Medium-chain acyl-CoA dehydrogenase deficiency (MCADD; OMIM 607008) is an autosomal recessive metabolic disorder of mitochondrial fatty acid β-oxidation. Medium-chain acyl-CoA dehydrogenase (MCAD) is a homotetramer and shows its highest activity with octanoyl-CoA. Clinical symptoms are variable, including hypoketotic hypoglycemia, hyperammonemia, gastritis, lethargy, seizure, coma, and sudden infant death syndrome (SIDS)-like illness. In about 20% of the patients, sudden death occurs at the first presentation of the disease [1]. Other affected individuals may remain without any symptoms for several years [2, 3]. The duration of the onset of the symptoms can range between 2 days to 6.5 yr, and the symptoms are triggered by catabolic stresses such as infection and prolonged fasting [1]. To prevent catabolic stress, patients are made to consume a carbohydrate-rich diet and carnitine supplements and prevented from fasting.

MCADD is the most frequently diagnosed clinical defect of fatty acid metabolism in Caucasians. The frequency of the disease ranges from 1:10,000 to 1:30,000 in the United States, and it is detected during newborn screening using tandem mass spectrometry [2, 4-6]. However, this disorder is relatively rare in Asian populations. Although the frequency of MCADD detection has increased after the introduction of neonatal newborn screening with tandem mass spectrometry, including acylcarnitine analysis, and with the increase of awareness of the disorder, it is still rare in Asians. The incidence of MCADD is 1:51,000 in the Japanese population, and there were no cases of the disorder among the 79,179 newborns screened in Korea [7, 8]. To date, 1 Korean and 11 Japanese patients have been confirmed to be positive for MCADD by molecular analysis [9-12].

The human ACADM gene is located on chromosome 1p31 and spans 44 kb. It contains 12 exons encoding a mature protein of 396 amino acids (43.6 kDa) [13]. The mutation frequency in the ACADM gene shows ethnic variation. In Caucasian patients with MCADD, a common c.985A>G mutation was identified in about 80-90% of the mutant alleles [2, 14]. However, the results of 2 previous reports indicate that c.449_452delCTGA is the most common disease-causing ACADM mutation identified in Asians [9, 10, 12]. In this report, we describe 2 Korean pediatric patients with MCADD that was detected during newborn screening tests by tandem mass spectrometry. Molecular studies revealed 2 genetic variations in each patient, and a novel ACADM mutation was identified.

To identify patients with fatty acid oxidation disorders, neonatal screening tests with tandem mass spectrometry, including acylcarnitine analysis and confirmatory mutation analysis are essential. The high levels of hexanoylcarnitine, octanoylcarnitine, and decanoylcarnitine and the octanoylcarnitine/decanoylcarnitine ratio can be used to differentiate of MCADD patients from healthy individuals and patients with other disorders [15]. Urine organic acid analysis of MCADD patients revealed peaks of medium-chain dicarboxylic acids, hexanoylglycine, and suberylglycine. In both our patients, significant elevations of medium-chain acylcarnitines, especially octanoylcarnitine, were observed, and hexanoylglycine and suberylglycine were detected in urine organic acid analysis. These findings are typical for patients with MCADD.

More than 70 mutations have been identified in the ACADM gene, and the most common type is a missense mutation. The most frequently detected mutation involves an A to G transition at nucleotide position 985 (c.985A>G), which results in the substitution of glutamate for lysine at amino acid position 329 (p.K329E). The distribution of the c.985A>G mutation is known to vary according to ethnicity and other factors. According to previous publications, a high frequency of homozygotes was observed in patients presenting with the clinical symptoms of MCADD, but a relatively lower frequency of homozygotes were found in patients identified through newborn screening [2, 6, 16]. In addition, the frequency of the c.985A>G mutation is much higher in Caucasians than in Asians [6, 10, 17, 18]. The c.449_452delCTGA mutation, which was identified in the 2 patients in the present report, was first reported in 2 of 3 Japanese patients with MCADD in 2005 and also detected in 1 Korean patient [10, 12]. Prior to this study, the same mutation was detected in 8 of 11 Japanese patients with MCADD and 1 Korean patient with MCADD and was not reported in Caucasian patients [9-12]. Thus, the allele frequency of the c.449_452delCTGA mutation, including the 2 present cases, is 46.2% (12/26 alleles), making this mutation the most common mutation associated with MCADD in East Asians. In the light of the novel mutation presented here and previous reports on MCADD in East Asians, we expect that the distribution of ACADM mutations will vary according to ethnic origin.

In the past, there have been several previous reports on the genotype-phenotype correlation in MCADD patients. The homozygosity of the c.985A>G mutation has been reported to indicate a high level of plasma octanoylcarnitine [14, 19] and an increased risk of developing of adverse events [20]. However, the pathogenic effects of the majority of mutations in the ACADM gene have not been assessed. A previous case report described a Japanese patient with the c.449_452delCTGA and c.1189T>A mutations, and these mutations were identical to that of Patient 2 in our present report. In this report, the patient experienced hypoglycemia, vomiting, and lethargy that seemed to have triggered due to infections after the age of 7 months and was diagnosed with the disorder after biochemical tests [11]. Therefore, a close follow-up is needed to check for the development of hypoglycemia and other symptoms in our patient.

The novel ACADM c.461T>G (p.L154W) mutation was not found in 170 healthy subjects. Although leucine at codon 154 is not conserved in all species, searches in the evolutionary annotation database (http://jbirc.jbic.or.jp/evola/search.html) revealed that it is conserved in 12 among 14 species. All the mutations registered in Human gene mutation database (HGMD, http://www.hgmd.cf.ac.uk/ac/index.php) are not conserved in all species. In addition, in-silico prediction analysis does not perfectly predict all mutations. Previous studies reported that scale-invariant feature transform (SIFT) predicted about 70% of substitutions annotated as involved in disease as damaging, and about 80% of the nonsynonymous SNPs found to affect protein function in patients. PolyPhen was also reported to have similar sensitivity [21, 22].

The relationship between genotype and phenotype of the c.449_452delCTGA mutation, which was detected in both our patients and in other Asian patients, has not been clearly defined. A previous report showed slight elevations of plasma octanoylcarnitine and/or decanoylcarnitine levels in the c.449_452delCTGA mutation carriers among the family members of the proband [10]. In the first case of this report, octanoylcarnitine level was moderately increased (2.19-2.59 µmol/L), and this was similar to the octanoylcarnitine level observed in infants with a single copy of the c.985A>G mutation (1.9-3.2 µmol/L). This was categorized as a severe mutation in previous reports [19, 23], and the patient did not present any clinical symptoms. Therefore, the novel c.461T>G mutation may be associated with a mild phenotype. However, we did not measure enzyme activity in our patients and did not perform acylcarnitine analysis in either of the parents of the first patient. Therefore, the apparent effect of the novel mutation on enzyme activity and acylcarnitine levels could not be assessed. It is also known that environmental factors, including diet and infections, can limit the evaluation of genotype effects. Two contrasting previous reports described one patient who had a homozygous frameshift mutation with no residual MCAD enzyme activity, but showed no severe clinical manifestations for several months after birth, and another compound heterozygote patient who had c.985A>G and another missense mutation and died unexpectedly a few days after birth [2, 3, 24]. Therefore, further data accumulation through clinical, biochemical, and molecular approaches is needed to evaluate the relationship between clinical phenotype and genotype.

In summary, we have described 2 Korean pediatric patients with MCADD that was diagnosed by molecular studies; 1 of the cases showed a novel mutation, c.461T>G, in the ACADM gene. We conclude that c.449_452delCTGA is a common mutation in Korean MCADD patients. We believe that this report will contribute to a better understanding of the genetic background in Asian MCADD patients. The detection of novel mutations and further molecular and biochemical studies are needed to establish genotype-phenotype correlations of MCADD and to manage the disease at an early stage.