNHL, similar to the role played by the p.R445H mutation in OPA1. Our findings suggest that patients who are heterozygous for WFS1 missense mutations should be carefully clinically examined for OA and other manifestations of Wolfram syndrome.

A gene for autosomal dominant hearing impairment (DFNA14) maps to a region on chromosome 4p16.3 that does not overlap the DFNA6 locus

Non-syndromic hearing impairment is one of the most heterogeneous hereditary conditions, with more than 40 reported gene localisations. We have identified a large Dutch family with autosomal dominant non-syndromic sensorineural hearing impairment. In most patients, the onset of hearing impairment is in the first or second decade of life, with a slow decline in the following decades, which stops short of profound deafness. The hearing loss is bilateral, symmetrical, and only affects low and mid frequencies up to 2000 Hz. In view of the phenotypic similarities of this family with an American family that has been linked to chromosome 4p16.3 (DFNA6), we investigated linkage to the DFNA6 region. Lod score calculations confirmed linkage to this region with two point lod scores above 6. However, as haplotype analysis indicated that the genetic defect in this family is located in a 5.6 cM candidate region that does not overlap the DFNA6 region, the new locus has been named DFNA14.