Molecular screening of patients with nonsyndromic hearing loss from Nanjing city of China

Hearing loss is the most frequent sensory disorder involving a multitude of factors, and at least 50% of cases are due to genetic etiology. To further characterize the molecular etiology of heating loss in the Chinese population, we recruited a total of 135 unrelated patients with nonsyndromic sensorineural hearing loss （NSHL） for mutational screening of GJB2, GJB3, GJB6, SLC26A4, SLC26A5 IVS2-2A〉G and mitochondrial 12SrRNA, tRNASer（t/CN） by PCR amplification and direct DNA sequencing. The carrier frequencies of deafness-causing mutations in these patients were 35.55% in GJB2, 3.70% in GJB6, 15.56% in SLC26A4 and 8.14% in mitochondrial 12SrRNA, respectively. The results indicate the necessity of genetic screening for mutations of these causative genes in Chinese population with nonsyndromic heating loss.

Genome-wide and Interaction Linkage Scan for Nonsyndromic Cleft Lip with or without Cleft Palate in Two Multiplex Families in Shenyang,China

Objectives To identify the loci involved in nonsyndromic cleft lip with or without cleft palate （NSCL/P） in Northern Chinese people in Shenyang by using genomewide and interaction linkage scan.Methods Two multiplex families in Shenyang from North China were ascertained through probands with NSCL/P.Blood of every member was drawn for DNA extraction and analysis.Genotypes were available for 382 autosomal short tandem repeat （STR） markers from the ABI Prism Linkage Mapping Set version 2.5.Linkage between markers and NSCL/P was assessed by 2-point parametric LOD scores,multipoint heterogeneity parametric LOD scores （HLODs）,and multipoint nonparametric linkage score （NPL）.Results The initial scan suggested linkage on Chromosomes 1,2,and 15.In subsequent fine mapping,1q32-q42 showed a maximum multipoint LOD score of 1.9（empirical P=0.013） and an NPL score of 2.35 （empirical P=0.053）.For 2p24-p25,the multipoint NPL increased to 2.94 （empirical P=0.007）.2-locus interaction analysis obtained a maximum NPL score of 3.73 （P=0.00078） and a maximum LOD score of 3 for Chromosome 1 （at 221 cM） and Chromosome 2 （at 29 cM）.Conclusion Both parametric and nonparametric linkage scores greatly increased over the initial linkage scores on 1q32-q42,suggesting a susceptibility locus in this region.Nonparametric linkage gave a strong evidence for a candidate region on chromosome 2p24-p25.The superiority of 2-locus linkage scores compared to single-locus scores gave additional evidence for linkage on 1q32-q42 and 2p24-p25,and suggested that certain genes in the two regions may contribute to NCSL/P risks with interaction.

A Case-control Study of Environmental Risk Factors for Nonsyndromic Cleft of the Lip and/or Palate in Xuzhou,China

In this study, we sought to determine the association between environmental factors and nonsyndromic cleft of the lip and/or palate （NSCLP） to understand the etiology of the disease. A total of 200 NSCLP cases and 327 controls were recruited at the Maternal and Child Health Hospital of Xuzhou City. We conducted face-to-face interviews with the mothers of both cases and controls. The factors increasing the risk of NSCLP were a positive family history [odds ratio （OR）=56.74], pesticide exposure （OR=8.90）, and indoor decoration pollution （OR= 4.32）. On the other hand, the factors decreasing the risk of NSCLP were a high education level （OR=0.22） and supplementation of folic acid （OR=0.23） and multivitamins （OR=0.16）. Positive family history, pesticide exposure, and indoor decoration pollution are associated with the risk of NSCLP. In contrast, high education level and folic acid and multivitamin supplementation are protective factors against NSCLP.

<i>GJB2</i>Gene Related Nonsyndromic Hearing Loss in Mazandaran Province, North of Iran

Introduction: Congenital hearing loss is the most common sensory deficit in the world and mutations in GJB2 gene are the most common cause of deafness in many populations. Frequency of GJB2 mutations is estimated about 16% in Iran and varies among different provinces with a decreasing trend from north to south. The aim of this study was to investigate the frequency of GJB2 mutations in Mazandaran province, north of Iran, among non-syndromic hearing loss patients. Methods: 262 patients from 204 families participated in this study. After genomic DNA extraction, GJB2 gene analysis was carried out using DNA sequencing of both coding and non-coding regions by ABI 3130XL genetic analyzer. Results: 30.15% of all subjects showed mutations in GJB2 gene. Four mutations, including c.35delG (Gly12Valfs*), IVSI-1 + 1G > A, c.95G > A (Arg32His) and c.224 G > A (Arg75Gln) comprises 69.89% of all mutations in this study c.35delG and IVSI-1 were the most common mutations among patients respectively. Codon 75 mutation (c.224G > A. p: Arg75Gln) with autosomal dominant inheritance was seen in 7 cases from 3 families. 22 patients showed only one mutation in GJB2 gene and in 126 (48.09%) individuals, parents had a consanguineous marriage. Discussion: Frequency of GJB2 gene related hearing loss among patients was higher than average (16%) in this province. This study also showed a dominant inheritance pattern of GJB2 gene in this area. Consanguineous marriage also showed highly frequent among parents. More investigation needs to clarify cause of hearing loss in those 22 patients with one mutation in GJB2 gene, either two gene inheritance or another gene may be responsible for hearing loss.

Association of Single Nucleotide Polymorphisms in IRF6 and TGFA Genes With Nonsyndromic Cleft Lip With Or Without Cleft Palate in Chinese Patients

Objective： Nonsyndromic cleft lip with or without cleft palate（NSCL/P） is a common birth defect with unclear etiology. Both genetic and environmental factors may contribute to NSCL/P. Many genes have been identified as candidate genes associated with this disease. Interferon regulatory factor 6（IRF6） gene and transforming growth factor-a（TGFA） gene seem to be crucial in the predisposition of NSCL/ P. Here we evaluated some single nucleotide polymorphisms（SNPs） loci of TGFA and IRF6 genes in Chinese nuclear families consisting of fathers, mothers and affected offspring with NSCL/P. Methods：Fifty patients of NSCL/P were confirmed by the plastic surgeons. They and their parents were included in the study, all with the informed consents. SNPs loci of TGFA and IRF6 genes were analyzed by microarray technology. Some PCR products were randomly chosen and sequenced to check microarray results. The distribution of gene type and allele frequency between patient group and parents group were compared. Then a Haplotype Relative Risk（HRR） and Transmission Disequilibrium Test（TDT） were performed. Results：The sequences of randomly selected PCR products were all consistent with the microarray results. All loci were in Hardy-Weinberg equilibrium. There were no significant differences in the distribution of genotypes and alleles between patients and their parents. Using HRR and TDT analyses the V274I of IRF6 was associated with NSCL/P, while another SNP locus oflRF6 was not. Strong evidence of linkage disequilibrium was found between the 2 SNP loci of TGFA and disease with the HRR analysis, but not with the TDT analysis. Conclusion：Our study confirms the contribution of IRF6 in the etiology of NSCL/P in populations of Asian ancestry. The association of TGFA with NSCL/P requires further research.

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 bearing 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, l：amily history, audiologic testing, and a high-resolution computed tomography scan were performed t＇or each patient. Bidirectional sequencing was carried out for all polymerase chain reaction products or＇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 bearing loss in this family. No mutation ofPOU3F4 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.

Identification of Two Disease-causing Genes TJP2 and GJB2 in a Chinese Family with Unconditional Autosomal Dominant Nonsyndromic Hereditary Hearing Impairment

Background： There are more than 300 genetic loci that have been found to be related to hereditary hearing impairment （HHI）, including 92 causative genes for nonsyndromic hearing loss, among which 34 genes are related to autosomal dominant nonsyndromic HHI （ADNSHH 1）. Traditional linkage analysis and candidate gene sequencing are not effective at detecting the ADNSHHI, especially for the unconditional families that may have more than one pathogenic cause. This study identified two disease-causing genes TJP2 and GJB2 in a Chinese family with unconditional ADNSHHI. Methods： To decipher the genetic code of a Chinese family （family 686） with ADNSHHI, different gene screening techniques have been performed, including linkage analysis, candidate genes screening, high-throughput sequencing and Sanger sequencing. These techniques were done on samples obtained from this family over a period of 10 years. Results： We identified a pathogenic missense mutation, c. 2081G〉A （p.G694E）, in TJP2, a gene that plays a crucial role in apoptosis and age-related hearing loss （ARHL）. The mutation was co-segregated in this pedigree in all, but not in the two patients who presented with different phenotypes from the other affected family members. In one of the two patients, we confirmed that the compound heterozygosity for p.Y136^＊ and p.G45E in the GJB2 gene may account for the phenotype shown in this patient. Conclusions： We identified the co-occurrence of two genetic causes in family 686. The possible disease-causing missense mutation of TJP2 in family 686 presents an opportunity for further investigation into ARHL. It is necessary to combine various genes screening methods, especially for some unconventional cases.

Genome-Wide Screening of Aberrant Methylation Loci for Nonsyndromic Cleft Lip

Background： The pathogenicity of cleft lip （CL） is pretty complicated since it is influenced by the interaction of environment and genetic factors. The purpose of this study was to conduct a genome-wide screening of aberrant methylation loci in partial lesion tissues of patients with nonsyndromic CL （NSCL） and preliminarily validate candidate dysmethylated genes associated with NSCL.Methods： Fifteen healthy and sixteen NSCL fetal lip tissue samples were collected. The Infinium HumanMethylation450 BeadChip was used to screen aberrant methylation loci in three NSCL and three healthy lip tissues. The differential methylation sites and functions of the annotated genes between NSCL and healthy lip tissues were analyzed using minfi package of R software, cluster analysis, Gene Ontology （GO） annotation, and metabolic pathway annotation. Gene expression was assessed in nine differentially methylated genes by real-time polymerase chain reaction （PCR）. The transcriptions mRNA levels of three out of nine candidate genes were downregulated remarkably in NSCL lip tissues, and these three genes＇ abnormal methylation loci were validated by pyrosequencing in 16 NSCL cases and 15 healthy cases.Results： In total, 4879 sites in the genes of NSCL odinopoeia fetuses showed aberrant methylation when compared with normal lip tissue genome. Among these, 3661 sites were hypermethylated and 1218 sites were hypomethylated as compared to methylation levels in healthy specimens. These aberrant methylation sites involved 2849 genes and were widely distributed among the chromosomes. Most differentially methylated sites were located in cytosine-phosphoric acid-guanine islands. Based on GO analysis, aberrantly methylated genes were involved in 11 cellular components, 13 molecular functions, and a variety of biological processes. Notably, the transcription of DAB1, REELIN, and FYN was significantly downregulated in lesion tissues of NSCL fetus （P 〈 0.05）. Pyrosequencing results validated that there were two loci in DABI with high methylation status in patient tissues （P 〈 0.05）. Conclusions： We detected numerous aberrantly methylated loci in lesion tissues of NSCL fetus. Aberrant gene expression in the REELIN signaling pathway might be related with NSCL. Decreased transcription of DAB1, a member of REELIN signal pathway, resulted from its abnormal high methylation, which might be one of the factors underlying the occurrence of NSCL.

Whole-exome sequencing identifies novel mutations in genes responsible for retinitis pigmentosa in 2 nonconsanguineous Chinese families

AIM: To detect the pathogenetic mutations responsible for nonsyndromic autosomal recessive retinitis pigmentosa(RP) in 2 nonconsanguineous Chinese families. METHODS: The clinical data, including detailed medical history, best corrected visual acuity(BCVA), slit-lamp biomicroscope examination, fundus photography, optical coherence tomography, static perimetry, and full field electroretinogram, were collected from the members of 2 nonconsanguineous Chinese families preliminarily diagnosed with RP. Genomic DNA was extracted from the probands and other available family members;wholeexome sequencing was conducted with the DNA samples provided by the probands, and all mutations detected by whole-exome sequencing were verified using Sanger sequencing in the probands and the other available family members. The verified novel mutations were further sequenced in 192 ethnicity matched healthy controls.RESULTS: The patients from the 2 families exhibited the typical symptoms of RP, including night blindness and progressive constriction of the visual field, and the fundus examinations showed attenuated retinal arterioles, peripheral bone spicule pigment deposits, and waxy optic discs. Whole-exome sequencing revealed a novel nonsense mutation in FAM161 A(c.943 A>T, p.Lys315*) and compound heterozygous mutations in RP1 L1(c.56 C>A, p.Pro19 His;c.5470 C>T, p.Gln1824*). The nonsense c.5470 C>T, p.Gln1824* mutation was novel. All mutations were verified by Sanger sequencing. The mutation p.Lys315* in FAM161A co-segregated with the phenotype, and all the nonsense mutations were absent from the ethnicity matched healthy controls and all available databases.CONCLUSION: We identify 2 novel mutations in genes responsible for autosomal recessive RP, and the mutation in FAM161A is reported for the first time in a Chinese population. Our result not only enriches the knowledge of the mutation frequency and spectrum in the genes responsible for nonsyndromic RP but also provides a new target for future gene therapy.

BHMT Gene Polymorphisms as Risk Factors for Cleft Lip and Cleft Palate in a Chinese Population

Objective Convincing evidence suggests a link between increased risk of nonsyndromic cleft lip with or without cleft palate （NSCL/P） and low intake of folic acid by the mother during pregnancy. The present study was designed to explore if genetic variation in the betaine‐homocysteine methyltransferase （BHMT） gene contributes to NSCL/P. Methods DNA was obtained from 166 individuals with NSCL/P and 285 healthy subjects. Three known single nucleotide polymorphisms （SNPs） present in the BHMT gene （rs651852, rs3797546, and rs3733890） were investigated by real‐time PCR‐based TaqMan genotyping. Results Neither allelic nor genotypic association was found between NSCL/P and SNPs rs651852 and rs3733890. SNP rs3797546 did not show allelic association with NSCL/P; however, a higher proportion of NSCL/P patients carry the CC genotype compared with the TT＋CT genotype （P=0.020, OR=2.10, 95% CI=1.11‐3.95）. Conclusion Our study suggests that polymorphism rs3797546 in the BHMT gene may confer genetic risk of NSCL/P in a recessive manner.