A Novel Mutation in CPT1A Resulting in Hepatic CPT Deficiency.

The present work presents a "from gene defect to clinics" pathogenesis study of a patient with a hitherto unreported mutation in the CPT1A gene. In early childhood, the patient developed a life-threatening episode (hypoketotic hypoglycemia, liver cytolysis, and hepatomegaly) evocative of a mitochondrial fatty acid oxidation disorder, and presented deficient fibroblast carnitine palmitoyltransferase 1 (CPT1) activity and homozygosity for the c.1783 C > T nucleotide substitution on exon 15 of CPT1A (p.R595W mutant). While confirming CPT1A deficiency, whole blood de novo acylcarnitine synthesis and the levels of carnitine and its esters formally linked intracellular free-carnitine depletion to intracellular carnitine esterification. Sequence alignment and modeling of wild-type and p.*R595W CPT1A proteins indicated that the Arg595 targeted by the mutated codon is phylogenetically well conversed. It contributes to a hydrogen bond network with neighboring residues Cys304 and Met593 but does not participate in the catalysis and carnitine pocket. Its replacement by tryptophan induces steric hindrance with the side chain of Ile480 located in α-helix 12, affecting protein architecture and function. This hindrance with Ile480 is also originally described with tryptophan 304 in the known mutant p.C304W CPT1A, suggesting that the mechanisms that invalidate CPT1A activity and underlie pathogenesis could be common in both the new (p.R595W) and previously described (p.C304W) mutants.

A novel mutation of the ACADM gene (c.145C>G) associated with the common c.985A>G mutation on the other ACADM allele causes mild MCAD deficiency: a case report.

A female patient, with normal familial history, developed at the age of 30 months an episode of diarrhoea, vomiting and lethargy which resolved spontaneously. At the age of 3 years, the patient re-iterated vomiting, was sub-febrile and hypoglycemic, fell into coma, developed seizures and sequels involving right hemi-body. Urinary excretion of hexanoylglycine and suberylglycine was low during this metabolic decompensation. A study of pre- and post-prandial blood glucose and ketones over a period of 24 hours showed a normal glycaemic cycle but a failure to form ketones after 12 hours fasting, suggesting a mitochondrial ß-oxidation defect. Total blood carnitine was lowered with unesterified carnitine being half of the lowest control value. A diagnosis of mild MCAD deficiency (MCADD) was based on rates of 1-14C-octanoate and 9, 10-3H-myristate oxidation and of octanoyl-CoA dehydrogenase being reduced to 25% of control values. Other mitochondrial fatty acid oxidation proteins were functionally normal. De novo acylcarnitine synthesis in whole blood samples incubated with deuterated palmitate was also typical of MCADD. Genetic studies showed that the patient was compound heterozygous with a sequence variation in both of the two ACADM alleles; one had the common c.985A>G mutation and the other had a novel c.145C>G mutation. This is the first report for the ACADM gene c.145C>G mutation: it is located in exon 3 and causes a replacement of glutamine to glutamate at position 24 of the mature protein (Q24E). Associated with heterozygosity for c.985A>G mutation, this mutation is responsible for a mild MCADD phenotype along with a clinical story corroborating the emerging literature view that patients with genotypes representing mild MCADD (high residual enzyme activity and low urinary levels of glycine conjugates), similar to some of the mild MCADDs detected by MS/MS newborn screening, may be at risk for disease presentation.

Deuterated palmitate-driven acylcarnitine formation by whole-blood samples for a rapid diagnostic exploration of mitochondrial fatty acid oxidation disorders.

BACKGROUND: The biochemical diagnosis of mitochondrial fatty acid oxidation defects (FAOD) currently rests on enzyme assays. A dynamic ex vivo exploration consisting of incubations of whole-blood samples with stable-labeled palmitate and determining leukocyte capacities to produce deuterated acylcarnitines was developed on healthy controls (n=52) and patients with very-long- (VLCADD) (n=2), medium- (MCADD) (n=6), or short- (SCADD) (n=1) chain acyl-CoA dehydrogenase deficiencies. METHODS: Incubations were optimized with L-carnitine and [16-(2)H(3), 15-(2)H(2)]-palmitate at 37 degrees C for various time periods on MCADD and control whole-blood samples. Labeled acylcarnitines were quantified by electrospray-ionization tandem mass spectrometry after thawing, extraction and derivatization to their butyl esters and the method was applied to patients with defects mentioned above. RESULTS: The production of acylcarnitines was linear until 6 h of incubation and optimal on 50 to 200 nmol deuterated substrate. A good discrimination between MCADD patient and control data was found, with median C8/C4 acylcarnitine production rate ratios of 81.0 (5th-95th percentile range: 16.6-209.9) and 0.21 (5th-95th percentile range: 0.06-0.79), respectively. The method also discriminated from controls the VLCADD and SCADD patients. Preliminary studies on a healthy control indicated that the storage at 4 degrees C does little or not alter capacities of whole-blood samples to generate labeled acylcarnitines over a period of 48 h. CONCLUSION: The rapid management afforded by the method, its abilities to characterize patients and to work on whole-blood samples after a stay of 24-48 h at 4 degrees C make it promising for the diagnostic exploration of FAOD.