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-ChainAcyl-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, Franceb Centre de Référence des Maladies Héréditaires du Métabolisme, INSERM U820, Centre de Biologie et de Pathologie Est, Hospices Civils de Lyon, 69677 Bron cedex, Francec Centre de reférence des Maladies Héréditaires du Métabolisme, Laboratoire de Biochimie-Hormonologie, Hôpital Robert-Debré, Assistance Publique-Hôpitaux de Paris, Paris, Franced Department of Biochemistry, Hôpital de Bicêtre, Assistance Publique-Hôpitaux de Paris, Paris, Le Kremlin-Bicêtre, Francee Centre de Référence Maladies Neuromusculaires Nantes-Angers, Département de Neurologie, CHU Angers, Francef Centre de Référence des maladies métaboliques de l’enfant, Unité de Médecine Infantile, CHU Timone Enfants, Assistance Publique-Hôpitaux de Marseille, Marseille, Franceg Clinique Ste-Odile, 6 rue Simonis, 67100 Strasbourg, Franceh Centre de Référence Neuropathies Rares et Maladies Musculaires Auvergne-Limousin, service de Génétique Médicale, CHU de Clermont-Ferrand, Francei Département de pédiatrie médicale, Hôpital Universitaire Dupuytren, CHU de Limoges, Francej Département de Neurologie, Hôpital Charles Nicolle, CHU de Rouen, Francek Service de Médecine Interne, CHU de Clermont-Ferrand, Francel INSERM U975, CRICM, Paris, Francem Research Unit for Molecular Medicine, Institute of Human Genetics, Aarhus University, Aarhus, Denmark

a r t i c l e i n f o

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

Keywords:Muscle lipidosisVery Long-Chain Acyl-Coenzyme AdehydrogenaseFatty acid oxidationMetabolic myopathy

0960-8966/$ - see front matter � 2009 Elsevier B.V. Adoi:10.1016/j.nmd.2009.02.007

* Corresponding author. Address: Institut de MGroupe Hospitalier Pitié-Salpêtrière, 47-83 boulevaCedex 13, France. Tel.: +33 142163776; fax: +33 1421

E-mail address: pascal.laforet@psl.ap-hop-paris.fr

a b s t r a c t

Very Long-Chain Acyl-CoA dehydrogenase (VLCAD) deficiency is an inborn error of mitochondrial long-chain 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, bio-chemical 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 exhibitedexercise intolerance and recurrent rhabdomyolysis episodes, which were generally triggered by strenu-ous exercise, fasting, cold or fever (mean age at onset: 10 years). Inaugural life-threatening general man-ifestations also occurred before the age of 3 years in four patients. Increased levels of long-chainacylcarnitines with tetradecenoylcarnitine (C14:1) as the most prominent species were observed in allpatients. Muscle biopsies showed a mild lipidosis in four patients. For all patients but two, molecularanalysis showed homozygous (4 patients) or compound heterozygous genotype (7 patients). For thetwo remaining patients, only one mutation in a heterozygous state was detected. This study confirms thatVLCAD deficiency, although being less frequent than CPT II deficiency, should be systematically consid-ered in the differential diagnosis of exercise-induced rhabdomyolysis. Measurement of fasting blood acyl-carnitines by tandem mass spectrometry allows accurate biochemical diagnosis and should therefore beperformed in all patients presenting with unexplained muscle exercise intolerance or rhabdomyolysis.

� 2009 Elsevier B.V. All rights reserved.

Disclosure: The authors report no conflicts of interest.

1. Introduction

Among the metabolic myopathies, mitochondrial fatty acid oxi-dation (FAO) defects are probably the most difficult to identify due

ll rights reserved.

yologie, Bâtiment Babinski,rd de l’Hôpital, 75651 Paris63793.

(P. Laforêt).

to their transient clinical and biological manifestations, as it hasbeen initially described in patients with carnitine palmi-toyltransferase (CPT II) deficiency [1]. Very Long-Chain Acyl-CoAdehydrogenase (VLCAD) is bound to the inner mitochondrial mem-brane, and catalyses the first step of the long-chain fatty acid b-oxidation spiral [2]. VLCAD deficiency is inherited as an autosomalrecessive trait, and three phenotypes have been described accord-ing to the age at onset of clinical manifestations [3–6]: (1) a severeinfantile form presenting in the neonatal period with hypertrophiccardiomyopathy and liver failure; (2) a childhood onset betweenages 1 and 13 years with hypoketotic hypoglycaemia as the main

P. Laforêt et al. / Neuromuscular Disorders 19 (2009) 324–329 325

presenting feature; and (3) a juvenile or adult-onset muscularform characterized by recurrent episodes of rhabdomyolysis trig-gered by prolonged exercise or fasting. The milder form of VLCADdeficiency is increasingly recognised due to the more wide spreaduse of tandem mass spectrometry (MS/MS), allowing the detectionof abnormal long-chain acylcarnitines in blood samples [7–10].However, clinical observations of patients with late-onset presen-tation of VLCAD deficiency remain somewhat rare [8–17]. We re-port here the clinical and biochemical results in 13 adultpatients all presenting with muscle complications of VLCADdeficiency.

2. Patients and methods

2.1. Patients

Thirteen patients originating from 10 different families havebeen examined by one of the authors. Only one case history waspreviously reported [3]. Clinical and biochemical data were retro-spectively collected, and 12 patients are still regularly followedin neuromuscular or metabolic French reference centres. One pa-tient died accidentally.

2.2. Plasma and urine biochemistry

Free and total carnitine levels in plasma were determined usingconventional methods. Urinary organic acid profiling was per-formed using gas chromatography–mass spectrometry (GC/MS)of methyl or trimethylsilyl derivatives. Acylcarnitines in driedblood spots or plasma were measured as methyl or butyl deriva-tives using tandem mass spectrometry with electrospray ionisa-tion (ESI–MS/MS). Plasma total long-chain fatty acids werestudied by gas chromatography–mass spectrometry of theirmethyl esters [18].

2.3. Muscle biopsy

Deltoid or quadriceps muscle biopsy was taken by open biopsyand processed for routine morphological techniques in 10 patients.Spectrophotometric analysis of the respiratory chain complexeswere performed using conventional methods on homogenatesfrom frozen samples of muscle in four patients.

2.4. Fatty acid oxidation studies and enzyme activities

[1-14C] palmitate oxidation and tritiated water release experi-ments from [9,10(n)-3H] palmitate and [9,10(n)-3H] myristatewere performed in intact cultured fibroblasts or lymphocytes aspreviously described [19,20]. Membrane-bound VLCAD activitywas assayed, with palmitoyl-CoA as substrate, in a membranepreparation from cultured skin fibroblasts (or muscle tissue orlymphocytes) [4].

2.5. DNA analysis

Patient’s DNA was extracted from whole blood or culturedfibroblasts using conventional methods. The 20 exons and exon–intron boundaries of the ACADVL gene were amplified from geno-mic DNA using standard PCR procedures with primers previouslydescribed [6] or in-house designed primers. Direct bidirectionalsequencing of purified PCR products was performed using the Big-Dye Terminator v3.1 Sequencing Kit (Applied Biosystems) and anABI 3100 genetic analyser. Sequences were analysed using Seq-scape Analysis Software (Applied Biosystems) in comparison withGenBank Reference genomic sequence (NC_000017). The mutation

nomenclature used in this work follows the guidelines of den Dun-nen and Antonarakis [21].

3. Results

3.1. Family history (Table 1a, b)

All patients but two (6,7) were born from non-consanguineousparents. Parents of patients 6 and 7 were Turkish, and parents ofpatients 5 and 11 originated from Philippine and Greece, respec-tively. In three sibships, family histories were remarkable by theoccurrence of sudden deaths of 8 siblings between 2 days and20 months of age (median: 6.5 months), often triggered by diar-rhea or vomiting.

3.2. Clinical features at onset (Table 1a, b)

Age at onset of the disease varied from birth to 13 years (med-ian: 7 years). In four patients the first symptoms occurred beforethe age of 3 years. Onset in patient 1 was a cardio-respiratory ar-rest at 36 h of life. Patient 3 was hospitalized at 6 and 14 monthsof age for hypoglycaemic episodes with rhabdomyolysis (CK level:6510 UI/L). Patient 4 had repeated hypoglycaemias and musclefatigability since the age of 1 year. Patient 5 presented a Reye-likesyndrome at 2.5 years of age. Hepatomegaly was also reported dur-ing childhood in all these four patients. In the nine other patientsthe first manifestations were exercise intolerance or myoglobinu-ria (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 was30.5 years (12–46 years). The main clinical manifestation wasexercise intolerance. Diffuse muscle pain and prolonged stiffnesslasting up to 48 h, generally occurred after 1 or 2 h of strenuousexercises. Other triggering factors were fasting (7 patients), cold(4 patients), and fever (3 patients). Nine patients experimentedepisodes of rhabdomyolysis after the practice of intensive leisurephysical activity like cycling, skiing, swimming, or playing tennis.Acute renal failure complicating a rhabdomyolysis episode oc-curred in four patients: twice after skiing (patients 5 and 10), onceafter carrying heavy weights (patient 7), and once after playingfootball (patient 13). A fasting test performed at age 9 in patient6, when diagnosis was not assessed, induced an acute episode ofrhabdomyolysis with hypoketotic hypoglycaemia, and massivedicarboxylic aciduria. All patients recovered without renal seque-lae. The frequency of rhabdomyolysis crises varied from less thanone to six attacks per year. CK levels were generally normal whenmeasured at rest, and patients did not show any sign of muscleweakness or atrophy at clinical examination. An echocardiographywas performed in all patients, revealing a mild ventricular hyper-trophy in patient 4, and a moderate septum hypokinesia in patient5. Conduction defects or arrhythmia were never detected.

A total of nine pregnancies occurred in five women. Two mis-carriages occurred in patient 6 before a normal pregnancy. Deliverywas carried out after caesarean section in patient 6 and 9, withoutany foetal or neonatal abnormality. Three women suffered fromdelayed myalgias after their first delivery (patients 5, 6, and 12),with concomitant elevation of CK levels up to 17,000 UI/L or myo-globinuria, but renal function always remained normal.

Various treatments were administrated, either alone or in asso-ciation: L-carnitine, riboflavin, coenzyme Q10, or dietary therapywith supplements of medium chain triglycerides. Patients 11 and13 experienced a subjective improvement, respectively, afterCoQ10 (300 mg/day) and riboflavin (80 mg/day) treatments.

Table 1aClinical features.

Pt I.D. Familyhistory

Sex Age atonset(years)

Age at diagnosis(years)

Current age(years)

Presentingsymptoms

Age at onsetof exerciseintolerance

Provocatingfactorsof myalgias

Frequency ofmyoglobinuriaepisodes per year

1

Sib1D1

F Birth Birth 16 Cardiac arrest 5 Exercise 1Hypotonia FeverHepatomegaly

2 F 5 0.2 12 Exercise intolerance 5 Exercise 1Fever

3 – M 0.5 23 23 Hypoglycaemia 20 Exercise 1Hepatomegaly FastingHigh CK level Cold

4 – M 1 12 21 Hypoglycaemia 1 Exercise <1Hepatomegaly FastingExercise intolerance

5 – F 2.5 22 31 Reye syndrome 9 Exercise 2Hepatomegaly Cold

Fasting6 Sib2D4 F 7 17 25 Exercise intolerance 7 Exercise 1

Myoglobinuria ColdFasting

7 M 11 11 � Exercise intolerance 11 Exercise <1Dizziness Cold

Fasting8 Sib1D3 M 9 21 29 Exercise intolerance 9 Exercise <1

Myoglobinuria

9 F 9 32 40 Exercise intolerance 9 Exercise <110 – M 10 40 42 Myoglobinuria Exercise 611 – F 10 43 46 Exercise intolerance 11 Exercise 2

Myoglobinuria Fasting12 – F 10 36 39 Exercise intolerance 11 Exercise 2

Myoglobinuria13 – M 13 34 41 Exercise intolerance 13 Exercise 3

Fasting

Table 1bClinical features.

Pt I.D. Higher recordedCK levels (UI/L)

Renal failure Number of pregnancies Echocardiography Treatment

1 13,400 � Normal Carnitine, riboflavin,MCT diet

2 2500 � Normal Carnitine, riboflavin,MCT diet

3 215,700 � Normal –4 10,000 � LV hypertrophy Carnitine, riboflavin,

MCT diet5 544,000 + 1 Septal hypokinesia –6 7720 � 2 Normal Carnitine, MCT diet7 N/a + Normal –8 141,500 � Normal Carnitine9 16,800 � 1 Normal Carnitine

10 28,000 + Normal Carnitine11 22,260 � 2 Normal CoQ1012 30,000 � 3 Normal Carnitine13 83,100 + Normal Riboflavin

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.

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

3.4. Muscle analysis

A muscle biopsy was performed in 10 patients. Histologicalanalysis was normal or showed non-specific abnormalities in sixpatients. A mild muscle lipidosis was observed in only four pa-tients (patients 3, 10, 11, 12). In addition, oxidative staining de-tected a subsarcolemmal mitochondrial accumulation in patient11, with respiratory chain analysis showing a combined defect ofcomplexes I + III, and II + III (respectively, 5 nmol/min/mg prot,controls 18 ± 12; and 8 nmol/min/mg prot, controls 22 ± 8). The le-vel 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 pa-tients 8, 12 and 13.

3.5. Biochemical studies (Table 2)

Free carnitine levels were lowered in seven out of 10 untreatedpatients, while esterified carnitine level was significantly increasedin three patients. Organic acid profile assessed in urine of five pa-tients between attacks of myoglobinuria was always normal, bycontrast with the detection of massive dicarboxylic aciduria atthe time of acute general manifestations during childhood in twopatients (patients 2 and 6). Repeated measurements of plasma

Table 2Results of laboratory investigations.

Pt I.D. Musclemorphology

Plasmacarnitine(lmol/L)

PlasmaC14:1cis-5(lmol/L)

Acylcarnitineprofile:C14:1(lmol/L)

Fatty acidoxidationstudies inlymphocytes orfibroblasts(% controls)

VLCAD activityin fibroblasts(nmol ETF/min/mg prot)

ACADVL gene substitutions

FC EC cDNAsubstitutions

Predicted aminoacid change

1 ND 30 38 16–560 1.6–8.5 CPF 56% 0.29 c.779C>T p.Thr260Metc.842C>A p.Ala281Asp

2 ND 63 15 46–433 3.3 CPF 35% 0.15 c.779C>T p.Thr260Metc.842C>A p.Ala281Asp

3 Regenerating fibres,lipidosis

42 18 ND 1.5 ND ND c.535G>T p.Gly179Trpc.1613G>C p.Arg538Pro

4 ND 4 13 119–414 2.3–9.3 CPF 28% 0.50 c.1837C>T p.Arg613Trpc.1837C>T p.Arg613Trp

5 Necrosis, 16 4 50 3 HPF 38% 0.06 c.1505T>C p.Leu502ProInflammation HMF 32% ? ?

6 Size inequality type II Cfibres

35 23 1.3; 273 0.7; 8.5 CPF N 0.50 c.1500_1502del p.501delLeuc.1500_1502del p.501delLeu

7 Atrophic fibres 30 10 <1; 20 0.4; 1.4 CPF N 0.68 c.1500_1502delc.1500_1502del

p.501delLeup.501delLeu

8 N ND ND 72 2.5 CPF N 0.69 c.779C>T p.Thr260Metc.1500_1502del p.501delLeu

9 N ND ND 4.8 4.0 ND ND c.779C>T p.Thr260Metc.1500_1502del p.501delLeu

10 Mild lipidosis in type Ifibres

ND ND 101 3.1 ND 0.28 c.779C>T p.Thr260Met? ?

11 Mild lipidosis 24 16 ND 1.3 ND 0.28 (lymphocytes) c.833_835del p.278delLysMitochondrialaccumulation

c.1376G>A p.Arg459Gln

12 Central nuclei 20 22 ND 1.8 CPL N 0.24 (lymphocytes) c.455G>A p.Gly152AspMild lipidosis c.833_835del p.278delLys

13 Necrotic andregenerating fibres

10 5 234 3.1 CPF 49% 0.14 c.779C>T p.Thr260Metc.779C>T p.Thr260Met

Controls 40 ± 10 14 ± 6 <2 <0.2 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 infibroblasts. 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.

P. Laforêt et al. / Neuromuscular Disorders 19 (2009) 324–329 327

fatty acids showed increased levels of cis-5-tetradecenoic acid(C14:1 cis-5) even when they were clinically asymptomatic, exceptfor one assessment performed under glucose infusion in patients 6and 7. Evaluation of acylcarnitine profiles in blood also showed anincrease of long-chain acylcarnitines with tetradecenoylcarnitine(C14:1) as predominant acylcarnitine in all assays. Reduced palmi-tate oxidation was observed in cultured fibroblasts from five pa-tients out of nine patients. VLCAD activity was assayed infibroblasts or lymphocytes from 11 patients and was significantlyreduced in all of them.

3.6. Molecular analysis

ACADVL gene analysis conducted to the identification of 10 dif-ferent sequence variations, all where missense mutations or inframe 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 buttwo, molecular analysis showed homozygous (3 families) or com-pound heterozygous genotype (5 families). For the two remainingfamilies, only one mutation in a heterozygous state was detected.The c.779C>T mutation was found in 5 out of 14 mutated allelesin patients of French origin (7 families).

4. Discussion

We report the largest series of patients with manifestations ofVLCAD deficiency persisting until adult age. Clinical symptoms

resemble closely to what is observed in the adult form of CPT IIdeficiency, with recurrent episodes of rhabdomyolysis induced byexercise, fasting or infections. However, in contrast to adult CPTII deficiency in which the clinical features are limited to skeletalmuscle, early-onset extra-muscular symptoms were observed inone-third of the patients with VLCAD deficiency. These symptomsmay be potentially life-threatening and were probably responsiblefor the early deaths in many of the siblings of our patients. Thisstudy also shows that despite the severity of the neonatal episodes,the clinical course of the patients may be favourable if they over-come the first years of life, thus widening the clinical spectrumof this disease.

Muscle symptoms are the main clinical features after the age of10, consisting in exercise intolerance. Muscle pain occurred a fewhours after starting of physical activity, and episodes of rhabdomy-olysis could be precipitated by prolonged exercise, fasting, cold orfever. A cumulative effect of exercise and cold could be also ob-served, with triggering of rhabdomyolysis after skiing in at leastthree patients. As previously reported in patients with metabolicrhabdomyolysis [22], acute renal failure rarely occurred despitethe high incidence of myoglobinuria episodes with major raisingof CK levels (only one patient necessitated intensive care).Although VLCAD deficiency may present with severe cardiacinvolvement during childhood, we did not find symptomatic car-diac abnormalities at adult age. However, a recent report oflife-threatening disease with cardiomyopathy and hypoketotichypoglycaemia in a previously healthy 32-year-old woman under-scores 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 deficiencyhad not been reported before. In our series we observed a total ofnine pregnancies in five women without any neonatal complica-tion, but three patients experienced myoglobinuria after delivery.Thus prolonged labour should be avoided, and we also recommendthe intravenous administration of glucose during the delivery.

The diagnosis of fatty acid oxidation disorder was generally dif-ficult to establish and often delayed, up to 33 years after onset offirst manifestations (median age at diagnosis: 22 years), since stan-dard biochemical analyses were frequently normal. Orientation to-wards a FAO disorder was possible in rare cases because of lowplasma carnitine levels, or massive dicarboxylic aciduria duringthe course of a metabolic crisis in two patients during childhood.The most important biochemical test which allowed an accuratediagnosis was the measurement of plasma or blood spotted ontofilter paper acylcarnitines by tandem mass spectrometry. Thismethod has always been decisive for the diagnosis of VLCAD defi-ciency in our patients, showing an accumulation of long-chainacylcarnitines with tetradecenoylcarnitine (C14:1) as predominantacylcarnitine. These abnormalities were even observed at distanceof rhabdomyolysis or acute metabolic decompensation. Interest-ingly, distinctive abnormal acylcarnitine profile are also observedin other fatty acid oxidation disorders such as mitochondrial tri-functional protein and multiple acyl-CoA dehydrogenase deficien-cies which may be revealed by muscle weakness orrhabdomyolysis in adults.

Flux of b-oxidation measured in cultured skin fibroblasts or inlymphocytes, was reduced in only 60% of cases, showing the lim-ited sensibility of these methods for the detection of adult formof VLCAD deficiency. However most oxidation studies have beenperformed using 14C-labelled fatty acids, which have probably alower sensitivity than those using 3H-labelled fatty acids [4]. Thesetime-consuming techniques are probably no more longer useful tothe diagnosis of VLCAD deficiency due to the reliability of C14:1acylcarnitine assessment with tandem mass spectrometry.

As previously reported [13,23], pathologic findings in musclebiopsies are most often non-specific. A moderate lipid storagewas present in only one-third of cases, predominating in type I fi-bres. Of particular importance is the possibility to mask the diag-nosis of a FAO defect due to the presence of mitochondrialabnormalities which may be erroneously considered as causativeof the disease. The mild mitochondrial proliferation, associatedwith a decrease of respiratory chain activity and coenzyme Q levelsthat we observed in one patient, were probably secondary abnor-malities. Similar observations have been recently made in patientswith ETF-QO deficiency whose muscle lipidosis was first consid-ered to be due to coenzyme Q deficiency [24].

ACADVL gene analysis of the patients confirmed the wide muta-tional spectrum of VLCAD deficiency [6,25] with the identificationof 10 different mutations. The unidentified mutations in patients5 and 10 may be located outside the examined regions, for instancein regulatory domains or in intronic regions not included in ouramplified products and essential for ACADVL gene expression. Inter-estingly, the c.779C>T mutation was found in 4/10 families and con-sequently seems to be prevalent in our population. It has also beenreported in German, Italian, Dutch and US patients [5,6,25]. All pa-tients have at least one missense mutation, meaning that they arelikely to have some residual enzyme activity. Moreover none ofthe mutations except one (which is present in heterozygous statein patient 11) affects the residues directly responsible for bindingto the matrix side of the inner mitochondrial membrane [26].

Current treatments for VLCAD deficiency are based on low long-chain fat diet supplemented with medium chain triglycerides(MCT), and carnitine supplementation. A study of dietary supple-mentation with triheptanoin, a seven-carbon medium chain fattyacid, also showed a remarkable improvement of cardiac and mus-

cular symptoms in three children [27]. The beneficial effect of car-nitine supplementation or MCT diet was difficult to evaluate in ourpatients due to the high variability of rhabdomyolysis episodes,and the retrospective nature of this study.

In conclusion, exercise intolerance and rhabdomyolysis are themain symptoms in adults with VLCAD deficiency, and our studyconfirms that plasma or blood acylcarnitine profile is the most reli-able diagnostic test allowing detection of this FAO disorder. There-fore we recommend that the diagnostic approach of patients withexercise intolerance or recurrent rhabdomyolysis systematicallyincludes acylcarnitine profile assessment in fasting state.

Acknowledgments

We thank Dr. Jacques Berthelot (CHU d’Angers) for referring pa-tient 8, and Pr. Olivier Bletry (Hôpital Foch) for referring patient 11.

References

[1] DiMauro S, Melis-DiMauro PM. Muscle carnitine palmitoyltransferasedeficiency and myoglobinuria. Science 1973;182:929–31.

[2] Izai K, Uchida Y, Orii T, Yamamoto S, Hashimoto T. Novel fatty acid beta-oxidation enzymes in rat liver mitochondria. I. Purification and properties ofvery-long-chain acyl coenzyme A dehydrogenase deficiency. J Biol Chem1992;267:1027–33.

[3] Bertrand C, Largillière C, Zabot MT, Mathieu M, Vianey-Saban C. Very longchain acyl-CoA dehydrogenase deficiency: identification of a new inborn errorof mitochondrial fatty acid oxidation in fibroblasts. Biochim Biophys Acta1993;1180:327–9.

[4] Vianey-Saban C, Divry P, Brivet M, et al. Mitochondrial very-long-chain acyl-coenzyme A dehydrogenase deficiency: clinical characteristics and diagnosticconsiderations in 30 patients. Clin Chim Acta 1998;269:43–62.

[5] Andresen BS, Vianey-Saban C, Bross P, et al. The mutational spectrum in verylong-chain acyl-CoA dehydrogenase deficiency. J Inherit Metab Dis1996;19(2):169–72.

[6] Andresen BS, Olpin S, Poorthuis BJHM, et al. Clear correlation of genotype withdisease phenotype in very-long-chain Acyl-CoA dehydrogenase deficiency. AmJ Hum Genet 1999;64:478–94.

[7] Boneh A, Andresen BS, Gregersen N, et al. VLCAD deficiency: Pitfalls innewborn screening and confirmation of diagnosis by mutation analysis. MolGenet Metab 2006;88(2):166–70.

[8] Minetti C, Garavaglia B, Bado M, et al. Very-long-chain acyl-coenzyme Adehydrogenase 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 myopathicpresentation in two sisters with very-long-chain acyl-CoA dehydrogenasedeficiency. 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 chainacyl-CoA dehydrogenase deficiency. Am J Med 2006;119:176–83.

[11] Ogilvie I, Pourfarzam M, Jackson S, et al. Very long-chain acyl coenzyme Adehydrogenase deficiency presenting with exercise-induced myoglobinuria.Neurology 1994;44:467–73.

[12] Smelt AHM, Poorthuis JHM, Onkenhout W, et al. Very long-chain acylcoenzyme A dehydrogenase deficiency with adult onset. Ann Neurol1998;43:540–4.

[13] Scholte HR, Van Coster RNA, de Jonge PC, et al. Myopathy in very-long-chainacyl-CoA dehydrogenase deficiency: clinical and biochemical differences withthe fatal cardiac phenotype. Neuromuscul Disord 1999;9:313–9.

[14] Pons R, Cavadini P, Baratta S, et al. Clinical and molecular heterogeneity invery-long-chain acyl-CoA dehydrogenase deficiency. Pediatr Neurol2000;22:98–105.

[15] Kluge S, Kühnelt P, Block A, et al. A young woman with persistenthypoglycemia, rhabdomyolysis, and coma: recognizing fatty acid oxidationdefects 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 dehydrogenasedeficiency in a pair of mildly affected monozygotic twin sister in their latefifties. J Inherit Metab Dis 2007;30(5):817.

[18] Divry P, Vianey-Saban C, Mathieu M. Determination of total fatty acids inplasma: cis-5-tetradecenoic acid (C14:1 omega-9) in the diagnosis of long-chain 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 Ideficiency presenting as a Reye-like syndrome without hypoglycemia. Eur JPediatr 1993;152:334–8.

[20] Brivet M, Slama A, Saudubray JM, Legrand A, Lemonnier A. Rapid diagnosis oflong chain fatty acid oxidation disorders using lymphocytes. Ann Clin Biochem1995;35:154–9.

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

[21] den Dunnen JT, Antonarakis SE. Mutation nomenclature extensions andsuggestions to describe complex mutations: a discussion. Hum Mutat2000;15(1):7–12.

[22] Melli G, Chaudhry V, Cornblath Rhabdomyolysis D. An evaluation of 475hospitalized patients. Medicine 2005;84:377–85.

[23] Ohashi Y, Hasegawa Y, Murayama K, et al. A new diagnostic test for VLCADdeficiency using immunohistochemistry. Neurology 2004;62:2209–13.

[24] Gempel K, Topaloglu H, Talim B, et al. The myopathic form of coenzyme Q10deficiency is caused by mutations in the electron-transferring-flavoproteindehydrogenase (ETFDH) gene. Brain 2007;130:2037–44.

[25] Mathur A, Sims HF, Gopalakrishnan D, et al. Molecular heterogeneity in very-long-chain acyl-CoA dehydrogenase deficiency causing pediatriccardiomyopathy and sudden death. Circulation 1999;99(10):1337–43.

[26] McAndrew RP, Wang Y, Mohsen A-W, et al. Structural basis for substrate fatty-Acyl chain specificity: crystal structure of human very-long-chain acyl-coenzyme A dehydrogenase. J Biol Chem 2008;283(14):9435–43.

[27] Roe CR, Sweetman L, Roe DS, David F, Brunengraber H. Treatment ofcardiomyopathy and rhabdomyolysis in long-chain fat oxidation disordersusing an anaplerotic odd-chain triglyceride. J Clin Invest 2002;110:259–69.