P.L.M. Huygen

GJB2 Mutations and Degree of Hearing Loss: A Multicenter Study

Hearing impairment (HI) affects 1 in 650 newborns, which makes it the most common congenital sensory impairment. Despite extraordinary genetic heterogeneity, mutations in one gene, GJB2, which encodes the connexin 26 protein and is involved in inner ear homeostasis, are found in up to 50% of patients with autosomal recessive nonsyndromic hearing loss. Because of the high frequency of GJB2 mutations, mutation analysis of this gene is widely available as a diagnostic test. In this study, we assessed the association between genotype and degree of hearing loss in persons with HI and biallelic GJB2 mutations. We performed cross-sectional analyses of GJB2 genotype and audiometric data from 1,531 persons, from 16 different countries, with autosomal recessive, mild-to-profound nonsyndromic HI. The median age of all participants was 8 years; 90% of persons were within the age range of 0-26 years. Of the 83 different mutations identified, 47 were classified as nontruncating, and 36 as truncating. A total of 153 different genotypes were found, of which 56 were homozygous truncating (T/T), 30 were homozygous nontruncating (NT/NT), and 67 were compound heterozygous truncating/nontruncating (T/NT). The degree of HI associated with biallelic truncating mutations was significantly more severe than the HI associated with biallelic nontruncating mutations (P<.0001). The HI of 48 different genotypes was less severe than that of 35delG homozygotes. Several common mutations (M34T, V37I, and L90P) were associated with mild-to-moderate HI (median 25-40 dB). Two genotypes--35delG/R143W (median 105 dB) and 35delG/dela(GJB6-D13S1830) (median 108 dB)--had significantly more-severe HI than that of 35delG homozygotes.

A genotype-phenotype correlation for GJB2 (connexin 26) deafness

Introduction: Mutations in GJB2 are the most common cause of non-syndromic autosomal recessive hearing impairment, ranging from mild to profound. Mutation analysis of this gene is widely available as a genetic diagnostic test. Objective: To assess a possible genotype-phenotype correlation for GJB2. Design: Retrospective analysis of audiometric data from people with hearing impairment, segregating two GJB2 mutations. Subjects: Two hundred and seventy seven unrelated patients with hearing impairment who were seen at the ENT departments of local and university hospitals from Italy, Belgium, Spain, and the United States, and who harboured bi-allelic GJB2 mutations. Results: We found that 35delG homozygotes have significantly more hearing impairment, compared with 35delG/non-35delG compound heterozygotes. People with two non-35delG mutations have even less hearing impairment. We observed a similar gradient of hearing impairment when we categorised mutations as inactivating (that is, stop mutations or frame shifts) or non-inactivating (that is, missense mutations). We demonstrated that certain mutation combinations (including the combination of 35delG with the missense mutations L90P, V37I, or the splice-site mutation IVS1+1G>A, and the V37I/V37I genotype) are associated with significantly less hearing impairment compared with 35delG homozygous genotypes. Conclusions: This study is the first large systematic analysis indicating that the GJB2 genotype has a major impact on the degree of hearing impairment, and identifying mild genotypes. Furthermore, this study shows that it will be possible to refine this correlation and extend it to additional genotypes. These data will be useful in evaluating habilitation options for people with GJB2 related deafness.

Occupational Noise, Smoking, and a High Body Mass Index are Risk Factors for Age-related Hearing Impairment and Moderate Alcohol Consumption is Protective: A European Population-based Multicenter Study

A multicenter study was set up to elucidate the environmental and medical risk factors contributing to age-related hearing impairment (ARHI). Nine subsamples, collected by nine audiological centers across Europe, added up to a total of 4,083 subjects between 53 and 67 years. Audiometric data (pure-tone average [PTA]) were collected and the participants filled out a questionnaire on environmental risk factors and medical history. People with a history of disease that could affect hearing were excluded. PTAs were adjusted for age and sex and tested for association with exposure to risk factors. Noise exposure was associated with a significant loss of hearing at high sound frequencies (>1 kHz). Smoking significantly increased high-frequency hearing loss, and the effect was dose-dependent. The effect of smoking remained significant when accounting for cardiovascular disease events. Taller people had better hearing on average with a more pronounced effect at low sound frequencies (<2 kHz). A high body mass index (BMI) correlated with hearing loss across the frequency range tested. Moderate alcohol consumption was inversely correlated with hearing loss. Significant associations were found in the high as well as in the low frequencies. The results suggest that a healthy lifestyle can protect against age-related hearing impairment.

Diagnosis and treatment of the Pierre Robin sequence : results of a retrospective clinical study and review of the literature

Abstract We performed a retrospective study of all children with Pierre Robin sequence (PRS), admitted to our hospital from 1981–1998 in order to evaluate diagnosis, treatment and prognosis. Patients were divided into two categories: isolated PRS (group 1) and PRS plus, i.e. PRS as part of a more complex syndrome (group 2). A total of 74 patients with PRS were found, 29 (39%) males and 45 (61%) females of whom 47 (63.5%) could be categorised as isolated PRS and 27 (36.8%) as PRS plus. The most frequent diagnoses in patients with PRS plus were Stickler syndrome and the velocardiofacial syndrome. Ophthalmological and fluorescent in situ hybridisation of chromosome 22 investigations should therefore be performed in all patients, as soon as the diagnosis of PRS is established. Some form of airway treatment was necessary in the majority of patients (52 of 74), most could be treated conservatively with prone/lateral positioning and close observation. Endotracheal intubation was necessary in one child from group 1 versus five from group 2. Tracheostomy was performed in three children from group 1 and two from group 2. Feeding problems occurred in about 25% of all PRS patients and stunted growth was seen especially in boys with isolated PRS before the age of 10 months. Conclusion In our series, 33% of patients with Pierre Robin sequence plus had Stickler and velocardiofacial syndromes. Conservative airway management was a sufficient treatment for respiratory problems in the majority of patients. Feeding and growth need special attention in patients with Pierre Robin sequence.

Genotype–phenotype studies in nail-patella syndrome show that LMX1B mutation location is involved in the risk of developing nephropathy

Nail-patella syndrome (NPS) is characterized by developmental defects of dorsal limb structures, nephropathy, and glaucoma and is caused by heterozygous mutations in the LIM homeodomain transcription factor LMX1B. In order to identify possible genotype–phenotype correlations, we performed LMX1B mutation analysis and comprehensive investigations of limb, renal, ocular, and audiological characteristics in 106 subjects from 32 NPS families. Remarkable phenotypic variability at the individual, intrafamilial, and interfamilial level was observed for different NPS manifestations. Quantitative urinanalysis revealed proteinuria in 21.3% of individuals. Microalbuminuria was detected in 21.7% of subjects without overt proteinuria. Interestingly, nephropathy appeared significantly more frequent in females. A significant association was established between the presence of clinically relevant renal involvement in an NPS patient and a positive family history of nephropathy. We identified normal-tension glaucoma (NTG) and sensorineural hearing impairment as new symptoms associated with NPS. Sequencing of LMX1B revealed 18 different mutations, including six novel variants, in 28 families. Individuals with an LMX1B mutation located in the homeodomain showed significantly more frequent and higher values of proteinuria compared to subjects carrying mutations in the LIM domains. No clear genotype–phenotype association was apparent for extrarenal manifestations. This is the first study indicating that family history of nephropathy and mutation location might be important in precipitating individual risks for developing NPS renal disease. We suggest that the NPS phenotype is broader than previously described and that NTG and hearing impairment are part of NPS. Further studies on modifier factors are needed to understand the mechanisms underlying phenotypic heterogeneity.

A mutation in the gamma actin 1 (ACTG1) gene causes autosomal dominant hearing loss (DFNA20/26).

Linkage analysis in a multigenerational family with autosomal dominant hearing loss yielded a chromosomal localisation of the underlying genetic defect in the DFNA20/26 locus at 17q25-qter. The 6-cM critical region harboured the γ-1-actin (ACTG1) gene, which was considered an attractive candidate gene because actins are important structural elements of the inner ear hair cells. In this study, a Thr278Ile mutation was identified in helix 9 of the modelled protein structure. The alteration of residue Thr278 is predicted to have a small but significant effect on the γ 1 actin structure owing to its close proximity to a methionine residue at position 313 in helix 11. Met313 has no space in the structure to move away. Moreover, the Thr278 residue is highly conserved throughout eukaryotic evolution. Using a known actin structure the mutation could be predicted to impair actin polymerisation. These findings strongly suggest that the Thr278Ile mutation in ACTG1 represents the first disease causing germline mutation in a cytoplasmic actin isoform.

Progressive hearing loss, hypoplasia of the cochlea and widened vestibular aqueducts are very common features in Pendred’s syndrome

Long-term hearing threshold-on-age follow-up data, including non-linear regression analysis, are given for 12 consecutive Pendred patients. The clinical diagnosis of Pendreds syndrome was confirmed by a mutation analysis of the PDS gene in 11 out of the 11 cases tested. Recent imaging of the temporal bones in seven out of these 12 patients showed widened vestibular aqueducts in each case. The diagnostic perchlorate test was negative in one patient, but this test was positive in her affected sister. Mutation analysis of the PDS gene in these patients confirmed that Pendreds syndrome is a monogenetic disorder. Progressive sensorineural hearing loss and widened vestibular aqueducts are characteristic features of Pendreds syndrome, which provides the opportunity to diagnose Pendreds syndrome clinically in the first few years of life, as has recently been suggested in a case report (Cremers et al., Progressive sensorineural hearing loss and a widend vestibular aqueduct in Pendred syndrome, Arch. Otolaryngol. 124 (1998) 501-505). Mutation analysis of the involved gene can be used to confirm the clinical diagnosis.

Hearing impairment and neurological dysfunction associated with a mutation in the mitochondrial tRNA^{Ser(UCN)} gene

We studied a large Dutch family with maternally inherited, progressive, sensorineural hearing loss in 27 patients. Only in a single family member was the hearing loss accompanied by neurological symptoms including ataxia and dysarthria. DNA analysis of the mitochondrial genome revealed the insertion of a C at nucleotide position 7472 in the tRNASer(UCN) gene (7472insC mutation). We determined the percentage of mutant DNA (heteroplasmy) in blood from all family members, and found no correlation between hearing loss and leucocyte heteroplasmy. The 7472insC mutation was previously identified in a smaller family from Sicily with sensorineural hearing loss in 9 family members, six of them also presenting neurologically with ataxia and myoclonus. The presence of the 7472insC mutation in two different pedigrees strongly supports its pathogenicity. However, the interfamilial difference in penetrance of the neurologic abnormalities is most likely to be strongly influenced by secondary factors different from the 7472insC mutation, as heteroplasmy or age of the patients were similar in both families. This mutation should therefore be analysed in families with maternally inherited hearing loss, irrespective of whether the hearing loss is non-syndromic or accompanied by neurologic abnormalities.

Next-generation sequencing identifies mutations of SMPX, which encodes the small muscle protein, X-linked, as a cause of progressive hearing impairment

In a Dutch family with an X-linked postlingual progressive hearing impairment, a critical linkage interval was determined to span a region of 12.9 Mb flanked by the markers DXS7108 and DXS7110. This interval overlaps with the previously described DFNX4 locus and contains 75 annotated genes. Subsequent next-generation sequencing (NGS) detected one variant within the linkage interval, a nonsense mutation in SMPX. SMPX encodes the small muscle protein, X-linked (SMPX). Further screening was performed on 26 index patients from small families for which X-linked inheritance of nonsyndromic hearing impairment (NSHI) was not excluded. We detected a frameshift mutation in SMPX in one of the patients. Segregation analysis of both mutations in the families in whom they were found revealed that the mutations cosegregated with hearing impairment. Although we show that SMPX is expressed in many different organs, including the human inner ear, no obvious symptoms other than hearing impairment were observed in the patients. SMPX had previously been demonstrated to be specifically expressed in striated muscle and, therefore, seemed an unlikely candidate gene for hearing impairment. We hypothesize that SMPX functions in inner ear development and/or maintenance in the IGF-1 pathway, the integrin pathway through Rac1, or both.

Contribution of the N-acetyltransferase 2 polymorphism NAT2*6A to age-related hearing impairment

Background: Age-related hearing impairment (ARHI) is the most common sensory impairment in older people, affecting 50% of those aged 80 years. The proportion of older people is increasing in the general population, and as a consequence, the number of people affected with ARHI is growing. ARHI is a complex disorder, with both environmental and genetic factors contributing to the disease. The first studies to elucidate these genetic factors were recently performed, resulting in the identification of the first two susceptibility genes for ARHI, NAT2 and KCNQ4. Methods: In the present study, the association between ARHI and polymorphisms in genes that contribute to the defence against reactive oxygen species, including GSTT1, GSTM1 and NAT2, was tested. Samples originated from seven different countries and were combined into two test population samples, the general European population and the Finnish population. Two distinct phenotypes for ARHI were studied, Zlow and Zhigh, representing hearing in the low and high frequencies, respectively. Statistical analysis was performed for single polymorphisms (GSTM1, GSTT1, NAT2*5A, NAT2*6A, and NAT2*7A), haplotypes, and gene–environment and gene–gene interactions. Results: We found an association between ARHI and GSTT1 and GSTM1 in the Finnish population sample, and with NAT2*6A in the general European population sample. The latter finding replicates previously published data. Conclusion: As replication is considered the ultimate proof of true associations in the study of complex disorders, this study provides further support for the involvement of NAT2*6A in ARHI.