Molecular Biological Detection Of Leber Hereditary Optic Neuropathy

Objective Leber hereditary optic neuropathy (LHON) is a maternally inherited disease characterized by a bilateral optic neuropathy. Most often, male patients are affected in early adult life. It was reported firstly as an independent disease by Theodor Leber in 1871. In 1988, Wallace and his coworkers identified a mutation in the mitochondrial DNA firstly. They found that LHON patients carried a G to A point mutation at nucleotide position 11778 that converts the highly conserved 340th amino acid from arginine to histidine. Since then, more than 20 point mutations in mtDNA have been described in LHON patients. Based on genetic, clinical and biochemical parameters, mutations in the mitochondrial genome at nucleotide positions (np) 11778,14484,3460 are regarded as primary mutations which can cause LHON alone. The others regarded as secondary mutations. The majority of LHON cases around the world are caused by these three primary mutations, which result in amino acid substitutions in the ND4, ND6, NDi subunits of respiratory chain complex I . Usually, these three mutations are screened in suspect to the diagnosis but also as different LHON mutations lead to variations in expression, severity, and recovery of the disease. Molecular biological detection is more important if the patient is not seen during the acute phase or is a singleton case. Up to now, most studies in China are limited to detect primary mutations using mutation specific primer PCR or PCR-RFLP (restriction fragment length polymorfism). For advancing study onpathogenesis, mechanism, diagnosis, treatment of LHON, we investigated thirty maternal members from three pedigrees using single strand conformation polymorphism and DNA sequencing.Materials and Methods Five pedigrees were inquired and 30 maternal members in three of them were tested. Following informed consent, periphery blood samples were obtained. Thirty-two healthy persons without optic disease were included in control group. Total DNA was isolated using routine way (SDS-proteinase K-hydroxybenzene-chloroform method). Four fragments we amplified using four pairs of primers, which covered eleven nucleotide position mutations reported abroad. Through electrophoresis on 1.50% agarose gel, purity and quantity of amplified product were examed. Then analysed the product using SSCP: after denatured by heating at 95.00癈 for five minutes, 5 1 PCR amplified product was injected into loading hole mixed 5 1 2 X loading buffer. PCR products were separated by constant temperature (18.00癈) and constant presssure (220.00V) electrophoresis on 8.00% non-denaturation polyacrylamide gel mixed 5.00%glycerol solution for 10.00-13.00 hours. The DNA marker is used in judging the length of amplification fragments. Results can be acquired after silver straining. At last these fragments were sequenced and compared with Cambridge standard mtDNA complete sequence.Results There were 78 blood species members in the five pedigrees which included 36 male and 42 female. Twenty males and 11 females were affected. Penetrance of male is 55.55% and female is 26.91%. Compared with control group, SSCP analysis of PCR products amplified with primer pair 1 in the experiment group showed band shift in all lanes, only one lane showed band shift in products amplified with primer pair 2, and no band shift was found in products amplified with primer pair 3 and 4. DNA sequencing revealed npl!778 G to A mutation in all samples of the experiment group. The frequency of the npl!778 mtDNA mutation in experiment group is 100.00%. Npll719 G to A mutation was found in all samples of experiment group and control group. Thirty samples sequenced at nucleotide position 11696. No mutation was found in this position. Fragment2 were sequenced 23 sameples and fregment 3 were sequenced 8 samples, but no mutation were found in these fragments. Thereare three objective mutation points (np4136, np4160, np4216) in fragment 4. Thirty-one samples were sequenced, no mtutation was found at these three points. Np4164 (AG) was screened in a experiment sa...