Mao-Chang Su

Mutations of Cx26 gene (GJB2) for prelingual deafness in Taiwan.

Mutations in the Cx26 (GJB2) gene have been shown to be responsible for a major part of autosomal recessive non-syndromic inherited prelingual deafness. We have sequenced the coding region of GJB2 gene from 169 Taiwanese patients with prelingual deafness and 100 unrelated normal individuals. In the deaf patients, three mutations were found: two novel mutations, 551G→A, and 299-300delAT, and one previously described mutation, 235delC. Four previously reported polymorphisms, 79G→A, 109G→A, 341A→G, and 608T→C, were also found in both deaf patients and normal individuals and one new possible polymorphism, 558G→A, which was only found in a patient. Interestingly, we did not find the 35delG allele, which is commonly found in the Caucasian population, either in the patients or in normal individuals we examined. Our data also showed 235delC to be the most common type of mutation found in Cx26 mutants (approximately 57%). Therefore, based on our findings, we have developed a simple molecular test for the 235delC mutation and it should be of considerable help to those families to understand the cause of their children having the prelingual deafness.

Identification of Novel Mutations in the KCNQ4 Gene of Patients with Nonsyndromic Deafness from Taiwan

Ion channels play important roles in signal transduction and in the regulation of the ionic composition of intra- and extracellular fluids. Mutations in ion channels have long been thought to be responsible for some forms of hearing loss. Defects in KCNQ4, a voltage-gated potassium channel, are a cause of nonsyndromic sensorineural deafness type 2, an autosomal dominant form of progressive hearing loss. We present data of mutation analysis of KCNQ4 from 185 unrelated Taiwanese probands with nonsyndromic hearing loss. The analysis revealed three novel KCNQ4 mutations and many polymorphisms. The prevalence of KCNQ4 gene mutations in this study was 1.62% (3/185). The mutations include a missense mutation (F182L) and two silent mutations (R216R and T501T). The F182L missense mutation was located in the S3 domain of KCNQ4. The F182 residue of KCNQ4 is highly conserved in KCNQ4 among various species and is less conserved in all members of the KCNQ family. In addition, although R216R is a silent mutation and does not alter the content of amino acid residue, the neural network prediction system revealed that it can potentially create a novel splice donor site during transcription. This mutation might affect the protein structure of KCNQ4 and consequently the normal function of the K+ channel. Our data provide the first comprehensive analysis of the KCNQ4 gene in Taiwanese patients with nonsyndromic deafness.

Epithelial Cells of Nasal Mucosa Express Functional Gap Junctions of Connexin 43

Objective—Recent studies have suggested that inositol triphosphate is transmitted through the gap junction of adjacent epithelial cells and communicates the intracellular calcium wave that controls the beating of cilia. Therefore, gap junction-mediated intercellular communication may modulate the metachronal ciliary beating of respiratory mucosa. Material and Methods—The expression and function of gap junctions were evaluated in nasal mucosa using immunofluorescent staining, Western blotting and Lucifer Yellow dye coupling in both whole nasal mucosa and cultured nasal epithelial cells. Results—Among connexins (Cxs) 26, 32 and 43, only Cx43 was found in the nasal mucosa, in both the epithelial cells and submucosa tissue. Using Western blotting, Cx43 protein was found in protein extracts of both whole mucosa and cultured epithelial cells. The Cx43 protein content increased twofold between the 3rd and 6th days of culture. Lucifer Yellow dye, microinjected into single cultured epithelial cells, had spread to the adjacent cells. Conclusions—Cx43 is present in nasal mucosa. The expression of Cx43 on epithelial cells increases after culture. The dye-coupling study in epithelial cells indicated that the signal may be transmitted to neighboring cells via a gap junction composed of Cx43. Thus, Cx43 may be involved in the regulation of metachronal ciliary beating in ciliated epithelial cells and growth or differentiation in nasal tissue.

A novel missense mutation in the connexin30 causes nonsyndromic hearing loss.

Dysfunctional gap junctions caused by GJB2 (CX26) and GJB6 (CX30) mutations are implicated in nearly half of nonsyndromic hearing loss cases. A recent study identified a heterozygous mutation, c.119C>T (p.A40V), in the GJB6 gene of patients with nonsyndromic hearing loss. However, the functional role of the mutation in hearing loss remains unclear. In this study, analyses of cell biology indicated that a p.A40V missense mutation of CX30 causes CX30 protein accumulation in the Golgi body rather than in the cytoplasmic membrane. The tet-on protein expression system was used for further study of mutant proteins in CX30 and CX30A40V co-expressions and in CX26 and CX30A40V co-expressions. The p.A40V missense mutation exerted a dominant negative effect on both normal CX30 and CX26, which impaired gap junction formation. Moreover, computer-assisted modeling suggested that this p.A40V mutation affects the intra molecular interaction in the hydrophobic core of Trp44, which significantly alters the efficiency of gap junction formation. These findings suggest that the p.A40V mutation in CX30 causes autosomal-dominant nonsyndromic hearing loss. These data provide a novel molecular explanation for the role of GJB6 in hearing loss.

Mutation R184Q of connexin 26 in hearing loss patients has a dominant-negative effect on connexin 26 and connexin 30.

Hearing impairment is the most common sensory disorder worldwide. In a recent study, the authors have shown that a heterozygous missense mutation, p.R184Q, in the connexin 26 (Cx26) is causally related to hearing loss. However, the functional change in the Cx26R184Q mutant remains unknown. This study compared the intracellular distribution and assembly of mutant Cx26R184Q with that of the wild-type (WT) Cx26 and Cx30WT in tet-on HeLa cells and the effect that the mutant protein had on those cells. Fluorescent localization assay of WT Cx26 showed the typical punctuate pattern of gap junction channel between neighboring expression cells. Conversely, the p.R184Q missense mutation resulted in accumulation of the Cx26 mutant protein in the Golgi apparatus rather than in the cytoplasmic membrane. Cx26R184Q coexpressed with either Cx26WT or Cx30WT showed perinuclear localization by bidirectional tet-on expression system, suggesting the impairment of the ability of both WT proteins to intracellular trafficking and targeting to the plasma membrane. Therefore, we proposed that Cx26R184Q has a dominant-negative effect on the function of WT Cx26 and Cx30.

Tailor-made zinc-finger transcription factors activate FLO11 gene expression with phenotypic consequences in the yeast Saccharomyces cerevisiae.

Cys2His2 zinc fingers are eukaryotic DNA-binding motifs, capable of distinguishing different DNA sequences, and are suitable for engineering artificial transcription factors. In this work, we used the budding yeast Saccharomyces cerevisiae to study the ability of tailor-made zinc finger proteins to activate the expression of the FLO11 gene, with phenotypic consequences. Two three-finger peptides were identified, recognizing sites from the 5′ UTR of the FLO11 gene with nanomolar DNA-binding affinity. The three-finger domains and their combined six-finger motif, recognizing an 18-bp site, were fused to the activation domain of VP16 or VP64. These transcription factor constructs retained their DNA-binding ability, with the six-finger ones being the highest in affinity. However, when expressed in haploid yeast cells, only one three-finger recombinant transcription factor was able to activate the expression of FLO11 efficiently. Unlike in the wild-type, cells with such transcriptional activation displayed invasive growth and biofilm formation, without any requirement for glucose depletion. The VP16 and VP64 domains appeared to act equally well in the activation of FLO11 expression, with comparable effects in phenotypic alteration. We conclude that the functional activity of tailor-made transcription factors in cells is not easily predicted by the in vitro DNA-binding activity.

Identification of novel variants in the TMIE gene of patients with nonsyndromic hearing loss

OBJECTIVE To determine whether variants of the TMIE gene are causes of nonsyndromic deafness in Taiwan. METHODS A genetic survey was made from 370 individuals, with 250 nonsyndromic hearing loss and 120 normal hearing individuals. Genomic DNA was extracted from peripheral blood leukocytes and then subjected to PCR to amplify selected exons and flanking introns of the TMIE gene; the amplified products were screened for base variants by autosequence. Data from the two groups were then compared using Fishers two-tailed exact test and Armitages trend test. RESULTS The analysis revealed 7 novel variants in the TMIE gene. Of the 7 variants, 5 variants were found in both nonsyndromic hearing loss and normal hearing group. Both allelic and genotype frequencies of these sequence changes did not differ significantly between patients and controls (P>0.05). However, a missense variant (c.257G>A) and one promoter variant (g.1-219A>T) were found in two patients with nonsyndromic hearing loss. Family study and microsatellite analysis found that c.257G>A variant is not inherited from his parents. The c.257G>A variant encodes a protein with glutamine at position 86 instead of arginine (p.R86Q), a residue that is conserved in mammals but different in fish, and predicted to be extracellular. CONCLUSIONS Despite the fact that the frequency of TMIE variants in our study subjects was low, we suggested that c.257G>A (p.R86Q) variant is a de novo and may be as a risk factor for the development of hearing loss in Taiwanese.

Identification of novel variants in the Myosin VIIA gene of patients with nonsyndromic hearing loss from Taiwan.

OBJECTIVE To determine whether variants of exons 7, 11, 22 and 28 of the MYO7A gene are causes of nonsyndromic deafness in Taiwanese. METHODS We screened a total of 331 unrelated Taiwanese individuals (age range, 4-22 years), including 231 patients with severe to profound nonsyndromic hearing loss and 100 individuals with normal hearing. Genomic DNA was extracted from peripheral blood leukocytes and then subjected to PCR to amplify selected exons and flanking introns of the MYO7A gene; the amplified products were screened for base mutations by autosequence. Data from the two groups were then compared using the chi-square (chi(2)) test. RESULTS The analysis revealed six variants in 3 out of 4 screened exons and flanking intronic sequences of the MYO7A gene (exons 7, 11, and 22). Three missense variants were found only in patients with hearing loss and were heterozygous, including Arg206Cys, Arg206His and Thr381Met. A variant, c.IVS22+58G>A, was found in intron 22 of the MYO7A gene from both patients and control group. Allele frequencies of c.IVS22+58G>A were shown to be significant between the two groups using chi(2) test (P<0.05). CONCLUSION Our results indicate that Arg206 and Thr381 residues in the motor head region of MYO7A protein are critical sites and the mutations of these residues may lead to the development of nonsyndromic deafness.

Functional study of the effect of phosphatase inhibitors on KCNQ4 channels expressed in Xenopus oocytes

AbstractAim:KCNQ4 channels play an important part in adjusting the function of cochlear outer hair cells. The aim of this study was to investigate the effects of ser/thr phosphatase inhibitors on human KCNQ4 channels expressed in Xenopuslaevis oocytes.Methods:Synthetic cRNA encoding human KCNQ4 channels was injected into Xenopus oocytes. We used a two-electrode voltage clamp to measure the ion currents in the oocytes.Results:Wild-type KCNQ4 expressed in Xenopus oocytes showed the typical properties of slow activation kinetics and low threshold activation. The outward K+ current was almost completely blocked by a KCNQ4 blocker, linopirdine (0.25 mmol/L). BIMI (a PKC inhibitor) prevented the effects of PMA (a PKC activator) on the KCNQ4 current, indicating that PKC may be involved in the regulation of KCNQ4 expressed in the Xenopus oocyte system. Treatment with the ser/thr phosphatase inhibitors, cyclosporine (2 μmol/L), calyculin A (2 μmol/L) or okadaic acid (1 μmol/L), caused a significant positive shift in V1/2 and a decrease in the conductance of KCNQ4 channels. The V1/2 was shifted from −14.6±0.5 to −6.4±0.4 mV by cyclosporine, −18.8±0.5 to −9.2±0.4 mV by calyculin A, and −14.1±0.5 to −0.7±0.6 mV by okadaic acid. Moreover, the effects of these phosphatase inhibitors (okadaic acid or calyculin A) on the induction of a positive shift of V1/2 were augmented by further addition of PMA.Conclusion:These results indicate that ser/thr phosphatase inhibitors can induce a shift to more positive potentials of the activation curve of the KCNQ4 current. It is highly likely that the phosphatase functions to balance the phosphorylated state of substrate protein and thus has an important role in the regulation of human KCNQ4 channels expressed in Xenopus oocytes.

Mucociliary Transport Pathway on Laryngotracheal Tract and Stented Glottis in Guinea Pigs

We investigated the laryngotracheal mucociliary transport pathway of guinea pigs in vivo and immediately postmortem. Only intraperitoneal anesthesia was used during the procedure to avoid the disturbance of mucociliary function. Resin particles were used as the marking substance. A microcolpohysteroscope was placed at different levels in the laryngotracheal region for observing the marking particles and recording the transport pattern. The tracheal mucociliary transport flow primarily moved along the posterior wall and both lateral walls in a zigzag trace. Upon reaching the subglottis, the resin particles stayed underneath the vocal cords, and a whirlpool phenomenon developed. The majority of the particles were shifted and directed onto the posterior glottic area. With a short delay, some resin particles crossed over the free edge of the vocal cords and turned posteriorly along the medial upper cordal margin. No mucociliary transport could be observed on the entire upper surface of the true vocal cords, which is covered by squamous epithelium. Occasionally, a few resin particles in the vicinity of the epiglottic root traveled along the aryepiglottic folds toward the posterior commissure. All streams of mucociliary transport finally joined together in the interarytenoid area. After leaving the glottis, the resin particles traveled to the hypopharynx and entered the esophagus through the motion of deglutition. The pattern of mucociliary clearance in the laryngotracheal region was not delayed by stenting.