Mouse model for molybdenum cofactor deficiency type B recapitulates the phenotype observed in molybdenum cofactor deficient patients.

Molybdenum cofactor (MoCo) deficiency is a rare, autosomal-recessive disorder, mainly caused by mutations in MOCS1 (MoCo deficiency type A) or MOCS2 (MoCo deficiency type B) genes; the absence of active MoCo results in a deficiency in all MoCo-dependent enzymes. Patients with MoCo deficiency present with neonatal seizures, feeding difficulties, severe developmental delay, brain atrophy and early childhood death. Although substitution therapy with cyclic pyranopterin monophosphate (cPMP) has been successfully used in both Mocs1 knockout mice and in patients with MoCo deficiency type A, there is currently no Mocs2 knockout mouse and no curative therapy for patients with MoCo deficiency type B. Therefore, we generated and characterized a Mocs2-null mouse model of MoCo deficiency type B. Expression analyses of Mocs2 revealed a ubiquitous expression pattern; however, at the cellular level, specific cells show prominent Mocs2 expression, e.g., neuronal cells in cortex, hippocampus and brainstem. Phenotypic analyses demonstrated that Mocs2 knockout mice failed to thrive and died within 11 days after birth. None of the tested MoCo-dependent enzymes were active in Mocs2-deficient mice, leading to elevated concentrations of purines, such as hypoxanthine and xanthine, and non-detectable levels of uric acid in the serum and urine. Moreover, elevated concentrations of S-sulfocysteine were measured in the serum and urine. Increased levels of xanthine resulted in bladder and kidney stone formation, whereas increased concentrations of toxic sulfite triggered neuronal apoptosis. In conclusion, Mocs2-deficient mice recapitulate the severe phenotype observed in humans and can now serve as a model for preclinical therapeutic approaches for MoCo deficiency type B.

Determination of urinary alpha-aminoadipic semialdehyde by LC-MS/MS in patients with congenital metabolic diseases.

This paper describes a full detailed high performance liquid chromatography/tandem mass spectrometry method for the identification and quantification of human urine alpha-aminoadipic semialdehyde, biomarker of pyridoxine-dependent epilepsy. The ionization mode of the electrospray interface was negative and the metabolite was detected in the multiple reaction monitoring mode. Intra-day and inter-day laboratory precision were 4.64% and 7.30%, respectively, total run time was 3.5min. The calibration curve was linear between 0.25 and 10nmol with a correlation coefficient of the calibration line (R(2)≥0.9984); the limit of quantification was 0.25nmol within the control group. This simple, fast, high reproducible and robust procedure facilitates a rapid diagnosis of patients with pyridoxine-dependent epilepsy and can also be used to confirm the elevated urinary alpha-aminoadipic semialdehyde excretion in patients with other metabolic diseases as molybdenum cofactor and isolated sulphite oxidase deficiencies.

Molybdenum cofactor deficiency: metabolic link between taurine and S-sulfocysteine.

Molybdenum cofactor deficiency (MoCD) is a rare inherited metabolic disorder characterized by severe and progressive neurologic damage mainly caused by the loss of sulfite oxidase activity. Elevated urinary levels of sulfite, thiosulfate, and S-sulfocysteine (SSC) are hallmarks in the diagnosis of both MoCD and sulfite oxidase deficiency. Sulfite is generated throughout the catabolism of sulfur-containing amino acids cysteine and methionine. Accumulated sulfite reacts with cystine, thus leading to the formation of SSC, a glutamate analogue, which is assumed to cause N-methyl-D-aspartate receptor-mediated neurodegeneration in MoCD patients. Recently, we described a fast and sensitive HPLC method for diagnostic and treatment monitoring of MoCD patients based on SSC quantification. In this study, we extend the HPLC method to the analysis of hypotaurine and taurine in urine samples and no interference with other compounds was found. Besides the known elevation of SSC and taurine, also hypotaurine shows strong accumulation in MoCD patients, for which the molecular basis is not understood. SSC, hypotaurine, and taurine urinary excretion values from control individuals as well as MoCD patients are reported and over 20-fold increase in taurine urinary excretion was determined for MoCD patients demonstrating a direct link between sulfite toxicity and taurine biosynthesis in MoCD.

Pyridoxine-dependent epilepsy with elevated urinary α-amino adipic semialdehyde in molybdenum cofactor deficiency.

α-Amino adipic semialdehyde (α-AASA) accumulates in body fluids from patients with pyridoxine-dependent epilepsy because of mutations in antiquitin (ALDH7A1) and serves as the biomarker for this condition. We have recently found that the urinary excretion of α-AASA was also increased in molybdenum cofactor and sulfite oxidase deficiencies. The seizures in pyridoxine-dependent epilepsy are caused by lowered cerebral levels of pyridoxal-5-phosphate (PLP), the bioactive form of pyridoxine (vitamin B(6)), which can be corrected by the supplementation of pyridoxine. The nonenzymatic trapping of PLP by the cyclic form of α-AASA is causative for the lowered cerebral PLP levels. We describe 2 siblings with clinically evident pyridoxine-responsive seizures associated with increased urinary excretion of α-AASA. Subsequent metabolic investigations revealed several metabolic abnormities, all indicative for molybdenum cofactor deficiency. Molecular investigations indeed revealed a known homozygous mutation in the MOCS2 gene. Based upon the clinically evident pyridoxine-responsive seizures in these 2 siblings, we recommend considering pyridoxine supplementation to patients affected with molybdenum cofactor or sulfite oxidase deficiencies.

Urinary AASA excretion is elevated in patients with molybdenum cofactor deficiency and isolated sulphite oxidase deficiency.

Analysis of α-aminoadipic semialdehyde is an important tool in the diagnosis of antiquitin deficiency (pyridoxine-dependent epilepsy). However continuing use of this test has revealed that elevated urinary excretion of α-aminoadipic semialdehyde is not only found in patients with pyridoxine-dependent epilepsy but is also seen in patients with molybdenum cofactor deficiency and isolated sulphite oxidase deficiency. This should be taken into account when interpreting the laboratory data. Sulphite was shown to inhibit α-aminoadipic semialdehyde dehydrogenase in vitro.

Idiopathic hypercalciuria: O2(-)NO relationship and altered bone metabolism.

The pathogenesis of idiopathic hypercalciuria (IH) has not been elucidated yet, but a correlation between IH and altered bone metabolism has been proposed. Since nitric oxide (NO) regulates osteoclasts' bone resorption, a possible role for NO can be suggested. In this study we evaluated iNOS gene expression by reverse transcription of mRNA from monocytes, followed by polymerase chain reaction in patients with IH subdivided into fasting (FH) and absorptive (AH) hypercalciuria. Since superoxide (O2-), which metabolizes NO, is overproduced by osteoclasts during bone resorption, peroxynitrite plasma level was evaluated as index of O2-. Vertebral BMD in IH as a whole group was lower vs controls (C) (Z score=-1.78+/-0.2 vs 0.51+/-0.25, p<0.001), but only FH patients showed a reduced bone density (2.13+/-0.18 vs 0.51+/-0.25, p<0.0001). PTH and calcitriol were not different. FH showed an increase in b-ALP vs AH and C (41.1+/-2.6 vs 30.1+/-3.9 vs 26.6+/-3.6 U/l p<0.02), and higher uHP, either on NCD (17.7+/-1.6 vs 11.4+/-1.3 mg/g uCr, p<0.04) or after LCD (26.7+/-2.5 vs 16.7+/-1.9, p<0.01). Cells from FH patients, but not from both AH patients and C, expressed iNOS. Peroxynitrite plasma level was elevated in FH (0.30+/-0.07) pmol/l while not detectable in AH and C. This study confirms an altered bone metabolism only in FH which shows an abnormal NO system. The increased iNOS gene expression in FH, in fact, points toward an altered NO system's activity downstream the generation of NO. A possible interaction of NO with O2-, which breaks down NO, and the role of this interaction in the pathophysiology of IH is discussed.

Nephrocalcinosis in three siblings with idiopathic hypercalciuria.

Idiopathic hypercalciuria (IH) associated with nephrocalcinosis was found in three of six siblings. After the three affected children were maintained on a low-calcium diet, they demonstrated increasing hypercalciuria, parathyroid hormone, and vitamin D3 levels. An oral calcium loading test was not necessary to diagnose renal IH. During treatment with hydrochlorothiazide, the calcium excretion was normalized. These patients are remarkable because nephrocalcinosis is generally regarded as a rare complication of renal IH. Moreover, the fact that three of six siblings are affected raises the question of whether the renal form of IH is genetically distinct from other forms of IH.

Metabolic studies in primary tubular hypomagnesaemia-hypokalaemia.

13 1/2 year old boy with short stature and pubertal delay had infrequent episodes of tetany. Biochemical determinations demonstrated low plasma and high urinary magnesium and potassium levels, hypocalciuria, slightly increased plasma bicarbonate, slightly reduced fractional distal reabsorption of chloride and sodium, high plasma renin activity and high urinary excretion of prostaglandins (E2, F2 alpha). The other parameters of renal functions were normal. Endocrine evaluation of short stature and pubertal delay was normal. Intracellular magnesium and potassium levels in lymphocytes and erythrocytes were within normal limits. Cyclooxygenase blockade with Indomethacin 2.5 mg/kg daily during 4 weeks normalized urinary excretion of prostaglandins and corrected in part low plasma and high urinary potassium levels, but had no effect on magnesium, calcium, sodium and chloride handling. These data raise the possibility that tubular hypomagnesaemia-hypokalaemia could be solely explained by a low renal threshold for magnesium.

Inborn errors of molybdenum metabolism: combined deficiencies of sulfite oxidase and xanthine dehydrogenase in a patient lacking the molybdenum cofactor.

A patient suffering from a combined deficiency of sulfite oxidase (sulfite dehydrogenase; sulfite:ferricytochrome c oxidoreductase, EC 1.8.2.1) and xanthine dehydrogenase (xanthine:NAD+ oxidoreductase, EC 1.2.1.37) is described. The patient displays severe neurological abnormalities, dislocated ocular lenses, and mental retardation. Urinary excretion of sulfite, thiosulfate, S-sulfocysteine, taurine, hypoxanthine, and xanthine is increased in this individual, while sulfate and urate levels are drastically reduced. The metabolic defect responsible for loss of both enzyme activities appears to be at the level of the molybdenum cofactor common to the two enzymes. Immunological examination of a biopsy sample of liver tissue revealed the presence of the xanthine dehydrogenase protein in near normal amounts. Sulfite oxidase apoprotein was not detected by a variety of immunological techniques. The plasma molybdenum concentration was normal; however, hepatic content of molybdenum and the storage pool of active molybdenum cofactor present in normal livers were below the limits of detection. Fibroblasts cultured from this patient failed to express sulfite oxidase protein or activity, whereas those from the parents and healthy brother of the patient expressed normal levels of this enzyme.