Pou3f4 deficiency causes defects in otic fibrocytes and stria vascularis by different mechanisms.

DFN3, the most prevalent X-linked hearing loss, is caused by mutations in the POU3F4 gene. Previous studies in Pou3f4 knockout mice suggest that defective otic fibrocytes in the spiral ligament of the cochlear lateral wall may underlie the hearing loss in DFN3. To better understand the pathological mechanisms of the DFN3 hearing loss, we analyzed inner ears of Pou3f4-deficient mice during development. Our results indicate that compartmentalization of the spiral ligament mesenchyme setting up boundaries for specific otic fibrocytes occurs normally in Pou3f4-deficient cochlea. However, differentiation of the compartmentalized mesenchyme into specific otic fibrocytes was blocked in the absence of Pou3f4 function. In addition, we found that stria vascularis in the cochlear lateral wall was also affected in Pou3f4-deficient cochlea. Unlike the otic fibrocytes, differentiation of stria vascularis was completed in the absence of Pou3f4 function, yet expression of Kir4.1 channels in the strial intermediate cells, essential for the sound transduction, was lost afterwards. These results suggest that Pou3f4 deficiency causes defects in both otic fibrocytes and stria vascularis at different developmental stages and by different pathological mechanisms, which may account for the progressive nature of DFN3 hearing loss.

Application of Macro-Instrumented Indentation Test for Superficial Residual Stress and Mechanical Properties Measurement for HY Steel Welded T-Joints.

HY-80 and HY-100 steels, widely used in constructing large ocean vessels and submarine hulls, contain mixed microstructures of tempered bainite and martensite and provide high tensile strength and toughness. Weld integrity in HY steels has been studied to verify and optimize welding conditions. In this study, the T-joint weld coupons, HY80 and HY100, were fabricated from HY-80 and HY-100 steel plates with a thickness of 30 mm as base metals by submerged-arc welding. Flux-cored arc welding was performed on an additional welding coupon consisting of HY-100 to evaluate the effect of repair welds (HY100RP). Microstructures in the heat-affected zones (HAZ) were thoroughly analyzed by optical observation. Instrumented indentation testing, taking advantage of local characterization, was applied to assess the yield strength and the residual stress of the HAZ and base regions. The maximum hardness over 400 HV was found in the HAZ due to the high volume fraction of untempered martensite microstructure. The yield strength of the weld coupons was evaluated by indentation testing, and the results showed good agreement with the uniaxial tensile test (within 10% range). The three coupons showed similar indentation residual stress profiles on the top and bottom surfaces. The stress distribution of the HY100 coupon was comparable to the results from X-ray diffraction. HY100RP demonstrated increased tensile residual stress compared to the as-welded coupon due to the effect of the repair weld (323 and 103 MPa on the top and bottom surfaces). This study verifies the wide applicability of indentation testing in evaluating yield strength and residual stress.

A novel frameshift mutation of POU4F3 gene associated with autosomal dominant non-syndromic hearing loss.

Autosomal dominant mutations in the transcription factor POU4F3 gene are associated with non-syndromic hearing loss in humans; however, there have been few reports of mutations in this gene worldwide. We performed a mutation analysis of the POU4F3 gene in 42 unrelated Koreans with autosomal dominant non-syndromic hearing loss, identifying a novel 14-bp deletion mutation in exon 2 (c.662del14) in one patient. Audiometric examination revealed severe bilateral sensorineural hearing loss in this patient. The novel mutation led to a truncated protein that lacked both functional POU domains. We further investigated the functional distinction between wild-type and mutant POU4F3 proteins using in vitro assays. The wild-type protein was completely localized in the nucleus, while the truncation of protein seriously affected its nuclear localization. In addition, the mutant failed to activate reporter gene expression. This is the first report of a POU4F3 mutation in Asia, and moreover our data suggest that further investigation will need to delineate ethnicity-specific genetic background for autosomal dominant non-syndromic hearing loss within Asian populations.

Clinical and molecular characterizations of novel POU3F4 mutations reveal that DFN3 is due to null function of POU3F4 protein.

X-linked deafness type 3 (DFN3), the most prevalent X-linked form of hereditary deafness, is caused by mutations in the POU3F4 locus, which encodes a member of the POU family of transcription factors. Despite numerous reports on clinical evaluations and genetic analyses describing novel POU3F4 mutations, little is known about how such mutations affect normal functions of the POU3F4 protein and cause inner ear malformations and deafness. Here we describe three novel mutations of the POU3F4 gene and their clinical characterizations in three Korean families carrying deafness segregating at the DFN3 locus. The three mutations cause a substitution (p.Arg329Pro) or a deletion (p.Ser310del) of highly conserved amino acid residues in the POU homeodomain or a truncation that eliminates both DNA-binding domains (p.Ala116fs). In an attempt to better understand the molecular mechanisms underlying their inner ear defects, we examined the behavior of the normal and mutant forms of the POU3F4 protein in C3H/10T1/2 mesodermal cells. Protein modeling as well as in vitro assays demonstrated that these mutations are detrimental to the tertiary structure of the POU3F4 protein and severely affect its ability to bind DNA. All three mutated POU3F4 proteins failed to transactivate expression of a reporter gene. In addition, all three failed to inhibit the transcriptional activity of wild-type proteins when both wild-type and mutant proteins were coexpressed. Since most of the mutations reported for DFN3 thus far are associated with regions that encode the DNA binding domains of POU3F4, our results strongly suggest that the deafness in DFN3 patients is largely due to the null function of POU3F4.

Methylenetetrahydrofolate reductase (MTHFR) and methionine synthase reductase (MTRR) gene polymorphisms as risk factors for hepatocellular carcinoma in a Korean population.

Hepatocellular carcinoma (HCC) is the third most frequent cause of cancer death in South Korea, but genetic susceptibility factors of HCC have not been examined extensively. Methylenetetrahydrofolate reductase (MTHFR) and methionine synthase reductase (MTRR) play an essential role in both DNA synthesis and methylation and polymorphisms in the MTHFR gene, 677C>T, 1298A>C and the MTRR gene, 66A>G, are associated with several types of malignancy. In this study, the allelic frequencies and genotype distribution of three polymorphisms in the MTHFR and MTRR genes from 96 hepatocellular carcinoma (HCC) patients and 201 controls were examined to assess the association between these polymorphisms and the development of HCC in this Korean population. The 66AG+GG (G allele-bearing) genotype of the MTRR gene was significantly associated with an increased risk of HCC (odds ratio, OR, 1.687; 95% confidence interval, CI=1.022-2.787). Moreover, the combination of MTHFR 1298AA/MTRR 66AG+GG (OR=1.854, 95% CI=1.005-3.420) and MTHFR 1298AC+CC/MTRR 66AG+GG (OR=2.733, 95% CI=1.195-6.249) showed a significant association with HCC risk. In the data classified by age and etiology, MTRR 66A>G over the age of 65 years, MTHFR 1298A>C under the age of 65 years and the MTRR 66AG+GG genotype in the hepatitis B virus (HBV) patients were increased risk factors for the disease. The MTHFR 1298A>C and the MTRR 66A>G genotypes were associated with an increased risk of HCC in this Korean population. Further studies involving larger and varied populations could provide a potential tool for cancer risk assessment in patients who are at risk of developing HCC.

CHD7 mutational analysis and clinical considerations for auditory rehabilitation in deaf patients with CHARGE syndrome.

BACKGROUND: Otologic manifestations are one of the most consistent findings of CHARGE syndrome found in more than 90%. Since genetic analysis of the CHD7 gene has rarely been performed in previous reports dealing with ear abnormalities, the genotypic spectrum of CHD7 mutations was analyzed in deaf patients with CHARGE syndrome, and the clinical considerations concerning auditory rehabilitation were investigated. METHODS: Nine Korean patients with CHARGE syndrome showing profound hearing loss and semicircular canal aplasia were included. All 38 exons of CHD7 were analyzed by direct sequencing. For splice site variations, in silico and exon-trapping analyses were performed to verify the pathogenicity of nucleotide variations. Clinical features and the outcome of auditory rehabilitation were also analyzed. RESULTS: Eight of 9 patients revealed alterations of the CHD7 gene including 3 frameshift, 2 nonsense, 2 splice site, and 1 missense mutations. Five of 9 patients were clinically diagnosed as atypical CHARGE syndrome but demonstrated various mutations of the CHD7 gene. One familial case showed intra-familial variability. Radiologic findings suggesting cochleovestibular nerve deficiency were identified in most of the patients. Of the 8 patients who underwent cochlear implantation, 5 patients demonstrated favorable outcome. Larger diameter of the cochleovestibular nerve on imaging and absence of severe mental retardation were factors related to better outcome after cochlear implantation rather than the type of CHD7 mutations. Auditory brainstem implantation was performed in two patients who did not benefit from cochlear implantation. CONCLUSIONS: Genetic analysis of the CHD7 gene should be performed in cases with semicircular canal aplasia even when other typical features of CHARGE syndrome are absent. For auditory rehabilitation in CHARGE syndrome, cochlear implantation should be strongly recommended in selected cases with favorable prognostic factors. Auditory brainstem implantation may be a viable option in patients with CHARGE syndrome who have failed to benefit from cochlear implantation.