Novel and Recurrent ACADS Mutations and Clinical Manifestations Observed in Korean Patients with Short-Chain Acyl-Coenzyme a Dehydrogenase Deficiency

Available online at www.annclinlabsci.org 360 Annals of Clinical & Laboratory Science, vol. 46, no. 4, 2016 Novel and Recurrent ACADS Mutations and Clinical Manifestations Observed in Korean Patients with Short-chain Acyl-coenzyme a Dehydrogenase Deficiency

Yoo-Mi Kim1, Chong-Kun Cheon1,*, Kyung-Hee Park2, Sung Won Park3, Gu-Hwan Kim4, Han-WookYoo4, Kyung-A Lee5, and Jung Min Ko6,*

1Department of Pediatrics, Pusan National University Children’s Hospital, Pusan National University School of Medi- cine, Yangsan, 2Department of Pediatrics, Pusan National University Hospital, Pusan National University School of Medicine, Busan, 3Department of Pediatrics, Dankook University College of Medicine, Cheil General Hospital & Woman's Health care Center, Seoul, 4Medical Genetics Center, Asan Medical Center Children’s Hospital, University of Ulsan College of Medicine, Seoul, 5Department of Laboratory Medicine, Yonsei University College of Medicine, Seoul, 6Department of Pediatrics, Seoul National University Children's Hospital, Seoul, Korea

Abstract. Short-chain acyl-CoA dehydrogenase (SCAD) catalyzes the first step in mitochondrial short- chain β-oxidation, and its deficiency is caused by mutations in the ACADS. We sought to investigate the spectrum ACADS mutations and associated clinical manifestations in Korean patients with SCAD deficiency. The study included ten patients with SCAD deficiency from 8 unrelated families as diagnosed by biochemical profile and mutation analyses. Clinical features, biochemical data, growth, and neurodevelopmental state were reviewed retrospectively. Eight patients were found during newborn screening, and two were diagnosed by family screening. During follow-up ranging from 2 months to 4.5 years, no hypoglycemic event was noted, and the development and growth of the patients were normal, except in two siblings. One exhibited hypotonia and gross motor delay, while one girl showed cyclic vomiting until the age of two years. We identified seven different mutations of ACADS. Of these, p.E344G was the most frequent mutation with an allele frequency of 50%, followed by p.P55L with 18.8%. p.G108D and four novel mutations were identified: p.L93I, p.E228K, p.P377L, and p.R386H. Korean patients with SCAD deficiency showed heterogenous clinical features and ACADS genotype. Our data contributes to a better understanding of the distinct molecular genetic characteristics and clinical manifestations of SCAD deficiency.

Key words: Fatty acid beta-oxidation, Short-chain acyl-CoA dehydrogenase deficiency, ACADS, Newborn screening test.

Introduction autosomal-recessively inherited metabolic disorder and has shown a wide clinical spectrum of mani- Short-chain acyl-coenzyme A dehydrogenase (acyl- festation ranging from asymptomatic to severe CoA dehydrogenase, short-chain; SCAD) catalyzes symptoms and signs including hypotonia, failure the first step in mitochondrial short-chain to thrive, developmental delay, ketotic hypoglyce- β-oxidation, and its deficiency (OMIM No. mia, epilepsy, and behavioral disorders [1–4]. 606885) is caused by mutations in ACADS (the gene for acyl-CoA dehydrogenase, short-chain) on Conventionally, a diagnosis of SCAD deficiency chromosome 12q22 [1]. SCAD deficiency is an has been made biochemically through the recogni- tion of increased serum butyryl carnitine (C4) and *These two authors have contributed equally to the work and are co-corresponding authors. Address correspondence to Jung Min Ko urinary excretion of ethylmalonic acid (EMA) M.D., Department of Pediatrics, Seoul National University Children’s and/or methylsuccinate (MS) [1–2]. SCAD defi- Hospital, 101 Daehak-ro, Jongno-gu, Seoul 110-769, South Korea; phone: +82 2 2072 3570; fax: +82 2 743 3455; e mail: jmko@snu. ciency is usually confirmed by DNA analysis for ac.kr or Chong-Kun Cheon, M.D., Department of Pediatrics, Pusan National University Children’s Hospital, Pusan National University ACADS, and approximately 60 disease-causing School of Medicine, Geumo-ro, Yangsan-si, Gyeongnam 602- mutations have been reported to date (http://www. 739, Korea; phone: 82 55 360 3158; fax: 82 55 360 2181; e mail: [email protected] hgmd.org/).

- Because most newborn patients found by screening 0 de .0

t for elevated C4 remain symptom free, there has /0 no T been some debate as to whether SCAD deficiency is , 00 00 00 IF 0. 0. 0. S

is an appropriate candidate for newborn screening [1, ND ys ; 5–7]. However, SCAD deficiency remains a poten- al ed an

yz tial risk with lack of a long-term prognosis, and sev- 2 al co 98

n- eral patients having neurological symptoms and li an 0. si t high EMA associated with neurotoxic effects have phe 2/ In no ly 000 , 999 12 been reported [7–9]. This study was conducted to 1. 0. 0. Po

NA determine the molecular spectrum of the ACADS

e; mutations and to analyze clinical outcomes and at

in biochemical features in Korean patients with SCAD cc G G G G G K† L†

su deficiency. 2 44 44 44 44 44 28 77 5L yl le le th E3 E3 E3 E3 E3 E2 P5 P3 ge p. p. p. p. p. p. p. p. Al Materials and Methods me an , ch MS id

; Patients and biochemical profile. Ten patients from ac id

o eight families—eight boys and two girls—were enrolled H† G G G D ac † in 1 in this study from March 2009 to December 2015. ic 86 08 44 44 44 3I 5L 5L le Am on le R3 L9 E3 E3 E3 P5 P5 G1 al p. p. p. p. p. p. p. p. Al Diagnosis of SCAD deficiency was based on biochemical lm findings and molecular analysis of ACADS. Biochemical hy

et analyses included measuring serum levels of C4 and G G G G G T A, urine levels of EMA and MS. Clinical manifestations, >A >T C> A> A> A> A> A> 2

EM clinical courses, and results of biochemical and genetic 2G 4C le 31 31 31 31 31 30 e; le studies were retrospectively reviewed. Denver develop- ge 68 10 10 16 10 10 11 10 in c. c. c. c. c. c. c. c. Al an it mental scales were administered until the age of five ch rn years. The data were collected from two clinical genetics ca de

yl centers in Korea (Pusan National University Children’s ti G G. G yr A eo Hospital and Seoul National University Children’s 1 >A >A >T >T cl y. A> A> A> G> but le S. Hospital). , 7C 3G nc le 4C 4C 31 31 31 Nu 57 ie AD -C Al 27 32 10 11 16 16 10 10 fic c. c. c. c. c. c. c. c. ) Molecular analysis of ACADS. The institutional review AC C4 de .8 ; M of e boards of Yangan Pusan National Hospital and Seoul 8 <8 s ne 2 7 8 as , si M/ .2 .7 .9 .0 ge

81 National University Children’s Hospital approved muta- 76 91 en (n Cr MS e aly 37 37 6. 13 2. ND 7. 20 ND ND tion analysis and clinical review of the patients. Written , og as an dr ) 2 r en ml informed consent was obtained from both parents of all 42 7 8 2 4 4 2 28 4 A la hy og 6. .8 .5 .3 .6 .9 7. .1 4.6 .9 4.6 M/ patients. cu de dr 37 20 62 24 94 23 10 29 77 50 <1 (n EM le A hy mo L,

de Genomic DNA was extracted from peripheral blood leu- 3) d me A .9 M/ 26 zy kocytes using a DNA isolation kit (Qiagen, Helden, 89 69 an 47 14 78 79 93 , 1. 1. 1. 2. 1. 1. NA NA 1. 3. <0 (µ C4 en me

es Germany). Ten ACADS coding exons and their intronic s zy ur -co flanking regions were amplified by polymerase chain re- si at at en yl m d d d d d d d e d fe

no action (PCR) with specific primers (available on re- y ac -co al 50 15 18 36 14 19 40 8 12 10 Ag ag in

yl quest). Subsequently, PCR products were sequenced di- di ac mic

t rectly with the same primers using a 3130xl Genetic cha , y y y he S m t- m m m m en d e Analyzer (Applied Biosystems, Foster City, CA, USA) m 4 4 2 oc rr or 70 9 15 24 19 18 4. 9. 4. 11 ag AD bi

sh and Sequencing Analysis software, version 5.2. The se- , Cu AC al quence results were compared with established human D, ic t. s: x

in ACADS (NCBI Accession No. NM_000531.5). To pre- an F M M M M M M M F M Se AD on ri Cl ti dict the functional impact of novel amino acid changes, b SC ia va ct 1. je Su we conducted molecular analyses of ACADS from the ev e el 10 9 8 7 5 6 4 2 3 1 ed; br bl ly ov patients’ parents and 50 healthy controls. In addition, ct te †n Ab 8 7 6 5 4 3 2 1 mi Fa Ta we assessed novel missense alterations using two in silico 362 Annals of Clinical & Laboratory Science, vol. 46, no. 4, 2016 e e prediction algorithms, Polymorphism Phenotyping-2 n n n n n n n n on in in vi vi vi vi vi vi vi vi ti (PolyPhen-2, http://genetics.bwh.harvard.edu/pph2/) it it ca fla fla fla fla fla fla fla fla rn rn and Sorting Intolerant from Tolerance (SIFT, http://sift. di bo bo bo bo bo bo bo bo ca ca jcvi.org/). L- Ri L- Ri Ri Ri Ri Ri Ri Ri Me )

/L Results ol 6 .5 µm .3

54 Clinical characteristics at presentation. The clini- 49 l 4 9 7 84 → ta

→ cal phenotypes, biochemical findings at diagnosis, .9 .6 .7 .4 .3 .1 8- 36 57 74 63 73 ND 54 ND (2 50 ND To and results of molecular analysis of ACADS are

) summarized in Table 1. Eight patients were detect- /L 9

ol ed by newborn screening using tandem mass spec- .4 .5 e 46

µm trometry, and two boys (subjects 2 and 4) were 38 in → it 8 →

66 identified by familial screening after their siblings 7 2 rn .0 .9 .3 .2 .6 .5 .3 ee 4- were diagnosed at the age of 14.1±31.9 months 62 50 57 ND 30 47 ND (2 Ca 42 ND 47 Fr (range, 10 days–8 years). Their birth weight was

y 3.5±0.3 kg (range, 2.8–3.8 kg), and all patients la t t t t t t t e, e, ,

de were born at 39.5±0.8 weeks (range, 37 weeks 5 en en en en en en en gu gu ng is ti ti ch ti days–40 weeks 3 days) with uneventful delivery pm pm pm pm pm pm pm os fa fa ee lo lo lo lo lo lo lo mi id g, g, and had no maternal events, such as maternal hy- sp ve ve ve ve ve ve ve ac vo s in in t a, a c de de de de de de de pertension, maternal HELLP syndrome or fetal dis- li l l l l l l l ed ed ni ni en om fe fe bo rr to to tress, during pregnancy. ta rma rma rma rma rma rma rma cu or or po po mpt No No No Po No Re No hy Po hy Me No Sy No Biochemical findings at diagnosis in all patients revealed increased serum C4 level of 1.99±0.51 ) µmol/L (reference: <0.83 µmol/L) and a urinary 67 ) 1) 0. .8 EMA level of 105.2±109.2 mmol/mmol Cr (refer- 0.0 cm (1 (– (- , ence: <14.6 mmol/mmol Cr). Urinary excretion of .5 .5 y. .5 .5 DS) nc NA 37 NA 45 NA 52 NA NA NA HC 46 (S MS in six patients was found with a level of 18.1± ie

fic 4.4 mmol/mmol Cr (reference: not checked). There de

2 were no hypoglycemic events or severe metabolic 2) se 8) 7) 5) .7 /m .8 .0 na crises. Seven of the nine patients showed normal .2 (1 –0 ge kg (0 (1

6 development and growth patterns (Table 2). 4 7 3( 8 5 2 7 2 2 I, dro .0 .1 .4 .7 .8 .5 .4 .0 .3 .3 DS) hy 19 14 16 15 17 17 17 14 24 BM 16 (S de Notably, subject 3 suffered from recurrent episodes x. oA of vomiting until the age of two years. She was ad- de ) ) -C in ) yl mitted to the hospital several times because of poor 27 66 9) 8) 8) ss 35 ac 4) 0. 0. 7) 2) .7 .2 3)

.8 oral intake and mild metabolic acidosis. Biochemical 1. .4 ) .6 .8 (1 (1 .6 (– (– in ma kg -0 (– (2 , (1 (0 (0 y

.5 findings revealed increased excretion of urinary .3 .7 .9 DS 8( 0 cha 4. 8. 13 14 10 13 16 22 44 10 (S Wt t- EMA and MS levels when she presented with vom- bod or I,

sh iting (Figure 1). Subject 5 showed mild hypotonia th BM

) and complained of easy fatigability with a mildly ; 3) 1) wi 2) 4) 17 4) .3 .6 6) ht elevated creatine kinase level of 264 mg/dL (refer- ts 7) .8 1. .4 7) .3 .5 ig (0 (2 4) .0 ) en (0 (– .5 (0 (1 (0 .1 1 7 ence: 5–217 mg/dL). His older brother (subject 4), cm ti we (0 (1 , .7 6 6. .3 (0 .6 2. , .5 DS pa who shared the same mutations, also showed poor 57 70 81 90 87 84 10 11 13 78 (S Ht Wt of t ; oral intake and mild hypotonia during infancy, and es ht en y y y he could not climb stairs alone or run well at the m rr ig m m m m d com e m 4 4 2 he 70 9 15 24 18 19 4. 4. 9. 11 ag Cu age of 40 months. These siblings (subject 4 and 5) , out t al Ht could walk alone at the age of 15 months and 18 ec ic s: bj in months, respectively. on Cl 10 9 8 7 6 5 4 3 2 1 Su ti ia 2. ly ev e Mutation spectrum of ACADS. Molecular analysis br bl mi identified seven different mutations from eight Ab 8 7 6 5 4 3 2 1 Fa Ta ACADS mutations in Korean SCAD deficiency 363

(subject 4) showed poor oral intake and mild hypotonia. They also showed low body mass index (14.8 kg/m2 and 15.3 kg/m2, respectively). Gross motor retardation was identified by the Denver developmental scale.

However, there were no hypoglycemic events or developmental delay in the other patients during follow-up periods. All patients have shown a normal growth pattern to date (Table 2).

Discussion

Figure 1. Ethylmalonic acid and methylsuccinate levels in This study investigated ten patients with SCAD de- subject 3. The patient presented with recurrent vomiting ficiency from eight unrelated Korean families. The events (grey arrows) requiring admission and fluid deficiency was confirmed in all patients by molecu- therapy. lar analysis of ACADS, and three known mutations families. The mutations were located throughout and four novel mutations were detected in our coding exons from exon 2 to 10, without any do- study. There have been few reports of ACADS mu- main specificity. tations in Asian populations. The most frequently identified mutation in our study, p.E344G, was The p.E344G mutation was the most frequently first identified in Caucasian populations [10]. The observed mutation found with an allelic frequency first reported patient with p.E344G was compound of 50% (8/16 alleles), followed by p.P55I at 18.8% heterozygous with p.G209S in the other allele and (3/16 alleles). We found four novel variants: p.L93I, showed developmental delay and hypotonia with- p.E228K, p.P377L, and p.R386H. Each of these out seizures [10]. Interestingly, p.E344G was ob- variants was identified in each family, was not served in six out of eight families with an allelic found in the 1000 Genomes database (http:// frequency of 50% (8/16 alleles), indicating that this browser.1000genomes.org/), and was strictly con- mutation may be the most common among Koreans served among multiple species (Supplementary with SCAD deficiency [11]. The second most com- Figure 1). In silico analysis software programs, in- monly observed mutation with an allelic frequency cluding PolyPhen-2 and SIFT, predicted the vari- of 18.8% (3/16 alleles), p.P55L, was first found in ants to damage protein function (Table 1). an African-American patient [12]. In Asian populations, two Japanese children remaining asymptom- Clinical course during follow-up. The clinical atic until they were 4 years of age had the same mu- courses of all subjects with SCAD deficiency are tations: p.P55L and p.G108D for the first child summarized in Table 2. Eight patients were treated and p.E344G and p.P55L for the second child, as with riboflavin (100 mg/day) and by avoidance of shown in our study [13]. In China, the first patient both long-time fasting and a high-fat diet. Two pa- identified with SCAD deficiency showed two vari- tients (subjects 8 and 9) were treated with ants of ACADS, p.G108S and p.G209S [14]. In L-carnitine supplements (100 mg/kg/day). Subject Taiwan, five patients with SCAD deficiency were 3, who had compound heterozygous mutations found by newborn screening, and all of them were (p.L93I and p.E228K), had shown recurrent vom- asymptomatic; however, detailed information iting in infancy and early childhood. Because she about their genotypes was not reported [4]. could not consume anything, including water, during each episode, several admissions and a high Consistent with previous reports, most patients in level of dextrose fluid therapy were needed. Urinary the present study revealed asymptomatic and nor- EMA and MS were increased, and metabolic acido- mal development and growth. However, among ten sis (range, HCO3– 14.9–16.7 mM) was also noted patients, three had some neurological symptoms at each episode. Subject 5 and his older brother with cyclic vomiting and hypotonia. One girl, 364 Annals of Clinical & Laboratory Science, vol. 46, no. 4, 2016

Supplementary Figure 1. Alignment of the ACADS sequence with corresponding segments in diverse species is shown. P377L (A), L93I (B), E228K (C), and R368H (D) in vertebrates. The sequence was selected by the UCSC Genome Browser (GRCh37/hg19). having two novel heterozygous mutations, p.L93I homozygotic for p.E344G, the neonate’s clinical and p.E228K, showed cyclic vomiting episodes that course could not be assessed because there are no resolved after the age of two years. Cyclic vomiting clinical follow-up data [11]. The three patients pre- is not a common symptom, although it has been senting with neurological symptoms in our study described as an accompanying symptom in patients had no perinatal distress, and their symptoms ap- with SCAD deficiency and other fatty acid oxida- pear to be related to SACD deficiency. tion disorders [15,16]. Symptomatic patients with SCAD deficiency tend to present before the age of The mechanisms of pathogenesis in SCAD defi- five years, and some of them show transient symp- ciency are still poorly understood, but recent stud- toms, even though an explanation appears to be dif- ies revealed that EMA, a hallmark of SCAD defi- ficult [5,7,10,17]. Tw o brothers (subjects 4 and 5) ciency, might play a central role in the pathogenesis having homozygosity for E334G showed poor oral of SCAD deficiency though several mechanisms [9, intake, fatigue, gross motor retardation, and speech 18-20]. EMA inhibits creatinine kinase activity and delay. Hypotonia, food refusal, and speech delay are modulates Na-K-ATPase activity and mRNA levels also observed in patients with SCAD deficiency, in the cerebral cortex [9,19]. EMA might also dis- and developmental delay is the most common rupt mitochondrial homeostasis in the CNS symptom (51%–69%) in symptomatic SCAD defi- [19,20]. Indeed, most mutations of ACADS are ciency [1,5,7-10,17]. Although there is a previous missense mutations that could lead to intramito- report of an asymptomatic Korean neonate chondrial aggregation of misfolded SCAD proteins, ACADS mutations in Korean SCAD deficiency 365 and this abnormal SCAD protein aggregation may The present study provides some information about lead to neurotoxicity [1,21]. Some in vitro studies the genetic basis of SCAD deficiency in Korean pa- of the mitochondrial processing of variant SCAD tients together with novel and recurrent mutations proteins have been performed and have suggested in ACADS. Considering that SCAD deficiency is that most mutations increase the tendency to mis- transmitted as an autosomal recessive condition fold and to form abnormally aggregated SCAD and that variability of the clinical manifestations is proteins, especially at elevated temperature, and suggested, even in the same family, we recommend may lead to SCAD dysfunction during febrile ill- that family screening of the patients’ siblings should ness [22]. be performed using molecular genetic analysis, even if the patients’ siblings show a normal result There is no established consensus on the treatment on newborn biochemical screening. of SCAD deficiency. Our patients have managed by taking riboflavin daily and avoiding long-term fast- Acknowledgements ing. Because riboflavin is the precursor of flavin ad- We express our gratitude to the patient and the parents for enine dinucleotide, which plays a role as a chemical their participation in this study. This study was supported by a grant from the Korean Health Technology R&D Project, chaperone and stabilizes mutant enzymes, it might Ministry of Health & Welfare, Republic of Korea (A120030). be effective for treating SCAD deficiency. Indeed, some patients showed reduced urinary EMA excre- References tion and clinical improvement, but this improve- 1. Jethva R, Bennett MJ, Vockley J. Short-chain acyl-coenzyme A ment was sustained after stopping riboflavin, and dehydrogenase deficiency. Mol Genet Metab only patients having a combination of variant and 2008;95:195–200. 2. van Maldegem B. Clinical aspects of short-chain acyl-CoA de- mutation seemed to respond to riboflavin [23]. hydrogenase deficiency. J Inherit Metab Dis 2010;33:507–511. Moreover, the effects of riboflavin in SCAD defi- 3. 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