Commentary: What degree of hyperphenylalaninaemia requires treatment?

Despite some 50 years' experience in the treatment of phenylketonuria and numerous scientific publications on the subject there is no clear consensus as to what degree of hyperphenylalaninaemia will result in intellectual impairment. Studies of three main types, on untreated cases of moderate hyperphenylalaninaemia, on treated cases of phenylketonuria, and on the effects of current blood phenylalanine concentration on executive function, have lead to different conclusions. Overall, there appears to be a fairly strong case for limiting dietary treatment to individuals whose blood phenylalanine levels would otherwise exceed 600 µmol/L. This is now policy in some European countries but a formal large-scale study of long-term outcomes to validate the approach is urgently required.

Newborn blood spot screening: new opportunities, old problems.

Newborn screening is evolving very rapidly. Geographical coverage is expanding, particularly for common disorders such as congenital hypothyroidism. New technologies, particularly tandem mass spectrometry and high throughput mutation analysis, have increased greatly the range of disorders which could be covered. However, these new possibilities are being exploiting at very different rates in different countries. This is due in part to the different ways in which generally-accepted screening criteria, based on the ten principles of Wilson and Jungner, are being interpreted and applied to policy. The appropriate management of some of the conditions newly-detectable by screening also remains controversial and there is a pressing need to align screening policy and clinical practice. Critical analysis and careful collection of data on an international basis are required to resolve these issues.

Qualitative urinary organic acid analysis: methodological approaches and performance.

A programme for proficiency testing of biochemical genetics laboratories undertaking urinary qualitative organic acid analysis and its results for 50 samples examined for factors contributing to poor performance are described. Urine samples from patients in whom inherited metabolic disorders have been confirmed as well as control urines were circulated to participants and the results from 94 laboratories were evaluated. Laboratories showed variability both in terms of their individual performance and on a disease-specific basis. In general, conditions including methylmalonic aciduria, propionic aciduria, isovaleric aciduria, mevalonic aciduria, Canavan disease and 3-methylcrotonyl-CoA carboxylase were readily identified. Detection was poorer for other diseases such as glutaric aciduria type II, glyceric aciduria and, in one sample, 3-methylcrotonyl-CoA carboxylase deficiency. To identify the factors that allow some laboratories to perform well on a consistent basis while others perform badly, we devised a questionnaire and compared the responses with the results for performance in the scheme. A trend towards better performance could be demonstrated for those laboratories that regularly use internal quality control (QC) samples in their sample preparation (p = 0.079) and those that participate in further external quality assurance (EQA) schemes (p = 0,040). Clinicians who depend upon these diagnostic services to identify patients with these defects and the laboratories that provide them should be aware of the potential for missed diagnoses and the factors that may lead to improved performance.

Introducing new screens: why are we all doing different things?

The disease panels covered by newborn blood spot screening vary greatly from country to country. There are different interpretations of the Wilson and Jungner principles and of underlying data in the scientific literature, and great disparities between the value judgements applied in screening and in routine clinical practice.

Population quantile-quantile plots for monitoring assay performance in newborn screening.

Immunoreactive trypsinogen (IRT) used in screening for cystic fibrosis is heterogeneous, poorly characterized, and displays marked matrix effects when incorporated into blood spots, making long-term control of assay calibration difficult. The cut-off required to select a fixed proportion of samples (0.5% for the UK protocol) for second-tier mutation analysis varies over time, partly owing to slight differences in calibration of individual lots of assay kit. To investigate this and possible inter-laboratory differences in analytical performance, we developed a monitoring system based on the distribution of measured IRT concentrations in the screened samples. Results were collected fortnightly from five UK screening laboratories in the form of numbers of samples in histogram 'bins' of IRT concentration. These data were converted to cumulative percentages and the IRT concentrations at fixed centiles then approximated by triangulation. The quantile-quantile plot of any subset (by laboratory or by kit lot) of these data using pooled results (all-laboratories all-kit-lots, approximately 270,000 samples) as the reference distribution is analogous to a calibration curve and gives a measure of bias in terms of sensitivity (slope) and baseline (y-intercept). This allows a revised 99.5th centile cut-off for each subset to be calculated directly. A similar approach has allowed inter-laboratory comparison of tandem-mass spectrometric assays for free carnitine (with emphasis on low values) and phenylalanine and has demonstrated that apparently trivial differences in instrumentation and procedures have resulted in marked variation in resultant assay performance.

International perspectives on newborn screening.

The development of electrospray tandem mass spectrometry (MS-MS) has greatly increased the number of diseases that can be detected by newborn blood-spot screening. Different countries are introducing the technology at different rates and for different disease panels. Current policies in the United Kingdom, Germany and the United States are taken as examples. In the United Kingdom, many laboratories are using MS-MS for routine screening for phenylketonuria but, except for those participating in a two-year pilot study of screening for medium-chain acyl-CoA dehydrogenase deficiency, are forbidden use MS-MS to screen for other disorders. In Germany there has been considerable experience of MS-MS screening for a wide range of diseases, but recently the Federal Ministry for Health and Social Security prescribed a much more restricted disease panel, with the instruction that any other diagnostic results are to be suppressed and not reported. By contrast, a recent report from the American College of Medical Genetics, still being debated, recommends screening procedures that will detect an extremely broad range of disorders, including some that are very rare or of unproven clinical significance. The lack of even broad concordance at the level of national policy is extremely disturbing. Though all discussion is nominally founded on the ten principles laid down by Wilson and Jungner in 1968, there seems no generally accepted way of using these principles, or derived criteria, as objective decision tools. Alternative, less categorical, approaches are needed: the disorders concerned are not homogeneous entities and there may be advantages to screening other than reducing morbidity or mortality.

Mutation and biochemical analysis in carnitine palmitoyltransferase type II (CPT II) deficiency.

Carnitine palmitoyltransferase type II (CPT II) deficiency has three basic phenotypes, late-onset muscular (mild), infantile/juvenile hepatic (intermediate) and severe neonatal. We have measured fatty acid oxidation and CPT II activity and performed mutation studies in 24 symptomatic patients representing the full clinical spectrum of disease. Severe and intermediate phenotypes show a clear correlation with biochemical indices and genetic analysis revealed causative mutations in most patients. Studies of mild phenotypes suggest a more complex interaction, with higher residual fatty acid oxidation, a wider range of CPT II activity (10-60%) but little evidence of genotype-phenotype correlation. Residual CPT II mutant protein from myopathic patients shows thermal instability at 41 degrees C. The common 'polymorphisms' V3681 and M647V are strikingly overrepresented in the myopathic patients, the implication being that they may significantly influence the manifestation of clinical disease and could therefore potentially be considered as a susceptibility variants. Among myopathic individuals, males comprised 88% of patients, suggesting increased susceptibility to clinical disease. A small number of symptomatic patients appear to have significant residual CPT II activity (42-60%) The synergistic interaction of partial deficiencies of CPT II, muscle adenosine monophosphate deaminase and possibly other enzymes of muscle energy metabolism in the aetiology of episodic myopathy deserves wider consideration.

2-methyl-3-hydroxybutyryl-CoA dehydrogenase deficiency in a 23-year-old man.

2-Methyl-3-hydroxybutyryl-CoA dehydrogenase (EC 1.1.1.178) deficiency is a recently described defect of isoleucine catabolism. The disorder is characterized by normal early development followed by a progressive loss of mental and motor skills. Deterioration may be rapid or may follow a slower decline with a possible stabilization of the disorder on a low-protein diet and appropriate medication. We report a 23-year-old man with 2-methyl-3-hydroxybutyryl-CoA dehydrogenase deficiency with a very mild clinical course. He had apparently normal early development and remained relatively well until the age of 6 years, when he contracted measles. Following this illness, his motor skills and school progress deteriorated. At 15 years he had significant dysarthria, and generalized rigidity with some dystonic and unusual posturing. He was then treated with a low-protein high-carbohydrate diet with a good response in terms of balance and gait. At 18 years he was given benzhexol (Artane), increased slowly from 2 mg to 6 mg daily, resulting in improvement in tremor and dystonia. At 23 years he can dress himself and works in sheltered employment but remains severely dysarthric.

Newborn mass screening versus selective investigation: benefits and costs.

Cost-benefit analysis of newborn screening has an unimpressive record and yet it is still regarded as an important decision tool. This workshop surveyed ongoing research into the costs and benefits of systematic whole-population screening, as opposed to selective investigation of symptomatic patients, for inherited metabolic disease. Much current interest is focused on newborn screening by tandem mass spectrometry, which can replace current methods for detecting phenylketonuria and cover a much wider range of diseases. Two observational studies are comparing cost-effectiveness of tandem mass spectrometry screening versus symptomatic diagnosis in either concurrent or historical control populations. A number of other studies are assessing screening performance against predetermined criteria but without any formal control group. Medium-chain acyl-CoA dehydrogenase deficiency is the most common of the additional diseases being detected and it seems that octanoylcarnitine in blood is a particularly sensitive indicator: some of the cases detected by screening have genotypes suggesting a relatively low risk of serious metabolic decompensation. Ongoing studies should provide further quantitative and qualitative data but will not in themselves define the optimum balance between screening sensitivity and specificity.

Features of carnitine palmitoyltransferase type I deficiency.

Carnitine palmitoyltransferase type I (CPT I) is unique among long-chain fatty acid oxidation enzymes in that there are two tissue-specific isoforms, 'hepatic' and 'muscle', which are encoded by two separate genes. The 'hepatic' isoform is expressed in liver, kidney and fibroblasts and at low levels in the heart, while the other isoform occurs in skeletal muscle and is the predominant form in heart. Reported patients with CPT I deficiency lack activity of the hepatic isoform and present before 30 months of age with hypoketotic hypoglycaemia, hepatomegaly with raised transaminases, seizures and coma. We discuss four new cases in three families showing, variously, renal tubular acidosis, transient hyperlipidaemia and, paradoxically, myopathy with elevated creatinine kinase or cardiac involvement in the neonatal period as additional features that deserve wider recognition.

Molecular characterization of methylmalonate semialdehyde dehydrogenase deficiency.

Three patients have been reported with (putative) methylmalonic semialdehyde dehydrogenase (MMSDH) deficiency. The urine metabolic pattern was strikingly different in all, including beta-alanine, 3-hydroxypropionic acid, both isomers of 3-amino- and 3-hydroxyisobutyric acids in one and 3-hydroxyisobutyric and lactic acids in a second, and mild methylmalonic aciduria in a third patient. In an effort to clarify these disparate metabolite patterns, we completed the cDNA structure, and characterized the genomic structure of human MMSDH gene in order to undertake molecular analysis. Only the first patient had alterations in the MMSDH coding region, revealing homozygosity for a 1336G > A transversion, which leads to substitution of arginine for highly conserved glycine at amino acid 446. No abnormalities of the MMSDH cDNA were detected in the other patients. These data provide the first molecular characterization of an inborn error of metabolism specific to the L-valine catabolic pathway.

Population newborn screening for inherited metabolic disease: current UK perspectives.

Some of the generally accepted criteria for screening programmes are inappropriate for newborn metabolic screening as they ignore the family dimension and the importance of timely genetic information. Uncritical application of such criteria creates special difficulties for screening by tandem mass spectrometry, which can detect a range diseases with widely different natural histories and responsiveness to treatment. Further difficulties arise from increasing demands for direct proof of the effects of screening on long-term morbidity and mortality. The randomized controlled trial is held to be the gold standard, but for ethical and practical reasons it will be impossible to achieve for such relatively rare diseases. This approach also oversimplifies the complex matrix of costs and benefits of newborn metabolic screening. A more workable approach could involve Bayesian synthesis, combining quantitative performance data from carefully designed prospective pilot studies of screening with existing experience of the natural history, diagnosis, and management of the individual disorders concerned.

Tandem mass spectrometry screening: proving effectiveness.

Despite the considerable literature now emerging on screening by tandem mass spectrometry there are many points of uncertainty about test performance and much work to do in formalizing and validating follow-up protocols. The relative sensitivities of tandem mass spectrometry and previously-used diagnostic methods may differ for some diseases but not others, with the possibility of some metabolites being over-sensitive and diagnosing mild variants or non-disease. Cut-offs should be set using clinical rather than analytical criteria and at this early stage it is important that presumptive positive cases diagnosed through acylcarnitine profiling are compared as thoroughly as possible with symptomatic cases diagnosed using other techniques. Caution should be used in accepting mutation analysis as the ultimate diagnostic criterion. Consideration of incidence and careful examination of family history will all contribute to evidence of the equivalence or otherwise of screen-detected and symptomatic disease. Finally, there should be registration and long-term surveillance of all cases detected.