Newborn Screening for Homocystinuria Revealed a High Frequency of MAT I/III Deficiency in Iberian Peninsula.

Homocystinuria due to cystathionine ß-synthase deficiency or "classical homocystinuria" is a rare autosomal recessive condition resulting in altered sulfur metabolism with elevated methionine and homocysteine in plasma and homocystine in urine. This condition is characterized by a high clinical heterogeneity, which contributes to late clinical diagnosis, usually only made after irreversible damage has occurred. Treatment is effective if started before clinical symptoms. The analysis of methionine levels by tandem mass spectrometry (MS/MS) allows the newborn screening for homocystinuria, but false-positive results can be frequently obtained and lead to the unwanted identification of methionine adenosyl transferase (MAT I/III) deficiency. This latter condition is biochemically characterized by isolated persistent hypermethioninemia, accompanied in some individuals with slightly elevated levels of homocysteine in plasma. A dominant form of MAT I/III deficiency, associated with mutation p.R264H, seems to be very frequent in the Iberian Peninsula and usually has a clinically benign course. Both these metabolic disorders are screened in Galicia and Portugal since the introduction of the MS/MS technology, in 2000 and 2004, respectively, resulting in the identification of three patients with classical homocystinuria and 44 patients with MAT I/III deficiency. All but one heterozygous parent of MAT I/III patients, identified with the p.R264H mutation, are healthy adults around the age of 30/40. The implementation of a second-tier test for homocysteine in dried blood spots would considerably reduce the number of MAT I/III-deficient patients identified and improve the specificity and positive predictive value for classical homocystinuria screening.

Clinical and metabolic findings in patients with methionine adenosyltransferase I/III deficiency detected by newborn screening.

Persistent hypermethioninemia due to mutations in the MAT1A gene is often found during newborn screening (NBS) for homocystinuria due to cystathionine beta-synthase deficiency, however, outcomes and optimal management for these patients are not well established. We carried out a multicenter study of MAT I/III-deficient patients detected by NBS in four of the Spanish regional NBS programs. Data evaluated during NBS and follow-up for 18 patients included methionine and total homocysteine levels, clinical presentation parameters, genotypes, and development quotients. The birth prevalence was 1:1:22,874. At detection 16 of the 18 patients exhibited elevations of plasma methionine above 60 µmol/L (mean 99.9 ± 38 µmol/L) and the mean value in confirmation tests was 301 µmol/L (91-899) µmol/L. All patients were asymptomatic. In four patients with more markedly elevated plasma methionines (>450 µmol/L) total homocysteine values were slightly elevated (about 20 µmol/L). The average follow-up period was 3 years 7 months (range: 2-123 months). Most patients (83%) were heterozygous for the autosomal dominant Arg264His mutation and, with one exception, presented relatively low circulating methionine concentrations (<400 µM). Additional mutations identified in patients with mean confirmatory plasma methionines above 400 µM were Arg199Cys, Leu355Arg, and a novel mutation, Thr288Ala. During continued follow-up, the patients have been asymptomatic, and, to date, no therapeutic interventions have been utilized. Therefore, the currently available evidence shows that hypermethioninemia due to heterozygous MAT1A mutations such as Arg264His is a mild condition for which no treatment is necessary.

Risk factors for developing mineral bone disease in phenylketonuric patients.

There is a compromised bone mass in phenylketonuria patients compared with normal population, but the mechanisms responsible are still a matter of investigation. In addition, tetrahydrobiopterin therapy is a new option for a significant proportion of these patients and the prevalence of mineral bone disease (MBD) in these patients is unknown. We conducted a cross-sectional observational study including 43 phenylketonuric patients. Bone densitometry, nutritional assessment, physical activity questionnaire, biochemical parameters, and molecular study were performed in all patients. Patients were stratified by phenotype, age and type of treatment. The MBD prevalence in phenylketonuria was 14%. Osteopenic and osteoporotic (n=6 patients) had an average daily natural protein intake significantly lower than the remaining (n=37) patients with PKU (14.33 ± 8.95 g vs 21.25 ± 20.85 g). Besides, a lower body mass index was found. There were no statistical differences in physical activity level, calcium, phosphorus and fat intake, and in phenylalanine, vitamin D, paratohormone, docosahexaenoic and eicosapentaenoic acid blood levels. Mutational spectrum was found in up to 30 different PAH genotypes and no relationship was established among genotype and development of MBD. None of the twelve phenylketonuric patients treated with tetrahydrobiopterin (27.9%), for an average of 7.1 years, developed MBD. Natural protein intake and blood levels of eicosapentaenoic acid were significantly higher while calcium intake was lower in these patients. This study shows that the decrease in natural protein intake can play an important role in MBD development in phenylketonuric patients. Therapy with tetrahydrobiopterin allows a more relaxed protein diet, which is associated with better bone mass.

Development of electrospray ionization tandem mass spectrometry methods for the study of a high number of urine markers of inborn errors of metabolism.

RATIONALE: Rapid and specific screening methods to detect abnormal metabolites in biological fluids are important for the diagnosis of many Inborn Errors of Metabolism (IEM). In Galicia (N.W. Spain), where newborn screening (NBS) has long used both blood and urine dried samples, an expanded NBS by tandem mass spectrometry (MS/MS) begun in July 2000 analyzing amino acids and acylcarnitines in blood. The purpose of this study is the development of methods to widen and to complement the present NBS with the study of the selected metabolites in urine. METHODS: We studied and optimized the fragmentation of a total of 96 marking compounds of IEM, as well as 34 isotopically labeled internal standards (IS). The isobaric interferences were resolved with the use of alternative fragmentation in 14 of the 28 groups found. The methods were validated for 68 compounds following the recommendations of the NCCLS. RESULTS: We have developed electrospray ionization (ESI)- MS/MS methods in positive and negative ionization modes to detect selected metabolites in urine. The study was performed by direct injection of amino acids and acylcarnitines in positive mode, and organic acids, acylglycines, purines and pyrimidines in negative mode. Run times were 2.5 and 2.6 min, respectively, allowing the daily analysis of a high number of samples. CONCLUSIONS: The validated methods were proved effective for the simultaneous study of a large number of metabolites which are commonly present in urine samples and are used for detecting IEM. The evaluation was done by searching diagnostic profiles with multiple markers to increase sensitivity and specificity (e.g., acylcarnitines plus amino acids) or with specific urine markers (cystine, homogentisic acid, sialic acid, N-acetylaspartic acid, etc.).

Comparison between the Cobas Integra immunoassay and high-performance liquid chromatography for therapeutic monitoring of carbamazepine.

OBJECTIVES: Carbamazepine (CBZ) is metabolized by the cytochrome P450 into carbamazepine-10,11-epoxide (CBZepox), a metabolite with similar pharmacological activity to the parent drug. Recently it has been indicated that most current immunoassays for the determination of CBZ are unable to quantitatively measure its active epoxide. An evaluation of the Cobas Integra immunoassay for the determination of CBZ was carried out, and the results are compared with those obtained for CBZ+CBZepox using high-performance liquid chromatography (HPLC) in patients on monotherapy and polytherapy. DESIGN AND METHODS: Steady-state serum trough concentrations of CBZ were determined in 119 epileptic patients using the Cobas Integra immunoassay and HPLC. In 91 cases CBZ was administered in monotherapy and in 28 cases in polytherapy with other anticonvulsant drugs. RESULTS: The study of within- and between-run imprecisiom for the Cobas Integra immunoassay led to clinically acceptable coefficients of variation. A high correlation was found between the concentrations of CBZ obtained using the immunoassay and HPLC (r = 0.981, p < 0.001). In both the group of patients on monotherapy and those on polytherapy, the levels of CBZepox were greater than the clinically acceptable error for CBZ; consequently, there is a clinically significant difference between the total of CBZ+CBZepox concentrations (HPLC) and the concentrations of CBZ (immunoassay). In the group of patients under monotherapy, a high correlation coefficient was obtained between the levels of CBZ and CBZ+CBZepox (r = 0.975, p < 0.001) with an standard error of the estimate similar to the clinically acceptable value. CONCLUSIONS: For the patients on monotherapy, it is possible to make a clinically valid estimation of CBZ+CBZepox from the concentration of CBZ obtained by means the immunoassay. In patients on polytherapy, the analytical determination of CBZepox could be of interest in cases where CBZ+CBZepox would be higher than the critical level of CBZ.