GJB2, SLC26A4 and mitochondrial DNA A1555G mutations in prelingual deafness in Northern Chinese subjects.

CONCLUSION: This genetic epidemiological study demonstrated that 26.65% of the prelingual deafness in Northern Chinese patients can be detected at younger ages by genetic testing of three common hearing loss genes (GJB2, SLC26A4 and mtDNA A1555G), and thus, early intervention measures could be undertaken to help them in language acquisition. OBJECTIVES: The GJB2, SLC26A4 and mtDNA A1555G mutations are the prevalent causes of prelingual deafness worldwide. Numerous studies have revealed that the forms and frequencies of the mutations in the three genes are largely dependent on the ethnic or geographic origins. Hence, this study aimed to characterize the mutation profiles of the three genes in prelingual deafness in Northern Chinese patients. SUBECTS AND METHODS: An investigation of 514 patients with prelingual deafness and 117 controls with normal hearing was conducted. Bidirectional sequencing (or enzyme digestion) was applied to identify sequence variations. RESULTS: This study revealed that 26.65% patients had two mutated alleles (homozygote or compound heterozygote) of GJB2 (9.14%) or SLC26A4 (8.95%) and/or an mtDNA A1555G (8.56%) mutation. In detail, 19.26% patients carried GJB2 mutations including 10.12% single mutant carriers. 235delC was the most common type, making up 69.18% of all mutants for GJB2. The mutant carrier rate for SLC26A4 was 15.2%, including 6.23% single mutant carriers. The two most common types (IVS7-2A > G and H723R) accounted for 51.61% and 33.06% mutations, respectively. Forty-five patients had mtDNA A1555G, giving a frequency of 8.75%. In the control group with normal hearing, 2.56%, 1.71% and 0% of the subjects carried a single mutant for GJB2, SLC26A4 and mtDNA A1555G, respectively.

A POU3F4 Mutation Causes Nonsyndromic Hearing Loss in a Chinese X-linked Recessive Family.

BACKGROUND: The molecular genetic research showed the association between X-linked hearing loss and mutations in POU3F4. This research aimed to identify a POU3F4 mutation in a nonsyndromic X-linked recessive hearing loss family. METHODS: A series of clinical evaluations including medical history, otologic examinations, family history, audiologic testing, and a high-resolution computed tomography scan were performed for each patient. Bidirectional sequencing was carried out for all polymerase chain reaction products of the samples. Moreover, 834 controls with normal hearing were also tested. RESULTS: The pedigree showed X-linkage recessive inheritance pattern, and pathogenic mutation (c.499C>T) was identified in the proband and his family member, which led to a premature termination prior to the entire POU domains. This mutation co-segregated with hearing loss in this family. No mutation of POU3F4 gene was found in 834 controls. CONCLUSIONS: A nonsense mutation is identified in a family displaying the pedigree consistent with X-linked recessive pattern in POU3F4 gene. In addition, we may provide molecular diagnosis and genetic counseling for this family.

Phenotype-genotype correlation in 295 Chinese deaf subjects with biallelic causative mutations in the GJB2 gene.

AIMS: The connexin 26 coding gene (GJB2) is the primary causative gene for nonsyndromic sensorineural hearing impairment (NSSHI). More than 100 mutations in this gene have been reported to be linked to hearing impairment (HI), from mild to profound hearing loss. To precisely estimate the impact of GJB2 mutations in the Chinese population, a cross-sectional study was performed to analyze the auditory data of Chinese patients with NSSHI. RESULTS: Two hundred ninety-five unrelated patients with NSSHI with biallelic mutations in GJB2 were recruited from seven provinces in Northern China from 2004 to 2008. The levels of HI and average pure tone audiometry were compared across different genotypes by χ(2) testing. The subjects with the genotypes of combined truncating mutations had more cases of severe HI than the subjects with a genotype of several nontruncating mutations. It was also revealed that subjects carrying either c.[79G>A; 341A>G]+[79G>A; 341A>G] or c.[109G>A]+[79G>A; 341A>G] had significantly fewer cases of severe HI than the reference group of homozygous c.235delC, whereas the subjects carrying c.[235delC]+[176_191del16] had more cases of severe HI than the homozygous c.235delC group. CONCLUSIONS: This is the first study to clarify the correlations between different GJB2 biallelic genotypes and NSSHI phenotype in the Chinese population. The Chinese subjects with two truncating mutations in GJB2 were shown to correlate with more severe HI.

The large Chinese family with Y-linked hearing loss revisited: clinical investigation.

CONCLUSION: The DFNY1 phenotypes shared many characteristics with some autosomal dominant hearing loss, in the aspects of age of onset, severity and audiometric configuration. However, the typical, outstanding feature of this trait was its remarkable pattern of inheritance. Similar traits, if ever encountered, can be most easily identified by discerning this exceptional and rare pattern of inheritance. OBJECTIVES: To analyze the audiological features in Chinese Y-linked non-syndromic hearing impairment, the extended DFNY1 family. SUBJECTS AND METHODS: A nine-generation Chinese family (DFNY1) was ascertained and expanded from the year of 2000 to 2006. The audiometric evaluations included pure-tone audiometry, tympanometry, and auditory brainstem responses. Some subjects received computerized tomography scan of the temporal bone. RESULTS: 52 out of 276 members in this family received clinical examinations. 24 live subjects had hearing impairment consisting of 23 patrilineal males and one female. In the affected lineage, 92% patrilineal males were well characterized as having hearing loss and 2 children remained to be diagnosed. Based on the audiological examinations on the male members, the degree of hearing loss was from mild (3 patients), moderate (7 patients) to severe (11 patients). The audiometry displayed 48% subjects with sloping in high frequencies, 38% flat in all frequencies, and the rest (14%) the U-shape. The age of onset ranged from 5-27 years with the average of 11.5 years.

[Correlation between phonetically balanced maximum and pure tone auditory threshold among 106 auditory neuropathy patients].

OBJECTIVE: To estimate correlation between phonetically balanced maximum (PB max) and pure tone auditory threshold in auditory neuropathy (AN) patients. METHODS: One hundred and six AN patients were identified using multiple criteria including PB max, a metric for speech recognition, pure tone auditory threshold, acoustic emission test, distortion products otoacoustic emission (DPOAE) and auditory brainstem response (ABR). SPSS statistical software was used to estimate the Pearson's correlation between PB max and pure tone auditory threshold and to test whether pure tone auditory threshold, or auditory configuration had a significant impact on PB max. RESULTS: Even the patients had the same or similar values for pure tone auditory threshold or auditory configuration, varied values of PB max were found in two hundreds and twelve ears for 106 patients. Analysis of the data for 106 patients revealed a negative correlation (r = -0. 602, P <0. 01) between PB max and pure tone auditory threshold, i. e. hearing loss at a mild relates to a lower PB max. By using analysis of variance (ANOVA) method, it was found that both pure tone auditory threshold and auditory configuration had a significant (P <0.01) impact on the patients' PB max. CONCLUSIONS: This analysis implicated the promise and potential of pure tone auditory threshold and auditory configuration for predicting PB max of the AN patients, and improving the diagnosis of AN.

[Studies of the strategy for newborn gene screening].

OBJECTIVE: To discuss and analyze the feasibility and strategy for perform the newborn gene screening in the process of newborn hearing screening in order to supply the defects or limitation in the hearing screening. METHODS: Four hundreds and sixty newborn babies from December 2006 to April 2007 accepted the simultaneous hearing and gene screening. Otoacoustic emission (OAE) was used for the first step hearing screening and OAE combined with auto auditory brainstem response (AABR) detection for the second step screening. Newborn genetic disease screening cards were used for collecting the blood spot from the umbilical cord within the moment of newborn. The cards could be directly performed the polymerase chain reaction (PCR) for screening the mitochondrial 12SrRNA 1555G and GJB2 as well as SLC26A4 genes mutations. The restriction enzyme Alw26I was used to recognize the point mutation of 12SrRNA A1555G. The samples with the possible 12SrRNA A1555G mutation were then sequenced to verify. The PCR products from the GJB2 coding region and SLC26A4 IVS7-2A > G hot spot region were sequenced directly. The software of DNAStar was used to analysis the sequence. RESULTS: The first step of hearing screening of 460 newborn babies showed " refer" on the left ear of nine babies and on the right ear of three babies. Seven showed "refer" on bilateral with the the total of babies 19. After 42 days, they accepted the second step for hearing screening. 16 of the 19 were showed "pass" with OAE and AABR. One baby showed "pass" on the left ear, "refer" on the right ear with the OAE detection but bilateral "pass" with AABR. Two babies failed to accept the re-examination. The newborn gene screening showed five of the 460 babies had the positive response on the A1555G restriction enzyme assay. Of the five babies, one was proved to be the 12SrRNA A1555G mutation and three were the C1556T mutations and one sequence was normal. For the SLC26A4 gene screening, five were the heterozygote of IVS7-2A > G mutation were found and one was carrier the polymorphism of IVS7-18T > G and another was IVS6-62_63insGT heterozygote carrier. For the GJB2 gene screening, eight were 235delC heterozygote carriers, four were G109A heterozygote carriers. All the gene screening found 23 newborn babies of the 460 harbored the changes in the three genes. Of those, one was the 12SrRNA A1555G. pathogenic mutation and 13 were pathogenic heterozygote carriers, nine were the polymorphisms. It was worth to pay more attentions that A1555G mutation was found in the baby whose hearing screening was "pass" in the hearing screening as well as the 13 heterozygote carrier for GJB2 and SLC26A4 gene. CONCLUSIONS: It might be one of the powerful strategy for adding the concept of newborn gene screening into the hearing screening for the purpose of early diagnosis and discovery the prelingual or late-onset or the high risk as well as the pathogenic carriers. On the basis of the research progress, it was necessary to develop the national newborn gene screening into the process of newborn hearing screening.

[Construction of an expression vector under the control of hTERT promoter].

AIM: To construct a luciferase expression vector driven by hTERT(human telomerase reverse transcriptase) core promoter and identify the transcriptional activity of the vector in tumor cells and normal cells. METHODS: hTERT gene core promoter was amplified by PCR using the total genomic DNA from HeLa cells as template. The amplified gene fragment was subsequently cloned into PGL3-basic vector. Then the expression vector was confirmed by restriction enzyme digestion and PCR analysis. The luciferase activity driven by the hTERT gene core promoter was identified after transient transfection of the expression vector into tumor cells and normal cells. RESULTS: The luciferase activity was high in the transfected tumor cells, and very low in transfected normal cells. CONCLUSION: hTERT gene core promoter is tumor-specific and may be useful in gene therapy of tumor.

[Apoptosis of laryngeal carcinoma cell line Hep2 induced by tumor-specific expression of TRAIL gene].

AIM: To explore targeted gene therapy of tumor by using the combination of TRAIL gene with the telomerase promoter. METHODS: TRAIL gene with an IL-2 signal peptide was constructed by PCR and cloned into vector pGL3-181hTERT downstream of hTERT promoter to form an eukaryotic expressing vector. Hep2 cells were transfected by the recombinant vector and apoptosis of the transfected cells was evaluated by trypan-blue exclusion and the agarosegel electrophoresis of DNA. RESULTS: We successfully constructed a recombinant eukaryotic expression vector for TRAIL gene.The expressed product significantly induced the apoptosis of Hep2 cells. CONCLUSION: The recombinant eukaryotic expression vector pGL3-181hTERT/TRAIL was successfully constructed, which provides the possibility for gene therapy of tumor.