István Sziklai

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.

Sensorineural hearing loss in chronic otitis media.

Objective To determine whether chronic suppurative otitis media may cause sensorineural hearing loss. Methods The files of 121 patients with unilateral chronic suppurative otitis media were reviewed in a retrospective study. Air conduction and bone conduction threshold averages were calculated over the speech frequencies (500 Hz, 1,000 Hz, and 2,000 Hz). Thresholds at 4 kHz were examined separately but in a similar way. Multiple linear regression models were used to clarify the relationships between sensorineural hearing loss and chronic otitis media. Results Chronic suppurative otitis media was seen to be associated with sensorineural hearing loss. When age and normal side were corrected for, pure-tone threshold and bone conduction threshold at either the speech frequencies or at 4 kHz increased gradually according to the duration of the chronic suppurative otitis media. The threshold shift was more accentuated as age increased. The sensorineural hearing loss at 4 kHz seemed to be higher than that at the speech frequencies. Conclusions The inner ear is vulnerable against chronic suppurative otitis media. Older age increases this vulnerability. The proximity of the sensory cells to the potential source of harm (inflamed middle ear) may mean higher exposure, as reflected by the fact that sensory cells processing higher frequencies are more seriously damaged.

The characteristics of human papillomavirus DNA in head and neck cancers and papillomas.

Aim: To determine the prevalence, type, physical state, and viral load of human papillomavirus (HPV) DNA in cases of head and neck cancer and recurrent respiratory papillomatosis (RRP). Methods: The prevalence and type of HPV DNA was determined in 27 fresh frozen tissue specimens from patients with head and neck cancers and 16 specimens from 10 patients with RRP by MY09/MY11 and GP5+/GP6+ nested polymerase chain reaction (PCR) and subsequent restriction enzyme cleavage. The physical state of HPV DNA was analysed by E1, E2, and E1E2 specific PCRs and Southern blot hybridisation (SBH). Results: HPV DNA was detected in 13 of 27 cancers and 10 of 10 papillomas. Both low risk HPV-6 and HPV-11 and high risk HPV-16 were present in cancers in low copy numbers, whereas papillomas exclusively harboured low risk HPV-6 and HPV-11. E1E2 PCRs failed to determine the physical state of HPV in cancers except one case where HPV-6 DNA was integrated. In contrast to cancers, all papillomas showed the episomal state of HPV DNA and a relatively higher viral load. Conclusions: Based on the prevalence, type, physical state, and copy number of HPV DNA, cancers and papillomas tend to show a different HPV DNA profile. The 100% positivity rate of low risk HPV types confirms the role of HPV-6 and HPV-11 in the aetiology of RRP.

Time-related alteration of endolymph composition in an experimental model of endolymphatic hydrops

The electrochemical changes of the inner ear fluids were studied in the guinea pig during the development of endolymphatic hydrops in an experimental model of Menieres disease obtained by the blockage of the vestibular aqueduct. The endocochlear potential (first and third turns) was recorded, and the sodium, potassium, and chloride concentrations, and osmolality of the endolymph (first and third turns) and perilymph were determined at different intervals from 2 to 24 weeks after the induction of the hydrops. The development of hydrops was monitored by the compound action potential once a week during the observation period. In normal, nonoperated guinea pigs, longitudinal endolymphatic gradients of endocochlear potential, potassium and chloride concentrations, and osmolality, increasing from the apex to the base of the cochlea, were observed. After 2 weeks of hydrops, no alteration of this pattern was detected. After 6 and 9 weeks of hydrops, a progressive decrease of endocochlear potential, potassium and chloride concentrations, and osmolality was noticed at the first turn (6 and 9 weeks) and then at the third turn (9 weeks) which resulted in the disappearance of longitudinal gradients. At 24 weeks, the endocochlear potential was still diminished by 60%, whereas potassium and chloride concentrations and osmolality increased as compared to 9‐week values but remained lower than in controls. The changes in composition of endolymph induced by the development of the hydrops could be related to the progressive alteration of the ionic permeability of the cochlear epithelium, which should be localized at the distended Reissners membrane.

Effect of acetylcholine and GABA on the transfer function of electromotility in isolated outer hair cells

Outer hair cells (OHC) from high- and low-frequency regions were separately isolated from guinea pig cochleas. The cells were inserted with their ciliary pole first into a partitioning microchamber so that only 20-50% of the cell length was excluded. Somatic length changes due to transcellular electrical stimulation were measured at the cuticular plate in the inserted portion of the cells. Transfer curves of electromotility of the OHCs were obtained by both a series of brief (2.5 ms) and longer (30 ms) square pulses with opposite polarity and linearly increasing size from 40 to 280 mV in both negative and positive directions. Alterations in the transient and steady-state electromotility transfer curves were examined by application of acetylcholine (ACh) and gamma-aminobutyric acid (GABA) to the synaptic pole. ACh, in the concentration range of 10-30 microM, evoked a significant magnitude and gain increase of electromotility in both transient and steady-state responses without a measurable shift in the operating point of the displacement-voltage transfer curve. A tonotopic response magnitude difference is found for ACh challenge. Basal turn OHCs responded with greater magnitude increase (+90% increase from control) than apical turn OHCs (+40%). GABA exerted an opposite effect, again in a location-dependent manner. Magnitude response decreased about 30% for long cells and 14% for short ones. Atropin, a muscarinic receptor antagonist, completely blocked the increase in electromotility response due to ACh. However, D-tubocurarine, a nicorinic receptor antagonist, while not blocking the ACh effect, altered the cells apparent operating point. Bicuculline methiodide, a GABAA-receptor antagonist, completely arrested GABA influences on the electromotility response. These results suggest that both ACh and GABA can change the electromotile activity of OHCs, in a tonotopically biased manner. ACh challenge evokes greater magnitude responses in basal turn OHCs, whereas GABA induces greater motility response decrease in apical turn OHCs. The control of the gain and magnitude of electromotility by the transmitter substances appear to involve at least two mechanisms. One is probably related to conformational changes of the voltage-to-movement converter molecules and a change in their number in an effective operational pool, the other operates via changing the electrical resistance of the basolateral cell membrane.

Phenotypic variability of patients homozygous for the GJB2 mutation 35delG cannot be explained by the influence of one major modifier gene

Hereditary hearing loss (HL) is a very heterogeneous trait, with 46 gene identifications for non-syndromic HL. Mutations in GJB2 cause up to half of all cases of severe-to-profound congenital autosomal recessive non-syndromic HL, with 35delG being the most frequent mutation in Caucasians. Although a genotype–phenotype correlation has been established for most GJB2 genotypes, the HL of 35delG homozygous patients is mild to profound. We hypothesise that this phenotypic variability is at least partly caused by the influence of modifier genes. By performing a whole-genome association (WGA) study on 35delG homozygotes, we sought to identify modifier genes. The association study was performed by comparing the genotypes of mild/moderate cases and profound cases. The first analysis included a pooling-based WGA study of a first set of 255 samples by using both the Illumina 550K and Affymetrix 500K chips. This analysis resulted in a ranking of all analysed single-nucleotide polymorphisms (SNPs) according to their P-values. The top 250 most significantly associated SNPs were genotyped individually in the same sample set. All 192 SNPs that still had significant P-values were genotyped in a second independent set of 297 samples for replication. The significant P-values were replicated in nine SNPs, with combined P-values between 3 × 10−3 and 1 × 10−4. This study suggests that the phenotypic variability in 35delG homozygous patients cannot be explained by the effect of one major modifier gene. Significantly associated SNPs may reflect a small modifying effect on the phenotype. Increasing the power of the study will be of greatest importance to confirm these results.

Acetylcholine controls the gain of the voltage-to-movement converter in isolated outer hair cells.

Sensitivity of the electromotility of isolated guinea pig outer hair cells (OHCs) to acetylcholine (ACh) was examined. OHCs were held in a partitioning microchamber in a position so that their ciliary poles were inserted and their somatic length changes measured. Transcellular square-wave stimuli were delivered to record voltage-to-movement conversion of the cells. ACh was applied to the synaptic poles. The transfer function of electromotility intimated shifts of the membrane potential with concomitant gain changes: gain decreases when the membrane potential is shifted in hyperpolarization direction and gain increases when the membrane potential is shifted in the opposite direction due to application of ACh in 100-500 microM concentration. Clear gain increase of the electromotility in basal turn OHCs to ACh was observed, whereas inconsistent results for apical turn OHCs were found. The latter is probably due to the pharmacological dose of ACh used. This possibility is further supported by results in which ACh abolished the sensitivity of the magnitude response of electromotility to DC depolarizing bias.

Phosphorylation Mediates the Influence of Acetylcholine upon Outer Hair Cell Electromotility

Isolated guinea-pig outer hair cells (OHCs) (n = 52) were inserted into a partitioning microchamber and electromotility was measured by a calibrated optoelectronic apparatus. Acetylcholine (ACh), and ACh together with different protein kinase inhibitors, were applied to OHCs through a puffer pipette. ACh produced a magnitude increase of electromotility. This magnitude increase was inhibited by co-application of KN-62, a calcium/calmodulin-dependent protein kinase II (CAMKII) inhibitor. Simultaneous application of ACh and H-89, a selective protein kinase A (PKA) inhibitor, did not antagonize the ACh response. Further support for the CAMKII-mediated ACh influence on electromotility is that the magnitude increase is also inhibited by the calmodulin antagonist trifluoperazine (TFP) and by the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) inhibitor thapsigargin. The results suggest an essential role of calcium in the ACh-mediated increase of the magnitude of electromotility. Elevation of the intracellular calcium concentration apparently activates CAMKII which, in turn, phosphorylates membrane or cytoskeletal substrate(s). This molecular modification probably leads to reduced axial cell stiffness and subsequent increase of the electromotile response.Isolated guinea-pig outer hair cells (OHCs) (n=52) were inserted into a partitioning microchamber and electromotility was measured by a calibrated optoelectronic apparatus. Acetylcholine (ACh), and ACh together with different protein kinase inhibitors, were applied to OHCs through a puffer pipette. ACh produced a magnitude increase of electromotility. This magnitude increase was inhibited by co-application of KN-62, a calcium/calmodulin-dependent protein kinase II (CAMKII) inhibitor. Simultaneous application of ACh and H-89, a selective protein kinase A (PKA) inhibitor, did not antagonize the ACh response. Further support for the CAMKII-mediated ACh influence on electromotility is that the magnitude increase is also inhibited by the calmodulin antagonist trifluoperazine (TFP) and by the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) inhibitor thapsigargin. The results suggest an essential role of calcium in the ACh-mediated increase of the magnitude of electromotility. Elevation of the intracellular calcium concentration apparently activates CAMKII which, in turn, phosphorylates membrane or cytoskeletal substrate(s). This molecular modification probably leads to reduced axial cell stiffness and subsequent increase of the electromotile response.

Ionic currents determining the membrane characteristics of type I spiral ganglion neurons of the guinea pig

Enzymatically isolated type I spiral ganglion neurons of the guinea pig have been investigated in the present study. The identity of the cells was confirmed by using anti‐neuron‐specific enolase immunostaining. The presence and shredding of the myelin sheath was also documented by employing anti‐S100 immunoreaction. The membrane characteristics of the cells were studied by using the whole‐cell patch‐clamp technique. The whole‐cell capacitance of the cells was 9 ± 2 pF (n = 51), while the resting membrane potential of the cells was −62 ± 9 mV (n = 19). When suprathreshold depolarizing stimuli were applied, the neurons fired a single action potential at the beginning of the stimulation. It was confirmed in this study that type I spiral ganglion cells possess a hyperpolarization‐activated nonspecific cationic current (Ih). The major characteristics of this current component were unaffected by the enzyme treatment. Type I spiral ganglion cells also expressed various depolarization‐activated K+ current components. A high‐threshold outward current was sensitive to 1–10 mm TEA+ application. The ganglion cells also expressed a relatively small, but nevertheless present, transient outward current component which was less sensitive to TEA+ but could be inhibited by 100 µm 4‐aminopyridine. A DTX‐I‐sensitive current was responsible for some 30% of the total outward current (at 0 mV), showed rapid activation at membrane potentials positive to −50 mV and demonstrated very little inactivation. However, inhibition of the highly 4‐AP‐ or DTX‐I‐sensitive component did not alter the rapidly inactivating nature of the firing pattern of the cells.

Measles virus prevalence in otosclerotic stapes footplate samples

Hypothesis: The cause of otosclerosis is still unknown. Persistent measles virus infection of the otic capsule is supposed to be one of the etiologic factors in otosclerosis. Chronic viral antigen expression on the surface of infected cells can induce a secondary autoimmune reaction against the otic capsule. Background: In the past 15 years, some reports proposed the possible etiologic role of measles virus in otosclerosis. The presence of measles virus was shown in otosclerotic patients by reverse-transcriptase polymerase chain reaction amplification of the viral RNA, detecting the viral proteins by immunohistochemistry and detecting antimeasles immunoglobulin G in the perilymph samples. Many concerns were elicited by these results. Methods: Nucleic acid was extracted from pulverized, frozen stapes footplate samples of otosclerotic patients. Measles virus RNA was amplified by reverse-transcriptase polymerase chain reaction: reverse transcription and the first round polymerase chain reaction amplification was performed by heat stable recombinant Thermus thermophilus polymerase, whereas in the nested round, polymerase chain reaction Taq-polymerase was used. Measles virus nucleoprotein RNA-specific oligonucleotide primers were used in these reactions. An Edmonston-type measles virus served as a positive control and cortical bone fragments or stapes superstructures served as negative controls. Results: Among 34 otosclerotic patients, 20 stapes footplate samples contained measles virus RNA. Measles virus RNA was not detected in other bone specimens of the patients. Conclusion: The etiologic role of measles virus in the pathogenesis of otosclerosis should be considered. The 14 negative samples may be genetically determined otosclerotic cases.