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Isolated mitochondrial long-chain ketoacyl-CoA thiolase deficiency resulting from mutations in the HADHB gene.

The mitochondrial trifunctional protein (MTP) [4] is a multienzyme complex involved in the (3-oxidation of fatty acids with chain lengths of C12 to C18. It is a heterooctamer of 4 [alpha] subunits (McKusick #600890) and 4 [beta] subunits (McKusick #143450). The a subunits (HADHA) and the [beta] subunits (HADHB) are encoded by different nuclear genes, consisting of 20 and 16 exons, respectively. Both genes are located on chromosome 2p23 (1,2). The 3 enzymes involved are long-chain enoyl-CoA hydratase (LCEH), long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD), and long-chain 3-ketoacyl-CoA thiolase (LCTH). The HADHB gene encodes the LCTH activity, whereas the HADHA gene contains the information for the other 2 enzymatic functions. The first step of the [beta]-oxidation cycle is catalyzed by acyl-CoA dehydrogenases, whereas the enzymes of MTP catalyze the last 3 steps of [beta]-oxidation.

Two biochemical phenotypes of defects concerning the MTP complex have been described: isolated LCHAD deficiency and generalized MTP deficiency, with decreased activities of all 3 enzymes.

LCHAD deficiency is the most common defect of the MTP complex, and hundreds of cases have been identified. More than 60% of cases with LCHAD deficiency are associated with the E474Q (1528G>C) mutation in the [alpha] subunit (3, 4), giving rise to heterogeneous phenotypes. The typical acylcarnitine profile in blood is characterized by increased concentrations of the acylcarnitines hydroxypalmitoylcarnitine (C160H), hydroxyhexadecenoylcarnitine (C16:1OH), hydroxyoctadecenoylcanitine (C18OH), and hydroxyoctadecenoylcarnitine (C18:1OH) (5). As acylcarnitine analysis by tandem mass spectrometry (MS/MS) cannot differentiate among different defects of the MTP complex, the metabolic defect must be confirmed and specified by additional enzyme analysis in cultured fibroblasts or lymphocytes and by mutation analysis.

LCHAD activity is deficient in cases of isolated LCHAD and MTP deficiency. Differentiation between the 2 disorders requires measurement of LCTH activity.

Urinary 3-hydroxydicarboxylic acids, with chain lengths of 6 to 16 carbon atoms, can be detected in patients with MTP and LCHAD deficiencies during spells of acute illness, but they may be absent during clinical remission (6).

In contrast to the large number of LCHAD-deficient patients, only a few MTP [alpha]--and [beta]-subunit--deficient patients have been identified since its first description in 1992 (7, 8). Molecular studies in MTP-deficient patients have shown a wide range of private mutations in both the a and [beta] subunits, in contrast to the common E474Q mutation in LCHAD deficiency. Spiekerkbtter et al. (9) investigated the pathophysiologic mechanisms in MTP deficiency. They concluded that [alpha]--and [beta]-subunit mutations have similar effects on the stability of the MTP complex and cannot be distinguished by clinical and biochemical criteria.

Impaired degradation of fatty acids leads to hypoketotic hypoglycemia, which is the biochemical hallmark of [beta]-oxidation disorders. The clinical spectrum in MTP deficiency is broader than in isolated LCHAD deficiency, usually with a first manifestation earlier in life. This includes cardiomyopathy with arrhythmia and heart failure, hepatomegaly with compromised liver function and hepatic steatosis, and capillary leakage with edema, followed by progressive neuropathy, retinopathy (as this is a late manifestation of the disease, it is found only in individuals with mild phenotypes who survive longer), and myopathy. A lethal outcome is common.

To date, isolated LCTH deficiency associated with HADHB mutations has not been reported. This is the first report of isolated LCTH deficiency in a newborn associated with mutations in the HADHB gene coding for MTP.

Materials and Methods


The male patient was born at 35 weeks of gestation to healthy, nonconsanguineous parents of German extraction. The child was delivered by caesarian section because of fetal distress presumably caused by placental dysfunction. The pregnancy was uncomplicated, with no indication of the HELLP syndrome (hemolysis, increased liver enzymes, and low platelets) in the mother. Birth weight was 1900 g (3rd percentile), length was 46 cm (10th percentile), head circumference was 30 cm (3rd percentile), and the Apgar scores were 4, 6, and 9 at 1, 5, and 10 min, respectively. One sibling is healthy; another sibling was born prematurely after 28 weeks of gestation and died at the age of 8 days, with no definitive diagnosis established. Soon after birth, our patient developed tachypnea and dyspnea and had to be put on mechanical ventilation. Because of respiratory distress syndrome, he was given surfactant twice. An ophthalmologic examination was unrevealing.

Lactic acidosis developed (maximum lactic acid concentration, 14 mmol/L; reference values <2.5 mmol/L) that could not be explained by cardiovascular dysfunction. We found no laboratory signs supporting a diagnosis of septicemia. Serum creatine kinase activity was within reference values, whereas the transaminases were slightly increased. On the 3rd day of life, a blood specimen for routine newborn screening of acylcarnitines by MS/MS was obtained that showed increased concentrations of tetradecenoylcarnitine [(C14:1); 0.28 [micro]mol/L; reference values <0.18 [micro]mol/L], hexadecenoylcamitine [(C16:1); 0.65 [micro]mol/L; reference values <0.39 [micro]mol/L], C160H (1.32 [micro]mol/L; reference values <0.06 [micro]mol/L), and C18:1OH (0.69 [micro]mol/L; reference values <0.04 [micro]mol/L), suggesting MTP or LCHAD deficiency. Free carnitine was at the low end of the reference interval (19 [micro]mol/L; reference interval, 20-60 [micro]mol/L). There was no appreciable difference in the acylcarnitine pattern between catabolic and anabolic spells. Subsequent analysis of organic acids in urine revealed increased concentrations of dicarboxylic and 3-hydroxydicarboxylic acids, which was consistent with the presumptive diagnosis.

Glucose administration was decreased, and a medium-chain triglyceride-based diet was started. Under this regimen, the lactate concentrations in the blood and the organic acid concentrations in the urine decreased to within reference values, and the boy stabilized clinically. He was discharged at the age of 4 weeks. A few days after discharge, the parents noticed that he had feeding difficulties, muscular hypotonia, and dyspnea; pneumonia was suspected, based on radiologic examination. The boy had to be readmitted to the hospital, and he was intubated and ventilated mechanically because of pulmonary edema. Because oxygen saturation was insufficient under conventional ventilation, high-frequency oscillation had to be instituted. His cardiac function deteriorated rapidly, right and left ventricular functions were severely compromised, with a shortening fraction of 11% and an ejection fraction of 30%. No dilatation or ventricular hypertrophy could be found initially by echocardiography, although ventricular hypertrophy was detected later. Because of cardiac dysfunction, he developed severe edema and ascites despite administration of catecholamines, an angiotensin-converting enzyme inhibitor, and a phosphodiesterase inhibitor. At the age of 6 weeks, the boy died from cardiorespiratory failure. Autopsy revealed severe myocardial hypertrophy and hyaline membrane disease of the lungs as well as bronchopneumonia. We found no other structural defects, in particular, no fatty infiltration of the liver.


We carried out enzyme activity measurements, as described in detail previously (7), originally in lymphocytes and later in fibroblasts. We performed quantitative acylcarnitine profiling by electrospray MS/MS in fibroblast monolayers, using [U-[sup.13]C]palmitic acid as described in detail elsewhere (10).


Measurements of the enzymes involved in fatty acid oxidation demonstrated a marked deficiency of the long-chain ketoacyl-CoA thiolase component of MTP as measured with 3-ketopalmitoyl-CoA as substrate. Measurement of LCEH and LCHAD activities showed no marked abnormalities. Subsequent immunoblot analysis in fibroblasts revealed the presence of both the [alpha] and [beta] subunits of MTP. These results are summarized in Table 1.

Quantitative acylcarnitine profiling in fibroblasts showed accumulation of [[sup.13]C]palmitoylcarnitine [(C16); 13.7 nmol x [(96 h).sup.-1] [(mg protein).sup.-1]; controls <6.0 nmol x [(96 h).sup.-1] x [(mg protein).sup.-1]; as well as [[sup.13]C]-C160H [1.2 nmol x [(96 h).sup.-1] x [(mg protein).sup.-1]; controls <0.20 nmol x [(96 h).sup.-1] x [(mg protein).sup.-1]], whereas the formation of [13C]acetylcarnitine (C2) was decreased [0.8 nmol (96 h)-1 x (mg protein)-1; controls, 7-43 nmol x [(96 h).sup.-1] x [(mg protein).sup.-1]]. These findings of the loading test were in line with disturbed MTP function (Table 2).

To confirm the diagnosis of isolated LCTH deficiency, we analyzed the HADHA and HADHB genes coding for MTP in cultured fibroblasts. The patient was compound heterozygous for a 185G>A (R62H) mutation in exon 4 and a 1292T>C (F431S) mutation in exon 15 of the HADHB gene. Mutation analysis of genomic DNA isolated from his father's blood cells showed carrier status for the 185G>A (R62H) mutation. In blood cells from his mother, however, both mutations were absent. The sister of our patient carries the mutation 1292T>C (F431S). The absence of mutations in the patient's mother is most likely the result of mosaicism, but this could not be confirmed because she refused any biopsies. A sample mix-up was excluded. Acylcarnitine profiles in dried blood spots and organic acids in urine were normal in both parents and in the patient's sister.


We report the first case of isolated LCTH deficiency resulting from mutations of the HADHB gene in a male newborn. The disease could be identified by the routine newborn-screening program using MS/MS that is now in force in Germany and is performed at the age of 36-72 h. However, the pattern of metabolites was not specific and indicated only a possible defect in the MTP complex.

In MTP deficiency, hepatic production of ketone bodies, which are important alternative fuels for the brain during catabolism, as well as fatty acid oxidation, especially in skeletal and heart muscle, are severely compromised, leading to energy depletion. Furthermore, toxic metabolites accumulate proximal to the enzyme block, leading to dysfunction in several organs. Clinically, patients may suffer from cardiomyopathy or skeletal myopathy, Reye-like syndrome (with brain and liver dysfunction), or a pathology mimicking sudden infant death syndrome.

To date, 3 different biochemical phenotypes of defects in the MTP complex with similar clinical features have been described. In isolated LCHAD deficiency, HADHA function is abnormal, but the amounts of HADHA and HADHB proteins are not decreased. In generalized MTP deficiency, all 3 enzyme activities of the complex are deficient, with a lack of both HADHA and HADHB proteins. Many patients belong to the group with isolated LCHAD deficiency, with homozygosity for the 1528G>C mutation in the HADHA gene (11). Generalized MTP deficiency as a result of mutations in the HADHA gene has been reported in 15 individuals (3, 8,12-19), whereas generalized MTP deficiency as a result of a mutation in the HADHB gene has been reported in 19 patients. Until 2003, only 4 patients with this kind of MTP deficiency had been reported (15, 20). In a recent study (21), 15 patients from 13 families with MTP deficiency resulting from a mutation in the HADHB gene were reported and investigated for genotype /phenotype correlations. Only 1 patient, with presumably isolated thiolase deficiency based on enzyme data associated with a splice-site mutation in the HADHA gene, was found by that group (9). The lack of HADHA was supposed to cause a secondary loss of HADHB function. It is not clear why this patient had LCEH and LCHAD activities within reference values.

Here we describe the first patient with isolated thiolase deficiency caused by HADHB mutations. Similar to the patient with presumably isolated thiolase deficiency attributable to HADHA mutations described previously (9), our patient presented with a lethal cardiac phenotype. The severe phenotype may be more common in isolated thiolase deficiency because this affects the last step of the [beta]-oxidation spiral. 3-Ketoacyl derivatives accumulating proximal to the blocked thiolase reaction may lead to greater toxicity, whereas in general MTP deficiency, the flux of metabolites to thiolase is limited by the deficiency of the other 2 enzymes of the complex.

It was previously assumed that [beta]-subunit mutations decrease the stability of the MTP complex (9,15); however, our findings demonstrate that this is not always the case. Some mutations of HADHB may affect the cooperativity and stability of both subunits, whereas others do not. The R62H mutation was previously reported as an R28H mutation, a different nomenclature, and is located in the dimerization domain of the protein (21). Because this mutation has been found in patients with severe presentations, it is assumed that the arginine at position 62 is critical for protein function or stability (21). The F431S mutation has not been described before and is located in a domain that is conserved between the human protein and yeast peroxisomal thiolase, which share considerable sequence similarities (21).

The phenylalanine at position 431 is not conserved in yeast peroxisomal thiolase but is located near the catalytic histidine at position 429 and apparently affects the catalytic activity of LCTH rather than the stability.

Genotyping was performed in our patient's family. The 185G>A mutation was of paternal origin, and the patient's sister is a carrier of the 1292T>G mutation, which is supposed to be of maternal origin. However, this mutation could not be detected in the mother's blood cells. Apparently, the 1292T>G mutation must be present in the mother's germ cells; we therefore assume genetic mosaicism for this mutation. This could not be confirmed because the mother refused any biopsies. Acylcarnitine profiles in blood and organic acids in urine were within reference intervals in both parents and in their daughter, which shows that the enzyme function is sufficient in individuals heterozygous for mutations.

As we have shown recently in a sample of 1.3 million newborns (22), the acylcarnitine profile from dried blood spots does not distinguish among generalized MTP deficiency, isolated LCHAD deficiency, and isolated thiolase deficiency. Similarly, the acylcarnitine profiles in fibroblasts from patients with these disorders were not diagnostic (results not shown). As in our patient, the clinical outcome is generally poor in isolated thiolase and generalized MTP deficiency, as shown in our previous study (22).


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(18.) Hintz SR, Matern D, Strauss AW, Bennett MJ, Hoyme E, Schelley S, et al. Early neonatal diagnosis of long-chain 3-hydroxy acyl coenzyme A dehydrogenase and mitochondrial and mitochondrial trifunctional protein deficiencies. Mol Genet Metab 2002;75: 120-7.

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(22.) Sander J, Sander S, Steuerwald U, Janzen N, Peter M, Wanders RJA, et al. Neonatal screening for defects of the mitochondrial trifunctional protein. Mol Genet Metab 2005;85:108-14.


[1] Department of Paediatrics, Hannover Medical School, Hannover, Germany.

[2] Screening Laboratory Hannover, Hannover, Germany.

[3] Department of Paediatrics and Clinical Chemistry, Laboratory Genetic Metabolic Diseases, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.

[4] Nonstandard abbreviations: MTP, mitochondrial trifunctional protein; HADHA and HADHB, hydroacyl-CoA dehydrogenase-[alpha] and -[beta] subunits, respectively; LCEH, long-chain enoyl-CoA hydratase; LCHAD, long-chain 3-hydroxyacyl Co-A dehydrogenase; LCTH, long-chain ketoacyl-CoA thiolase; C160H, hydroxypalmitoylcarnitine; C16:1OH, hydroxyhexadecenoylcarnitine; C180H, hydroxyoctadecanoylcamitine; C18:1OH, hydroxyoctadecenoylcarnitine; MS/MS, tandem mass spectrometry; C14:1, hexadecenoylcarnitine; C16, palmitoylcarnitine; C16:1, hexadecenoylcarnitine; and C2, acetylcamitine.

* Address correspondence to this author at: Department of Paediatrics, Hannover Medical School, Carl-Neuberg Strasse 1, D-30623 Hannover, Germany. Fax 49-511-5328073; e-mail

Received October 13, 2005; accepted December 22, 2005.

Previously published online at DOI: 10.1373/clinchem.2005.062000
Table 1. MTP enzyme activities, mutation analysis, and
immunoblotting results in fibroblasts from our patient and controls.

 Patient Controls

Enzymes in HSF, (a)
nmol x [min.sup.-1]
(mg protein) (1)
 LCTH (C16) 0.8 20.6 (7.8) (b) (n = 47)
 LCHAD (C16) 56.4 81.8 (22.8) (b) (n = 102)
 LCEH (C12:1) 75 78 (25) (b) (n = 59)
 HBDHA No mutation found No mutation
 HBDHB 185G A (R62H), No mutation
 exon 4
 1292T C (F431S),
 exon 15

Western blotting
 [alpha] subunit +
 [beta] subunit +

(a) HSF, human skin fibroblasts; C12:1, dodecenoylcarnitine.

(b) Mean (SD).

Table 2. Acylcarnitine profiles after [U-[13.sub.C]]palmitate
loading test in cultured fibroblasts.

 Acylcarnitines (a)

 C16 C16OH C2 C16/C2 ratio

Patient 13.7 1.2 0.8 17.1
Controls < 6 < 0.2 7-43 0.05 (0.05) (b)

(a) Values are given as nmol x
[(96 h).sup.-1] x [(mg protein).sup.-1].

(b) Mean (SD).
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Title Annotation:Case Report
Author:Das, Anibh M.; Illsinger, Sabine; Lucke, Thomas; Hartmann, Hans; Ruiter, Jos P.N.; Steuerwald, Ulrik
Publication:Clinical Chemistry
Date:Mar 1, 2006
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