| Objective:Basal ganglia calcification is a common sign on cranial computer tomography. It was thought that basal ganglia calcification (especially globus pallidus calcification) on cranial CT belonged to physiological calcification like pineal body and tela chorodea calcification. Most patients with basal ganglia calcification have no symptoms, especially those beyond 40 years old. At present, study of mitochondrial diseases is not much in our country. Many of such patients were misdiagnosed because of lacking of useful clues for selection and diagnosis clinically. In our study, PCR/RFLP and PCR-DNA sequence analysis were used to detect the mitochondrial point mutation of patients with basal ganglia calcification on cranial CT, so as to probe the relationship between abnormal mitochondrial DNA and basal ganglia calcification and to inquire into whether or not basal ganglia calcification can be regarded as the target of selection and diagnosis of mitochondrial diseases.Material and methods:20 cases of basal ganglia calcification (especially globus pallidus) were collected. They were all inpatients or outpatients from department of neurology of Qilu hospital, Shandong university. Among them, 3 cases were studied with their blood specimens and 17 cases received muscle biopsy, 10 of which were diagnosed as MELAS syndrome. Genomic DNA was of 17cases was from specimen of muscle. Genomic DNA was extracted from those specimens. The mutation hot spot -tRNALeu(UUR)region was amplified by PCR, and then the products were purified with reagent of PCR. Some of them were digested with the restriction enzyme Apa I and Afl II ,and then lectrophoresed through a 1.2% agarose gel (with EB 8ul), others were transmitted to Shanghai to analyse the sequence of the DNA fragment. Results:1. MELAS group: (1) Results of digestion of mtDNA: Point mutation at A3243G of mtDNA was found in 6 of the 10 patients of MELAS. There was no mitochondrial point mutation at T3271C. (2) Analysis of mtDNA sequence: Point mutation at A3243G of mtDNA was found in 7 of the 10 patients of MELAS, 6 of which were those discovered in digestion of mtDNA and 1 has A3 243 G point mutation only in analysis of mtDNA sequence, companied by C3524G and C3527T. There was no mitochondria point mutation at T3271C. (3) Clinical manifestation features: In MELAS group clinical manifestations varied. 3 cases had stroke; 4 cases had epilepsy, 2 of which had the state of epilepsy; 1 couldn't see clearly when epilepsy broke out, but without disorders of consciousness; Intelligence declined in 5 cases, vision declined in 2 cases, and hearing declined in 7 cases, headache in 3 cases, vomiting in 4 cases, emaciating in 9 cases, short stature in 4 cases, diabetes in 4 cases, tendon reflex in 2 cases. The relationship between case 1 and 2 is the same as case 3 and 4— mother and son. Local brain lesion of CT or MRI was found in 4 cases and cerebral atrophy in 2 cases; Lactic acidosis in blood was tested in 7 cases , with 6 cases rising. (4) Muscle biopsy features: The 10 cases all had typical RRFs, but the rates were different. 6 cases had blue fiber in COX/SDH and 4 cases had COX deficiency. Abnormal mitochondrial was found in all 10 cases by electroscope.2. Non-MELAS group: (1) Results of digestion of mtDNA: There was no mitochondrial point mutation at A3243G and T3271C. (2) Analysis of mtDNA sequence: Point mutation at A3243G and T3271C of mtDNA was not found in 10 patients of this group, but C3524G was found in 2 cases, C3527T in 3 cases, C3527A in 1 case and C3527G in 1 case. (3) Clinical manifestation features: clinical manifestations varied. 1 case had stroke; 7 cases had headache as chief symptom; Memory declined in 6 cases and epilepsy in 2 cases; There was no diabetes and lossof hearing in this group; Lactic acidosis was tested in 4 cases, all normal. (4) Muscle biopsy features: 7 of the 10 cases received muscle biopsy, none of which had typical RRFs and blue fiber in COX/SDH, and only one had fatty increase lightly. Conclusion:1. PCR/RFLP can be widely applied to the detection of known mtDNA point mutations, with the advantages of sensitive, repetitive, simple, convenient, rapid, effective, and easily observed. The procedure is especially applicable for mass samples detection. PCR-DNA sequence analysis is the most direct and believable method of gene detection. PCR-DNA sequence analysis doesn't need many specimens, and can easily be standardized and automated. Its efficiency is high, but the cost is high comparatively too.2. The results of the test show that: 7 of the 10 cases of MELAS with basal ganglia calcification had A3 243 G point mutation and 4 of the above 7 cases were mother-son relationship. This indicates that: (1) The ratio of point mutation at 3243 is high in the patients of MELAS with basal ganglia calcification on brain CT and it is the commonest point mutation of MELAS syndrome; (2) Mitochondrial diseases have the specific property of maternal inheritance; (3) Different point mutations of mtDNA can lead to same clinical manifestation; (4) Basal ganglia calcification on brain CT can be regarded as an important clue of the selection of mitochondrial disorders.3. Clinical and pathological characteristics of our patients with MELAS show that: Clinical manifestations can be the same or similar in the patients with basal ganglia calcification on brain CT. But they have different features on muscle pathology. In the group of MELAS, variation of COX can be seen, typical RRFs can be seen by MGT and abnormal mitochondria can be found under the electron-scope. So muscle biopsy should be done for the patients with basal ganglia calcification and suspicious mitochondrial disorders. Lactic acidosis should be tested too because 6 of the 7 cases with MELAS in which lactic acidosis was tested were higher than normal.4. Our researches show that: The results of PCR/RFLP and PCR-DNA sequence analysis are not completely the same. There was 1 case that had A3243G pointmutation in PCR—DNA sequence analysis, but there was no in PCR/RFLP. This indicates that PCR/RFLP has the limitation that it needs high peculiar sequence around the site of mutation, if not, false negative results would be found.
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