The most common mutation causing medium-chain acyl-CoA dehydrogenase deficiency is strongly associated with a particular haplotype in the region of the gene

Summary RFLP haplotypes in the region containing the medium-chain acyl-CoA dehydrogenase (MCAD) gene on chromosome 1 have been determined in patients with MCAD deficiency. The RFLPs were detected after digestion of patient DNA with the enzymes BanII, PstI and TaqI and with an MCAD cDNA-clone as a probe. Of 32 disease-causing alleles studied, 31 possesed the previously publised AG point-mutation at position 985 of the cDNA. This mutation has been shown to result in inactivity of the MCAD enzyme. In at least 30 of the 31 alleles carrying this G985 mutation a specific RFLP haplotype was present. In contrast, the same haplotype was present in only 23% of normal alleles (P3.4×10^-18). These findings are consistent with the existence of a pronounced founder effect, possibly combined with biological and/or sampling selection.     

A rare disease-associated mutation in the medium-chain acyl-CoA dehydrogenase (MCAD) gene changes a conserved arginine, previously shown to be functionally essential in short-chain acyl-CoA dehydrogenase (SCAD)

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is a serious and potentially fatal inherited defect in the β-oxidation of fatty acids. Approximately 80% of patients with MCAD deficiency are homozygous for a single disease-causing mutation (G985). The remaining patients (except for a few cases worldwide) are compound heterozygous with G985 in one allele. By sequencing of cloned PCR-amplified MCAD cDNA from a G985 compound heterozygous patient, we identified a C-to-T transition at position 157 as the only change in the entire coding sequence of the non-G985 allele. The presence of the T157 mutation was verified in genomic DNA from the patient and her mother by a PCR-based assay. The mutation changes a conserved arginine at position 28 (R28C) of the mature MCAD protein. The effect of the T157 mutation on MCAD protein was investigated by expression of mutant MCAD cDNA in COS-7 cells. On the basis of knowledge about the three-dimensional structure of the MCAD protein, we suggest that the mutation destroys a salt bridge between arginine²⁸ and glutamate⁸⁶, thereby affecting the formation of enzymatically active protein. Twenty-two additional families with compound heterozygous patients were tested in the PCR-based assay. The T157 mutation was identified in one of these families, which had an MCAD-deficient child who died unexpectedly in infancy. Our results indicate that the mutation is rare. It is, however, noteworthy that a homologous mutation has previously been identified in the short-chain acyl-CoA dehydrogenase (SCAD) gene of a patient with SCAD deficiency, suggesting that the conserved arginine is crucial for formation of active enzyme in the straight-chain acyl-CoA dehydrogenases.     

Identification of a mutation cluster in mevalonate kinase deficiency, including a new mutation in a patient of Mennonite ancestry.

Mevalonate kinase (MKase) deficiency (MKD) is a rare autosomal recessive disorder in the pathway of cholesterol and nonsterol isoprenoid biosynthesis. Thus far, two disease-causing missense alleles have been identified, N301T and A334T. We report four additional mutations associated with MKD: L264F, T243I, L265P, and I268T, the last found in a patient of Mennonite ancestry. Electrophoretic analysis of bacterially expressed wild-type and mutant MKase indicated that I268T and T243I mutants produced normal or somewhat reduced amounts of MKase protein; conversely, L264F and L265P mutations resulted in considerably decreased, or absent, MKase protein. Immunoblot analysis of MKase from all patients suggested that the MKase polypeptide was grossly intact and produced in amounts comparable to control levels. Three mutations resulted in significantly diminished MKase enzyme activity (<2%), whereas the I268T allele yielded approximately 20% residual enzyme activity. Our results should allow more-accurate identification of carriers and indicate a mutation "cluster" within amino acids 240-270 of the mature MKase polypeptide.