In conclusion, our findings are consistent with the severe MADD phenotype of the patient and underscore the importance of testing mRNA splicing consequences when predicting the effect of exonic sequence changes. Background: Carnitine-acylcarnitine translocase (CACT) deficiency is a life-threatening, rare autosomal recessive disease often presenting in early infancy with seizures, hypoketotic hypoglycemia, hyperammonemia, cardiomyopathy, liver failure, muscle weakness, and apnea, similar to other defects of long chain fatty acid beta-oxidation, with indistinguishable acylcarnitine profile form infantile CPTII deficiency. Previously, diagnosis of CACT deficiency relied on in vitro enzymatic assay that is tedious and not practical. Objective: To develop Molecular methods for definitive diagnosis of CACT deficiency. Method: Sanger sequencing and oligonucleotide aCGH were used for the analysis of the SLC25A20 gene. Literatures were reviewed. Results: Among 100 individuals suspected of having CACT deficiency, mutations were identified in 7 unrelated families. Eight novel mutations were found, including a large 25.9 kb deletion encompassing exons 5 to 9 of the gene detected by aCGH. All patients presented clinical features and acylcarnitine profiles at early life consistent with CACT or CPTII deficiency. However, sequence analysis of CPT2 gene did not find mutations. Conclusion: CACT deficiency is pan-ethnic. It may not be as rare as originally thought. Patients with infantile form of CPTII deficiency should be analyzed for mutations, including large deletions, in the SLC25A20 gene. We report our experience in managing three surviving children (7 years, 5 years and 22 months) with carnitine-acylcarnitine translocase deficiency, confirmed by mutation analysis of the SLC25A20 gene. Patient A, born prematurely at 32 weeks, developed ST elevation and ventricular tachycardia on cardiac monitor on day 17 when feeding was reintroduced after recovery from necrotizing enterocolitis. Patient B, who was a term infant already discharged from hospital, presented on day 3 with cardiorespiratory failure requiring cardiopulmonary resuscitation (CPR) in the Accident and Emergency Department. Patient C presented on day 1 with cardiorespiratory arrest successfully revived with CPR. All 3 children were put on frequent feeding with a high carbohydrate / low fat diet, mainly with medium-chain triglycerides, supplemented with walnut oil rich in essential fatty acids. Patients A & C had significant feeding problems with food refusal requiring gastrostomy feeding and central venous line to maintain adequate glucose infusion, whilst Patient B was on a less meticulous diet with infrequent decompensations and no feeding difficulty. Patient B (aged 5 years) suffered from hypoxic brain damage resulting in mild grade mental retardation. The other 2 children have normal development despite frequent episodes of decompensation with hyperammonaemia and rhabdomyolysis. Background: To evaluate the relationship between the blood acylcarnitine profiles and clinical picture in patients with late-onset muscular phenotype of carnitine palmitoyltransferase (CPT II) deficiency (MIM 255110). CPT II deficiency is an autosomal recessive disorder of carnitine dependent transport of long-chain fatty acids into mitochondrial matrix. Methods: We analysed clinical data and the blood acylcarnitine profiles [C16, C18:1, the (C16+C18:1)/C2 ratios] in 32 samples of two adult male patients with the muscular phenotype of CPT II deficiency. Results: Patients presented with myalgia/weakness in 12/32 examinations. The elevation of the (C16+C18:1)/C2 ratios was found in 10/12 symptomatic and 9/20 asymptomatic cases. The levels of C16 acylcarnitine were elevated in 3/12 symptomatic and 2/20 asymptomatic cases. The elevation of C18:1 acylcarnitine concentrations was observed in 5/12 symptomatic and 5/20 asymptomatic cases. Higher levels of C18:1 acylcarnitine concentrations were accompanied by the elevation of C16 acylcarnitine concentrations in 3/5 symptomatic and 2/5 asymptomatic cases. Conclusions: Our patients had no correlation between the absolute values of the (C16+C18:1)/C2 ratios, C16 and C18:1 acylcarnitine concentrations and clinical symptoms. The (C16+C18:1)/C2 ratio was the most sensitive marker for the diagnosis of CPT II deficiency. Supported by the grant MZ0VFN2005 of Ministry of Health of Czech Republic. Since valproic acid interferes with mitochondrial energy metabolism, carnitine imbalance may occur in liver, the major target of VPA-toxicity. Aim: To investigate the acylcarnitine profile in animal liver tissues. Methods: Wistar rats were treated with one single injection of sodium valproate (100, 500 mg/kg) or subjected to a subchronic regimen (100 mg/kg/day for two weeks). Carnitine and acylcarnitines were quantified in liver tissues using ESI-MS/MS. Results: There was a significant dose-related increase of free carnitine and total acylcarnitine levels in the liver of rats treated with a single injection of valproate as compared with controls. Acylcarnitine levels were normal in livers of rats submitted to subchronic treatment except for malonyl-, isovaleryl-, 3-hydroxyisovaleryl-, adipoyl-, nonanoyl-, cis-4-decenoyl-and decanoylcarnitine which remained significantly increased. Discussion: The results clearly show an accumulation of acylcarnitines in liver at the onset of valproate administration. However, the disturbances in carnitine homeostasis appeared to recover after a subchronic regimen, except for specific individual acylcarnitines involved in the metabolism of branched-chain amino acids and medium-chain fatty acids. Endogenous biosynthesis of carnitine or its redistribution from peripheral organs may play an important role in the rescue of free carnitine levels in liver after a drug induced stress on mitochondrial metabolism. Background: Primary systemic carnitine deficiency (PSCD) is caused by mutation in the SLC22A5 gene encoding carnitine transporter OCTN2. The disorder is characterized by cardiomyopathy, weakness, hypoglycemic hypoketotic encephalopathy and highly responsive to L-carnitine therapy. Objectives: The aim was to investigate identification of clinical phenotype and genotypes with emphasis to cardiac manifestation and response to L-carnitine therapy in 14 patients in order to provide genotype-phenotype correlations. Material and Methods: Mutations for SLC22A5 gene in 14 patients with PSCD followed at Istanbul Medical Faculty was confirmed by direct sequence analysis. Clinical phenotype; ECG, echocardiography, 24-hours holter-ECG; genotype; dosage, duration of L-carnitine therapy was evaluated. Results: Age at diagnosis was 5.7+10.7 years (range:0.2-41.0). Follow-up duration was 4.4+2.2 years (range:2-9). L-carnitine 178.8+63.7 mg/day (range:75-300) was recommended. Symptoms at referral were Reye-like syndrome(4), cardiac insufficiency(4), convulsions(1), psychomotor retardation(1) and family history ( Plasma acylcarnitines are important biomarkers in the diagnosis of mitochondrial fatty acid beta-oxidation disorders (mFAOD). Although regarded as a reflection of the acyl-CoAs accumulating intramitochondrially in mFAOD patients, the true etiology of plasma acylcarnitines still requires clarification. It is generally assumed that these intermediates are formed within mitochondria via the activity of carnitine palmitoyltransferase 2 (CPT2). However, their transport across the mitochondrial and plasma membrane into the extracellular space remains undefined. Herein we aimed to resolve the mechanism by which intramitochondrial acylcarnitines are synthesized and further transported from the mitochondrial matrix to the extracellular space. Using lentiviral shRNA, medium-chain acyl-CoA dehydrogenase (MCAD) gene was silenced in control, CPT2 and OCTN2 deficient human fibroblasts. Transfected cell lines were incubated with 120 μM of n-decanoic acid plus Lcarnitine. In shMCAD transfected cell lines beta-oxidation proceeds until the formation of C8-CoA which will accumulate intramitochondrially. Extracellular acylcarnitine profiles were analyzed by ESI-MS/MS. Preliminary results show the absence of C8-carnitine in the medium of CTP2 deficient cells, pointing towards the involvement of this enzyme in the production of medium-chain acylcarnitines. In OCTN2 deficient cell lines however, extracellular accumulation of C8-carnitine is observed suggesting that the cellular export of acylcarnitines does not depend on OCTN2. Background: Short/branched-chain acyl-CoA dehydrogenase deficiency (SBCAD or 2-methylbutyryl dehydrogenase) is a very uncommon autosomic recessive disorder of the isoleucine catabolic pathway, with variable and not well defined phenotype. We describe a case with clinical features not reported previously. Case Report: A 14 months old boy was referred for evaluation on arrival to our country. He is the first son of healthy consanguineous parents from Pakistan, with a normal psychomotor development until the age of 8 months. During pneumonia he suffered an acute neurological regression with seizures and feeding difficulties. On admission, the patient showed visual contact, severe generalized hypotonia, hyperreflexia, oral and upper limb dyskinesias. Metabolic screening revealed hyperlactacidemia and increase in 2-methylbutyrylglycine, 2ethylhydracrylic, ethylmalonic and methylsuccinic acids, all consistent with a diagnosis of SBCAD deficiency. Genetic testing is ongoing. Brain MRI shows bilateral striatal lesions. The patient is on a low-protein (lowisoleucin) diet. Discussion: The clinical spectrum of SBCAD deficiency is not well defined because of the small number of reported cases and their different phenotype. While it may be asymptomatic in some patients, in others it manifests as a severe neurological disorder. To our knowledge our patient is the first presenting with bilateral striatal involvement. The patient presented with brief hypoglycaemic seizures at the age of 3 and 4 days. Further diagnostic work-up revealed medium chain acylCoA dehydrogenase (MCAD)-deficiency. In the following years, she developed normally, liver and muscle function tests were normal. At the age of 11 years, isolated AST elevation was first noticed. AST values remained elevated ever since at 600-800 U/l (normal <35 U/l), all other liver enzymes, coagulation, CK and carnitine were normal. An infection with hepatotropic viruses could be excluded. Impaired fatty acid oxidation in MCAD-deficiency can compromise skeletal muscle, heart and liver function, especially in adolescents and adults. AST-elevation was considered to be related to MCAD-deficiency, further tests like liver ultrasound, echocardiography and ECG were unrevealing. At the age of 15 years, the girl presented first in our metabolic clinic. As organ functions were normal and there was isolated elevation of AST we considered macro-AST. The presence of macro-AST was confirmed by polyethylene glycol precipitation. Macro-AST is usually a harmless finding due to complex formation with immunoglobulins which cannot be excreted via urine. Conclusion: AST-elevation in this patient was unrelated to MCADdeficiency but due to macro-AST. IgE-elevation associated with pollenallergy may play a role. It has been estimated that 15-20% of the children with MCAD deficiency (MCADD) died suddenly during a first metabolic decompensation. Data from analysis of clinical symptoms preceeding sudden death are still rare. Case report: A 9 months old gypsy boy was found dead at home in the morning. A postmortal acylcarnitine profile from a dry blood spot raised the suspicon of MCADD. Mutation analysis revealed homozygosity for the 985A>G mutation in the ACADM gene. Retrospective analysis of the clinical course revealed that on the day preceeding his death the patient had developed vomiting and diarrhea without fever and any signs of respiratory infection. A consulted primary care paediatrician did not recommend hospitalization and suggested only oral rehydratation after vomiting had reiterated. Autopsy revealed fatty liver as well as severe cerebral edema. Microscopic examination of lungs revealed signs of acute viral infection. Virologic tests did not detect virus of seasonal influenza or pandemic influenza type A (H1N1). Sudden death in four children with MCADD have been described by Yusupov et al. despite newborn screening and in all vomiting was the dominant symptom preceeding sudden death (Molecular Genetics and Metabolism, 101, 2010, p. 33-39) . Clinical analysis of our case confirmed this observation. Introduction: MCADD is the most prevalent deficiency of mitochondrial fatty acid Beta-oxidation defects (FAOD) and is included in newborn screening program (NSP) of various occidental countries. France would implement MCADD in its NSP in future. Methods: through a retrospective study, we analysed 66 paediatric cases affected with MCADD, confirmed by enzymatic or molecular analysis by 3 French laboratories. Results: since the 1980s, 66 French cases have been collecte. According to these results, French MCADD's prevalence would be 1.1/million illustrating a probably large number of undiagnosed cases. Diagnosis was performed during acute decompensation in 86%, as sibling of a newly diagnosed case in 14%. Median age at diagnosis was 14 months. Clinical presentation was mostly hypoketotic hypoglycemia, liver involvement, and Reye syndrome. After 165months mean follow-up, 72% present full recovery from attack, 10% have neurological impairment, 18% died. Death rate was 63% before 3 months, 6% between 3 months and 3 years and 0% after 3 years. Death was always observed during the first metabolic attack. All patients screened after a diagnosis in sibship remained asymptomatic after 118-months mean follow-up. Conclusion: Ay present in France, MCADD is a severe under diagnosed disease, with high mortality and neurological sequelae. More than 60% of clinically manifested MCADD patients are homozygous for a single mutation the p. K304E (c. 985A>G). We have recently genotyped a cohort of 60 MCADD patients. During this study we found in heterozygosity with the p.K304E mutation a previously unreported variant p.G377V (c.1205 G>T). In order to infer the diseasecausing effect of this novel mutant we developed an E.coli expression system to produce a recombinant form of the p.G377V protein. This novel mutant showed 27% residual activity when compared with the hMCADwt recombinant protein. These results are consistent with the close proximity of residue 377 to the enzyme's substrate (Glu376) and FAD binding sites, the latter essential for dimerisation, and thus prove the disease-causing effect of the novel mutation. The developed expression system constitutes a powerful tool for the characterization of novel hMCAD mutants and open the way to the study of potential activity modulators of hMCAD variants aiming at the development of effective therapeutic approaches for this inherited metabolic disease. Background: MCADD deficiency is the most common disorder of fatty acid oxidation (1 in 15,000 live births in Western Europe). A single gene change, K304E, accounts for 90% of MCADD cases. The frequency of LCHADD is 1 in 70,000 live births in Western Europe. 60-86% of reported patients with isolated LCHAD deficiency have a prevalent E474Q mutation. Objectives: Following the recent diagnosis of 25 patients with b-oxidation defects (17 among them with LCHADD and 4 with MCADD) within 2 years of selective screening in our laboratory, we investigated the molecular features of LCHADD and MCADD in Russian patients. Patients and Methods: DNA extracted from dried blood spots of 21 patients and 792 randomized derived from newborns from Moscow city were studied. Results: All patients with LCHADD except one were homozygous for the mutation E474Q and all patients with MCADD were homozygous for the K304E mutation. 5/792 blood spots from newborns were heterozygous for the E474Q mutation , 2/722 blood spot heterozygous for the K304E and gives estimated newborn incidence of 1/84000 for LCHADD and 1/ 640000 for MCADD. Conclusion: LCHAD deficiency is prevalent among other disorders of mitochondrial b-oxidation and MCADD has unexpectedly low incidence in the Russian population. Background: Very long chain acyl-CoA dehydrogenase deficiency (VLCADD) is a disorder of long chain fatty acid beta-oxidation, that compromises energy homeostasis and leads to accumulation of long-chain fatty acids and derivatives. Patients may develop hypoglycemia, rhabdomyolysis, hepatomegaly and cardiomyopathy. The main goal of treatment is to avoid catabolism. In addition, many patients use a long chain fatty acid restricted diet. Because essential fatty acids (EFA) are long chain fatty acids, those patient may be at risk for developing EFA deficiency. Methods: We retrospectively studied EFA measurements performed in erythrocytes of Dutch VLCADD patients on long chain fatty acid restricted diet, via gas-chromatography. Results: 25 blood samples of 11 patients were available for analysis. Eight patients (18/25 samples) had decreased levels of linoleic acid (C18:2n-6). Alpha-linoleic acid (C18:3n-3) was not decreased in any of the patients (0/ 25). Elevated mead acid (C20:3n-9), a putative marker for EFA deficiency, was found in only one sample. Conclusion: VLCADD patients are more prone to become EFA deficient, especially for linoleic acid. Metabolic consultants should be aware of this possibility and supplement EFA's. Furthermore, although patients were EFA deficient, mead acid did not increase. Mead acid might therefore not be a good marker for EFA deficiency. In our centre we have been providing preimplantation genetic diagnosis (PGD) since 2007. Up to now we have accomplished 117 IVF cycles followed by PGD in 40 genetic diseases. The success rate of the procedure (calculated on the fetal heart beat pregnancy) is around 43%. The case report descibes PGD in a 39 year old woman. Her son from a consanguineous marriage (r=1/8) has LCHAD deficiency. Molecular analysis in the child showed homozygosity for the 1528 G-C mutation in the HADHA gene which maps to 2p23 (MIM ID 609016). Both parents are heterozygous for the 1528 G-C mutation and the risk of recurrence for their children is 25%. We used genetic haplotyping technique by multiplex PCR on products of MDA (multiple displacement amplification) from 1 blastomere biopsied from the embryo. 9 embryos were biopsied, 4 of them were wild homozygotes, 4 of them were heterozygotes and in 1 embryo monosomy X was confirmed. 8 of the embryos were recommended to transfer, only 1 embryo was actually transferred. The pregnancy was confirmed by ultrasound investigation. The karyotype of the foetus from the chorionic biopsy was 46,XX. Molecular analysis of HADHA gene did not confirm 1528 G-C mutation. Background: Bacillus cereus occasionally causes diarrheic and emetic food poisoning. It was reported that cereulide, emetic toxin of Bacillus cereus, is associated with encephalopathy and Reye-like syndrome that resemble fatty acid oxidation (FAO) disorders. However, the underlying mechanism for Reye-like syndrome associated with Bacillus cereus is not known. Herein, the effect of cereulide on FAO was determined. Materials and Methods: Control fibroblasts were cultured in the presence or absence of cereulide along with octanoic or palmitic acid as substrate. FAO capacity was determined by in vitro probe assay and tandem-massspectrometry. Results: Octanoic or palmitic acid led to an increase of acetylcarnitine (C2) without causing accumulation of medium (C4, C6, C8 and C10) and long (C12, C14 and C16) chain acylcarnitines in the cells cultured without cereulide. In contrast, cereulide decreased C2 and markedly increased C4, C6, C8 or C16 compared to cells treated with octanoic or palmitic acid alone. Conclusion: The acylcarnitine profile in the presence of cereulide was similar to neonatal multiple acyl-CoA dehydrogenase deficiency. The results indicate that cereulide severely inhibits mitochondrial FAO toward a broad range of acyl-CoAs. Malfunction of FAO resulting from intoxication of cereulide may be responsible for Reye-like syndrome associated with Bacillus cereus. Investigation of a fat oxidation disorder by acylcarnitine analysis is common practice in the differential diagnosis of sudden unexpected death in infancy (SUDI). Taking samples as soon as possible after death minimises post mortem artefactual changes. We describe three recent cases that illustrate the benefit of a clear and well implemented SUDI policy by contrasting findings with samples taken at autopsy: Case-1: A male infant collapsed and died on day 2. Bloodspots taken immediately after death showed increased long chain acylcarnitines; those at autopsy were far less suggestive of a fat oxidation defect. Carnitine acylcarnitine translocase deficiency was later confirmed by fibroblast studies. Case-2: A 16 month old girl, initially found to have an isolated increase of urinary suberylglycine, but with normal bloodspot acylcarnitines, died unexpectedly of respiratory failure. Bloodspots taken at the time of death show increases in short and medium chain acylcarnitines but samples collected at autopsy were difficult to interpret. Investigations into a possible riboflavin transporter defect are ongoing. Case-3: A one month old male infant died suddenly. Bloodspot acylcarnitine analysis showed a deficiency of long chain acylcarnitines (notably C16, C18:1 and C18) and normal free carnitine. Subsequent fibroblast studies confirmed carnitine palmitoyl transferase type 1 deficiency.