| Summary: | 碩士 === 國立陽明大學 === 遺傳學研究所 === 89 === Methylmalonic acidemia (MMA) is a common autosomal recessive disease of organic acid metabolism caused by deficiency of mitochondrial methylmalonyl CoA mutase (MCM, EC 5.4.99.2), which using adenosylcobalamine as a cofactor. MMA patient will accumulate great mount of methylmalonate in blood and urine during acute stage, and will manifest clinical symptoms varying from benignancy to death. MMA can be divided into two classes, cbl MMA and mut MMA. The former is caused by deficiency of the cofactor, adenosylcobalamin (MIM 251100, MIM 251120) or by deficiency of the adenosylcobalamin and methylcobalamin (MIM 277400, MIM 277410) ; the latter is caused by deficiency of the MCM apoenzyme (MIM 251000). Two types of mut MMA, namely mut0 and mut－, can be distinguished depending on the MCM activity of the patients, and are correlated with the clinical manifestation. No mutase activity could be detected in the lymphocytes of mut0 patients while residual mutase activity (2%~75% of normal) could be detected in mut－ patients. At present, the molecular study of the MUT gene has been carried out in Blacks, Caucasians and Japanese, but not in Chinese. In this study, we analyzed the mutation of the MUT gene in five Chinese MMA families.
Each of the 13 exons and their relevant exon/intron junction of the MUT gene were amplified by polymerase chain reaction (PCR) from genomic DNA of MCM deficient MMA families and sequenced directly. Four mutations, namely 316A>C, 682C>T, 1280G>A, and [1630G>T+1631G>A], were identified in the MUT gene of these Chinese MMA patients. The 316A>C transversion results in a replacement of Thr for Pro at codon 106 (T106P). The 316A>C alteration was found in a mut－ MMA patient originated from northern Chinese. The 1280G>A transition leads to a substitution of Gly for Asp at codon 427 (G427D). The 1280G>A substitution was found both in mut0 and mut－ patients originated from southern Chinese. Two adjacent alterations, namely 1630G>T and 1631G>A, were found in three MMA patients originated from both northern and southern Chinese. These two alterations were found to occur in the same allele (in cis) and lead to a codon change from GGA to TAA at 544 resulting in replacement of Gly for termination (G544X). The [1630G>T+1631G>A] mutation might be a common mutation in the MUT gene of Chinese MMA patients. The 682C>T transition results in an exchange from Arg to termination at codon 228 (R228X). The 682C>T was found in a southern Chinese MMA patient, and it had been reported in a Caucasian patient. To evaluate the occurrence of the nucleotide changes, 50 normal individuals from northern and southern Chinese corresponding to the population of mutant alleles were screened for these four alternations by restriction fragment analysis and sequencing. The results showed none of the normal controls were found to have these alterations. These data suggested that the 316A>C, 682C>T, 1280G>A, and [1630G>T+1631G>A] substitutions might be disease causing mutations in MMA patients.
In this study, five single nucleotide polymorphisms in the coding sequence (cSNPs) of MUT gene were identified in both MMA patients and normal individuals. Three of them, namely 636A/G (K/K 212), 1595A/G (H/R 532) and 2011G/A (V/I 671), had been reported in the Caucasian and the Blacks. Two of them, designated 461G/A (R/H 154) and 1992G/A (A/A 664), were novel polymorphisms found in the MUT gene. The allele frequency of these five polymorphisms in Chinese was determined to be: G/A, 0.99/0.01 for 461G/A, A/G, 0.52/0.48 for 636A/G, G/A, 0.18/0.82 for 1595A/G, G/A, 0.93/0.07 for 1992G/A, G/A, 0.52/0.48 for 2011G/A, respectively. The 636A/G, 461G/A, 1595A/G and 2011G/A polymorphisms were in Hardy-Weinberg equilibrium among Chinese population, while the 1992G/A polymorphism was not. The 636A/G and 2011G/A were found to be tightly linked.
Six haplotypes were deduced from 636A/G, 461G/A, 1595A/G and 1992G/A polymorphism in Chinese population. Haplotype 2 and 4 appeared to be most frequent in both normal population and MMA families. The haplotype frequency showed no significant difference between normal population and MMA patients. However, the [1630G>T+1631G>A] and 1280G>A appeared to link to haplotype 2. These data indicated that [1630G>T+1631G>A] and 1280G>A mutations might have founder effects in Chinese MMA patients.
The mutations identified in this study could be applied to carrier detection and prenatal diagnosis for families suffering from MCM deficient MMA. The mutations identified in MUT gene could also help to better understand the structure-function relationship of the MCM protein.
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