Newborn screening for homocystinurias: Recent recommendations versus current practice.

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.

Brain and spine MRI features of Hunter disease: frequency, natural evolution and response to therapy.

BACKGROUND: Hunter disease is a rare X-linked mucopolysaccharidosis. Despite frequent neurological involvement, characterizing the severe phenotype, neuroimaging studies are scarce. OBJECTIVES: To determine frequency and severity of neuroradiological mucopolysaccharidosis-related features; to correlate them with clinical phenotype; to evaluate their natural evolution and the impact of intravenous enzymatic replacement therapy (ERT). METHODS: Sixty nine brain MRI examinations of 36 Italian patients (mean-age 10.4 years; age-range 2.2-30.8; severe phenotype in 22 patients) were evaluated. Twenty patients had multiple MRIs (median follow-up 3.1 years, range 1-16.9): among them 15 had MRIs before and after ERT, six had repeated MRIs without being on ERT and five while on ERT. Perivascular, subarachnoid and ventricle space enlargement, white matter abnormality (WMA) burden, pituitary sella/skull/posterior fossa abnormalities, periodontoid thickening, spinal stenosis, dens hypoplasia, myelopathy, vertebral and intervertebral disc abnormalities were graded by means of dedicated scales. RESULTS: Perivascular spaces enlargement (89%), WMAs (97%), subarachnoid space enlargement (83%), IIIrd-ventricle dilatation (100%), pituitary sella abnormalities (80%), cranial hyperostosis (19%), craniosynostosis (19%), enlarged cisterna magna (39%), dens hypoplasia (66%), periodontoid thickening (94%), spinal stenosis (46%), platyspondylia (84%) and disc abnormalities (79%) were frequently detected. WMAs, IIIrd-ventricle dilatation and hyperostosis correlated with the severe phenotype (p < 0.05). Subarachnoid spaces and ventricle enlargement, WMAs and spinal stenosis progressed despite ERT, while other MR features showed minimal or no changes. CONCLUSIONS: The spectrum of brain and spine MRI abnormalities in Hunter disease is extremely wide and requires a thorough evaluation. WMAs, atrophy/communicating hydrocephalus and spinal stenosis progress over time and might represent possible disease severity markers for new treatment efficacy assessment.

Treatment of organic acidurias and urea cycle disorders.

Three steps are extremely important in emergency management (awaiting diagnosis) of organic acidurias and urea cycle disorders. The first is to obtain adequate samples (blood and urine) before any treatment, parenteral and/or enteral nutrition with protein-free solution and toxin removal procedures are the second step. Additional therapies with cofactors and vitamins are the last step. It is very important to quickly carry out this strategy every time we suspect an inborn error of metabolism.

Molecular analysis of glycogen storage disease type Ib in Sardinian population: evidence for a founder effect.

We describe epidemiological, genetic, and clinical data of the 1124-2del mutation in the G6PT gene, detected in homozygosity in three glycogen storage disease type Ib patients of Sardinian origin. This mutation was found to be associated with four sequence variations: c.593 A>T (p.N198I), c.625+19 C>T, c.1062 C>T (N354N), and c.1224 G>A (p.T408T) in the G6PT gene. RNA studies were performed for c.1124-2del and c.625+19 C>T. The c.1124-1del2 acceptor splicing mutation showed skipping of 31 nucleotides of exon 9 due to the activation of a downstream cryptic acceptor splice site in 1154-1155 nucleotide positions, resulting in a downstream stop codon at aa position 402. RNA analysis of c.625+19 C>T variation showed a small amount of alternative splicing with skipping of exon 4, resulting in a stop codon at aa position 211. Our cases present most of features of the severe form of disease, including early onset with chronic neutropenia, frequent infections, and inflammatory bowel disease. Our results suggest a founder effect for glycogen storage disease type Ib that facilitates diagnosis using mutation analysis, sparing patients from liver biopsy. DNA-based diagnosis will enable us to make accurate determination of carrier status and prenatal diagnosis, thus improving genetic counseling.

Increase in urinary purines and pyrimidines in patients with methylmalonic aciduria combined with homocystinuria.

BACKGROUND: Methylmalonic aciduria combined with homocystinuria (MMA-HC) is the biochemical trait of a metabolic disorder resulting from impaired conversion of dietary cobalamin (cbl, or vitamin B12) to its two metabolically active forms. Effects on urinary purine and pyrimidine levels have not been described for this condition. METHODS: Urine samples were collected from three patients with methylmalonic aciduria combined with homocystinuria and from 70 healthy subjects. Urinary purine and pyrimidine levels were quantitated by the use of LC/UV-Vis and LC/ESI/MS. RESULTS: Higher urine levels of pyrimidines were detected with both methods in patients compared to controls. CONCLUSION: Methylmalonic aciduria with homocystinuria is due to deficiency of the enzyme, cobalamin reductase. The enzyme defect leads to altered hepatic metabolism, which appears to modify circulating pyrimidine levels.

Kinetic and expression analyses of seven novel mutations in mitochondrial acetoacetyl-CoA thiolase (T2): identification of a Km mutant and an analysis of the mutational sites in the structure.

Mitochondrial acetoacetyl-CoA thiolase (T2) deficiency is an inborn error of metabolism that affects isoleucine catabolism and ketone body metabolism. We identified 7 novel and 2 previously reported mutations in six T2-deficient patients. Transient expression analysis of wild-type and eight mutant cDNAs was performed at 40, 37 and 30 degrees C. Although no significant residual activity was detected, mutant proteins were detected in the N158D, N158S, R208Q, Y219H and N282H mutants. Accumulation of these mutant proteins was temperature-sensitive with the highest expression levels at lower temperatures. Expression of Q73P and N353K cDNAs yielded neither residual T2 protein nor enzyme activity. An E252del mutant T2 was detected with a relative protein amount and enzyme activity of 30% and 25%, respectively, in comparison to the wild-type at 37 degrees C. The E252del mutant protein was more stable at 30 degrees C expression than 37 degrees C, but was essentially undetectable at 40 degrees C, indicating its temperature-sensitive instability. Kinetic studies revealed a twofold K(m) elevation for substrates coenzyme A and acetoacetyl-CoA in the E252del mutant, while V(max) was comparable to the wild-type. We conclude that the E252del is a temperature-sensitive K(m) mutant. This correlates well with the effect predicted from the T2 tertiary structure analysis, using the crystal structure of the human T2 homotetramer. The probable effect of the other mutations on the T2 tertiary structure was also evaluated.