Balkan Journal of Medical Genetics

GENETIC AND CLINICAL ANALYSIS OF NONSYNDROMIC HEARING IMPAIRMENT IN PEDIATRIC AND ADULT CASES
Xing J, Liu X, Tian Y, Tan J, Zhao H
*Corresponding Author: Mr. Xinguo Liu, Ear, Nose and Throat Department, The Central Hospital of Zhumadian, No. 747, Zhonghua Road, Zhumadian City, Henan Province, People’s Republic of China, 463000. Tel: +86-396-292-6205. Fax: +86-396-272-6530. E-mail: xingglsci@163.com
page: 35

PATIENTS AND METHODS

Patients. This prospective study included 263 NSHI patients who first visited the Ear, Nose and Throat (ENT) Department at the Central Hospital of Zhumadian, Zhumadian City, Henan Province, People’s Republic of China (PRC) between March 2008 and March 2013. The 168 males (63.9%) and 95 females (36.1%) had a mean age of 13.1 + 10.8 years (range: 2 months to 60 years). The mean age of NSHI onset was 5.72 years. The study excluded SHL patients and patients with complications resulting from causes such as tympanitis, meningocephalitis, late-stage Meniere’s disease, trauma, acoustic neuroma and ototoxic drugs. The subjects were divided into age groups according to their ages at the first hospital visit and at onset of NSHI: adult group (>18 years old) and pediatric group (<18 years); the pediatric group was subdivided into infants (<3 years), preschool (3-6 years) and school-age (7-18 years) groups. The age of onset was defined as the age at which a patient or patient’s family discovered the patient’s deafness, or the age at which objective audiometry identified the condition. This study was approved by the Hospital Ethics Committee, and written informed consent was obtained from the patients or their families. Audiometry and Deafness Phenotype. All the patients were assessed by audiometry, including pure tone audiometry (PTA) or auditory brainstem response (ABR) tests and multiple-frequency auditory steady-state response (ASSR). The criteria for analysis and classification of deafness were in accordance with published guidelines [11] as follows: 1) hearing loss was classified by frequency measurements as full-frequency hearing loss (0.25-8.0 kHz), high-frequency hearing loss (2.0-8.0 kHz), mid-frequency hearing loss (0.5-2.0 kHz), or low-frequency hearing loss (0.25-0.5 kHz); 2) according to the stages of speech development, deafness was classified into post-linguistic deafness (>3 years) or pre-linguistic deafness (<3 years); and 3) the severity of hearing impairment was judged by the better-hearing ear as being mild [20-40 decibels (dB), hearing level (HL)], moderate (41-70 dB HL), severe (71-95 dB HL), or profound (>95 dB HL). GJB2 Gene Sequencing. Peripheral venous blood (4 mL) was collected from each subject. The Universal DNA Isolation Kit (BioTeke Corporation, Beijing, PRC) was used to isolate genomic DNA according to the manufacturer’s instructions. Polymerase chain reaction (PCR) was used to amplify the GJB2 gene for mutation analysis. The PCR primers, synthesized by Sangon Biotech (Shanghai, PRC) were as follows: downstream primer (5’-GGG CAA TGC TTA AAC TGG C-3’); upstream primer (5’- TAT GAC ACT CCC CAG CAC AG-3’) [12]. The PCR product was recovered for sequence determination. The sequencing results were compared with the published GJB2 gene sequence (GenBank accession number M86849) to determine the presence of deafness-related sequence variants. Mutation Analysis of the Mitochondrial Genome. Primers for PCR amplification covered mtDNA 1988-2007 and mtDNA 618-635, and PCR amplification involved all fragments of the mitochondrial 12S rRNA gene. Twenty-four sets of primers, covering the whole mitochondrial genome with partially overlapping fragments, were used to perform PCR amplification for mtDNA from patients with the A1555G/C1494T mutations [13]. After the PCR-amplified DNA was recovered from gel with the Agarose Gel DNA Purification Kit Ver. 2.0 (Code No. DV805; TaKaRa Biotech Co. Ltd., Dalin, PRC), the BigDye® Terminator Cycle Sequencing Kit (Microread Genetics, Beijing, PRC) was used to perform sequencing on the ABI PRISM™ 3700 DNA automated sequencer (Biocoen Biotechnology, Beijing, PRC). Sequencing results were compared with the Cambridge Reference Sequence (GenBank accession number NC_001807). Statistical Analysis. EpiData version 3.1 was used to create a data bank using double data entry, and logic checks were performed. SAS 9.2 (SAS Institute, Cary, NC, USA) was used to analyze data by the χ2 test. A value of p <0.05 was considered to indicate a statistically significant difference.

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