| Background and Objective: Leber's hereditary optic neuropathy (LHON) is a maternally inherited disease characterized by acute or subacute visual loss, with bad prognosis and associated with the degeneration of the retinal ganglion cell layer and optic nerve. The optic neuropathy involves a loss of central vision due to dysfunction of the retinal ganglion cells and optic nerve axons that subserve central vision. It commonly affects young adult males.It was first reported as an independent disease in 1871 by Leber and subsequently confirmed by numerous studies that the risk of vision loss in LHON families is inherited exclusively from the mother. An early change in the acute phase is papillitis,subsequently envolved optic neuropathy atrophy. In 1988, Wallace first found the mutation position in mtDNA11778 in LHON patients, Vikky and Noory confirmed this finding furthermore. From then on, the pathogenesis of LHON is due to point mutation of mtDNA which results in cell oxidative phosphorylation and respiratory chain dysfunction. With rapid development of molecular biologic technology, scientists found other mutations, mtDNA11778 , mtDNA 3460,mtDNA 14484 are known as primary point mutation. There are many secondary point mutations ,such as, mtDNA 4216, mtDNA9804, mtDNA13708 and so on. While these secondary point mutations may cause LHON lonely or associated with primary mutation and other secondary mutations. The secondary mutation plays an important role in the pathogenesis of LHON. The spectrum of mtDNA mutation is different in races and areawith LHON.The mtDNA3460, mtDNA14484 has rare mutation frequency in Chinese. Consequently, the study on secondary mutation has an important role in Chinese LHON. Especially, at present, we haven't found the study on secondary mutation.Our experiment investigate the spectrum of primary mutation: 11778 and secondary mutation :13708,13730,15257,9804,observe their roles and mutant frequency in our Han population of LHON.Methods: Five LHON pedigrees were studied, all came from Henan and Anhui proviences, only 3 pedigrees agree with checking, including 15 patients and 16 asymptomatic maternal relatives. In the patients with LHON, 12 were males 3 were females. Blood samples were obtained from consented LHON patients, their unaffected maternal relatives, and 47 healthy controls. One milliliter of whole blood was withdrawn from each subject and was kept in a glass tube containing EDTA-K2.MtDNA was extracted from cells of peripheral blood samples. Four pairs of primers were prepared with DNA synthesier. By way of PCR-SSCP and DNA sequencing, we performed mtDNA analyses for one primary mutation at npll778,and 4 secondary mutations at npl3708,npl3730,npl5257,np9804. The results were dealt with by SPSS11.0.ResultsrThere are 82 maternal relatives in 5 LHON pedigrees, including 31 patients(20 males, 11 females). The disease incidence is 25.2%, the mean age of onset is 29.8.The disease incidence of maternal lineages decreased followed with generations increased. The penetrance of males is higher than females.There was statistically significant difference in penetrance between males and females. The age of onset is younger than that of parental generation in three LHON pedigrees.Compared with control group, SSCP analysis of PCR products amplified with primer pair 1 in the experiment group showed band shift in all lanes,and no band shift was found in PCR products amplified with primer pair 4, some lanes appeared band shift in products amplified with primer 2 and 3. DNA sequencing revealed npll778 G to A in all samples of the experiment group. The frequency of npll778 mutation in experiment group is100%.At npll719 G to A mutation was found in all samples.Fragment 2,3,4 were sequenced by 42 samples, including 30 experimental controls and 12 healthy controls, no mutation were found in these fragments at npl3708,npl3730,npl5257 and np9804. But at npl3759, we found 26 samples had G to A point mutation. The penetrance of LHON in npl3759 positive pedigree is lower than in 13759 negative pedigree. It probable decreases the penetrance of LHON.The np 13928(G to Qmutation were found in fifteen samples, and we found a new mutation at np 13942 in a control sample. At fragment 3, npl5326 A to G mutation was found in all samples. At npl5323(G to A) in two normal controls. In fragment 4, there is a novel mutation at np9738(G to A) in a normal control.Conclusionsid) The mutation with LHON has significant difference in race and area.(2) Primary mutation at npll778 is predominant in China.In our test,the ratio of mutation is 100%.It should be regarded as a routine test in diagnosis of LHON.(3) A new secondary mutation at npl3759 has been found, it has statistically significant difference(i><0.05). The penetrance of LHON in npl3759 positive pedigree is lower than in 13759 negative pedigree. It probable decreases the penetrance of LHON.(4) Npll719 and npl5326 are probable specific sequence of mtDNA in our Han population.(5) In part of LHON pedigrees, the age of onset is younger than that of parental generation.(6) Two novel mutations at np9738 and npl3942 are mtDNA polymorphism in normal person?... |
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