| Objective: Through the entire sequence amplification and sequencing of mitochondrial DNA in 97 patients with nonsyndromic hearing loss in Northwest china,we further analyzed the possible mitochondrial DNA mutation sites.Methods: Collected blood samples relevant clinical and pedigree datas from 97 cases with moderate to profound non syndrome deafness,who from five provinces in Northwest China and had a family history of deafness,another 376 healthy people were collected as control group.Genomic DNA was extracted from the case group and the control group by conventional phenol chloroform method,the entire mitochondrial DNA sequence of these two groups were PCR-amplified with 24 overlapping fragments primers,then sequencing,the results were analyzed by using biological software,and then found all of the mitochondrial DNA mutation sites,determined the mitochondrial haplotype,analysed the conservation and frequency of the mutation sites and the effect of gene structure by mutation sites,so as to find the mitochondrial candidate genes that induced deafness.Results:1.Analysed of 97 probands showed that the proportion of male and female patients was 0.9:1,with an average detection age was 22±0.10 years,the average age of oneset was 3±0.83 years,and the hearing loss ranged from moderate to profound,among them,10 probands had a history of using aminoglycosides.2.A total of 706 mitochondrial mutations were sequenced in case group,including 180 variants in the D-loop region,27 variants(1 novel and 26 known)in the 12 S r RNA genes,29 variants(7 novel and 22 known)in the 16 S r RNA genes,122 missense mutations(4 novel and 108 known)and 312 silent variants(20 novel and 292 known)in the protein encoding genes,29 variants(3 novel and 26 known)in the t RNA genes and 7 variants in the non-coding region.3.In the case group,the frequency of haplogroup D,G,M7,M8,M10,M11,M12,A,B4,F,H,N and R were 24.74%,7.22%,5.15%,10.31%,1.03%,1.03%,1.03%,8.25%,5.15%,13.40%,7.22%,7.22% and 8.25%,in the control group,the frequency of them were 21.54%,4.26%,6.91%,10.64%,1.33%,0.53%,0.00%,6.65%,18.62%,15.96%,0.80%,8.78%? 2.39%.The difference of haplogroup B,H,T in the case and control groups were statistically significant.Conclusion: In this study,we found five mitochondrial candidate genes related to deafness.1.The new mutation 1473C>T in the 12 S r RNA gene may alter the tertiary or quaternary structure of 12 S r RNA,may effect the synthesis of mitochondrial proteins and then markedly effect the function of mitochondrial.2.The new mutation 614A>C in the tRNA gene locates at the anticodon loop of t RNAPhe,may affect the fidelity of codon recognition and the structural stabilization and formation of functional t RNA.The new mutation 8339A>G locates at the T-stem of t RNALys,this mutation abolishs the A-U base pair,causing a failure in t RNA metabolism.3.Mutation 5656A>G lies at the junction of the t RNAAla and t RNAAsn,may affect the processing of the t RNA precursors,can contribute to clinical abnormalities.4.Mutation 3866T>C of the ND1,make the transition of the Polarity hydrophobicity isoleucine in the NADH dehydrogenase subunit1 to hydrophilic threonine,it can affect the activity of NADH dehydrogenase and destroy the normal function of mitochondria,and then damage the hair cells in the inner ear.5.The difference of haplogroup H and T in the case and control groups were statistically significant,considering to be associate with deafness. |
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