Molecular Genetic Studies of the MMAA and MMAB Genes in Chinese Methylmalonic Acidemia

Methylmalonic scidemia (MMA) is an autosomal recessive disease of organic acid metabolism caused by defects in mitochondrial methylmalonyl-CoA mutase (MCM, EC 5.4.99.2) or the synthesis deficiency of adenosylcobalamin that acts as a cofactor for MCM. Generally speaking, MMA patients often fall ill in infant stage and accumulate a lot of methylmalonate in blood and urine during acute illness. MMA is classified into mut and cbl type according to different complementation groups. Futhermore, mut type MMA can be classified into two sub-type, mut- or mut0, caused by partial or complete enzyme deficiency, respectively. Cbl type MMA caused by the synthesis deficiency of adenosylcobalamin can be classified into six complement groups, namely cblA, cblB, cblC, cblD, cblF, and cblH. Among which cblA, cblB and cblH are isolated MMA while cblC, cblD and cblF are combined MMA and homocystinuria.

Twelve patients with elevated urinary methylmalonate were included in this study and classified by clinical symptoms and biochemical findings for the MCM activity, accumulation of methylmalonate in urine and homocysteine in blood. One of these MMA patients was mut type and the others were cbl types. Two of these cbl type MMA patients were

differentiated as isolated cbl type MMA; seven were combined methylmalonic aciduria and homocystinuria and two were unable to classify. In this study, both the MMAA and MMAB genes were scanned to identify the genetic mutation for four unrelated Chinese cbl type MMA patients without homocystinuria. DNA sequence analysis of the 5'-UTR, 3'-UTR, coding region and the flanking regions of exons for the MMAA gene had identified twleve single nucleotide polymorphisms (SNPs) in the Chinese population.

Among which five SPS, termed c.-2233A/C, C.-1755A/G, c.-1391A/T, c-996C/T and c.1089G/C had been reported elsewhere while the others were novel SNPs, namely c.-1740 A/G, c.-713A/G, c.-469A/G, c.67+52C/T, c.734-264G/A, c.819+168C/G and c.884+2678C/T. For the MMAB gene, we had identified four published SNPs, 56-57GC/AA, c.520-96C/T, c.585-204A/G and c.716T/A, and three novel SNPs, namely c.-414C/T, c.520-159C/T and c.520-128C/T. The SNP identified in the Chinese population for the MMAA and MMAB genes were in H-W equilibrium except c.-2263a/C (allele frequency not determined) and c.-469A/G for MMAA and c.585-204A/G (allele frequency not determined) for the MMAB. These SNPs in MMAA and MMAB genes may assist in studying promoter, RNA stability and mRNA splicing mechanism of these genes.

Mutation analysis of the MMAA gene for four unrelated Chinese cbl type MMA patients without homocystinuria had identified a c.742C>T nd a c.-504C>T substitution. A homozygote of c.742C>T mutation (Q248X), which had been reported as a disease-causing mutation, was identified in the patient MMA001 from a consanguineous family. In the patient MMA021, we had found a c.-504C>T alteration in the predicted promoter region of the MMAA gene. RT-PCR combined sequencing analysis of the MMAA pre-mRNA for MMA021 had shown 40% decreased expression of the c.-504T allele as compare to the c.-504C allele. This data suggested the c.-504C>T mutation might affect MMAA expression and leads to the disease.

MRNA analysis of the MMAB gene for MMA010 patient had found to have some extent of intron 6 retention (r.519_520ins519+91_519+177) while the mRNA of the MMAA gene expressed normally. This retention of intron 6 affects the original reading frame of the protein leading to pre-mature translation stop and produces a truncated protein with seventy-seven amino acids deleted at its C-terminus. However, this intron 6 retention was also found in normal lymphoblast cells suggesting alternative splicing in lymphoblast cells. None of the variation other than

SNP in both the MMAA and MMAB genes (including the predicted promoter regions) was found in patient MMA011. RT-PCR analysis of the MMAA pre-mRNA suggested on of the MMAA allele did not expressed in this patient. The genetic mutation of MMA011 patient remains to be addressed further.