Miller (Genee-Wiedemann) syndrome represents a clinically and biochemically distinct subgroup of postaxial acrofacial dysostosis associated with partial deficiency of DHODH.

Biallelic mutations in the gene encoding DHOdehase [dihydroorotate dehydrogenase (DHODH)], an enzyme required for de novo pyrimidine biosynthesis, have been identified as the cause of Miller (Genée-Weidemann or postaxial acrofacial dysostosis) syndrome (MIM 263750). We report compound heterozygous DHODH mutations in four additional families with typical Miller syndrome. Complementation in auxotrophic yeast demonstrated reduced pyrimidine synthesis and in vitro enzymatic analysis confirmed reduced DHOdehase activity in 11 disease-associated missense mutations, with 7 alleles showing discrepant activity between the assays. These discrepancies are partly explained by the domain structure of DHODH and suggest both assays are useful for interpretation of individual alleles. However, in all affected individuals, the genotype predicts that there should be significant residual DHOdehase activity. Urine samples obtained from two mutation-positive cases showed elevated levels of orotic acid (OA) but not dihydroorotate (DHO), an unexpected finding since these represent the product and the substrate of DHODH enzymatic activity, respectively. Screening of four unrelated cases with overlapping but atypical clinical features showed no mutations in either DHODH or the other de novo pyrimidine biosynthesis genes (CAD, UMPS), with these cases also showing normal levels of urinary OA and DHO. In situ analysis of mouse embryos showed Dhodh, Cad and Umps to be strongly expressed in the pharyngeal arch and limb bud, supporting a site- and stage-specific requirement for de novo pyrimidine synthesis. The developmental sensitivity to reduced pyrimidine synthesis capacity may reflect the requirement for an exceptional mitogenic response to growth factor signalling in the affected tissues.

Neonatal jaundice: a critical review of the role and practice of bilirubin analysis.

Neonatal jaundice is common, and usually harmless, because of physiological jaundice or breast-feeding. In some neonates unconjugated bilirubin concentration, coupled with other risk factors, is sufficient to allow free bilirubin to cross the blood-brain barrier and cause kernicterus. Another subgroup of infants is jaundiced because of elevated conjugated bilirubin; a marker for a number of pathological conditions. Bilirubin measurement must identify those infants at risk. Transcutaneous bilirubin measurement is increasingly used in healthy infants, especially before early discharge or at home, to assess the need for laboratory bilirubin measurement. Transcutaneous measurements are not covered by laboratory quality assessment schemes. Guidelines on management of neonatal jaundice utilize age in hours and other risk factors to define bilirubin action thresholds, which may be as low as 100 micromol/L for sick premature infants, whereas early discharged babies may only present after bilirubin concentrations are extremely high. Hence, there is a requirement for accurate total bilirubin measurement from <100 to >500 micromol/L, with sufficient precision to assess the rate of bilirubin change with time. Babies presenting with late jaundice always require conjugated bilirubin measurement. It is of concern that many total and direct bilirubin automated kit methods suffer from haemolysis interference, while use of in-house methods or modification of commercial methods has virtually disappeared. External quality assessment has a vital role in providing data on different methods' performance, including accuracy, precision and susceptibility to interference. Laboratories should consider whether their adult bilirubin methods are suitable for neonates.

Guidelines for the performance of the sweat test for the diagnosis of cystic fibrosis.

A multidisciplinary group (representing various professional bodies and supported by the Cystic Fibrosis Trust) has developed evidence-based guidelines for the performance of the sweat test in the UK. The guidelines cover patient information, subject suitability, sweat collection, sweat analysis, quality, interpretation of results, and responsibility for testing and training. The guidelines were produced following a detailed literature search by the process described by the Royal College of Paediatrics and Child Health (RCPCH), using the Scottish Intercollegiate Guidelines Network 1 (SIGN 1) criteria to grade the evidence. Recommendations are graded A, B, or C, depending on the level of evidence. The grade B recommendations (there were no grade A recommendations) were subsequently appraised and endorsed as part of the RCPCH process, according to Appraisal of Guidelines for Research and Evaluation in Europe (AGREE). The recommendations are summarized in tabular form representing the final version incorporating the comments from the appraisal process. Both the appraisal comments and the full evidence base can be found on www.rcpch.ac.uk/publications/clinical_docs.html. The full guidelines can also be found on http://www.ukneqas.org.uk/guidelines.htm.

Sweat testing for cystic fibrosis: A review of New Zealand laboratories.

BACKGROUND: Evolving diagnostic criteria for cystic fibrosis, broadening of the populations being tested and the need to interpret intermediate sweat test results have imposed a much greater need to standardize the collection and analysis of sweat. AIM: To identify variations in sweat testing in New Zealand laboratories and compare these with guidelines from the UK and the USA. METHODS: All laboratories in New Zealand offering sweat testing were identified and data collected from these laboratories by structured questionnaire. RESULTS: There were no New Zealand laboratories that conformed to either set of guidelines. Inconsistencies were observed in minimum sweat quantities, the nature of the iontophoresis solution, the sweat electrolytes analysed, quoted reference ranges and recommendations made as a consequence of the result. CONCLUSIONS: Conformity to the guidelines would help to minimize variation in sweat testing in New Zealand. Performance of a sufficient number of tests to maintain expertise is critical, but geographical constraints make patient travel to distant centres difficult in a small, scattered population. A possible solution, where numbers permit, may be the collection of sweat locally, with referral to a major laboratory for analysis. This is only possible with adequate training in collection and follow-up audit of the sweat testing procedure both in the collection and in the analytical phase.

Folate-induced reversal of leukoencephalopathy and intellectual decline in methylene-tetrahydrofolate reductase deficiency: variable response in siblings.

Homocystinuria due to 5,10-methylenetetrahydrofolate reductase deficiency may present with variable neurological manifestations. Radiological features include white matter changes (leukoencephalopathy). Clinical, biochemical, and radiological response to treatment may again be variable. Here we present a 12-year follow-up of two siblings on the same treatment regimen, with contrasting long-term findings. The first patient, a female presenting at 15 years, showed a good clinical response, substantial intellectual gain, and complete reversal of leukoencephalopathy. Her brother presented at 13 years 9 months and showed limited clinical and cognitive improvement with persistence of the leukoencephalopathy. Both siblings showed a partial biochemical response to treatment.

Novel disease-causing mutations in the dihydropyrimidine dehydrogenase gene interpreted by analysis of the three-dimensional protein structure.

Dihydropyrimidine dehydrogenase (DPD) deficiency is an autosomal recessive disease characterized by thymine-uraciluria in homozygous deficient patients. Cancer patients with a partial deficiency of DPD are at risk of developing severe life-threatening toxicities after the administration of 5-fluorouracil. Thus, identification of novel disease-causing mutations is of the utmost importance to allow screening of patients at risk. In eight patients presenting with a complete DPD deficiency, a considerable variation in the clinical presentation was noted. Whereas motor retardation was observed in all patients, no patients presented with convulsive disorders. In this group of patients, nine novel mutations were identified including one deletion of two nucleotides [1039-1042delTG] and eight missense mutations. Analysis of the crystal structure of pig DPD suggested that five out of eight amino acid exchanges present in these patients with a complete DPD deficiency, Pro86Leu, Ser201Arg, Ser492Leu, Asp949Val and His978Arg, interfered directly or indirectly with cofactor binding or electron transport. Furthermore, the mutations Ile560Ser and Tyr211Cys most likely affected the structural integrity of the DPD protein. Only the effect of the Ile370Val and a previously identified Cys29Arg mutation could not be readily explained by analysis of the three-dimensional structure of the DPD enzyme, suggesting that at least the latter might be a common polymorphism. Our data demonstrate for the first time the possible consequences of missense mutations in the DPD gene on the function and stability of the DPD enzyme.