Mutations in the AGXT2L2 gene cause phosphohydroxylysinuria.

Phosphohydroxylysinuria has been described in two patients with neurological symptoms, but the deficient enzyme or mutated gene has never been identified. In the present work, we tested the hypothesis that this condition is due to mutations in the AGXT2L2 gene, recently shown to encode phosphohydroxylysine phospholyase. DNA analysis from a third patient, without neurological symptoms, but with an extreme hyperlaxicity of the joints, shows the existence of two mutations, p. Gly240Arg and p.Glu437Val, both in the heterozygous state. Sequencing of cDNA clones derived from fibroblasts mRNA indicated that the two mutations were allelic. Both mutations replace conserved residues. The mutated proteins were produced as recombinant proteins in Escherichia coli and HEK293T cells and shown to be very largely insoluble, whereas the wild-type one was produced as a soluble and active protein. We conclude that phosphohydroxylysinuria is due to mutations in the AGXT2L2 gene and the resulting lack of activity of phosphohydroxylysine phospholyase in vivo. The finding that the nul alleles of p.Gly240Arg and p.Glu437Val are present at low frequencies in the European and/or North American population suggests that this condition is more common than previously thought. The diversity of the clinical symptoms described in three patients with phosphohydroxylysinuria indicates that this is most likely not a neurometabolic disease.

Fatal cerebral edema associated with serine deficiency in CSF.

Two young girls without a notable medical history except for asthma presented with an acute toxic encephalopathy with very low serine concentrations both in plasma and cerebrospinal fluid (CSF) comparable to patients with 3-phosphoglycerate dehydrogenase (3-PGDH) deficiency. Clinical symptoms and enzyme measurement (in one patient) excluded 3-PGDH deficiency. Deficiencies in other serine biosynthesis enzymes were highly unlikely on clinical grounds. On basis of the fasting state, ketone bodies and lactate in plasma, urine and CSF, we speculate that reduced serine levels were due to its use as gluconeogenic substrate, conversion to pyruvate by brain serine racemase or decreased L-serine production because of a lack of glucose. These are the first strikingly similar cases of patients with a clear secondary serine deficiency associated with a toxic encephalopathy.

Proline affects brain function in 22q11DS children with the low activity COMT 158 allele.

The association between the 22q11.2 deletion syndrome (22q11DS) and psychiatric disorders, particularly psychosis, suggests a causal relationship between 22q11DS genes and abnormal brain function. The genes catechol-O-methyl-transferase (COMT) and proline dehydrogenase both reside within the commonly deleted region of 22q11.2. COMT activity and proline levels may therefore be altered in 22q11DS individuals. Associations of both COMT(158) genotype and elevated serum proline levels with abnormal brain function have been reported. Fifty-six 22q11DS children and 75 healthy controls were assessed on physiological measures of brain function, including prepulse inhibition (PPI) of startle, P50 auditory sensory gating and smooth pursuit eye movements (SPEM). COMT(158) genotype and plasma proline levels were determined in the 22q11DS children. We hypothesized an interaction between the COMT(158) genotype and proline, predicting the strongest negative effect of high proline on brain function to occur in 22q11DS children who are carriers of the COMT(met) allele. Of the three physiological measures, only SPEM and PPI were abnormal in the patient sample. With regard to the SPEM performance, there was a significant interaction between the COMT(158) genotype and proline level with significantly decreased SPEM performance in children with high plasma proline levels and the low activity COMT(met) allele. A similar interaction effect was not observed with regard to PPI. These findings are consistent with a model in which elevated proline negatively affects brain function by an increase in dopamine in the prefrontal cortex. 22q11DS patients with low dopamine catabolic capacity are therefore especially vulnerable to this functional disruption.

Plasma acylcarnitine and fatty acid profiles during exercise and training in Standardbreds.

OBJECTIVE: To evaluate alterations in skeletal muscle carnitine metabolism during exercise and training by measuring changes in plasma acylcarnitine concentrations in Standardbreds. ANIMALS: 10 Standardbred geldings with a mean +/- SD age of 20 +/- 2 months and weight of 384 +/- 42 kg. PROCEDURES: In a 32-week longitudinal study, training on a treadmill was divided into 4 phases as follows: phase 1, acclimatization for 4 weeks; phase 2, 18 weeks with alternating endurance and high-intensity exercise training; phase 3, increased training volume and intensity for another 6 weeks; and phase 4, deconditioning for 4 weeks. In phase 3, horses were randomly assigned to 2 groups as follows: control horses (which continued training at the same level as in phase 2) and high-intensity exercise trained horses. At the end of each phase, a standardized exercise test (SET) was performed. Plasma acylcarnitine, fatty acids, and lactic acid and serum beta-hydroxybutyric acid (BHBA) concentrations were assessed before and at different time points after each SET. RESULTS: Plasma lactic acid, total nonesterified fatty acids, 3-hydroxyisobutyric acid, and acetylcarnitine (C2-carnitine) concentrations significantly increased during SETs, whereas serum BHBA, plasma propionylcarnitine (C3-carnitine), and plasma butyryl- and isobutyrylcarnitine (C4-carnitine) concentrations decreased significantly, compared with those before SETs. CONCLUSIONS AND CLINICAL RELEVANCE: Our findings indicated that the plasma acylcarnitine profile in horses likely reflects skeletal muscle carnitine metabolism following exercise, thereby providing a possible practical method to investigate potential disorders in carnitine metabolism in horses with myopathy.

Quantification of free and total sialic acid excretion by LC-MS/MS.

BACKGROUND: The main purpose for measuring urinary free sialic acid (FSA) is to diagnose sialic acid (SA) storage diseases. Elevated amounts of conjugated sialic acid (CSA) are observed in several diseases indicating the need to quantify CSA as well. A LC-MS/MS method for quantification of FSA and total sialic acid (TSA) in urine is developed and validated. METHODS: FSA is analyzed directly after filtration of urine samples. For determination of TSA an enzymatic (neuraminidase) and a chemical (acid) hydrolysis were compared. 13C3-sialic acid was used as internal standard. LC-MS/MS was performed in negative electrospray ionisation mode with multiple reaction monitoring of transitions m/z 308.2-->87.0 (SA) and m/z 311.2-->90.0 (13C3-SA). CSA was calculated by subtracting FSA from TSA. RESULTS: Limit of detection for FSA and TSA was 0.3 and 1.7 micromol/L, respectively. Limit of quantification for FSA and TSA was 1.0 and 5.0 micromol/L. Intra- and inter-assay variations of FSA were 4.6% and 6.6% (n=10) for FSA and 6.5% and 3.6% (n=10) for TSA. Linearity was tested till 7800 micromol/L (r2=0.9998). Values of SA analyzed after neuraminidase- or acid hydrolysis treatment were comparable. Urine samples from patients with inborn errors of SA (related) metabolism were analyzed and compared with age-related reference values. CONCLUSION: A method has been developed for routine determination of urinary FSA and TSA. The method is rapid, specific, robust and sensitive. Age-related reference values for FSA, TSA and CSA were determined and improved diagnostic efficacy.

Ventricular fibrillation without overt cardiomyopathy as first presentation of organic cation transporter 2-deficiency in adolescence.

This case report describes ventricular fibrillation without overt cardiomyopathy as the presenting symptom of primary carnitine deficiency due to organic cation transporter 2 (OCTN2)-deficiency in a 15-year-old girl. Normally this disease presents early in life with hypoketotic hypoglycemia, muscle weakness, and/or cardiomyopathy. The patient fully recovered after carnitine supplementation. Recognition of this disease is important because its treatment is easy and effective.

Deficiency of GDP-Man:GlcNAc2-PP-dolichol mannosyltransferase causes congenital disorder of glycosylation type Ik.

The molecular nature of a severe multisystemic disorder with a recurrent nonimmune hydrops fetalis was identified as deficiency of GDP-Man:GlcNAc(2)-PP-dolichol mannosyltransferase, the human orthologue of the yeast ALG1 gene (MIM 605907). The disease belongs to the group of congenital disorders of glycosylation (CDG) and is designated as subtype CDG-Ik. In patient-derived serum, the total amount of the glycoprotein transferrin was reduced. Moreover, a partial loss of N-glycan chains was observed, a characteristic feature of CDG type I forms. Metabolic labeling with [6-(3)H]glucosamine revealed an accumulation of GlcNAc(2)-PP-dolichol and GlcNAc(1)-PP-dolichol in skin fibroblasts of the patient. Incubation of fibroblast extracts with [(14)C]GlcNAc(2)-PP-dolichol and GDP-mannose indicated a severely reduced activity of the beta 1,4-mannosyltransferase, elongating GlcNAc(2)-PP-dolichol to Man(1)GlcNAc(2)-PP-dolichol at the cytosolic side of the endoplasmic reticulum. Genetic analysis of the patient's hALG1 gene identified a homozygous mutation leading to the exchange of a serine residue to leucine at position 258 in the hALG1 protein. The disease-causing nature of the hALG1 mutation for the glycosylation defect was verified by a retroviral complementation approach in patient-derived primary fibroblasts and was confirmed by the expression of wild-type and mutant hALG1 in the Saccharomyces cerevisiae alg1-1 strain.

L-arginine supplementation improves function and reduces inflammation in renal allografts.

Recovery from ischemia/reperfusion and immune-mediated injury in the renal transplant is associated with reduced renal hemodynamics and increased leukocyte infiltration. In diverse models of renal failure, L-arginine supplementation improved hemodynamics and reduced inflammation. However in a proinflammatory environment, L-arginine can worsen renal injury. This study investigated the therapeutic potential of L-arginine supplementation in allogeneic renal transplantation: Brown Norway rat kidneys were transplanted into Lewis rat recipients, with one native kidney remaining. Recipients received low-dose cyclosporin A (2.5 mg/kg per d subcutaneously) to obtain moderate vascular and interstitial rejection, with or without 1% L-arginine in drinking water for 7 d posttransplantation. Transplantation increased renal vasoconstriction (from 16.9 +/- 1.33 to 35.1 +/- 8.6 units; P: < 0.01), thereby reducing GFR (from 0.96 +/- 0.09 to 0.48 +/- 0.10 ml/min; P: < 0.05). Treatment with L-arginine restored renal graft function to levels found in normal donors (renal vascular resistance, 15.7 +/- 1.69 units; GFR, 0.80 +/- 0.06 ml/min). L-arginine significantly reduced vascular occlusion because of less inflammation, endothelial disruption, and thrombosis. L-arginine also decreased tubulitis, interstitial injury, and macrophage infiltration. These protective effects suggest that L-arginine might be useful as additive therapy to conventional immune suppression.