Available online at www.annclinlabsci.org 84 Annals of Clinical & Laboratory Science, vol. 41, no. 1, 2011 Mutations of ACADS Gene Associated with Short-Chain Acyl-Coenzyme A Dehydrogenase Deficiency Se Hwa Kim,1* Hyung-Doo Park,2* Young Bae Sohn,1 Sung Won Park,1 Sung Yoon Cho,1 Suntae Ji,1 Su Jin Kim,3 Eun Wha Choi,4 Chi Hwa Kim,5 Ah-Ra Ko,5 Sunghee Yeau,6 Kyung-Hoon Paik,1 and Dong-Kyu Jin1 Departments of 1Pediatrics and 2Laboratory Medicine & Genetics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul; 3Center of Pediatric Oncology, National Cancer Center, Goyang; 4Laboratory Animal Research Center and 5Clinical Research Center, Samsung Biomedical Research Institute, Seoul; and 6Department of Science Education, Ewha Woman’s University, Seoul, Korea Abstract. Short-chain acyl-coenzyme A dehydrogenase deficiency (SCADD) is an autosomal recessive disorder of mitochondrial fatty acid oxidation associated with mutations in the ACADS gene (Acyl-CoA Dehydrogenase, Short- chain, OMIM #606885). SCADD is a heterogeneous condition that has been associated with various clinical phenotypes ranging from fetal metabolic decompensation in infancy to asymptomatic individuals. Here, the first Korean neonate diagnosed with SCADD by biochemical and genetic findings is reported. The patient has remained asymptomatic by avoiding hypoglycemia. An increased concentration of butylcarnitine was detected on newborn screening. Subsequent urine organic acid analysis showed increased urinary excretion of ethylmalonic acid. To confirm the presence of the genetic abnormality, all the coding exons of theACADS gene and flanking introns were amplified by the polymerase chain reaction (PCR). Sequence analysis of theACADS gene revealed novel homozygous missence mutations, c. 1031A>G (p.E344G) in exon 9. In summary, the first Korean patient with confirmed SCADD by genetic analysis is reported with novel mutation. Introduction tonia in infants, children, and adults SCAD deficiency. Overall, approx- [4,5]. Because of the enzyme defect, imately 60 mutations in ACADS are Short-chain acyl-CoA dehydrogen- increased level of C4-acylcarnitine known [9], with two common ase (SCAD) is member of the acyl- in the plasma and increased level of variants. The A 511C >T polymorph- CoA dehydroganase (ACAD) ethylmalonic acid (EMA) in the ism located in exon 5 leads to family of mitochondrial enzymes urine are observed and suggest the substitution of tryptophan for involved in the B-oxidation system. diagnosis of SCADD. However, arginine at position 147 of the It catalyzes the first step in mito- these findings are also found in mature enzyme (R147W; position chondrial B-oxidation of fatty acids individuals who carry one of two 171 in the precursor); the A 625G > four to six carbons in length [1,2]. common polymorphisms identified A variant in exon 6 substitutes Short-chain acyl-coenzyme A in the SCAD coding region [6,7]. serine for glycine at position 185 of dehydrogenase deficiency SCADD( ) Therefore, genetic analysis is helpful the mature protein (G185S; position is a rare autosomal recessive disorder in the diagnosis. 209 in the precursor protein). Both of mitochondrial fatty acid oxid- The SCAD gene is located on ACADS sequence variants are ation, first reported in 1987 [3]. The chromosome 12q22 and is approx- relatively common in the healthy clinical symptoms of primary imately 13kb long with 10 exons population [7]. SCADD include failure to thrive, and 1236 nucleotides in the coding Recently, with newborn metabolic acidosis, ketotic hypo- sequence [8]. Several inactivating screening (NBS) by tandem mass glycemia, developmental delay, mutations in the gene encoding spectrometry (MS/MS), several seizures, and neuromuscular symp- SCAD (ASCADS ; OMIM #606885) metabolic disorders are detected toms such as myopathy and hypo- have been identified in patients with early in life, which allows immediate Address correspondence to Dong-Kyu Jin, M.D., Department of Pediatrics, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Irwon-dong, Gangnam-gu, Seoul, 135-710, Korea; e-mail: [email protected]. *These authors equally contributed to this study. 0091-7370/11/0084-0088. $2.50. © 2011 by the Association of Clinical Scientists, Inc. Short-chain acyl-coenzyme A dehydrogenase deficiency gene mutation 85 initiation of treatment and monitor- Korean patient with SCADD is the hospital for evaluation of an ing, particularly during times of reported. The patient was found to abnormal tandem mass spectrometry illness [10]. Therefore, individuals have a novel homozygous missense analysis performed three days after with SCADD identified through mutation of the ACADS gene. birth at a local hospital. When the newborn screening have a chance to patient was 10 days of age, the Material and Methods remain asymptomatic [11]. following studies were performed: In this article, the first biochem- Clinical and biochemical analysis. liquid chromatography tandem ically and genetically confirmed A female neonate was admitted to mass spectrometry (LC-MS/MS), Fig. 1. Acylcarnitine profiling in a healthy newborn (A) and in the study patient (B). Elevated levels of C4 species are shown. 86 Annals of Clinical & Laboratory Science, vol. 41, no. 1, 2011 plasma amino acids, urine organic acids, and quantitative acylcarnitine profile. Mutation analysis. Genomic DNA was extracted from the peripheral blood leukocytes of the patient, using the Wizard Genomic DNA Purification Kit according to the manufacturer’s protocol (Promega, Madison, WI, USA). We failed to obtain the DNAs from the parents. The coding exons and exon-intron boundaries of the ACADS gene were amplified by the polymerase chain reaction (PCR) on a thermal cycler (Applied Biosystems, Foster City, CA, USA) using primer pairs designed by the authors (sequences available upon request). Direct sequencing was performed with the BigDye Terminator Cycle Sequen- cing Ready Reaction kit on an ABI Prism 3130 Genetic Analyzer (Applied Biosystems). The sequences were analyzed using the Sequencher program (Gene Codes Corp., Ann Fig.2. The patient had a homozygous c.1031A>G mutation of the ACADS gene. A Arbor, MI, USA) with comparison contol study did not reveal any carriers with the c.1031A>G mutation. to reference sequences. All number- ing of nucleotide positions was done alanine aminotransferase 31 (RR, Mutation findings. A homozygous according to the ACADS cDNA 0-40) U/L; alkaline phosphatase c.1031A>G mutation of the ACADS sequence (GenBank accession NM 223 (RR, 42-98) U/L; ammonia gene was identified. The c.1031A>G 000017.2). Sequence variation was 75.5 (RR, 11.2-48.2) mmol/L, and transition resulted in amino acid described according to the recom- lactate 6.07 (RR, 0.5–2.2) mmol/L. substitution of Glu to Gly at codon mendations by the Human Genome Assay of urine organic acids revealed 344 (p.E344G) in exon 9. Fifty Variation Society (www.hgvs.org/ a significant increase in the urinary healthy normal subjects were mutnomen). Novel mutations were excretion of ethylmalonic acid (98.0 screened by direct sequencing and confirmed by sequencing 50 control mmol/mol creatinine (RR, 1.7-14.6) none carried the c.1031A>G chromosomes. and mild elevations of 3-hydroxy- mutation of the ACADS gene (Fig. isobutyric acid, fumaric acid, 2). In silico analysis of the sequence Results 3-hydroxyisobaleric acid, aconitic variations was performed. The p. Clinical and biochemical findings. acid, and hippuric acid. Quantitative E344G variation showed a ‘possibly The patient was born at 39 weeks of acylcarnitine profiling in plasma damaging change’ in the PolyPhen gestation by vaginal delivery and showed prominent accumulation of prediction of the functional effects weighed 2.52 kg. Physical examin- C4, 3.59 µmol/L (RR, 0-1.2) (Fig. of human nsSNPs (http://genetics. ation revealed no abnormal findings. 1). The patient was suspected as bwh.harvard.edu/pph/) and ‘not A newborn screening test showed having SCADD based on the bio- tolerated change’ in the SIFT increased concentrations of plasma chemical results at 10 days after analysis (http://sift.jcvi.org/). The C4-acylcarnitine (C4): 2.85 µmol/L birth, without any symptoms. The novel mutation was a major amino (cut-off, 1.2). Serum tests of liver problems associated with SCADD acid substitution. function performed at 10 days of were explained to the parents and Discussion age gave the following results: they were instructed to keep the aspartate aminotransferase 44 patient from fasting for long A novel ACADS mutation was [reference range (RR) -40] U/L; durations. identified in a neonate affected by Short-chain acyl-coenzyme A dehydrogenase deficiency gene mutation 87 SCAD deficiency. SCADD illus- There have been no reports of Therefore, molecular analysis can trates several of the clinical and Korean patients with SCADD, thus aid tandem mass spectrometry in public policy issues currently facing this is the first case in Korea of the differential diagnosis and the detection of many inborn errors SCADD that was confirmed by confirmation of SCADD. of metabolism that are identified by genetic analysis and biochemical In summary, a Korean female expanded newborn screening with abnormalities. The mutation iden- patient who, as a result of MS/MS tandem mass spectrometry. Clinical tified in this study is a novel newborn screening, was suspected presentation of SCADD includes homozygous mutation, c. 1031A>G as having SCADD had the diagnosis failure to thrive, metabolic acidosis, (p.E344G) of exon 9. This confirmed by biochemical and developmental and cognitive delay, c.1031A>G transition results in genetic analysis. A novel ACADS seizures, and neuromuscular symp- amino acid substitution of Glu to gene mutation was detected. Further toms [4,5]. These symptoms have Gly. Glutamic acid, with a study may be needed to understand been attributed to intermittent hydropathy index of -3.5, is one of the functional and structural episodes of metabolic decompen- the most hydrophilic amino acids changes of the proteins involved in sation precipitated by fasting, [17].