,GHQWLÀFDWLRQRID1RYHOHADHB Gene Mutation Original Article ,GHQWL¿FDWLRQRID1RYHOHADHB Gene Mutation in an Iranian 3DWLHQWZLWK0LWRFKRQGULDO7ULIXQFWLRQDO3URWHLQ'H¿FLHQF\ Mahdiyeh Shahrokhi MSc10RKDPPDG6KD¿HL3K'1, Hamid Galehdari PhD1, Gholamreza Shariati PhDƔ Abstract Introduction:0LWRFKRQGULDOWULIXQFWLRQDOSURWHLQ 073 LVDKHWHURRFWDPHUFRPSRVHGRIHLJKWSDUWV VXEXQLWV IRXUĮVXEXQLWVFRQWDLQLQJ /&(+ ORQJFKDLQHQR\O&R$K\GUDWDVH DQG/&+$' ORQJFKDLQK\GUR[\DF\O&R$GHK\GURJHQDVH DFWLYLW\DQGIRXUȕVXEXQLWVWKDW SRVVHVV/&.7 ORQJFKDLQNHWRDF\O&R$WKLRODVH DFWLYLW\ZKLFKFDWDO\]HV three out of four steps in ȕoxidation spiral of longchain fatty acid. Its GH¿FLHQF\ is an autosomal recessive disorder that causes a clinical spectrum of diseases. Materials and Methods: A blood spot was collected from the patient’s original newborn screening card with parental informed consent. A newborn screening test and quantity plasma acylcarnitine SUR¿OH analysis by MSMS were performed. After isolation of '1$ and $PSOL¿FDWLRQ of all exons of the HADHA and HADHB, directly Sequence analyses of all exons and the ÀDQNLQJ introns both of genes were performed. Result: Here, we report a novel mutation in a patient with 073 GH¿FLHQF\ diagnosed with newborn screening test and quantity plasma acylcarnitine SUR¿OH analysis by MSMS and then FRQ¿UPHG by HQ]\PH analysis in cultured ¿EUREODVWV and direct sequencing of the HADHA and HADHB genes. Molecular analysis of causative genes showed a missense mutation p.43 c.$ > C in exon of HADHB gene. Conclusions: Since this mutation was not found in normal control cases; so it was concluded that c.$ > C mutation was a causative mutation. Phenotype analysis of this mutation predicted pathogenesis which reduces the stability of the 073 protein complex. Keywords: 'H¿FLHQF\, fatty acid oxidation, HADHA, HADHB, mitochondrial trifunctional protein 073 novel mutation Cite this article as: 6KDKURNKL06KD¿HL0*DOHKGDUL+6KDULDWL*,GHQWL¿FDWLRQRIDQRYHOHADHB gene mutation in an Iranian patient with mitochondrial WULIXQFWLRQDOSURWHLQGH¿FLHQF\Arch Iran Med. 2017; 20(1): 22 – 27. Introduction that causes a clinical spectrum of diseases.4 Symptoms of mitochondrial trifunctional protein GH¿FLHQF\ may present in atty acid oxidation is the main source of energy for heart infancy or later in life. Features that occur during infancy comprise and muscles. Mitochondrial Trifunctional Protein lack of energy (lethargy), feeding GLI¿FXOWLHV, hypoketotic F (M-FTP) is bound to the inner mitochondrial membrane hypoglycemia provoked by prolonged fasting, weak muscle tone which catalyzes three out of four steps in ȕ-oxidation of long- (hypotonia), lactic acidemia, acute renal failure, and fulminant chain fatty. This protein is a hetero-octamer composed of eight liver failure. That patients who present in infancy are also at high parts (subunits): four Į-subunits containing LCEH (long-chain risk for breathing GLI¿FXOWLHV, serious heart problems, coma, and 2,3-enoyl-CoA hydratase) and LCHAD (long-chain 3-hydroxyacyl sudden unexplained infant death. Some patients survive into CoA dehydrogenase) activity, and four ȕ-subunits that possess adolescence and adulthood with hypotonia, muscle aches, LCKT (long-chain 3-ketoacyl-CoA thiolase) activity.1 M-TFP damaged skeletal muscle tissue (Rhabdomyolysis) and impaired GH¿FLHQF\ is FODVVL¿HG into 2 phenotypes: the more prevalent sensation in the extremities (peripheral neuropathy).5 The most isolated LCHAD GH¿FLHQF\ (OMIM: 609016) with defects of the common defect of mitochondrial trifunctional protein is isolated Į-subunits encoded by the HADHA (OMIM: 600890) LCHAD GH¿FLHQF\ (LCHADD), and it occurs when the activity (hydroxyacyl-CoA dehydrogenase Į-subunit) gene and the less of the enzyme “long chain 3-hydroxyacyl-CoA dehydrogenase” common pattern of complete M-TFP GH¿FLHQF\ (OMIM: (LCHAD) is reduced with substantial preservation of the other 609015).2 Generally, all 3 enzyme activities of the M-FTP two MTP enzymatic activities.6 complex are undetectable in M-FTP GH¿FLHQF\ due to defects of The incidence of LCHAD GH¿FLHQF\ is unknown and this both HADHA and HADHB (hydroxyacyl-CoA dehydrogenase disorder can occur in anyone of any ethnic background.7 Based ß-subunit) genes (OMIM: 143450).3 HADHA and HADHB genes on an American population, it is estimated at approximately 1 are located in the same region on chromosome 2p23 (Respectively in 75,000, with a calculated carrier frequency of 1 in 138.8 In ENSG00000084754, ENSG00000138029). Mitochondrial Europe, the prevalence is probably much lower, approximately trifunctional protein GH¿FLHQF\ is an autosomal recessive disorder 1 in 100,000.8 More than 60% of patients with LCHADD have the E474Q (c.1528G > C) mutation in the gene encoding the $XWKRUV¶ DI¿OLDWLRQV 1Department of Genetics, Faculty of Science, Shahid alpha-subunit of the mitochondrial trifunctional protein. E474Q 2 Chamran University, Ahvaz, Iran, Narges Medical Genetic Laboratory, Ahvaz, mutation replaces a glutamic acid with the glutamine at position Iran, 3Department of Medical Genetics, Jundishapur University of Medical Science, Ahvaz, Iran. 474 in the active site of LCHAD that is located in the C-terminal •Corresponding author and reprints: Gholamreza Shariati PhD, Narges domain.9 The other defect of mitochondrial trifunctional protein Medical Genetic Laboratory, Ahvaz, Iran, Department of Medical Genetics, is complete M-TFP GH¿FLHQF\ in which the activities of all Jundishapur University of Medical Science, Ahvaz, Iran. Tel: 0661-3336681, E-mail: [email protected] three enzymes of the complex are disrupted. Complete M-TFP Accepted for publication: 23 November 2016 22 Archives of Iranian Medicine, Volume 20, Number 1, January 2017 06KDKURNKL06KDÀHL+*DOHKGDULHWDO GH¿FLHQF\ (TFPD) is a rare disorder and far less common than performed with reverse primers. Finally, to predict the possible the LCHADD.5 Despite the clinical and phenotypic similarities effects of the observed mutations, an online software was used. between these two disorders, there are some distinct differences Approved software of SIFT, Mutation Taster, Polyphen-2 and in data from screening patients by tandem mass spectrometry PANTHER were used to predict disease-causing mutation. Also, and biochemical and genetic analysis can distinguish between To predict the effects of mutations on protein stability based on LCHADD and TFPD when compared to each other.10 This article the sequence, I-Mutant and MuStab software were used. To verify describes the clinical and biochemical ¿QGLQJV of a patient with the effect of this mutation on tertiary structure of the protein, the M-TFP GH¿FLHQF\ and one novel mutation of the HADHB genes. Swiss model software was used and three-dimensional structure of proteins was rebuilt by Swiss model. Materials and Methods Results The male patient was the second born to consanguine parents in Iran. His parents were asymptomatic. They were heterozygous Acylcarnitine analysis of blood spots showed prominent in terms of mutation but without any pretense of decease. In fact, accumulation of long chain hydroxylated acylcarnitines, as based on all the studies, individuals heterozygous for this disease follows: 3-OH-palmitoylcarnitine (C16-OH), 0.76 μmol/L (RR < do not have any clinical symptoms. The newborn patient suffered 0.08 μmol/L); 3-OH-oleylcarnitine (C18:1-OH), 0.34 μmol/L (RR IURPDVSK\[LDWLRQZKLOHQXUVLQJLQWKH¿UVWPRQWKRIKLVELUWK < 0.1) (Figure 1a). In addition, the level of tetradecenoylcarnitine He eventually died of multi-organ failure at 25 days of age. His (C14:1), 0.32 μmol/L (RR < 0.25) was midly elevated. clinical symptoms included cardiomyopathy, hepatopathy and The concentration of free carnitine was normal: 34 μmol/L hypoketotic hypoglycemia. A blood spot from the patient was PHWKRG ZLWK GHULYLWLVDWLRQ 55 ± ȝPRO/ )LJXUH D collected from the patient’s original newborn screening card Therefore, a disorder of long chain hydroxylated fatty acid with parental informed consent. All ethical considerations such R[LGDWLRQ /&+$'7)3GH¿FLHQF\ /RQJ &KDLQ +\GUR[\ODWHG as parental and all case-control consent form to maintain patient $F\O&R$'HK\GURJHQDVH7ULIXQFWLRQDO3URWHLQGH¿FLHQF\ ZDV data were included in this study. A questionnaire was distributed suspected. to all controls and patient’s family. Acylcarnitines were extracted The results of electromyography and skeletal muscle biopsy with methanol and then derivatized with butanolic HCl. Butylated pathologic analysis in this study were not available. Since acylcarnitines were analyzed by tandem mass spectrometry. the newborn had a serious problems and died earlier, it was Urine organic acids and plasma amino acids were analyzed. For impossible to obtain this data and the parents were not available; DNA mutation analysis, a blood spot (5 mL) was collected from so that this Electroretinogram could be performed on them. the patient and his parents. After isolation of DNA by standard DNA analysis using genomic DNA revealed a novel homozygous salting out method from peripheral blood leukocytes, the quality missense mutation in the HADHB gene of the patient with MTP RIH[WUDFWHG'1$ZDVFRQ¿UPHGXVLQJJHOHOHFWURSKRUHVLV7KHQ GH¿FLHQF\EHORQJLQJWRWKHVHFRQGSKHQRW\SH7KHSDWLHQWKDGD the 20 exons and adjacent intronic consensus splice sites of the homozygous missense mutation“c.1154A > C - cDNA.1258A > HADHA and 16 exon of the HADHB JHQH ZHUH DPSOL¿HG E\ C - g.41718A > C” mutations in the HADHB gene (Figure 1b). polymerase chain reaction. Sixteen sets of primers for HADHB The c.1154A > C transition resulted