Diagnostic assessment and long-term follow-up of 13 patients with Very Long-Chain Acyl-Coenzyme A dehydrogenase (VLCAD) deficiency

Neuromuscular Disorders 19 (2009) 324–329

Contents lists available at ScienceDirect

Neuromuscular Disorders journal homepage: www.elsevier.com/locate/nmd

Diagnostic assessment and long-term follow-up of 13 patients with Very Long-Chain Acyl-Coenzyme A dehydrogenase (VLCAD) deficiency Pascal Laforêt a,*, Cécile Acquaviva-Bourdain b, Odile Rigal c, Michèle Brivet d, Isabelle Penisson-Besnier e, Brigitte Chabrol f, Denys Chaigne g, Odile Boespflug-Tanguy h, Cécile Laroche i, Anne-Laure Bedat-Millet j, Anthony Behin a, Isabelle Delevaux k, Anne Lombès l, Brage S. Andresen m, Bruno Eymard a, Christine Vianey-Saban b a

Centre de Référence de pathologie neuromusculaire Paris-Est, Groupe Hospitalier Pitié-Salpêtrière, Assistance Publique-Hôpitaux de Paris, Paris, France Centre de Référence des Maladies Héréditaires du Métabolisme, INSERM U820, Centre de Biologie et de Pathologie Est, Hospices Civils de Lyon, 69677 Bron cedex, France Centre de reférence des Maladies Héréditaires du Métabolisme, Laboratoire de Biochimie-Hormonologie, Hôpital Robert-Debré, Assistance Publique-Hôpitaux de Paris, Paris, France d Department of Biochemistry, Hôpital de Bicêtre, Assistance Publique-Hôpitaux de Paris, Paris, Le Kremlin-Bicêtre, France e Centre de Référence Maladies Neuromusculaires Nantes-Angers, Département de Neurologie, CHU Angers, France f Centre de Référence des maladies métaboliques de l’enfant, Unité de Médecine Infantile, CHU Timone Enfants, Assistance Publique-Hôpitaux de Marseille, Marseille, France g Clinique Ste-Odile, 6 rue Simonis, 67100 Strasbourg, France h Centre de Référence Neuropathies Rares et Maladies Musculaires Auvergne-Limousin, service de Génétique Médicale, CHU de Clermont-Ferrand, France i Département de pédiatrie médicale, Hôpital Universitaire Dupuytren, CHU de Limoges, France j Département de Neurologie, Hôpital Charles Nicolle, CHU de Rouen, France k Service de Médecine Interne, CHU de Clermont-Ferrand, France l INSERM U975, CRICM, Paris, France m Research Unit for Molecular Medicine, Institute of Human Genetics, Aarhus University, Aarhus, Denmark b c

a r t i c l e

i n f o

Article history: Received 13 January 2009 Received in revised form 3 February 2009 Accepted 13 February 2009

Keywords: Muscle lipidosis Very Long-Chain Acyl-Coenzyme A dehydrogenase Fatty acid oxidation Metabolic myopathy

a b s t r a c t Very Long-Chain Acyl-CoA dehydrogenase (VLCAD) deficiency is an inborn error of mitochondrial longchain fatty acid oxidation (FAO) most often occurring in childhood with cardiac or liver involvement, but rhabdomyolysis attacks have also been reported in adults. We report in this study the clinical, biochemical and molecular studies in 13 adult patients from 10 different families with VLCAD deficiency. The enzyme defect was demonstrated in cultured skin fibroblasts or lymphocytes. All patients exhibited exercise intolerance and recurrent rhabdomyolysis episodes, which were generally triggered by strenuous exercise, fasting, cold or fever (mean age at onset: 10 years). Inaugural life-threatening general manifestations also occurred before the age of 3 years in four patients. Increased levels of long-chain acylcarnitines with tetradecenoylcarnitine (C14:1) as the most prominent species were observed in all patients. Muscle biopsies showed a mild lipidosis in four patients. For all patients but two, molecular analysis showed homozygous (4 patients) or compound heterozygous genotype (7 patients). For the two remaining patients, only one mutation in a heterozygous state was detected. This study confirms that VLCAD deficiency, although being less frequent than CPT II deficiency, should be systematically considered in the differential diagnosis of exercise-induced rhabdomyolysis. Measurement of fasting blood acylcarnitines by tandem mass spectrometry allows accurate biochemical diagnosis and should therefore be performed in all patients presenting with unexplained muscle exercise intolerance or rhabdomyolysis. Ó 2009 Elsevier B.V. All rights reserved.

Disclosure: The authors report no conflicts of interest. 1. Introduction Among the metabolic myopathies, mitochondrial fatty acid oxidation (FAO) defects are probably the most difficult to identify due * Corresponding author. Address: Institut de Myologie, Bâtiment Babinski, Groupe Hospitalier Pitié-Salpêtrière, 47-83 boulevard de l’Hôpital, 75651 Paris Cedex 13, France. Tel.: +33 142163776; fax: +33 142163793. E-mail address: [email protected] (P. Laforêt). 0960-8966/$ - see front matter Ó 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.nmd.2009.02.007

to their transient clinical and biological manifestations, as it has been initially described in patients with carnitine palmitoyltransferase (CPT II) deficiency [1]. Very Long-Chain Acyl-CoA dehydrogenase (VLCAD) is bound to the inner mitochondrial membrane, and catalyses the first step of the long-chain fatty acid boxidation spiral [2]. VLCAD deficiency is inherited as an autosomal recessive trait, and three phenotypes have been described according to the age at onset of clinical manifestations [3–6]: (1) a severe infantile form presenting in the neonatal period with hypertrophic cardiomyopathy and liver failure; (2) a childhood onset between ages 1 and 13 years with hypoketotic hypoglycaemia as the main

P. Laforêt et al. / Neuromuscular Disorders 19 (2009) 324–329

presenting feature; and (3) a juvenile or adult-onset muscular form characterized by recurrent episodes of rhabdomyolysis triggered by prolonged exercise or fasting. The milder form of VLCAD deficiency is increasingly recognised due to the more wide spread use of tandem mass spectrometry (MS/MS), allowing the detection of abnormal long-chain acylcarnitines in blood samples [7–10]. However, clinical observations of patients with late-onset presentation of VLCAD deficiency remain somewhat rare [8–17]. We report here the clinical and biochemical results in 13 adult patients all presenting with muscle complications of VLCAD deficiency. 2. Patients and methods

325

nomenclature used in this work follows the guidelines of den Dunnen and Antonarakis [21]. 3. Results 3.1. Family history (Table 1a, b) All patients but two (6, 7) were born from non-consanguineous parents. Parents of patients 6 and 7 were Turkish, and parents of patients 5 and 11 originated from Philippine and Greece, respectively. In three sibships, family histories were remarkable by the occurrence of sudden deaths of 8 siblings between 2 days and 20 months of age (median: 6.5 months), often triggered by diarrhea or vomiting.

2.1. Patients Thirteen patients originating from 10 different families have been examined by one of the authors. Only one case history was previously reported [3]. Clinical and biochemical data were retrospectively collected, and 12 patients are still regularly followed in neuromuscular or metabolic French reference centres. One patient died accidentally. 2.2. Plasma and urine biochemistry Free and total carnitine levels in plasma were determined using conventional methods. Urinary organic acid profiling was performed using gas chromatography–mass spectrometry (GC/MS) of methyl or trimethylsilyl derivatives. Acylcarnitines in dried blood spots or plasma were measured as methyl or butyl derivatives using tandem mass spectrometry with electrospray ionisation (ESI–MS/MS). Plasma total long-chain fatty acids were studied by gas chromatography–mass spectrometry of their methyl esters [18]. 2.3. Muscle biopsy Deltoid or quadriceps muscle biopsy was taken by open biopsy and processed for routine morphological techniques in 10 patients. Spectrophotometric analysis of the respiratory chain complexes were performed using conventional methods on homogenates from frozen samples of muscle in four patients. 2.4. Fatty acid oxidation studies and enzyme activities [1-14C] palmitate oxidation and tritiated water release experiments from [9,10(n)-3H] palmitate and [9,10(n)-3H] myristate were performed in intact cultured fibroblasts or lymphocytes as previously described [19,20]. Membrane-bound VLCAD activity was assayed, with palmitoyl-CoA as substrate, in a membrane preparation from cultured skin fibroblasts (or muscle tissue or lymphocytes) [4]. 2.5. DNA analysis Patient’s DNA was extracted from whole blood or cultured fibroblasts using conventional methods. The 20 exons and exon– intron boundaries of the ACADVL gene were amplified from genomic DNA using standard PCR procedures with primers previously described [6] or in-house designed primers. Direct bidirectional sequencing of purified PCR products was performed using the BigDye Terminator v3.1 Sequencing Kit (Applied Biosystems) and an ABI 3100 genetic analyser. Sequences were analysed using Seqscape Analysis Software (Applied Biosystems) in comparison with GenBank Reference genomic sequence (NC_000017). The mutation

3.2. Clinical features at onset (Table 1a, b) Age at onset of the disease varied from birth to 13 years (median: 7 years). In four patients the first symptoms occurred before the age of 3 years. Onset in patient 1 was a cardio-respiratory arrest at 36 h of life. Patient 3 was hospitalized at 6 and 14 months of age for hypoglycaemic episodes with rhabdomyolysis (CK level: 6510 UI/L). Patient 4 had repeated hypoglycaemias and muscle fatigability since the age of 1 year. Patient 5 presented a Reye-like syndrome at 2.5 years of age. Hepatomegaly was also reported during childhood in all these four patients. In the nine other patients the first manifestations were exercise intolerance or myoglobinuria (median age at onset: 10 years, range 5–13). 3.3. Clinical course (Table 1a, b) Median age of patients at time of last examination was 30.5 years (12–46 years). The main clinical manifestation was exercise intolerance. Diffuse muscle pain and prolonged stiffness lasting up to 48 h, generally occurred after 1 or 2 h of strenuous exercises. Other triggering factors were fasting (7 patients), cold (4 patients), and fever (3 patients). Nine patients experimented episodes of rhabdomyolysis after the practice of intensive leisure physical activity like cycling, skiing, swimming, or playing tennis. Acute renal failure complicating a rhabdomyolysis episode occurred in four patients: twice after skiing (patients 5 and 10), once after carrying heavy weights (patient 7), and once after playing football (patient 13). A fasting test performed at age 9 in patient 6, when diagnosis was not assessed, induced an acute episode of rhabdomyolysis with hypoketotic hypoglycaemia, and massive dicarboxylic aciduria. All patients recovered without renal sequelae. The frequency of rhabdomyolysis crises varied from less than one to six attacks per year. CK levels were generally normal when measured at rest, and patients did not show any sign of muscle weakness or atrophy at clinical examination. An echocardiography was performed in all patients, revealing a mild ventricular hypertrophy in patient 4, and a moderate septum hypokinesia in patient 5. Conduction defects or arrhythmia were never detected. A total of nine pregnancies occurred in five women. Two miscarriages occurred in patient 6 before a normal pregnancy. Delivery was carried out after caesarean section in patient 6 and 9, without any foetal or neonatal abnormality. Three women suffered from delayed myalgias after their first delivery (patients 5, 6, and 12), with concomitant elevation of CK levels up to 17,000 UI/L or myoglobinuria, but renal function always remained normal. Various treatments were administrated, either alone or in association: L-carnitine, riboflavin, coenzyme Q10, or dietary therapy with supplements of medium chain triglycerides. Patients 11 and 13 experienced a subjective improvement, respectively, after CoQ10 (300 mg/day) and riboflavin (80 mg/day) treatments.

326

P. Laforêt et al. / Neuromuscular Disorders 19 (2009) 324–329

Table 1a Clinical features. Pt I.D.

Family history

1

Sex

Age at onset (years)

Age at diagnosis (years)

Current age (years)

Presenting symptoms

F

Birth

Birth

16

Cardiac arrest Hypotonia Hepatomegaly

5

Exercise Fever

1

F

5

0.2

12

Exercise intolerance

5

1

Hypoglycaemia Hepatomegaly High CK level Hypoglycaemia Hepatomegaly Exercise intolerance Reye syndrome Hepatomegaly

20

Exercise Fever Exercise Fasting Cold Exercise Fasting

Sib1D1 2

Age at onset of exercise intolerance

3

–

M

0.5

23

23

4

–

M

1

12

21

5

–

F

2.5

22

31

F

7

17

25

Exercise intolerance Myoglobinuria

7

M

11

11

 

Exercise intolerance Dizziness

11

M

9

21

29

Exercise intolerance Myoglobinuria

9

Exercise intolerance Myoglobinuria Exercise intolerance Myoglobinuria Exercise intolerance Myoglobinuria Exercise intolerance

9

Sib2D4

6

7

Sib1D3

8

9 10 11

– –

F M F

9 10 10

32 40 43

40 42 46

12

–

F

10

36

39

13

–

M

13

34

41

1

9

11 11 13

Provocating factors of myalgias

Exercise Cold Fasting Exercise Cold Fasting Exercise Cold Fasting Exercise

Exercise Exercise Exercise Fasting Exercise Exercise Fasting

Frequency of myoglobinuria episodes per year

1

<1

2

1

<1

<1

<1 6 2 2 3

Table 1b Clinical features. Pt I.D.

Higher recorded CK levels (UI/L)

Renal failure

Number of pregnancies

Echocardiography

Treatment Carnitine, riboflavin, MCT diet Carnitine, riboflavin, MCT diet – Carnitine, riboflavin, MCT diet – Carnitine, MCT diet – Carnitine Carnitine Carnitine CoQ10 Carnitine Riboflavin

1

13,400

Normal

2

2500

Normal

3 4

215,700 10,000

5 6 7 8 9 10 11 12 13

544,000 7720 N/a 141,500 16,800 28,000 22,260 30,000 83,100

Normal LV hypertrophy +

1 2

+ 1 + 2 3 +

Septal hypokinesia Normal Normal Normal Normal Normal Normal Normal Normal

Pt I.D., patient identification number. Sib, affected sibling (number of affected siblings in exponent). D, death of siblings early in infancy (number of early deaths in exponent).  , accidental death at 24 years of age. LV, left ventricular. MCT diet, diet with supplements of medium chain triglyceride oil.

3.4. Muscle analysis A muscle biopsy was performed in 10 patients. Histological analysis was normal or showed non-specific abnormalities in six patients. A mild muscle lipidosis was observed in only four patients (patients 3, 10, 11, 12). In addition, oxidative staining detected a subsarcolemmal mitochondrial accumulation in patient 11, with respiratory chain analysis showing a combined defect of complexes I + III, and II + III (respectively, 5 nmol/min/mg prot, controls 18 ± 12; and 8 nmol/min/mg prot, controls 22 ± 8). The level of coenzyme Q in muscle was slightly reduced in this patient (15.8 nmol/g prot; controls 15.9–36.8). Spectrophotometric analy-

sis of respiratory chain complexes in muscle was normal in patients 8, 12 and 13. 3.5. Biochemical studies (Table 2) Free carnitine levels were lowered in seven out of 10 untreated patients, while esterified carnitine level was significantly increased in three patients. Organic acid profile assessed in urine of five patients between attacks of myoglobinuria was always normal, by contrast with the detection of massive dicarboxylic aciduria at the time of acute general manifestations during childhood in two patients (patients 2 and 6). Repeated measurements of plasma

327

P. Laforêt et al. / Neuromuscular Disorders 19 (2009) 324–329 Table 2 Results of laboratory investigations. Pt I.D.

Muscle morphology

Plasma carnitine (lmol/L) FC

EC

Plasma C14:1 cis-5 (lmol/L)

Acyl carnitine profile: C14:1 (lmol/L)

Fatty acid oxidation studies in lymphocytes or fibroblasts (% controls)

VLCAD activity in fibroblasts (nmol ETF/min/ mg prot)

ACADVL gene substitutions

cDNA substitutions

Predicted amino acid change

c.779C>T c.842C>A c.779C>T c.842C>A c.535G>T c.1613G>C c.1837C>T c.1837C>T c.1505T>C ? c.1500_1502del c.1500_1502del c.1500_1502del c.1500_1502del c.779C>T c.1500_1502del c.779C>T c.1500_1502del c.779C>T ? c.833_835del c.1376G>A

p.Thr260Met p.Ala281Asp p.Thr260Met p.Ala281Asp p.Gly179Trp p.Arg538Pro p.Arg613Trp p.Arg613Trp p.Leu502Pro ? p.501delLeu p.501delLeu p.501delLeu p.501delLeu p.Thr260Met p.501delLeu p.Thr260Met p.501delLeu p.Thr260Met ? p.278delLys p.Arg459Gln

c.455G>A c.833_835del c.779C>T c.779C>T

p.Gly152Asp p.278delLys p.Thr260Met p.Thr260Met

1

ND

30

38

16–560

1.6–8.5

CPF 56%

0.29

2

ND

63

15

46–433

3.3

CPF 35%

0.15

3

Regenerating fibres, lipidosis ND

42

18

ND

1.5

ND

ND

4

13

119–414

2.3–9.3

CPF 28%

0.50

16

4

50

3

0.06

35

23

1.3; 273

0.7; 8.5

HPF 38% HMF 32% CPF N

0.50

7

Necrosis, Inflammation Size inequality type II C fibres Atrophic fibres

30

10

<1; 20

0.4; 1.4

CPF N

0.68

8

N

ND

ND

72

2.5

CPF N

0.69

9

N

ND

ND

4.8

4.0

ND

ND

Mild lipidosis in type I fibres Mild lipidosis Mitochondrial accumulation Central nuclei Mild lipidosis Necrotic and regenerating fibres

ND

ND

101

3.1

ND

0.28

24

16

ND

1.3

ND

0.28 (lymphocytes)

20

22

ND

1.8

CPL N

0.24 (lymphocytes)

10

5

234

3.1

CPF 49%

0.14

40 ± 10

14 ± 6

<2

<0.2

4 5 6

10 11

12 13 Controls

1.64 ± 0.57 (fibroblasts) 1.68; 2.35 (lymphocytes)

ND, not done. N, normal. FC, free carnitine; EC, esterified carnitine. C14:1 cis-5, cis-5-tetradecenoic acid. C14:1, tetradecenoylcarnitine. CPF, [1-14C] palmitate oxidation in fibroblasts. CPL, [1-14C] palmitate oxidation in lymphocytes. HPF, [9,10(n)-3H] palmitate oxidation in fibroblasts. HMF, [9,10(n)-3H] myristate oxidation in fibroblasts. Abnormal values are in bold characters.

fatty acids showed increased levels of cis-5-tetradecenoic acid (C14:1 cis-5) even when they were clinically asymptomatic, except for one assessment performed under glucose infusion in patients 6 and 7. Evaluation of acylcarnitine profiles in blood also showed an increase of long-chain acylcarnitines with tetradecenoylcarnitine (C14:1) as predominant acylcarnitine in all assays. Reduced palmitate oxidation was observed in cultured fibroblasts from five patients out of nine patients. VLCAD activity was assayed in fibroblasts or lymphocytes from 11 patients and was significantly reduced in all of them. 3.6. Molecular analysis ACADVL gene analysis conducted to the identification of 10 different sequence variations, all where missense mutations or in frame aminoacid deletions. Five mutations were already described: c.779C>T, c.842C>A, c.833_835del, c.1837C>T, and c.1505T>C [5,6]. The five others are new mutations, namely c.455G>A, c.535G>T, c.1376G>A, c.1500_1502del and c.1613G>C. For all families but two, molecular analysis showed homozygous (3 families) or compound heterozygous genotype (5 families). For the two remaining families, only one mutation in a heterozygous state was detected. The c.779C>T mutation was found in 5 out of 14 mutated alleles in patients of French origin (7 families). 4. Discussion We report the largest series of patients with manifestations of VLCAD deficiency persisting until adult age. Clinical symptoms

resemble closely to what is observed in the adult form of CPT II deficiency, with recurrent episodes of rhabdomyolysis induced by exercise, fasting or infections. However, in contrast to adult CPT II deficiency in which the clinical features are limited to skeletal muscle, early-onset extra-muscular symptoms were observed in one-third of the patients with VLCAD deficiency. These symptoms may be potentially life-threatening and were probably responsible for the early deaths in many of the siblings of our patients. This study also shows that despite the severity of the neonatal episodes, the clinical course of the patients may be favourable if they overcome the first years of life, thus widening the clinical spectrum of this disease. Muscle symptoms are the main clinical features after the age of 10, consisting in exercise intolerance. Muscle pain occurred a few hours after starting of physical activity, and episodes of rhabdomyolysis could be precipitated by prolonged exercise, fasting, cold or fever. A cumulative effect of exercise and cold could be also observed, with triggering of rhabdomyolysis after skiing in at least three patients. As previously reported in patients with metabolic rhabdomyolysis [22], acute renal failure rarely occurred despite the high incidence of myoglobinuria episodes with major raising of CK levels (only one patient necessitated intensive care). Although VLCAD deficiency may present with severe cardiac involvement during childhood, we did not find symptomatic cardiac abnormalities at adult age. However, a recent report of life-threatening disease with cardiomyopathy and hypoketotic hypoglycaemia in a previously healthy 32-year-old woman underscores the potential risk of severe complications even in adults [15].

328

P. Laforêt et al. / Neuromuscular Disorders 19 (2009) 324–329

Issues of pregnancies in women affected with VLCAD deficiency had not been reported before. In our series we observed a total of nine pregnancies in five women without any neonatal complication, but three patients experienced myoglobinuria after delivery. Thus prolonged labour should be avoided, and we also recommend the intravenous administration of glucose during the delivery. The diagnosis of fatty acid oxidation disorder was generally difficult to establish and often delayed, up to 33 years after onset of first manifestations (median age at diagnosis: 22 years), since standard biochemical analyses were frequently normal. Orientation towards a FAO disorder was possible in rare cases because of low plasma carnitine levels, or massive dicarboxylic aciduria during the course of a metabolic crisis in two patients during childhood. The most important biochemical test which allowed an accurate diagnosis was the measurement of plasma or blood spotted onto filter paper acylcarnitines by tandem mass spectrometry. This method has always been decisive for the diagnosis of VLCAD deficiency in our patients, showing an accumulation of long-chain acylcarnitines with tetradecenoylcarnitine (C14:1) as predominant acylcarnitine. These abnormalities were even observed at distance of rhabdomyolysis or acute metabolic decompensation. Interestingly, distinctive abnormal acylcarnitine profile are also observed in other fatty acid oxidation disorders such as mitochondrial trifunctional protein and multiple acyl-CoA dehydrogenase deficiencies which may be revealed by muscle weakness or rhabdomyolysis in adults. Flux of b-oxidation measured in cultured skin fibroblasts or in lymphocytes, was reduced in only 60% of cases, showing the limited sensibility of these methods for the detection of adult form of VLCAD deficiency. However most oxidation studies have been performed using 14C-labelled fatty acids, which have probably a lower sensitivity than those using 3H-labelled fatty acids [4]. These time-consuming techniques are probably no more longer useful to the diagnosis of VLCAD deficiency due to the reliability of C14:1 acylcarnitine assessment with tandem mass spectrometry. As previously reported [13,23], pathologic findings in muscle biopsies are most often non-specific. A moderate lipid storage was present in only one-third of cases, predominating in type I fibres. Of particular importance is the possibility to mask the diagnosis of a FAO defect due to the presence of mitochondrial abnormalities which may be erroneously considered as causative of the disease. The mild mitochondrial proliferation, associated with a decrease of respiratory chain activity and coenzyme Q levels that we observed in one patient, were probably secondary abnormalities. Similar observations have been recently made in patients with ETF-QO deficiency whose muscle lipidosis was first considered to be due to coenzyme Q deficiency [24]. ACADVL gene analysis of the patients confirmed the wide mutational spectrum of VLCAD deficiency [6,25] with the identification of 10 different mutations. The unidentified mutations in patients 5 and 10 may be located outside the examined regions, for instance in regulatory domains or in intronic regions not included in our amplified products and essential for ACADVL gene expression. Interestingly, the c.779C>T mutation was found in 4/10 families and consequently seems to be prevalent in our population. It has also been reported in German, Italian, Dutch and US patients [5,6,25]. All patients have at least one missense mutation, meaning that they are likely to have some residual enzyme activity. Moreover none of the mutations except one (which is present in heterozygous state in patient 11) affects the residues directly responsible for binding to the matrix side of the inner mitochondrial membrane [26]. Current treatments for VLCAD deficiency are based on low longchain fat diet supplemented with medium chain triglycerides (MCT), and carnitine supplementation. A study of dietary supplementation with triheptanoin, a seven-carbon medium chain fatty acid, also showed a remarkable improvement of cardiac and mus-

cular symptoms in three children [27]. The beneficial effect of carnitine supplementation or MCT diet was difficult to evaluate in our patients due to the high variability of rhabdomyolysis episodes, and the retrospective nature of this study. In conclusion, exercise intolerance and rhabdomyolysis are the main symptoms in adults with VLCAD deficiency, and our study confirms that plasma or blood acylcarnitine profile is the most reliable diagnostic test allowing detection of this FAO disorder. Therefore we recommend that the diagnostic approach of patients with exercise intolerance or recurrent rhabdomyolysis systematically includes acylcarnitine profile assessment in fasting state. Acknowledgments We thank Dr. Jacques Berthelot (CHU d’Angers) for referring patient 8, and Pr. Olivier Bletry (Hôpital Foch) for referring patient 11. References [1] DiMauro S, Melis-DiMauro PM. Muscle carnitine palmitoyltransferase deficiency and myoglobinuria. Science 1973;182:929–31. [2] Izai K, Uchida Y, Orii T, Yamamoto S, Hashimoto T. Novel fatty acid betaoxidation enzymes in rat liver mitochondria. I. Purification and properties of very-long-chain acyl coenzyme A dehydrogenase deficiency. J Biol Chem 1992;267:1027–33. [3] Bertrand C, Largillière C, Zabot MT, Mathieu M, Vianey-Saban C. Very long chain acyl-CoA dehydrogenase deficiency: identification of a new inborn error of mitochondrial fatty acid oxidation in fibroblasts. Biochim Biophys Acta 1993;1180:327–9. [4] Vianey-Saban C, Divry P, Brivet M, et al. Mitochondrial very-long-chain acylcoenzyme A dehydrogenase deficiency: clinical characteristics and diagnostic considerations in 30 patients. Clin Chim Acta 1998;269:43–62. [5] Andresen BS, Vianey-Saban C, Bross P, et al. The mutational spectrum in very long-chain acyl-CoA dehydrogenase deficiency. J Inherit Metab Dis 1996;19(2):169–72. [6] Andresen BS, Olpin S, Poorthuis BJHM, et al. Clear correlation of genotype with disease phenotype in very-long-chain Acyl-CoA dehydrogenase deficiency. Am J Hum Genet 1999;64:478–94. [7] Boneh A, Andresen BS, Gregersen N, et al. VLCAD deficiency: Pitfalls in newborn screening and confirmation of diagnosis by mutation analysis. Mol Genet Metab 2006;88(2):166–70. [8] Minetti C, Garavaglia B, Bado M, et al. Very-long-chain acyl-coenzyme A dehydrogenase deficiency in a child with recurrent myoglobinuria. Neuromuscul Disord 1998;8:3–6. [9] Merinero B, Pascual Pascual SI, Pérez-Cerda C, et al. Adolescent myopathic presentation in two sisters with very-long-chain acyl-CoA dehydrogenase deficiency. J Inherit Met Dis 1999;22:802–10. [10] Voermans NC, van Engelen BG, Kluijtmans LA, Stikkelbroeck NM, Hermus AR. Rhabdomyolysis caused by an inherited metabolic disease: very long chain acyl-CoA dehydrogenase deficiency. Am J Med 2006;119:176–83. [11] Ogilvie I, Pourfarzam M, Jackson S, et al. Very long-chain acyl coenzyme A dehydrogenase deficiency presenting with exercise-induced myoglobinuria. Neurology 1994;44:467–73. [12] Smelt AHM, Poorthuis JHM, Onkenhout W, et al. Very long-chain acyl coenzyme A dehydrogenase deficiency with adult onset. Ann Neurol 1998;43:540–4. [13] Scholte HR, Van Coster RNA, de Jonge PC, et al. Myopathy in very-long-chain acyl-CoA dehydrogenase deficiency: clinical and biochemical differences with the fatal cardiac phenotype. Neuromuscul Disord 1999;9:313–9. [14] Pons R, Cavadini P, Baratta S, et al. Clinical and molecular heterogeneity in very-long-chain acyl-CoA dehydrogenase deficiency. Pediatr Neurol 2000;22:98–105. [15] Kluge S, Kühnelt P, Block A, et al. A young woman with persistent hypoglycemia, rhabdomyolysis, and coma: recognizing fatty acid oxidation defects in adults. Crit Care Med 2003;31:1273–6. [16] Hoffman JD, Steiner RD, Paradise L, et al. Rhabdomyolysis in the military: recognizing late-onset very long-chain acyl Co-A dehydrogenase deficiency. Mil Med 2006;171:657–8. [17] Zia A, Kolodny EH, Pastores GM. Very long chain acyl-CoA dehydrogenase deficiency in a pair of mildly affected monozygotic twin sister in their late fifties. J Inherit Metab Dis 2007;30(5):817. [18] Divry P, Vianey-Saban C, Mathieu M. Determination of total fatty acids in plasma: cis-5-tetradecenoic acid (C14:1 omega-9) in the diagnosis of longchain fatty acid oxidation defects. J Inherit Metab Dis 1999;22:286–8. [19] Vianey-Saban C, Mousson B, Bertrand C, et al. Carnitine palmitoyl transferase I deficiency presenting as a Reye-like syndrome without hypoglycemia. Eur J Pediatr 1993;152:334–8. [20] Brivet M, Slama A, Saudubray JM, Legrand A, Lemonnier A. Rapid diagnosis of long chain fatty acid oxidation disorders using lymphocytes. Ann Clin Biochem 1995;35:154–9.

P. Laforêt et al. / Neuromuscular Disorders 19 (2009) 324–329 [21] den Dunnen JT, Antonarakis SE. Mutation nomenclature extensions and suggestions to describe complex mutations: a discussion. Hum Mutat 2000;15(1):7–12. [22] Melli G, Chaudhry V, Cornblath Rhabdomyolysis D. An evaluation of 475 hospitalized patients. Medicine 2005;84:377–85. [23] Ohashi Y, Hasegawa Y, Murayama K, et al. A new diagnostic test for VLCAD deficiency using immunohistochemistry. Neurology 2004;62:2209–13. [24] Gempel K, Topaloglu H, Talim B, et al. The myopathic form of coenzyme Q10 deficiency is caused by mutations in the electron-transferring-flavoprotein dehydrogenase (ETFDH) gene. Brain 2007;130:2037–44.

329

[25] Mathur A, Sims HF, Gopalakrishnan D, et al. Molecular heterogeneity in verylong-chain acyl-CoA dehydrogenase deficiency causing pediatric cardiomyopathy and sudden death. Circulation 1999;99(10):1337–43. [26] McAndrew RP, Wang Y, Mohsen A-W, et al. Structural basis for substrate fattyAcyl chain specificity: crystal structure of human very-long-chain acylcoenzyme A dehydrogenase. J Biol Chem 2008;283(14):9435–43. [27] Roe CR, Sweetman L, Roe DS, David F, Brunengraber H. Treatment of cardiomyopathy and rhabdomyolysis in long-chain fat oxidation disorders using an anaplerotic odd-chain triglyceride. J Clin Invest 2002;110: 259–69.