Medium-chain acyl-CoA dehydrogenase (MCAD) mutations identified by MS/MS-based prospective screening of newborns differ from those observed in patients with clinical symptoms: identification and characterization of a new, prevalent mutation that results in mild MCAD deficiency

Abstract

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is the most frequently diagnosed mitochondrial beta-oxidation defect, and it is potentially fatal. Eighty percent of patients are homozygous for a common mutation, 985A-->G, and a further 18% have this mutation in only one disease allele. In addition, a large number of rare disease-causing mutations have been identified and characterized. There is no clear genotype-phenotype correlation. High 985A-->G carrier frequencies in populations of European descent and the usual avoidance of recurrent disease episodes by patients diagnosed with MCAD deficiency who comply with a simple dietary treatment suggest that MCAD deficiency is a candidate in prospective screening of newborns. Therefore, several such screening programs employing analysis of acylcarnitines in blood spots by tandem mass spectrometry (MS/MS) are currently used worldwide. No validation of this method by mutation analysis has yet been reported. We investigated for MCAD mutations in newborns from US populations who had been identified by prospective MS/MS-based screening of 930,078 blood spots. An MCAD-deficiency frequency of 1/15,001 was observed. Our mutation analysis shows that the MS/MS-based method is excellent for detection of MCAD deficiency but that the frequency of the 985A-->G mutant allele in newborns with a positive acylcarnitine profile is much lower than that observed in clinically affected patients. Our identification of a new mutation, 199T-->C, which has never been observed in patients with clinically manifested disease but was present in a large proportion of the acylcarnitine-positive samples, may explain this skewed ratio. Overexpression experiments showed that this is a mild folding mutation that exhibits decreased levels of enzyme activity only under stringent conditions. A carrier frequency of 1/500 in the general population makes the 199T-->C mutation one of the three most prevalent mutations in the enzymes of fatty-acid oxidation.

Figure 1

LightCycler assay for the 985A→G mutation and the 199T→C mutation. A, Assay-melting curves for the 985A→G mutation, for 985A homozygotes (two samples), 985A/985G heterozygotes (five samples), and 985G homozygotes (two samples). B, Assay-melting curves for the 199T→C mutation, for 199T homozygotes (three samples) and a 199T/199C heterozygote (one sample).

Figure 2

Enzyme activity of mutant MCAD proteins. Transformed JM109 cells harboring expression plasmid encoding the mature part of either wild-type (WT) MCAD protein or one of the mutant MCAD proteins and either the plasmid pGroESL, encoding the chaperonins GroEL and GroES (unshaded bars), or the control plasmid pCaP, which lacks the GroESL chaperonin genes (shaded bars), grown and induced as described by Andresen et al. (1997). Extracts were investigated by measurement of enzyme activity. The designations of the mutant are indicated below the corresponding bars; + and − respectively indicate whether the chaperonins GroEL and GroES were co-overexpressed. The culture temperature is indicated. MCAD enzyme activity in the soluble fraction was measured by the ferricenium assay (Lehman et al. 1990). Columns represent the average from two to five independent experiments in which enzyme-activity measurements were performed in duplicate in each experiment. The vertical lines on top of the bars indicate the ranges of the values obtained in the experiments.

Figure 3

Thermal inactivation profiles of the mutant proteins Y42H, S220L, and G285R and of wild-type (WT) MCAD protein. Investigation of the thermal-inactivation profiles of the mutant proteins Y42H, S220L, and G285R and of wild-type MCAD was performed as described elsewhere (Bross et al. ; Andresen et al. 1997). Lysed samples from cells overexpressing the mutant proteins were incubated for 10 min at the temperatures indicated, before MCAD enzyme activity in the soluble fraction was measured by the ferricenium assay. Each value represents the mean of four samples, and the bars represent the ranges of the highest and lowest measurements.