Clinical Generics 1991: 40: 283-286

Heterogeneity for mutations in medium chain acyl-CoA dehydrogenase deficiency in the UK population

DIANA CURTIS, ALEXANDRA 1. F. BLAKEMORE, PAUL C. ENGEL’, DONALD MACGREGOR’, G w BESLEY’, SEEN KOLVRAA‘ A N D NIELS GREGERSEN’

Sub-Department of Human Genetics and ‘Department of Molecular Biology and Biotechnology, University of Shefield, Shefield; ZDepartment of Paediatnc Neurology and ’Department of

Paediatric Biochemistry, Royal Hospital for Sick Children, Edinburgh, UK. The Institute of Human Genetics, University of Aarhus and Laboratory of Molecular Genetics, Skejby Sygehus, Aarhus,

Denmark

MCAD is the commonest inherited disorder of fatty acid oxidation. We have sought for and studied 21 affected children from 18 families within the UK. In 14 families the children are homozygous for the G985 mutation. In three families the children are compound heterozygotes for (3985 and thus carry another and unknown mutation. In one family the child does not carry the G985 mutation on either allele. The carrier incidence of the G985 mutation is 1 in 68, which suggests that the natural history of MCAD deficiency deserves further study.

Received 18 February, accepted for publication 12 April 1991

Key worak common mutation; heterogeneity; MCAD deficiency; population incidence.

Medium chain acyl-CoA dehydrogenase (MCAD) deficiency is an autosomal reces- sive disorder which has been implicated as one cause of sudden unexplained death in young children (Bennett et al. 1990). The symptoms of MCAD deficiency are caused by decreased oxidation of fatty acids. There- fore affected children suffer from recurrent attacks of hypoglycaemia and lethargy, often leading to coma (Vianey-Liaud et al. 1987)). Some children have been described as having a Reye’s syndrome-like illness be- fore thorough investigation. The hypoglyca- emic attacks can be fatal, with reports of 40% fatality when the first attack occurs, in children between 1 and 2 years of age (Roe & Coates 1989). Traditional tests. in suspected cases of MCAD deficiency, com- prise a hierarchy of biochemical analyses;

these range from routine screening by gas chromatography combined with mass spec- trometry for urinary organic acids to meas- urement of MCAD activity in cells grown in tissue culture (Vianey-Liaud et al. 1987).

Recently the gene has been cloned (Kelly et al. 1987) and a common mutation, involv- ing an A to G nucleotide replacement at position 985 of the MCAD cDNA (Matsub- ara et al. 1990, Gregersen et al. 1990), has been identified. The first reports suggested that the “common” G985 mutation was present in most cases tested. More recent reports (Yokota et al. 1990) have indicated that other mutations in the MCAD gene are present in children with an undoubted MCAD phenotype.

As part of our studies of MCAD de- ficiency, we have sought to document fam-

284 CURTIS ET A L .

ilies and screen MCAD-affected offspring for the G985 mutation, in order to gain some insight into the frequency of the dis- order in the UK population.

Patients

We sought for MCAD families from col- leagues in Clinical Genetics and Biochemis- try. To date, we have been informed of 25 MCAD families resident within the UK. One of these families is of Asian origin; the remainder, like all previously reported cases, are of Caucasian origin. Not all of these families have been available for study and we are well aware that our list of known families is still incomplete.

Laboratory Tests

DNA samples from the affected children were routinely prepared from blood samples, fibroblast samples and from dried blood spots (Guthrie spots). A polymerase chain reaction (PCR) was used to amplify a 199bp DNA fragment which includes the G985 mutation (Gregersen et al. 1990). A mismatch to produce a StyI (NcoI) restric- tion site at the mutation point and at the 3’ end of the amplified sequence was intro- duced via the PCR primers. Thus. following PCR amplification of StyI restriction, the normal allele fragment is cut to 178 bp com- pared with the mutant allele fragment which is cut to 154 bp. This difference was resolved on a 12% polyacrylamide gel and visualised following ethidium bromide staining. All routine controls and precautions against cross-contamination for PCR tests were em- ployed.

Results

We have used this PCR test to screen 21 affected children from 18 families. In 14 families the common G985 mutation was

present in homozygous form in 16 affected offspring. In 3 families, 4 affected children were heterozygous for the G985 mutation. These 4 children, who are therefore com- pound heterozygotes, must carry a different mutation on one of their alleles. In the final family studied, we have identified an MCAD deficient child who is homozygous for the normal A nucleotide at position 985.

Case Report This affected child presented at 16 months with a Reye’s syndrome-like illness. He was vomiting and had become drowsy following a few days of feeling unwell. A year pre- viously, he had had a similar episode of drowsiness following 2 to 3 days of vomit- ing. At the time of presentation, he was drowsy with signs of raised ICP, hypoglyca- emia and with raised levels of urinary dicar- boxylic acids. MCAD deficiency was con- firmed by ETF assay, which showed abnor- mally reduced levels of medium chain acyl- CoA dehydrogenase activity in cultured fibroblasts. Since diagnosis, he has had 2 further emergency hospital admissions con- current with infections. This child had a sibling who died in 1981 at 15 months of age with a Reye’s syndrome-like illness (Fig.

T I

* Homozygous for A985

MCAD affected phenotype

FIg. 1. Pedigree of family who do not carry the common G985 mutation.

ACYL-COA DEHYDROGENASE D E F I C I E N C Y I N U K 285

1). In follow-up studies, a PCR test on the Guthrie spot obtained from the dead sib- ling’s records showed that he too was homo- zygous for the normal nucleotide at position 985. The family are not consanguineous back to the great-grandparental generation, at which time the maternal and paternal branches of the family came from different regions of Lowland Scotland.

Discussion

We have screened 21 children affected with MCAD deficiency for the common G985 mutation. Sixteen of these children were homozygous for G985. Four children were compound heterozygotes for G985. One child did not carry the common G985 mu- tation on either allele. We do not know whether this will be a single second mu- tation event in the UK population or whether there will be a collection of rare mutations, perhaps limited to single fam- ilies. The final case is, we believe, the first report of a child with an undoubted MCAD phenotype who has no apparent abnor- mality detected by PCR for the G985 mu- tation.

In our UK study the G985 mutation ac- counts for 86% of MCAD alleles detectable with the G985 assay. However, some 2.4% or up to 1 in 42 affected children will not carry the G985 mutation and will not be detected by the present test. MCAD is the commonest inherited disorder of fatty acid oxidation. Others (Bennett et al. 1990) have speculated that the occurrence of MCAD deficiency may be rather high. Recently, we have reported the frequency of the G985 mutation in a consecutive series of neonatal blood spots to be 1 in 68 (Blakemore et al. 1991).

It would therefore be convenient to have both a screening programme for the MCAD mutation@) and a rapid and reliable test for children with suspected MCAD disease. At

present, such a test must await the delin- eation of other molecular defects respon- sible for MCAD deficiency, since the current PCR test for G985 will miss children such as the case described here.

Acknowledgements

This work is funded by a grant from the Welcome Trust to support AIFB. We are grateful to J. J. Cogswell (Poole), N. R. Dennis (Southampton), George Gray (Bir- mingham), P. Johnston (Southampton) R. W. Logan (Glasgow), Alan McDermott (Bristol), J. P. Osborne (Bath) and Rodney Pollitt (Shefield) for sending us samples from their patients.

References

Bennett, M. J., F. Allison, R. J. Pollitt & S. Vari- end (1990). Fatty acid oxidation defects as causes of unexpected death in infancy. In Farty Acid Oxidation: Clinical, Biochemical and Mol- ecular Aspects. K. Tanaka & P. M. Coates eds. New York, Alan R. Liss Inc, pp. 349-364.

Blakemore, A. I. F., H. Singleton. R. J. Pollitt, P. C. Engel, S. Kolvraa, N. Gregersen & D. Curtis (1991). Frequency of the G985 mutation in the general population Lancer i, 298-299.

Gregersen, N., B. S. Andresen, P. Bross, V. Win- ter, s. Engst, N. Rudinger, E. Christensen, D. Kelly, A. W. Strauss. S. Kolvraa, L. Bolund & S. Ghisla (1990). Molecular characterisation of medium chain acyl-CoA dehydrogenase (MCAD) deficiency: identification of a lys 329 - glu mutation in the MCAD gene and ex- pression of inactive mutant protein in E. coli. Hum. Genet. (in press).

Kelly, D. P., J. J. P. Kim, J. J. Billadelo, B. E. Hainline, T. W. Chu & A. W. Strauss (1987). Nucleotide sequence of medium chain acyl- CoA dehydrogenase mRNA and its expression in enzyme-deficient human tissue. Proc. Natl. Acad. Sci. 84 40684072.

Matsubara, Y., K. Narisawa, S. Miyabayashi, K. Tada & P. M. Coates (1990). Molecular lesion in patients with medium chain acyl-CoA de- hydrogenase deficiency. Lancet i, 1589.

286 C U R T I S E T A L .

Roe, C. R. & P. M. Coates (1989). Acyl-CoA dehydrogenase deficiencies. In The Metabolic Basis of Inherited Disease, C . R. Scriver, A. L. Beaudet, W. S. Sly & D. Valle, eds. New York, McGraw-Hill, pp. 889-914.

Vianey-Liaud, C., P. Divry, N. Gregersen & M. Mathieu (1987). The inborn errors of mito- chondrial fatty acid oxidation. J . Znher. Metab. Dis. 10, suppl 1 , pp. 159-198.

Yokota, I., K. Tanaka, P. Coates & M. Ugarte

(1990). Mutations in medium chain acyl-CoA dehydrogenase deficiency. Lancet ii, 148.

Address: Dr Diana Curtis. PhD Sub-Department of Human Genetics 1 17, Manchester Road Sheffleld SIO SDN UK