Molecular and enzymatic characterization of a unique carnitine palmitoyltransferase 1A mutation in the Hutterite community.

Hepatic carnitine palmitoyltransferase 1 (CPT1A) deficiency is a rare disorder of mitochondrial fatty acid oxidation inherited as an autosomal recessive trait. Symptomatology comprises attacks of hypoketotic hypoglycemia with risk of sudden death or neurological sequelae. Only one CPT1A mutation has been reported so far. Identification of the disease-causing mutations allows both insights into the structure-function relationships of CPT1A and management of the patients and their relatives. The molecular analysis of CPT1A deficiency in a large Hutterite kindred illustrates this point. Both cDNA and genomic DNA analysis demonstrate that the affected patients are homozygous for a 2129G>A mutation predicting a G710E substitution. Studies in fibroblasts from one patient as well as heterologous expression of the mutagenized CPT1A in yeast show that the G710E mutation alters neither mitochondrial targeting nor stability of the CPT1A protein. By contrast, kinetic studies conclusively establish that the mutant CPT1A is totally inactive, indicating that the G710E mutation dramatically impairs the catalytic function of CPT1A. Finally, due to a strongly suspected founder effect for the origin of CPT1A deficiency in this Hutterite kindred, identification of this disease-causing mutation allows the setup of a targeted DNA-based newborn screening in this at-risk population.

Liver transplantation in propionic acidaemia.

Despite the improvement in dietary therapy during the past 20 years, the overall outcome of severe forms of propionic acidaemia (PA) remains often disappointing. Good results can be obtained at a very high price in terms of medical attention, family burden and high cost. In most early onset forms of PA, the intake of natural protein must be rigidly restricted to 8-12 g/day for the first 3 years of life, and then slowly increased to 15-20 g/day by the age of 6-8 years. Supplementation with a precursor-free aminoacid mixture to provide 1.5 g/kg protein per day is generally recommended, although remains controversial. From the age of 1 year onward, these children are often severely anorectic and most of the diet must be delivered by nocturnal gastric drip feeding or gastrostomy. Metronidazole is very effective in reducing the excretion of propionate metabolites derived from the gut. L-carnitine (50 to 100 mg/kg) is systematically given to promote propionylcarnitine synthesis and excretion. We report here a retrospective study of 33 patients with PA diagnosed during the last 20 years in our hospital. Of them, 2 have been liver transplanted. In these two patients who presented frequent severe and unexpected metabolic decompensations despite good compliance with the dietary therapy, orthotopic liver transplantation (OLT) was done at 7 and 9 years respectively. One child died 15 months after transplantation due to a severe lymphoproliferative disorder; the other child now aged 13.5 years is doing well. Despite a persistent methylcitrate excretion, she is under normal moderate daily protein intake (40-50 g/day) and still on carnitine supplementation. Interestingly, another patient who filled the criteria for OLT (very frequent and severe decompensations leading to frequent admissions to the intensive care unit despite excellent dietary management) was also placed on the list for OLT. From the time he was registered onward, he experienced no further episodes of metabolic decompensation, there was almost no interruption in his daily intake and he gained height and weight and developed well. He was finally removed from the list and is still doing very well 2 years thereafter. Correction of propionylCoA carboxylase deficiency restricted to hepatic tissues seems to induce a change towards clinical normalisation and a milder biochemical phenotype. Liver transplanted PA patients still require slight protein restriction and carnitine treatment. We consider that at the moment OLT should only be performed in severe forms of PA, mostly characterised by frequent and unexpected episodes of metabolic decompensation despite good dietary therapy. However, a strict appreciation of these criteria is difficult. A more generalised indication for OLT in PA will require more information about the long-term outcome of transplanted patients. We should also await other alternatives like auxiliary partial OLT from living donors or transplantation of isolated allogenic hepatocytes, genetically modified or not.

Recognition and management of fatty acid oxidation defects: a series of 107 patients.

In a personal series of 107 patients, we describe clinical presentations, methods of recognition and therapeutic management of inherited fatty acid oxidation (FAO) defects. As a whole, FAO disorders appear very severe: among the 107 patients, only 57 are still living. Including 47 siblings who died early in infancy, in total 97 patients died, of whom 30% died within the first week of life and 69% before 1 year. Twenty-eight patients presented in the neonatal period with sudden death, heart beat disorders, or neurological distress with various metabolic disturbances. Hepatic presentations were observed in 73% of patients (steatosis, hypoketotic hypoglycaemia, hepatomegaly, Reye syndrome). True hepatic failure was rare (10%); cholestasis was observed in one patient with LCHAD deficiency. Cardiac presentations were observed in 51% of patients: 67% patients presented with cardiomyopathy, mostly hypertrophic, and 47% of patients had heart beat disorders with various conduction abnormalities and arrhythmias responsible for collapse, near-miss and sudden unexpected death. All enzymatic blocks affecting FAO except CPT I and MCAD were found associated with cardiac signs. Muscular signs were observed in 51% of patients (of whom 64% had myalgias or paroxysmal myoglobinuria, and 29% had progressive proximal myopathy). Chronic neurologic presentation was rare, except in LCHAD deficiency (retinitis pigmentosa and peripheral neuropathy). Renal presentation (tubulopathy) and transient renal failure were observed in 27% of patients. The diagnosis of FAO disorders is generally based on the plasma acylcarnitine profile determined by FAB-MS/MS from simple blood spots collected on a Guthrie card. Urinary organic acid profile and total and free plasma carnitine can also be very helpful, mostly in acute attacks. If there is no significant disturbance between attacks, the diagnosis is based upon a long-chain fatty acid loading test, fasting test, and in vitro studies of fatty acid oxidation on fresh lymphocytes or cultured fibroblasts. Treatment includes avoiding fasting or catabolism, suppressing lipolysis, and carnitine supplementation. The long-term dietary therapy aims to prevent periods of fasting and restrict long-chain fatty acid intake with supplementation of medium-chain triglycerides. Despite these therapeutic measures, the long-term prognosis remains uncertain.

[Hyperhomocysteinemia in coronary artery diseases. Apropos of a study on 102 patients]. / Hyperhomocystéinémie chez des patients coronariens. A propos d'une étude portant sur 102 sujets.

Homocystein is at the crossroads of the metabolic pathways of sulphuric amino acids. Homocystinuria is a congenital autosomal recessive disease, usually related to cystathionine beta-synthetase deficiency. Children with homozygotic forms of the disease have early vascular complications which represent the main cause of death. Moderately elevated serum homocystein levels are related to two major genetic factors (heterozygotic cystathionine beta-synthetase deficiency and mutation of the 5-10 methylene tetrahydrofolate reductase) and several minor, genetic and non-genetic factors (folic acid, vitamins B6 and B12 and betain deficiencies). Previous studies have suggested that hyperhomocysteinaemia could be a cardiovascular risk factor. This study was based on 222 subjects including 102 consecutive patients with angiographically documented coronary artery disease and 120 control subjects without vascular disease. No relationship was observed between serum homocystein concentrations and the classical cardiovascular risk factors. Coronary patients had higher average homocystein concentrations than control subjects (11.27 +/- 0.52 vs 8.77 +/- 0.31 mumol/l); p < 0.0001): moreover, the prevalence of hyperhomocysteinaemia (> 15.67 mumol/l) was higher in the coronary group (15.7%) than in the controls (2.5%). A significant relationship was also observed between homocystein concentrations and the severity of the coronary disease (defined by a coronary score) and the number of diseased vascular territories. These results underline the relationship between homocystein and vascular risk, especially that of coronary artery disease. The treatment of hyperhomocysteinaemia by folic acid supplements is effective in correcting plasma levels, without side effects and at a relatively low cost.

Clinical outcome and long-term management of 17 patients with propionic acidaemia.

UNLABELLED: A retrospective study was performed on the clinical outcome and long-term treatment of 17 patients with propionic acidaemia diagnosed during the last 20 years in our hospital. The study group consisted of 12 patients with early onset type of disease and 5 patients with late onset. Seven (41%) patients died, five with early onset and two with late onset. The deceased early onset patients had a median survival of 0.4 years while the deceased late onset patients died at the age of 2.8 and 4 years respectively. Median age of the living early onset patients was 5.2 (1-9.25) years, the late onset patients were 4, 7 and 23 years old. Patients were all treated with natural protein restriction and in most cases carnitine and metronidazole were added. The early onset patients were almost all treated with daily home tube feeding. The mean natural protein intake of early onset patients (6.3 +/- 1.5 g/day) was significantly lower than the natural protein intake of late onset patients (17.6 +/- 5.3 g/day). Supplemental protein intake was higher in early onset patients. The general neurological outcome of our study group was satisfactory with a better outcome for early onset patients. As to growth, many patients showed a failure to thrive, this was particularly for height. The strong protein restriction during the first years of life probably contributed to this. CONCLUSION: The prognosis for patients with propionic acidaemia appeared to be satisfactory in terms of survival and outcome characteristics such as neurological and mental development. Despite these results the authors feel that the prognosis and quality of life of these patients might be improved with liver transplantation or possibly somatic gene therapy in the future.

Long-term folic acid (but not pyridoxine) supplementation lowers elevated plasma homocysteine level in chronic renal failure.

Moderate hyperhomocysteinemia, a risk factor for premature atherosclerosis, is present in chronic uremic patients. We prospectively evaluated the effects of sequential supplementation with pyridoxine (70 mg/day) and folic acid (10 mg/day) for two 3-month periods in 37 nondialyzed patients (29 males) with creatinine clearance (CCr) ranging from 10 to 80 ml/min, whose plasma vitamin B12 and folate level was in the normal range. Mean (+/- SD) baseline plasma total homocysteine (Hcy) was 14.9 +/- 5.2, 16.5 +/- 5.1 and 26.1 +/- 12.1 mumol/l (upper limit in 45 healthy controls 14.1 mumol/l) in patients with CCr 40-80, 20-40 and < 20 ml/min, respectively. Following pyridoxine Hcy did not significantly decrease whereas following folic acid Hcy decreased significantly to 9.9 +/- 2.9 (-33% vs. baseline), 10.3 +/- 3.4 (-37%) and 15.4 +/- 5.5 (-40%), respectively (Student's paired t test, p < 0.001) in the 3 groups. We conclude that folate (but not pyridoxine) pharmacologic supplementation is effective in lowering elevated plasma Hcy in chronic renal failure patients, thus suggesting that enhancing the Hcy remethylation pathway may overcome hyperhomocysteinemia in such patients. In view of the potential atherogenic effects of hyperhomocysteinemia, long-term folate supplementation should be considered in uremic patients.

Hyperhomocysteinemia, a risk factor for atherosclerosis in chronic uremic patients.

Hyperhomocysteinemia has been shown to constitute an independent risk factor for premature occlusive arterial disease (N Engl J Med 324:1149), a frequent complication in chronic uremic patients in whom homocysteine (Hcy) accumulation has been reported to occur. We prospectively determined fasting plasma level of total, protein-bound Hcy in 118 adult chronic uremic patients, either dialyzed or not. In 79 non-dialyzed patients (47 male) with various degrees of chronic renal failure (RF) assessed by creatinine clearance (CCr), none receiving folate, B6 or B12 vitamin supplementation, mean (+/- 1 SD) plasma Hcy level was 16.2 +/- 8.1 mumol/liter in 28 patients with mild RF (CCr 30 to 75 ml/min), 23.3 +/- 14.7 in 29 patients with moderate RF (CCr 10 to 29.9), and 29.5 +/- 14.4 in 22 patients with advanced RF (CCr < 10), a significant difference (P < 0.01 for all groups) compared to 45 healthy controls (8.2 +/- 2.2 mumol/liter). Linear regression analysis showed a significant correlation between plasma creatinine and Hcy concentrations (r = 0.49, P < 0.0001). Hcy was significantly higher in 20 patients (16 males) who had past histories of occlusive arterial disease than in the 59 (31 males) who did not (30.9 +/- 19.1 vs. 19.6 +/- 9.7 mumol/liter, P < 0.001) and all of the former had Hcy level > 14.1 mumol/liter (the upper limit in healthy controls) versus 35 of 59 in the latter.(ABSTRACT TRUNCATED AT 250 WORDS)

Localization of copper-zinc superoxide dismutase mRNA in human hippocampus by in situ hybridization.

The cellular localization of copper-zinc superoxide dismutase (CuZn SOD) mRNA was determined in the human hippocampus by in situ hybridization with a 35S-labelled DNA probe complementary to human CuZn SOD mRNA. A positive hybridization signal was detected in pyramidal cell layers CA1-CA4 of Ammon's horn (CA), pyramidal cells of subiculum and in the granule cells of the dentate gyrus. The fact that CuZn SOD gene expression is important in neurones which are preferentially vulnerable in neurodegenerative processes such as Alzheimer's disease, suggests a role played by oxygen free radicals in the mechanism of nerve cell death.