Mutation Analysis Of Mtdna D-loop Hypervariable Region In Huntington's Disease Pedigrees

Objects:Huntington's disease(HD) is an autosomal dominant inherited neurodegen erative disorder,which is taken cerebral cortex and new corpus striatum as main morbidity spot.In clinic,it is characterized by a triad of symptoms:involuntary choreiform movements,cognitive decline,psychiatric impediment.This sickness occurs secretly and the handicapped,lethal ratio is high.Predilection age is mainly in the middle age,the natural course of an illness will be 15～20 years.The disease incidence rate,race,the male and female difference are not obvious.There is no effective treatment to prevent or palliate the progress of HD.The mechanism of HD pathogenesis remains unclear now.The IT15 gene is the pathogenesis gene.The dynamic mutation of three nucleotide(CAG)n repetition sequence is the HD's genetic foundation.Moreover,the patient's origin age,the state and course of the illness have the close relationship with repetition times of(CAG)n.Many researches indicated that HD was connected with the polymorphism of mitochondrial DNA(mtDNA).The mitochondrial is the unique organelle which has DNA besides the nucleus and it's the important place of cell metabolism.Its function barrier is following the energy metabolism disorder,which may cause the neuron lack and the proliferation of neuroglia cell,at last it can induce many kinds of nervous system degrade diseases.The D-loop is located at the mitochondrial non-encode region,which has the important regulative function to the duplication and the transcription of mtDNA.The mtDNA's mutation may affect the function of mitochondrial and induce the occurrence of HD.This article analyses the relationship between the two hypervariable region and HD by detecting the mutation of D-loop in order to study the HD pathogenesis and provide the theory and experiment basis for genetic counseling,gene diagnose and the establishment of prevention system.Methords:7 peripheral blood samples(1 patient,6 normal phenotype relatives) from A pedigree,21 peripheral blood samples(4 patient,16 normal phenotype relatives,1 normal phenotype mate) from B pedigree and 20 healthy control blood samples were extracted respectively.The two hypervariable region of mtDNA were amplified by PCR, the products were purified after gel electrophoresis and detected by sequencing to find the long fragment deletion,insertion and mutation.Results:This article will pedigree outside the normal control group compared with the Cambridge sequence,there are differences in several sites of HVR I,such as: T16030A,A16039G,A16065G,T16102A,CT16114GG,C16082A,T16165A,C16236T,A16384T,Suggesting that the locus of the sequence of the Chinese Han ethnic differences exist with the Cambridge sequence.For all(48 cases) Samples Hypervariable Region I conducted multiple sequence comparison,Found in mtDNAD Hypervariable Region I,16061bp～16171bp is the nucleotide concentration of regional.there were no long fragment deletion and insertion in HVRⅠand HVRⅡof the mitochondrial control region.There are multiple sites or the existence of single-strand 2 bp -3 bp changes when comparing HD patients and normal unrelated individuals:TCT 16027-16029 CTC;T 16141 A;C 16356 A;T C 16444,16445 A T;A 16384 C.Suggesting that these sites may be related to variation in susceptibility to related HD.There are single locus changes in 16284,16356,16116 between the HD patients and normal unrelated individuals,and the sequence in healthy offspring of the patients is different from the patients also.These loci may be related to the progress of HD.Conclusions:1.There is no significant difference in mtDNA D-loop between the patients and normal proved that the change of polymorphism in HD patients' mtDNA D-loop may has no correlation with the HD.2.some changes of HD patients in mtDNA D-loop may be related to HD is related to expandding the sample to be confirmed in further.