Annals of Clinical & Laboratory Science 39:399-404 (2009)
© 2009 Association of Clinical Scientists
Two Novel HADHB Gene Mutations in a Korean Patient with Mitochondrial Trifunctional Protein Deficiency
Hyung-Doo Park1,a,
Suk Ran Kim1,a,
Chang-Seok Ki1,
Soo-Youn Lee1,
Yun Sil Chang2,
Dong-Kyu Jin2 and
Won Soon Park2
Departments of 1 Laboratory Medicine & Genetics and 2 Pediatrics, Sungkyunkwan University School of Medicine, Samsung Medical Center, Seoul, Korea
Address correspondence to Won-Soon Park, M.D., Ph.D., Department of Pediatrics, Samsung Medical Center, 50 Ilwon-dong, Gangnam-gu, Seoul, 135-710, Korea; tel 82 2 3410 3523; fax 82 2 3410 0043; e-mail wonspark{at}skku.edu.
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Abstract
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Mitochondrial trifunctional protein (MTP) is a heterocomplex composed of 4 
-subunits containing LCEH (long-chain 2,3-enoyl-CoA hydratase) and LCHAD (long-chain 3-hydroxyacyl CoA dehydrogenase) activity, and 4 β-subunits that harbor LCKT (long-chain 3-ketoacyl-CoA thiolase) activity. MTP deficiency is an autosomal recessive disorder that causes a clinical spectrum of diseases ranging from severe infantile cardiomyopathy to mild chronic progressive polyneuropathy. Here, we report the case of a Korean male newborn who presented with severe lactic acidosis, seizures, and heart failure. A newborn screening test and plasma acylcarnitine profile analysis by tandem mass spectrometry showed an increase of 3-hydroxy species: 3-OH-palmitoylcarnitine, 0.44 nmol/ml (reference range, RR <0.07); 3-OH-linoleylcarnitine, 0.31 nmol/ml (RR <0.06); and 3-OH-oleylcarnitine, 0.51 nmol/ml (RR <0.04). These findings suggested either long-chain 3-hydroxyacyl-coA dehydrogenase deficiency or complete MTP deficiency. By molecular analysis of the HADHB gene, the patient was found to be a compound heterozygote for c.358dupT (p.A120CfsX8) and c.1364T>G (p.V455G) mutations. These 2 mutations of the HADHB gene were novel and inherited. Although the patient was treated by reduction of glucose administration and supplementation of a medium-chain triglyceride-based diet with L-carnitine, he died 2 mo after birth due to advanced cardiac failure.
Keywords: mitochondrial trifunctional protein (MTP) deficiency, HADHB gene mutations
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Introduction
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Fatty acid oxidation is a major source of energy for skeletal and cardiac muscle. Mitochondrial trifunctional protein (MTP) is bound to the inner mitochondrial membrane and is a heterocomplex of 4 -subunits containing LCEH (long-chain 2,3-enoyl-CoA hydratase) and LCHAD (long-chain 3-hydroxyacyl CoA dehydrogenase) activity, and 4 β-subunits that harbor LCKT (long-chain 3-ketoacyl-CoA thiolase) activity [1]. Mitochondrial β-oxidation of fatty acids, which consists of multiple transport steps, is initiated by a catalytic reaction mediated by a long-chain acyl-CoA dehydrogenase, followed by MTP [2].
MTP deficiency, an autosomal recessive disorder, leads to a spectrum of diseases ranging from severe infantile cardiomyopathy, inducing early death, to mild chronic progressive sensorimotor polyneuropathy with episodic rhabdomyolysis [1]. Deficiency of either MTP or LCHAD in association with fetal MTP defects occurs at a rate of 1/ 38,000 pregnancies, calculated from a molecular screening study of 351 normal subjects [3]. MTP complex disorders are classified into 2 phenotypes: isolated LCHAD deficiency and general MTP deficiency.
Different nuclear genes, namely, HADHA and HADHB, consisting of 20 and 16 exons, respectively, encode each subunit of MTP. Both genes are located on chromosome 2p23. More than 60% of cases associated with LCHAD deficiency have the E474Q (c.1528G>C) mutation in the -subunit [4,5]. The remaining cases consist of complete MTP deficiency that is caused by defects in either the - or β-subunits encoded by the HADHB gene [5,6]. Generally, all 3 enzyme activities of the MTP complex are undetectable in MTP deficiency due to a lack of both HADHA and HADHB proteins [7]. In contrast to many patients with LCHAD deficiency, only a few patients with MTP deficiency have been reported. Here, we present a Korean patient with complex MTP deficiency confirmed by clinical, biochemical, and molecular findings.
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Materials and Methods
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The male patient in this study was born at 36 weeks of gestation to healthy, non-consanguineous Korean parents. The weight of the patient at birth was 2600 g (25–50th percentile), the length was 48.5 cm (50–75th percentile), and the head circumference was 33 cm (50th percentile). The patient’s Apgar score was 8 at 1 min and 10 at 5 min. The family history was unremarkable. Chest retractions with grunting sounds were observed 14 hr after birth. Blood gas analysis showed pH 6.98, pO2 56.1 mmHg, bicarbonate 6.8 mmol/L, and base excess –23.9 mmol/L, indicating severe metabolic acidosis. Thereafter, hypotension (BP, 22/15 mmHg) and oliguric renal failure developed and the patient was treated with intravenous dopamine, dobutamine, epinephrine, sodium bicarbonate, glucose, and vasopressin. The clinical status of the patient did not improve following onset of these symptoms, and he exhibited a pale appearance, decreased activity, and tachypnea.
Four days after birth, a blood spot was collected from the patient’s original newborn screening card and butylated acylcarnitines were analyzed by tandem mass spectrometry (Waters, Manchester, UK). Initial laboratory findings included serum urea, 31.4 mg/dl (reference range, RR 8–22); creatinine, 1.25 mg/dl (RR 0.7–1.3); AST, 128 U/L (RR <40); ALT, 57 U/L (RR <40); and glucose 63 mg/dl (RR 70–110). The serum ammonia level was 358 µmol/L (RR 56–92), and the lactic acid level was 24.8 mmol/L (RR 0.7–2.5). Plasma amino acids and urine organic acids were analyzed. Quantitative acylcarnitine profile testing in plasma (Mayo Medical Lab, Rochester, MN) was also performed.
Five days after birth the patient developed frequent seizures, and electroencephalography showed abnormal findings. Postnatal echocardiography revealed a significant left ventricular dilatation, reduced cardiac dysfunction (ejection fraction, 25%), moderate mitral regurgitation, and tricuspid regurgitation. Brain MRI analysis suggested liquefaction of hemorrhages in both frontal lobes. The serum levels of cardiac markers were as follows: CK-MB, 127.6 ng/ ml (RR <5); cardiac troponin I, 1.21 ng/ml (RR <0.78); and N-terminal pro-BNP, 35,000 pg/ml (RR <88).
Molecular defects in the HADHA and HADHB genes were investigated to confirm the diagnosis of MTP deficiency. After obtaining informed consent from the parents, blood samples were collected from the patient and parents. Genomic DNA was isolated from peripheral blood leukocytes using a Wizard genomic DNA purification kit according to the manufacturer’s instructions (Promega, Madison, WI). The patient’s HADHA and HADHB genes were amplified by PCR using primers designed by the authors (Table 1
) and a Thermal Cycler 9700 (Applied Biosystems, Foster City, CA). Sequence analyses of all coding exons and the flanking introns of the HADHA and HADHB genes were performed using the BigDye Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems) on an ABI Prism 3130 genetic analyzer (Applied Biosystems).
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Results
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A newborn screening test by tandem mass spectrometry revealed elevation of 3-hydroxydicarboxylic derivatives of the C16:0 and C18:1 species (C16-OH: 1.17 µmol/L, cut-off <0.07; C18:1-OH: 0.44 µmol/L, cut-off <0.07) (Fig. 1). Plasma amino acid profiling produced nonspecific findings, and urine organic acids revealed increases of adipic acid and 3-hydroxysebacic acid. Quantitative acylcarnitine profiling in plasma showed prominent accumulation of 3-hydroxy species levels as follows: 3-OH-palmitoylcarnitine (C16-OH), 0.44 nmol/ml (RR <0.07); 3-OH-linoleylcarnitine (C18:2-OH), 0.31 nmol/ml (RR <0.06); and 3-OH-oleylcarnitine (C18:1-OH), 0.51 nmol/ml (RR <0.04).
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Fig. 1. Acylcarnitine profiling in a healthy newborn (A) and in the study patient (B). Elevated levels of 3-hydroxydicarboxylic derivatives of the C16:0 and C18:1 species are shown.
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We identified 2 novel mutations in the HADHB gene of the patient with MTP deficiency belonging to the second phenotype as described above. Specifically, the patient was a compound heterozygote for c.358dupT and c.1364T>G mutations of the HADHB gene (Table 2). An exonic single T duplication at c.358 in exon 7 created a novel early termination codon (p.A120CfsX8) while the c.1364T>G transition resulted in an amino acid substitution of Val to Gly at codon 455 (p.V455G) in exon 15 (reference sequence from NM_000183.2
[GenBank]
). The 2 molecular defects in the HADHB gene originated from the parents. Specifically, the patient’s father was a carrier of the c.358dupT mutation while his mother was heterozygous for the c.1364T>G mutation (Fig. 2). We looked for these mutations in 100 normal Korean alleles, but neither mutation was identified.
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Fig. 2. Direct sequencing of the HADHB gene in the patient revealed 2 novel mutation: c.358dupT (p.A120CfsX8) and c.1364T>G (p.V455G). The patient’s father was heterozygous for the c.358dupT mutation and his mother was a carrier for the c.1364T>G mutation.
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Because his laboratory findings were suggestive of LCHAD or MTP deficiency, glucose administration was reduced and the patient was started on a medium chain triglyceride-based diet with an L-carnitine supplement. However, the patient died 2 mo after birth, due to advanced cardiac failure.
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Discussion
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We have identified 2 novel HADHB mutations in a neonate affected by MTP complex deficiency. Valine, with a hydropathy index of 4.2, is one of the most hydrophobic amino acids [8]. Glycine is much less hydrophobic compared to valine, and as a result the p.V455G mutation of HADHB may have influenced the MTP structure because hydrophilic amino acids tend to be located closer to protein surfaces. In addition, the c.358dupT (p. A120CfsX8) frameshift mutation resulted in a novel internal termination codon and possible nonsense-mediated RNA decay. Although the enzymatic activities of each component of MTP were not determined in the patient, we assume that the HADHB mutations influenced protein stability and function. Clinically, many patients with 2 missense HADHB mutations exhibit more mild myopathic phenotypes [9,10]. The patient in the present study had a severe neonatal phenotype with hypoketotic hypoglycemia, cardiomyopathy, and Reye-like syndrome. He died 2 mo after birth due to cardiac failure, and it was not possible to perform an autopsy. It has been reported that mice can exhibit a defect in the cardiac conduction system caused by a mutation of Hadhb, with a reduction of both LCHAD and LKAT enzyme activities [11]. Likewise, Spiekerkoetter et al [12] reported that fatty acid oxidation plays a significant role during intrauterine development with special regard to the heart and severe cardiac mitochondrial proliferation in MTP deficiency.
There have been 2 reports of Korean patients with MTP deficiency diagnosed by acylcarnitine profiling and DNA analysis [9,13]. A heterozygous 2 bp deletion (bp 1793_4) in the HADHA gene was first identified in a Korean patient with MTP deficiency [13]. Table 2
compares the HADHB mutations found in our patient with those detected by Choi et al [9] in 2 other Korean patients. Considering that 5 different HADHB mutations have been found in 3 patients, including 2 from the patient in this study, there do not yet appear to be any mutational hot-spots among Korean patients with MTP deficiency, even though the number of subjects is small. Presently, >20 unique mutations have been described by studies of the HADHB genes in MTP deficient patients, as reviewed in the HADHB gene mutation database (http://www.hgmd.org/). Most cases are compound heterozygotes for β-subunit mutations, with missense or nonsense mutations comprising a large majority [5,7,10,14]. Approximately 69% of mutant alleles are located on exons 4, 9, and 10 in the HADHB gene [10], suggesting genetic heterogeneity of MTP deficiency due to β-subunit mutations.
MTP deficiency disease is rare, but newborn screening by tandem mass spectrometry may be required because early diagnosis and treatment can help to improve clinical outcomes in the patients. Sander et al [15] recommended that neonatal screening for MTP deficiency is necessary on the basis of specified criteria. Typical acylcarnitine profiles in blood are characterized by increased concentrations of 3-hydroxy-palmitoylcarnitine (C16-OH), 3-hydroxy-oleylcarnitine (C18:1-OH), tetradecenoylcarnitine (C14:1), and 3-hydroxy-myristoylcarnitine (C14-OH). Because acylcarnitine analysis by tandem mass spectrometry cannot differentiate among different defects of the MTP complex, metabolic defects must be confirmed and specified by additional enzyme analysis in cultured fibroblasts as well as by mutation analysis of causative genes. Enzyme assays performed for MTP-deficient patients tend to show a lack of 1 or more enzymes in cultured lymphocytes and fibroblasts. Although determination of enzyme deficiency is a valuable tool for the diagnosis of the disease, direct sequencing of the HADHA and HADHB genes should also be performed to confirm the diagnosis. We were unable to measure enzyme activities in the patient and his family, and thus genetic-based investigation was helpful for determining the diagnosis of the patient and understanding the mutation inheritance pattern from the parents. Molecular analysis can be an important approach for the differential diagnosis and confirmation of MTP deficiency. Indeed, the identification of genetic mutations makes prenatal molecular diagnosis feasible for at-risk family members.
In conclusion, we screened a patient with MTP deficiency by tandem mass spectrometry and confirmed the diagnosis by biochemical and genetic analysis. We envision that future work on the novel HADHB mutations identified in this study will confirm their effects on the functional or structural aberration of the protein.
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Footnotes
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a Hyung-Doo Park and Suk Ran Kim contributed equally to the work. 
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Abstract
Introduction
