Mutation analysis of common deafness-causing genes among 506 patients with nonsyndromic hearing loss from Wenzhou city, China.

OBJECTIVES: The frequency and spectrum of mutations in deafness-causing genes differs significantly according to the ethnic population and region under investigation. The molecular etiology of nonsyndromic hearing loss (NSHL) in Wenzhou, China, has not yet been systematically elucidated. To provide accurate genetic testing and counseling in this area, we investigated the molecular etiology of NSHL in a deaf population from Wenzhou. METHODS: A total 506 unrelated patients with NSHL were enrolled in this study. Nine hotspot mutations in four major deafness genes were investigated by sequencing (Group I: 187 patients enrolled between 2011 and 2015) or allele-specific PCR-based universal array (Group II: 319 patients enrolled between 2016 and 2017). The investigated genes included GJB2 (c.35delG, c.176_191del16, c.235delC, c.299-300delAT), SLC26A4 (c.2168A > G, c.919-2A > G), mtDNA 12SrRNA (m.1555A > G, m.1494C > T), and GJB3 (c.538C > T). Furthermore, whole coding region sequencing or improved multiplex ligation detection reaction (IMLDR) were performed for patients who carried mono-allelic variants of GJB2 and SLC26A4, in order to detect other mutations among these patients. RESULTS: GJB2 mutations were detected in 22.92% (116/506) of the entire cohort and SLC26A4 mutations were found in 6.52% (33/506) of the cohort. GJB3 mutations were detected in 0.79% (4/506) of the cohort. The mutation rate of mitochondrial DNA 12SrRNA in our patients was 17.40% (88/506), including 17.00% (86/506) with the m.1555A > G mutation and 0.40% (2/506) with the m.1494C > T mutation. The allelic frequency of the c.235delC mutation was 14.62% (148/1012), which is significantly higher than that of c.109G > A (33/1012, 3.26%), c.299_300delAT (13/1012, 1.28%), and c.176_191del16 (6/1012, 0.59%). The most common pathogenic mutation of SLC26A4 was the c.919-2A > G mutation (37/1012, 3.66%), followed by c.2168A > G (6/1012, 0.59%), and c.1229C > T (4/1012, 0.40%). Moreover, five rare pathogenic variants of GJB2 and eight rare pathogenic variants of SLC26A4 were identified. CONCLUSION: GJB2 is the primary deafness-causing gene in deaf patients from Wenzhou, China; this is consistent with what is observed in most Chinese populations. However, the surprisingly high rate of the m.1555A > G mutation (17.00%) in patients from Wenzhou was significantly higher than in other populations in China. These findings highlight the specificity of the common deafness-causing gene mutation spectrum in the Wenzhou area. This information may be of benefit for genetic counseling and risk assessment for deaf patients from this area.

Interaction of serum proteins with CYP isoforms in human liver microsomes: inhibitory effects of human and bovine albumin, alpha-globulins, alpha-1-acid glycoproteins and gamma-globulins on CYP2C19 and CYP2D6.

The effects of serum proteins on the in vitro hydroxylation pathways of mephenytoin (CYP2C19) and debrisoquine (CYP2D6) were studied to enhance the predictability of in vivo drug metabolism from in vitro assays. Both CYP substrates are known to be weakly bound to albumin and the applicability of the free drug hypothesis was further appraised. Since bovine serum albumin (BSA) is used widely in in vitro assays, a comparison between human and bovine proteins was made. Four major serum proteins were studied: albumin, alpha1-acid glycoprotein (AGP), alpha- and gamma-globulins. Human serum albumin (HSA) inhibited both CYP activities about 20% more than BSA. The addition of human alpha-globulins, but not the bovine protein, resulted in marked reduction of 86% and 41% in CYP2C19 and CYP2D6 activities, respectively. This reduction of activity was strikingly greater than the fraction bound (14 and 22%, respectively). The inhibition was of the competitive type and the Ki values of human alpha-globulins on CYP2C19 and CYP2D6 were found to be 0.45% (4.5 mg/ml) and 3.5% (35 mg/ml), respectively. The effect of both human and bovine gamma-globulins on CYP isoforms was negligible. The Ki values of human and bovine AGP for CYP2C19 were 1.84% (420 microM) and 0.93% (210 microM), respectively. For HSA, human alpha-globulins and human and bovine AGP, the strongly decreased CYP activities in vitro cannot be explained by the free drug hypothesis. A direct interaction of these serum proteins with CYP enzymes is postulated. Differential effects of bovine and human serum proteins and CYP specific inhibition were observed.