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J Inherit Metab Dis ; 42(1): 128-139, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30740731


PURPOSE: To assess how the current practice of newborn screening (NBS) for homocystinurias compares with published recommendations. METHODS: Twenty-two of 32 NBS programmes from 18 countries screened for at least one form of homocystinuria. Centres provided pseudonymised NBS data from patients with cystathionine beta-synthase deficiency (CBSD, n = 19), methionine adenosyltransferase I/III deficiency (MATI/IIID, n = 28), combined remethylation disorder (cRMD, n = 56) and isolated remethylation disorder (iRMD), including methylenetetrahydrofolate reductase deficiency (MTHFRD) (n = 8). Markers and decision limits were converted to multiples of the median (MoM) to allow comparison between centres. RESULTS: NBS programmes, algorithms and decision limits varied considerably. Only nine centres used the recommended second-tier marker total homocysteine (tHcy). The median decision limits of all centres were ≥ 2.35 for high and ≤ 0.44 MoM for low methionine, ≥ 1.95 for high and ≤ 0.47 MoM for low methionine/phenylalanine, ≥ 2.54 for high propionylcarnitine and ≥ 2.78 MoM for propionylcarnitine/acetylcarnitine. These decision limits alone had a 100%, 100%, 86% and 84% sensitivity for the detection of CBSD, MATI/IIID, iRMD and cRMD, respectively, but failed to detect six individuals with cRMD. To enhance sensitivity and decrease second-tier testing costs, we further adapted these decision limits using the data of 15 000 healthy newborns. CONCLUSIONS: Due to the favorable outcome of early treated patients, NBS for homocystinurias is recommended. To improve NBS, decision limits should be revised considering the population median. Relevant markers should be combined; use of the postanalytical tools offered by the CLIR project (Collaborative Laboratory Integrated Reports, which considers, for example, birth weight and gestational age) is recommended. tHcy and methylmalonic acid should be implemented as second-tier markers.

Urolithiasis ; 43(1): 61-7, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25370766


Hereditary xanthinuria (HX) is a rare inherited disorder caused by a deficiency of xanthine dehydrogenase/oxidase (XDH/XO). Missing XDH/XO activity leads to undetectable levels of uric acid excessively replaced by xanthine in serum/urine. The allopurinol loading test has been traditionally used to differentiate between HX types I and II. Final confirmation of HX has been based on the biopsy finding of the absent XDH/XO activity in the small intestine or liver. We present the clinical, biochemical, ultrasound and molecular genetics findings in three new patients with HX and suggest a simple three-step approach to be used for diagnosis, typing and confirmation of HX. In the first step, the diagnosis of HX is determined by extremely low serum/urinary uric acid excessively replaced by xanthine. Second, HX is typed using urinary metabolomics. Finally, the results are confirmed by molecular genetics. We advocate for this safe and non-invasive diagnostic algorithm instead of the traditional allopurinol loading test and intestinal or liver biopsy used in the past.

Erros Inatos do Metabolismo/diagnóstico , Xantina Desidrogenase/deficiência , Alopurinol/metabolismo , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Metabolômica , Ácido Úrico/sangue , Ácido Úrico/urina
Clin Chim Acta ; 437: 211-7, 2014 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-25086281


BACKGROUND: Disorders of homocysteine and B-vitamin metabolism represent a significant problem in clinical practice. Establishing the diagnosis requires specialized tests with demanding preanalytical requirements. To advance the detection of patients with these disorders, we developed a method for the simultaneous determination of cystathionine (Cysta), methionine (Met) and total homocysteine (tHcy) in dried blood spots (DBSs). METHODS: A punch from a DBS sample was mixed with a solution of isotopically labeled internal standards, and analytes were extracted using methanol/0.1% formic acid/0.5mol/L dithiothreitol. The extract was injected into an LC-MS/MS system operating in MRM mode. RESULTS: The analytical performance of the method employing DBS is adequate for its purpose and the type of sample. Compared with Cysta, tHcy and Met plasma levels, our method exhibited a negative bias between -3.8% and -42.2% due to the lower concentrations of these analytes in erythrocytes. The tHcy level and the Met/Cysta ratio in DBS enabled the clear detection of 12 patients with disorders of transsulfuration and with genetic and nutritional remethylation defects. CONCLUSIONS: The ease of collecting and transporting DBS samples may advance diagnostic procedures in patients with neuropsychiatric disorders and thromboembolism. Consequently, this approach may facilitate detection and simplify the monitoring of patients with homocystinuria.

Cistationina/sangue , Teste em Amostras de Sangue Seco/métodos , Homocisteína/sangue , Homocistinúria/sangue , Metionina/sangue , Espectrometria de Massas em Tandem/métodos , Adolescente , Criança , Pré-Escolar , Cromatografia Líquida/métodos , Cromatografia Líquida/estatística & dados numéricos , Gerenciamento Clínico , Teste em Amostras de Sangue Seco/estatística & dados numéricos , Feminino , Homocistinúria/diagnóstico , Humanos , Lactente , Recém-Nascido , Masculino , Espectrometria de Massas em Tandem/estatística & dados numéricos
Clin Chim Acta ; 413(1-2): 93-9, 2012 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-21963464


BACKGROUND: The article describes the clinical, biochemical, enzymological and molecular genetics findings in two patients from two families with xanthinuria type I. METHODS: Biochemical analysis using high performance liquid chromatography, allopurinol loading test and analysis of xanthine oxidase activity in plasma and of uromodulin excretion in urine were performed. Sequencing analysis of the xanthine dehydrogenase gene and the haplotype and statistical analyses of consanguinity were performed. RESULTS: Probands showed extremely low concentrations of uric acid, on seven occasions under the limit of detection. The concentration of uric acid in 38-year-old female was 15 µmol/L in serum and 0.04 mmol/L in urine. Excretion of xanthine in urine was 170 mmol/mol creatinine. The concentration of uric acid in 25-year-old male was 0.03 mmol/L in urine. Excretion of xanthine in urine was 141 mmol/mol creatinine. The allopurinol loading test confirmed xanthinuria type I. The xanthine oxidase activities in patients were 0 and 0.4 pmol/h/mL of plasma. We found three nonsense changes: p.P214QfsX4 and unpublished p.R825X and p.R881X. CONCLUSIONS: We found two nonconsanguineous compound heterozygotes with xanthinuria type I caused by three nonsense changes. The methods used did not confirm consanguinity in the probands, thus there might be an unconfirmed biological relationship or mutational hotspot.

Mutação , Ácido Úrico/sangue , Xantina Desidrogenase/genética , Xantina Oxidase/genética , Xantina/urina , Adulto , Sequência de Bases , Western Blotting , República Tcheca , Primers do DNA , Eletroforese em Gel de Poliacrilamida , Feminino , Haplótipos , Humanos , Masculino , Repetições de Microssatélites
Nucleosides Nucleotides Nucleic Acids ; 30(12): 1112-6, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22132965


Primary renal hypouricemia is a genetic disorder characterized by defective renal uric acid (UA) reabsorption with complications such as nephrolithiasis and exercise-induced acute renal failure. The known causes are: defects in the SLC22A12 gene, encoding the human urate transporter 1 (hURAT1), and also impairment of voltage urate transporter (URATv1), encoded by SLC2A9 (GLUT9) gene. Diagnosis is based on hypouricemia (<119 µmol/L) and increased fractional excretion of UA (>10%). To date, the cases with mutations in hURAT1 gene have been reported in East Asia only. More than 100 Japanese patients have been described. Hypouricemia is sometimes overlooked; therefore, we have set up the flowchart for this disorder. The patients were selected for molecular analysis from 620 Czech hypouricemic patients. Secondary causes of hyperuricosuric hypouricemia were excluded. The estimations of (1) serum UA, (2) excretion fraction of UA, and (3) analysis of hURAT1 and URATv1 genes follow. Three transitions and one deletion (four times) in SLC22A12 gene and one nucleotide insertion in SLC2A9 gene in seven Czech patients were found. Three patients had acute renal failure and urate nephrolithiasis. In addition, five nonsynonymous sequence variants and three nonsynonymous sequence variants in SLC2A9 gene were found in two UK patients suffering from acute renal failure. Our finding of the defects in SLC22A12 and SLC2A9 genes gives further evidence of the causative genes of primary renal hypouricemia and supports their important role in regulation of serum urate levels in humans.

Técnicas e Procedimentos Diagnósticos , Erros Inatos do Transporte Tubular Renal/diagnóstico , Cálculos Urinários/diagnóstico , Adolescente , Adulto , Idoso , Proteínas Facilitadoras de Transporte de Glucose/genética , Humanos , Pessoa de Meia-Idade , Mutação/genética , Transportadores de Ânions Orgânicos/genética , Proteínas de Transporte de Cátions Orgânicos/genética , Erros Inatos do Transporte Tubular Renal/genética , Cálculos Urinários/genética , Adulto Jovem