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Background: In classical homocystinuria (HCU, MIM# 236200) due to the deficiency of cystathionine β-synthase (EC 4.2.1.22) there is a clear evidence for the success of early treatment. The aim of this study was to develop and evaluate a two-tier strategy for HCU newborn screening., Methods: We reevaluated data from our newborn screening programme for Qatar in a total number of 125,047 neonates including 30 confirmed HCU patients. Our hitherto existing screening strategy includes homocysteine (Hcy) measurements in every child, resulting in a unique dataset for evaluation of two-tier strategies. Reevaluation included methionine (Met) levels, Met to phenylalanine (Phe) ratio, and Hcy. Four HCU cases identified after database closure were also included in the evaluation. In addition, dried blood spot samples selected by Met values >P97 in the newborn screening programs in Austria, Australia, the Netherlands, and Taiwan were analyzed for Hcy., Results: Met to Phe ratio was found to be more effective for first sieve than Met, sorting out nearly 90% of normal samples. Only 10% of the samples would have to be processed by second-tier measurement of Hcy in dried blood spots. As no patient with HCU was found neither in the samples investigated for HCU, nor by clinical diagnosis in the other countries, the generalization of our two-tier strategy could only be tested indirectly., Conclusion: The finally derived two-tier algorithm using Met to Phe ratio as first- and Hcy as second-tier requires 10% first-tier positives to be transferred to Hcy measurement, resulting in 100% sensitivity and specificity in HCU newborn screening.

Background: Congenital hypothyroidism causes intellectual delay unless identified and effectively treated soon after birth. Newborn screening has almost eliminated intellectual disability associated with congenital hypothyroidism. However, clinical uncertainty remains about infants with thyroid-stimulating hormone (TSH) concentrations less than the newborn screening cutoffs. We assessed the association between neonatal TSH concentrations and educational and developmental outcomes., Methods: We did a population-based record-linkage study of all liveborn infants undergoing newborn screening from 1994 to 2008 in New South Wales, Australia, with assessments of childhood development or school performance. Very-low-birthweight babies (<1500 g) were excluded. Developmental and educational outcomes were obtained and these were linked to individual records by the New South Wales Centre for Health Record Linkage. The primary educational outcome was the proportion of students with National Assessment Program Literacy and Numeracy (NAPLAN) results lower than the national minimum standard in reading or numeracy measured at all ages, and the primary developmental outcome was the proportion of children who were classified as being developmentally high risk (vulnerable in two or more of the five developmental domains assessed by the Australian Early Development Census) at age 4-6 years. The proportions of infants with each outcome were calculated per percentile (0-100) of TSH concentration. Multivariable logistic regression was used to account for potential confounding by maternal and fetal variables known to affect neonatal TSH concentrations or neurodevelopmental outcomes., Findings: 503 706 infants had a neonatal TSH result that linked to a developmental or educational outcome. 149 569 infants born between 2002 and 2008 were linked to an Australian Early Development Census developmental outcome and 354 137 were linked to a NAPLAN educational outcome. Median follow-up for educational outcome was 10 years (IQR 8-12) and for developmental outcome was 5 years (5-6). 5·5% (14 137 of 257 752) of infants scored less than the national minimum standard for numeracy in percentiles lower than the 75th percentile and this increased with each increase of percentile group to 11·3% (15 of 133) of infants with a TSH concentration between the 99·90th and 99·95th percentile. Infants with a neonatal TSH concentration in the 99·95th percentile or higher (above newborn screening cutoff) and likely to have diagnosed and treated congenital hypothyroidism had similar results to infants with a TSH concentration lower than the 75th percentile for both educational and developmental outcomes. Infants with a neonatal TSH concentration between the 99·5th and 99·9th percentile were more likely to have special needs (adjusted odds ratio [aOR] 1·68, 95% CI 1·23-2·30), poor numeracy performance (aOR 1·57, 1·29-1·90), and developmentally high risk (aOR 1·52, 1·20-1·93)., Interpretation: We found an association between neonatal TSH concentrations lower than the present newborn screening thresholds and poor educational and developmental outcomes. This association needs further investigation to assess whether assessment and treatment of these infants might improve their long-term cognitive outcomes., Funding: Australian National Health and Medical Research., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Classical homocystinuria is caused by mutations in the cystathionine β-synthase (CBS) gene. Previous experiments in bacterial and yeast cells showed that many mutant CBS enzymes misfold and that chemical chaperones enable proper folding of a number of mutations. In the present study, we tested the extent of misfolding of 27 CBS mutations previously tested in E. coli under the more folding-permissive conditions of mammalian CHO-K1 cells and the ability of chaperones to rescue the conformation of these mutations. Expression of mutations in mammalian cells increased the median activity 16-fold and the amount of tetramers 3.2-fold compared with expression in bacteria. Subsequently, we tested the responses of seven selected mutations to three compounds with chaperone-like activity. Aminooxyacetic acid and 4-phenylbutyric acid exhibited only a weak effect. In contrast, heme arginate substantially increased the formation of mutant CBS protein tetramers (up to sixfold) and rescued catalytic activity (up to ninefold) of five out of seven mutations (p.A114V, p.K102N, p.R125Q, p.R266K, and p.R369C). The greatest effect of heme arginate was observed for the mutation p.R125Q, which is non-responsive to in vivo treatment with vitamin B(6). Moreover, the heme responsiveness of the p.R125Q mutation was confirmed in fibroblasts derived from a patient homozygous for this genetic variant. Based on these data, we propose that a distinct group of heme-responsive CBS mutations may exist and that the heme pocket of CBS may become an important target for designing novel therapies for homocystinuria.

Newborn screening has evolved fast following recent advances in diagnosis and treatment of disease, particularly the development of multiplex testing and applications of molecular testing. Formal evidence of benefit from newborn screening has been largely lacking, due to the rarity of individual disorders. There are wide international differences in the choice of disorders screened, and ethical issues in both screening and not screening are apparent. More evidence is needed about benefit and harm of screening for specific disorders and renewed discussion about the basic aims of newborn screening must be undertaken., (© 2015 The Authors. Journal of Paediatrics and Child Health © 2015 Paediatrics and Child Health Division (Royal Australasian College of Physicians).)

There have been few reports of cases missed by expanded newborn screening. Tandem mass spectrometry was introduced in New South Wales, Australia in 1998 to screen for selected disorders of amino acid, organic acid and fatty acid metabolism. Of 1,500,000 babies screened by 2012, 1:2700 were diagnosed with a target disorder. Fifteen affected babies were missed by testing, and presented clinically or in family studies. In three cases (cobalamin C defect, very-long-chain acyl-CoA dehydrogenase deficiency and glutaric aciduria type 1), this led to modification of analyte cut-off values or protocols during the first 3 years. Two patients with intermittent MSUD, two with β-ketothiolase deficiency, two with citrin deficiency, two siblings with arginosuccinic aciduria, two siblings with homocystinuria, and one with cobalamin C defect had analyte values and ratios below the action limits which could not have been detected without unacceptable false-positive rates. A laboratory interpretation error led to missing one case of cobalamin C defect. Reference ranges, regularly reviewed, were not altered. For citrin deficiency, while relevant metabolites are detectable by tandem mass spectrometry, our cut-off values do not specifically screen for that disorder. Most of the missed cases are doing well and with no acute presentations although eight of 15 are likely to have been somewhat adversely affected by a late diagnosis. Analyte ratio and cut-off value optimisations are important, but for some disorders occasional missed cases may have to be tolerated to maintain an acceptable specificity, and avoid harm from screening.

We report the case of an 8-year-old boy with pyridoxamine 5'-phosphate oxidase (PNPO) deficiency. He developed seizures at 24 h of age that were refractory to standard anticonvulsant therapy and a trial of pyridoxine but responded to pyridoxal phosphate (PLP) at 28 days of life. Genetic testing identified compound heterozygous mutations in the PNPO gene. Management of encephalopathic episodes required escalation of PLP dose to 100 mg/kg/day by 2 years of age. Routine blood tests at this time showed significantly deranged liver function tests (LFTs). A wedge liver biopsy showed early cirrhosis with marked elevation of pyridoxal and pyridoxic acid levels in the liver sample. Despite extensive investigation, no cause other than PLP therapy could be identified for the cirrhosis. The PLP dose was weaned to 50 mg/kg/day before episodes of encephalopathy recurred. Concurrent with the reduction of his PLP dose, LFTs showed improvement. However, at 8 years of age, there is persistent evidence of hepatic fibrosis and early portal hypertension. We hypothesise that hepatic toxicity due to PLP or its degradation products is the cause of cirrhosis in this boy. Until further evidence becomes available, we would suggest that people with PNPO deficiency are treated with the minimum dose of PLP required to prevent episodes of encephalopathy.

Phenylketonuria (PKU) is an autosomal recessive inborn error of phenylalanine metabolism predominantly caused by mutations in the phenylalanine hydroxylase (PAH) gene. Mutation screening was carried out in a large cohort of PKU patients from New South Wales, Australia. Pathogenic mutations were identified in 99% of the alleles screened, with the two most common mutations (p.R408W and IVS12+1G>A) accounting for 30.7% of alleles. Most individuals were compound heterozygotes for previously reported mutations, but four novel mutations (c.163+1G>T, c.164-2A>G, c.461A>T [p.Y154F], and c.510-1G>A) and a novel polymorphism (c.60+62C>T) were also identified. A number of patients have been previously tested for their response to dietary supplementation of tetrahydrobiopterin (BH4), the cofactor of PAH. Correlation between genotype and the responses revealed that although genotype is a major determinant of BH4 responsiveness, patients with the same genotype may also show disparate responses to this treatment. A clinical and biochemical evaluation should be undertaken to determine the effectiveness of PKU treatment by supplementation of BH4.

Although fragile X syndrome (FXS) is the commonest cause of inherited intellectual disability the mean age of diagnosis in Australia is 5.5 years. Newborn screening for FXS can provide an early diagnosis, preventing the "diagnostic odyssey", allowing access to early interventions, and providing reproductive information for parents. Parents of affected children support newborn screening, but few clinical studies have evaluated community attitudes. A pilot study in 2009-2010 was performed in a tertiary hospital to explore feasibility and maternal attitudes. FXS testing of male and female newborns was offered to mothers in addition to routine newborn screening. Mothers were provided with information about FXS, inheritance pattern, carrier status, and associated adult-onset disorders. One thousand nine hundred seventy-one of 2,094 mothers (94%) consented to testing of 2,000 newborns. 86% completed the attitudinal survey and 10% provided written comments. Almost all parents (99%) elected to be informed of both premutation and full mutation status and there was little concern about identification of carrier status or associated adult-onset disorders. Most mothers (96%) were comfortable being approached in the postnatal period and supported testing because no extra blood test was required. Mothers considered an early diagnosis beneficial to help prepare for a child with additional needs (93%) and for reproductive planning (64%). Some were anxious about the potential test results (10%) and others felt their feelings towards their newborn may change if diagnosed with FXS (16%). High participation rates and maternal attitudes indicate a high level of maternal acceptance and voluntary support for newborn screening for FXS., (Copyright © 2013 Wiley Periodicals, Inc.)

This paper reviews the evidence of benefit resulting from newborn screening in Australia as well as for some of those disorders not yet included in the Australian panels, and discusses briefly disorders under active consideration for inclusion in the screening panels.There is solid evidence of benefit from newborn screening for phenylketonuria, congenital hypothyroidism, cystic fibrosis, and overall for the disorders included in tandem mass spectrometry screening. There is also some evidence of benefit for several disorders not screened for in Australia, including congenital adrenal hyperplasia. Harms resulting from screening include anxiety related to false positive results; adverse effects of unwarranted treatment for mild variants; unwanted genetic information; and the costs (opportunity costs) of screening. For well-run programs these harms are relatively small.Screening could become more effective with the development of good systems for rational consideration of disorders to be included, with the extended use of second tier testing to reduce the false positive rate, and with research on the most effective way to deal with mild variants. The most important aspect of increasing effectiveness is the full integration of the screening program, diagnostic laboratories, and the clinical service. This is already in place in Australasia.

Disorders of cobalamin and folate intracellular metabolism that result in defective remethylation of homocysteine to methionine are associated with leukodystrophy, whereas disorders of cobalamin transport generally are not. Cobalamin derivatives are needed for only two reactions in man; remethylation of homocysteine to methionine, with methylcobalamin as a cofactor for methionine synthase, and the conversion of methylmalonyl-coenzyme A to succinyl coenzyme A by methylmalonyl-CoA mutase, with adenosylcobalamin as a cofactor. Mutations at various metabolic steps affect the synthesis of adenosylcobalamin (CblA, CblB, and CblD2), methylcobalamin (CblE, CblG, and CblD1), or both of these (CblF, CblD, and CblC). The most common disorder of folate metabolism, 5,10-methylenetetrahydrofolate deficiency, also affects remethylation and presents with leukodystrophy. Pathways of cobalamin and folate metabolism intersect at one site, methionine synthase. Patients with the remethylating disorders present acutely or chronically with significant neurologic, hematologic, vascular, and other symptoms. Circulating levels of cobalamin and folate are usually normal in these disorders, and initial diagnosis is aided by measurement of homocysteine and methylmalonic acid in blood or urine, together with hematologic tests. Current diagnosis is often by newborn screening. These disorders all show autosomal recessive inheritance, and all are treatable, although with variable outcome., (Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.)

Expanded newborn screening (NBS) leads to an increased number of false positive results, causing parental anxiety, greater follow-up costs, and the need for further metabolic investigations. We developed and validated a second-tier approach for NBS of homocystinurias by measuring the total homocysteine (tHcy) on the initial dried blood spot (DBS) samples to reduce the need for further investigation, and investigated newborn DBS homocysteine values in patients with homocystinuria. Total DBS homocysteine was measured in normal newborns, and retrospectively in newborns with established disorders, using liquid chromatography tandem mass spectrometry (LC-MS/MS) with stable isotope-labelled internal standards for homocysteine. Analytes were separated using reverse phase chromatography with a total run time of 3 min. The method was linear over the range of 10-100 μmol/L of tHcy and showed excellent precision; intra-batch CV was 4% and inter-batch precision 6.5%. Comparison of 59 plasma values with DBS for tHcy taken at the same time showed excellent correlation, (r (2)>0.97). The reference range for current neonatal samples was 5.4-10.7 μmol/L (n=99), and for the stored neonatal samples (stored dry, sealed in plastic at room temperature for 10 years) was 1.7-5.5 μmol/L, (n=50), both being normally distributed. The clinical utility of this method was checked by retrospective analysis of stored NBS samples from patients with different forms of homocystinuria, including four different remethylating disorders. All had clear elevations of tHcy.

Genetic disorders are caused by abnormalities in genes and chromosomes and for the most part have implications for family members, affecting such matters as confidentiality and disclosure to third parties. Genetic testing can be not only diagnostic but also predictive, raising issues of the need for pre-test counselling, protection of children from unwanted testing, and most importantly, the imprecision of interpretation of future risk. The rise in availability of direct-to-consumer testing is a fresh cause for concern, as are the new possibilities in reproductive medicine. New technologies and the falling cost of whole genome sequencing ensure that ethics will be a prominent concern for clinical genetics., (© 2011 The Author. Journal of Paediatrics and Child Health © 2011 Paediatrics and Child Health Division (Royal Australasian College of Physicians).)

In general, newborn screening is now a highly successful enterprise. The introduction of tandem mass spectrometry in the mid-1990s changed the pace of screening, raising its profile and increasing its relevance to a wider range of health professionals. The clinical effectiveness is not in doubt for some conditions, but is lacking for others. Evaluation has major difficulties for the rarer disorders and has been sadly neglected. Partly because clinical effectiveness has not been enthusiastically addressed, but also because of undue caution on the part of regulators, who often seem to ignore available evidence, there are huge differences in the adoption of screening programmes in different jurisdictions. New treatments, especially mutation-specific treatments, and technological advances in diagnostic testing are being rapidly developed, and this will further change the face of newborn screening and probably magnify these differences. The challenges will be considerable, especially with the increasing availability of DNA testing at modest cost. It is likely that there will be pressure to change the aims of newborn screening to encompass "personalised medicine". We must all prepare in a thoughtful way for these future challenges.

Glutaric aciduria type I (synonym, glutaric acidemia type I) is a rare organic aciduria. Untreated patients characteristically develop dystonia during infancy resulting in a high morbidity and mortality. The neuropathological correlate is striatal injury which results from encephalopathic crises precipitated by infectious diseases, immunizations and surgery during a finite period of brain development, or develops insidiously without clinically apparent crises. Glutaric aciduria type I is caused by inherited deficiency of glutaryl-CoA dehydrogenase which is involved in the catabolic pathways of L-lysine, L-hydroxylysine and L-tryptophan. This defect gives rise to elevated glutaric acid, 3-hydroxyglutaric acid, glutaconic acid, and glutarylcarnitine which can be detected by gas chromatography/mass spectrometry (organic acids) or tandem mass spectrometry (acylcarnitines). Glutaric aciduria type I is included in the panel of diseases that are identified by expanded newborn screening in some countries. It has been shown that in the majority of neonatally diagnosed patients striatal injury can be prevented by combined metabolic treatment. Metabolic treatment that includes a low lysine diet, carnitine supplementation and intensified emergency treatment during acute episodes of intercurrent illness should be introduced and monitored by an experienced interdisciplinary team. However, initiation of treatment after the onset of symptoms is generally not effective in preventing permanent damage. Secondary dystonia is often difficult to treat, and the efficacy of available drugs cannot be predicted precisely in individual patients. The major aim of this revision is to re-evaluate the previous diagnostic and therapeutic recommendations for patients with this disease and incorporate new research findings into the guideline.

Background: Very-low-birth-weight babies (VLBW) with hypothyroidism may show a delayed postnatal rise in thyroid stimulating hormone (TSH), mainly due to immaturity of the hypothalamic-pituitary-thyroid axis. Transient hypothyroidism is prevalent in VLBW babies and some affected babies are considered to need treatment. There is disagreement about whether a second screening test is needed in VLBW babies to detect all cases that need treatment., Methods: We included in the study all babies with a birth weight ≤ 1,500 g born in New South Wales and the Australian Capital Territory between January 2006 and December 2008. Newborn screening samples for TSH measurement were taken in the first days of life and again at 1 month. During week 1, a blood-spot TSH level of  ≥20 mIU/L was considered positive, and at 1 month a positive level was  ≥7 mIU/L, and triggered full investigation., Results: In the cohort of 301,000 babies, 2,313 VLBW babies survived for testing, and 2,117 repeat screening samples were received. Forty-three babies had transient hypothyroidism, with thyroid function normalising before 2 months of age, usually without treatment. Eighteen babies required treatment beyond 2 months of age (1:128 of surviving babies), 16 having had normal TSH results on initial testing, and 12 having levels below 6 mIU/L., Conclusion: Significant hypothyroidism, transient or permanent, but persisting beyond 2 months of age is common in VLBW babies. There is a delayed rise in TSH in some, and secondary screening at 1 month of age detects babies deemed by local paediatric endocrinologists as needing treatment.

Achieving the goals of newborn screening is, as for any screening, a balancing act: getting the maximum benefit from screening while producing the minimum harm. The advent of "expanded" newborn screening, with a large number of disorders detectable using a single test, has also posed problems, not new, but now more obvious. One is the finding of many more cases by screening, the extra cases being largely patients who have attenuated phenotypes and may remain asymptomatic for many years, even throughout life. These may or may not require active management in the short term, but do need lifelong awareness. Additionally, disorders have been included that are now thought benign or largely so. Babies risk being unnecessarily medicalized. Assessing outcome has also proved difficult because of the rarity of some disorders and the impracticality of randomized controlled trials. The requirements for valid studies include the need for case definitions, comparable comparison groups and probably assessment on a whole-population basis. An Australia-wide study of tandem mass spectrometry newborn screening involving 2 million screened and unscreened babies has demonstrated benefits overall to screened patients at age 6 years. The study was too small to provide conclusions for individual disorders other than for medium-chain acyl-CoA dehydrogenase deficiency.

Assessing the outcome of fatty acid oxidation disorders is difficult, as most are rare. For diagnosis by newborn screening, the situation is compounded: far more cases are diagnosed by screening than by clinical presentation, representing a somewhat different cohort. The literature on outcome was reviewed. For disorders other than medium-chain acyl-coenzyme A (CoA) dehydrogenase (MCAD) deficiency there was insufficient evidence to make many firm statements. In MCAD deficiency, risk of death in the first 72 h is around 4%, with a further approximately 5-7% fatality rate in the first 6 years but very low subsequent risk in previously undiagnosed patients. The risk of death after diagnosis is very low at any age, with good management. The long-term outcome is good nowadays. Very-long-chain acyl-CoA dehydrogenase deficiency poses a risk of death in early infancy, but the condition is generally treatable, with a good outcome after diagnosis. Approximately 10-20% of patients diagnosed by newborn screening and treated nevertheless suffer episodic rhabdomyolysis. Some patients never become symptomatic. Isolated long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency is treatable, but most patients suffer episodic hypoketotic hypoglycaemia and rhabdomyolysis. Generalised mitochondrial tri-functional protein deficiency has high early mortality rate. A more insidious presentation also occurs, with symptoms sometimes confined to progressive axonal neuropathy. Among carnitine cycle disorders, carnitine transporter deficiency, potentially lethal, is uniformly successfully treated orally with carnitine. Carnitine-acylcarnitine translocase and early-onset carnitine palmitoyl transferase type II (CPT II) deficiencies have an extremely high neonatal mortality rate. Late-onset CPT II is characterised only by episodic rhabdomyolysis on severe exercise. CPT type IA deficiency may often be benign, although early presentation with hypoketotic hypoglycaemia certainly occurs.

Treatment recommendations in mitochondrial fatty acid oxidation (FAO) defects are diverse. With implementation of newborn screening and identification of asymptomatic patients, it is necessary to define whom to treat and how strictly. We here discuss critical questions that are currently under debate. For some asymptomatic long-chain defects, long-chain fat restriction plays a minor role, and a normal diet may be introduced. For patients presenting only with myopathic symptoms, e.g., during exercise, treatment may be adapted to energy demand. As a consequence, patients with exercise-induced myopathy may be able to return to normal activity when provided with medium-chain triglycerides (MCT) prior to exercise. There is no need to limit participation in sports. Progression of retinopathy in disorders of the mitochondrial trifunctional protein complex is closely associated with hydroxyacylcarnitine accumulation. A strict low-fat diet with MCT supplementation is recommended to slow or prevent progression of chorioretinopathy. Additional docosahexanoic acid does not prevent the decline in retinal function but does promote nonspecific improvement in visual acuity and is recommended. There is no evidence that L-carnitine supplementation is beneficial. Thus, supplementation with L-carnitine in a newborn identified by screening with either a medium-chain or long-chain defect is not supported. With respect to the use of the odd-chain medium-chain triglyceride triheptanoin in myopathic phenotypes, randomized trials are needed to establish whether triheptanoin is more effective than even-chain MCT. With increasing pathophysiological knowledge, new treatment options have been identified and are being clinically evaluated. These include the use of bezafibrates in myopathic long-chain defects.

Tyrosine hydroxylase deficiency is an autosomal recessive disorder resulting from cerebral catecholamine deficiency. Tyrosine hydroxylase deficiency has been reported in fewer than 40 patients worldwide. To recapitulate all available evidence on clinical phenotypes and rational diagnostic and therapeutic approaches for this devastating, but treatable, neurometabolic disorder, we studied 36 patients with tyrosine hydroxylase deficiency and reviewed the literature. Based on the presenting neurological features, tyrosine hydroxylase deficiency can be divided in two phenotypes: an infantile onset, progressive, hypokinetic-rigid syndrome with dystonia (type A), and a complex encephalopathy with neonatal onset (type B). Decreased cerebrospinal fluid concentrations of homovanillic acid and 3-methoxy-4-hydroxyphenylethylene glycol, with normal 5-hydroxyindoleacetic acid cerebrospinal fluid concentrations, are the biochemical hallmark of tyrosine hydroxylase deficiency. The homovanillic acid concentrations and homovanillic acid/5-hydroxyindoleacetic acid ratio in cerebrospinal fluid correlate with the severity of the phenotype. Tyrosine hydroxylase deficiency is almost exclusively caused by missense mutations in the TH gene and its promoter region, suggesting that mutations with more deleterious effects on the protein are incompatible with life. Genotype-phenotype correlations do not exist for the common c.698G>A and c.707T>C mutations. Carriership of at least one promotor mutation, however, apparently predicts type A tyrosine hydroxylase deficiency. Most patients with tyrosine hydroxylase deficiency can be successfully treated with l-dopa.

Carnitine is necessary for transport of long-chain fatty acids into mitochondria, to enter the beta-oxidation cycle. Four carnitine cycle defects have been described. The carnitine transporter mediates carnitine transport across the plasma membrane. Symptoms include hypoketotic hypoglycaemia and cardiomyopathy. Some affected subjects are asymptomatic. Newborn screening detects very low levels of free carnitine in some but not all. Carnitine palmitoyltransferase type IA (CPTI) transports long-chain fatty acyl-CoAs across the outer mitochondrial membrane. Affected infants have hypoketotic hypoglycaemia with catabolic stress, but otherwise remain well. Newborn screening tests reveal elevated free carnitine, (elevated C0/C16+C18). Sensitivity is unclear and confirmation needs leukocyte or fibroblast assays. Carnitine-acylcarnitine translocase transfers fatty acylcarnitines across the inner mitochondrial membrane. The most common presentation is sudden death in the first days. Carnitine palmitoyltransferase type II (CPTII) converts long-chain acylcarnitines to long-chain acylCoAs for beta-oxidation. Severe deficiency is lethal. Newborn screening for both disorders reveals elevated palmitoylcarnitine and enzymology or mutation analysis is needed for diagnosis. Late-onset CPTII is the most common disorder, presenting as muscle pain and rhabdomyolysis on severe exercise. All 4 disorders can be detected by newborn screening, with variable sensitivity. Late-onset CPTII probably cannot be detected. Carnitine transporter, CPTI and late-onset CPTII have proven treatment strategies.

Comprehensive newborn screening for cystic fibrosis has occurred for more than 25 years in some regions and the results of randomised controlled trials reporting the outcomes have been published. Testing protocols for CF have recently been reviewed and the sensitivity and specificity of these protocols are high. In spite of this, many remain sceptical in respect of the advantages conferred by newborn screening for CF. Every study of newborn screening has shown that diagnosis occurs at a significantly younger age. While this alone is sufficient to justify newborn screening, the clinical course of those diagnosed via newborn screening indicates that many additional advantages accrue. These include a decreased morbidity and mortality in early life, facilitation of better growth and prevention of vitamin deficiency in early infancy, as well as some indication of an advantage in terms of pulmonary status later in life. This review summarises the arguments in favour of newborn screening for CF.

Tandem mass spectrometry has become increasingly popular as the preferred technology for detecting inborn errors of metabolism in newborn screening programmes. Its sensitivity and specificity for detecting numerous inborn errors has been well documented. However, there are continuing questions about whether the technology should be used to the fullest when such usage may mean detecting and reporting analytical findings that could lead to diagnosing conditions for which clinical outcome is unclear and treatment may not be needed, or treatment efficacy may not yet be proven and cost effectiveness is unlikely. As part of a friendly debate to educate conference attendees on both sides of somewhat controversial issues, these 2 papers at the conference presented some of the information supporting or questioning the cost effectiveness of full scan usage and reporting in tandem mass spectrometry newborn screening. Reported here are some of the questioning arguments.

The aim of newborn screening is to detect newborns with serious, treatable disorders so as to facilitate appropriate interventions to avoid or ameliorate adverse outcomes. Mass biochemical testing of newborn babies was pioneered in the 1960s with the introduction of screening for phenylketonuria, a rare inborn error of metabolism, tested by using a dried blood spot sample. The next disorder introduced into screening programs was congenital hypothyroidism and a few more much rarer disorders were gradually included. Two recent advances have greatly changed the pace: modification of tandem mass spectrometry and DNA extraction and analysis from newborn screening dried blood spot. These two technologies make the future possibilities of newborn screening seem almost unlimited. Newborn screening tests are usually carried out on a dried blood spot sample, for which there are special analytical considerations. Dried blood spot calibrators and controls, prepared on the same lot number of filter paper, are needed. Methods have a co-efficient of variation of about 10% due to the increased variability of a dried filter paper sample compared with other biochemical samples. The haematocrit is an additional variable not able to be measured. Also of importance is obtaining a balance between the sensitivity and specificity of each assay. Fixing cut-off points for action needs consideration of what is an acceptable percentage of the population to recall for further testing. Few assays are 100% discriminatory. Programs in Australasia currently screen for at least 30 disorders. Detection of these requires not only the assay of a primary marker but often determination of a ratio of that marker with another, or possibly an alternative assay, for example a DNA mutation. The most important disorders screened for are described briefly: phenylketonuria, primary congenital hypothyroidism, cystic fibrosis, the galactosaemias, medium-chain acyl-CoA dehydrogenase deficiency, glutaryl-CoA dehydrogenase deficiency and congenital adrenal hyperplasia, together with several other disorders detectable by tandem mass spectrometry. Newborn screening deals with rare disorders and benefit cannot be shown easily without very large pilot studies. There have been randomised controlled trials of screening for cystic fibrosis, and now several studies are beginning to establish the benefit of tandem mass spectrometry screening for disorders of fatty acid and amino acid metabolism. Two things will influence the new directions for newborn screening: the development of effective treatments for hitherto untreatable disorders, and advancing technology, enabling new testing strategies to be developed. There are novel treatments on the horizon for many discrete disorders. Susceptibility testing has recently been considered for newborn screening application, but is more controversial. Newborn screening has entered a new and exciting phase, with an explosion of new treatments, new technologies, and, possibly in the future, new preventive strategies.

Aims: Expanded newborn screening uses a new technology, tandem mass spectrometry, to diagnose an additional 20 plus rare treatable inborn errors of metabolism based on the further analysis of the current newborn Guthrie card blood sample. The purpose of this study was to investigate the incidence of these disorders in New Zealand, based on clinical diagnosis rates, and compare these to the incidence, based on the established expanded newborn screening programme, in New South Wales, Australia., Methods: Over a 3-year period, the cases of inborn errors of metabolism notified to the New Zealand Paediatric Surveillance Unit and/or identified by the relevant metabolic laboratories were recorded and compared to the incidence rates during the same period in New South Wales., Results: There were 175,000 and 270,000 births in New Zealand and New South Wales respectively during the study period. Eight cases of treatable inborn errors (potentially diagnosable by newborn screening) were diagnosed in New Zealand compared to 41 (including two prior to screening) in New South Wales. The disorder medium chain acyl Co-A dehydrogenase deficiency was diagnosed twice in New Zealand and in 24 newborn infants in New South Wales., Conclusions: Without expanded newborn screening, inborn errors of metabolism are under-diagnosed in New Zealand. This study supports the recent establishment of screening in New Zealand.

Objective: To describe and analyze the use and costs of hospital services for children diagnosed with medium-chain acyl-CoA dehydrogenase (MCAD) deficiency either with newborn screening or clinical diagnosis in Australia between 1994 and 2002. MCAD deficiency is a potentially lethal disorder of fatty-acid oxidation., Study Design: We conducted a retrospective audit of medical records supplemented by a parental survey., Results: A total of 59 children with MCAD deficiency were identified, 24 by using newborn screening. In the first 4 years of life, screening children cost an average of A$1676 (US$1297) per year for inpatient, emergency department, and outpatient visits, compared with A$1796 (US$1390) for children in whom a clinical diagnosis was made. Forty-two percent of the children who underwent screening were admitted to the hospital, compared with 71% of children who did not undergo screening. Children who did not undergo screening used significantly more inpatient services and cost significantly more in emergency services. There were also some significant differences in use on a year-by-year basis., Conclusions: Children who do not undergo screening may be more likely to be admitted to the hospital and to incur higher emergency department costs than children who underwent screening, and children seem more likely to attend hospital outpatient clinics. Screening does not result in higher costs from a hospital perspective.

Newborn screening for cystic fibrosis has been carried out for over 25 years, and clinical and cost benefits have been documented. There is still much variation in the methods and strategies adopted. All current screening programmes use a measurement of immunoreactive trypsin as a primary screening test, and in most, a second tier test involves analysing DNA mutations. The choice of DNA mutations depends on the genetic background in the region, and considerations of cost. Using DNA analysis as part of a screening procedure has introduced unwanted carrier detection, and protocols have now been devised in an attempt to avoid this. There are at least seven distinct protocols in use, all of which have different advantages and disadvantages, and no method or strategy will suit every region. Further careful study of performance and costs of various strategies is needed.

Objectives: We present the first prenatal diagnosis of familial hepatic veno-occlusive disease with immunodeficiency (VODI). Homozygous mutations in the gene SP110 are the genetic basis of VODI. The proband in this report presented at three months of age with hepatomegaly hepatic failure and was found to have hypogammaglobulinemia. He died one month after hepatic transplant at eight months of age due to hemophagocytic syndrome. DNA testing detected a homozygous truncating mutation in exon 5; SP110 c.642delC. Prenatal testing was offered to this family in a subsequent pregnancy., Methods: Chorion villus was sampled at 12 weeks' gestation. DNA was extracted using standard techniques, and sequencing of SP110 exon 5 was performed using flanking primers. Maternal contamination was excluded by examining STR markers in CVS and maternal DNA., Results: A heterozygous SP110 c.642delC mutation was detected in exon 5. This mutation was present in heterozygous form in both parents., Conclusions: The prenatal test result is predictive of a child with a normal immune and hepatic phenotype. This report presents the first prenatal molecular diagnosis for VODI and shows the importance of molecular genetic research in not only defining the aetiology of syndromes but also in assisting reproductive choices through the collaboration of genetic and feto-maternal services.

Homozygosity or compound heterozygosity for the c.833T>C transition (p.I278 T) in the cystathionine beta-synthase (CBS) gene represents the most common cause of pyridoxine-responsive homocystinuria in Western Eurasians. However, the frequency of the pathogenic c.833C allele, as observed in healthy newborns from several European countries (q(c.833C) approximately equals 3.3 x 10(-3)), is approximately 20-fold higher than expected on the basis of the observed number of symptomatic homocystinuria patients carrying this mutation (q(c.833C) approximately equals 0.18 x 10(-3)), implying clinical underascertainment. Intriguingly, the c.833C mutation is also present in combination with a 68-bp insertion, c.[833C; 844&#95;845ins68], in a substantial proportion of chromosomes from nonhomocystinuric individuals worldwide. We have sought to study the relationship between the pathogenic and nonpathogenic c.833C-bearing chromosomes and to determine whether the pathogenic c.[833C; -] chromosomes are identical-by-descent or instead arose by recurrent mutation. Initial haplotype analysis of 780 randomly selected Czech and sub-Saharan African wild-type chromosomes, employing 12 intragenic markers, revealed 29 distinct CBS haplotypes, of which 10 carried the c.[833C; 844&#95;845ins68] combination; none carried an isolated c.833C or c.844&#95;845ins68 mutation. Subsequent examination of 69 pathogenic c.[833C; -] chromosomes, derived from homocystinuria patients of predominantly European origin, disclosed three unrelated haplotypes that differed from their wild-type counterparts by virtue of the presence of c.833C, thereby indicating that c.833T>C transition has occurred repeatedly and independently in the past. Since c.833T does not reside within an obvious mutational hotspot, we surmise that the three pathogenic and comparatively prevalent c.[833C; -] chromosomes may have originated by recurrent gene conversion employing the common nonpathogenic c.[833C; 844&#95;845ins68] chromosomes as templates.

Background: Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is the disorder thought most to justify neonatal screening by tandem-mass spectrometry because, without screening, there seems to be substantial morbidity and mortality. Our aim was to assess the overall effectiveness of neonatal screening for MCAD deficiency in Australia., Methods: We identified MCAD-deficient patients from a total population of 2,495,000 Australian neonates (810,000 screened) born between April 1, 1994, and March 31, 2004. Those from a cohort of 1,995,000 (460,000 screened) were followed up for at least 4 years, and we recorded number of deaths and severe episodes, medical and neuropsychological outcome, and hospital admissions within the screened and unscreened groups., Findings: In cohorts aged at least 4 years there were 35 MCAD-deficient patients in those not screened (2.28 per 100,000 total population) and 24 in the screened population (5.2 per 100,000). We estimated that patients with this disorder in the unscreened cohort remained undiagnosed. Before 4 years of age, three screened patients had an episode of severe decompensation (including one neonatal death) versus 23 unscreened patients (including five deaths). At the most conservative estimate, relative risk of an adverse event was 0.44 (95% CI 0.13-1.45). In the larger cohort the relative risk (screened vs unscreened) of an adverse event by age 2 years was 0.26 (95% CI 0.07-0.97), also a conservative estimate. 38 of 52 living patients had neuropsychological testing, with no suggestions of significant differences in general cognitive outcome between the groups., Interpretation: Screening is effective in patients with MCAD deficiency since early diagnosis reduces deaths and severe adverse events in children up to the age of 4 years.

Objectives: To determine whether pregnant women and their newborns show evidence of iodine deficiency, and to examine the correlation between maternal urine iodine concentration (UIC) and newborn thyroid-stimulating hormone (TSH) level., Design: A cross-sectional study., Setting: Hospital antenatal care services (March-May 2004) and private obstetrician clinics (June 2004) in the Central Coast area of New South Wales., Participants: 815 pregnant women (> or = 28 weeks' gestation) and 824 newborns., Main Outcome Measures: World Health Organization/International Council for the Control of Iodine Deficiency Disorders criteria for assessing severity of iodine deficiency (recommended levels: < 20% of urine samples in a population with UIC < 50 microg/L; and < 3% of newborns with whole-blood TSH level > 5 mIU/L)., Results: The median UIC for pregnant women was 85 microg/L, indicating mild iodine deficiency. Almost 17% of pregnant women had a UIC < 50 microg/L, and 18 newborns (2.2%) had TSH values > 5 mIU/L. There was no statistically significant linear correlation between neonatal whole-blood TSH level and maternal UIC (r = - 0.03; P = 0.4). Mothers with a UIC < 50 microg/L were 2.6 times (relative risk = 2.65; 95% CI, 1.49-4.73; P = 0.01) more likely to have a baby with a TSH level > 5 mIU/L., Conclusion: The pregnant women surveyed were mildly iodine deficient. TSH values for their newborns were mostly within acceptable limits. Ongoing surveillance of the iodine status of NSW communities to establish trends over time is recommended.

'Classical organic acidurias' comprise isovaleric aciduria, propionic aciduria and methylmalonic aciduria. Available data from the literature suggest that the use of 'new' therapeutic strategies has improved survival but has not modified neurodevelopment. Progressive neurocognitive deterioration is almost invariably present in propionic and methylmalonic acidurias, while large-scale studies on the long-term outcome of patients with isovaleric aciduria are still lacking. In order to answer to some of the questions suggested by Wilson and Jungner in 1968 about the criteria of disease screening, we compared the natural history of patients with 'classical' organic acidurias diagnosed on clinical bases to those diagnosed through neonatal mass screening using tandem mass spectrometry. Decreased early mortality, less severe symptoms at diagnosis, and more favourable short-term neurodevelopmental outcome were recorded in patients identified through expanded newborn screening. The short duration of follow-up so far does not allow us to draw final conclusions about the effects of newborn screening on long-term outcome. The evaluation of the effect of neonatal screening on the detection rate of these three diseases showed that the incidence of isovaleric aciduria was significantly higher in the screening population than in clinically detected cases, with no changes for propionic and methylmalonic acidurias. Further multicentre longitudinal studies are needed to assess the usefulness of expanded newborn screening for 'classical' organic acidurias and to better understand the clinical spectrum of these diseases. This paper describes the long-term outcome and the impact of expanded newborn screening on the so-called 'classical' organic acidurias (propionic aciduria, methylmalonic aciduria and isovaleric aciduria).

With the application of tandem mass spectrometry, newborn screening has become an important topic in inborn metabolic disease. The aim of newborn screening is to produce an improved clinical outcome by early detection of disease, but it has been difficult to measure clinical effectiveness. Good evidence of clinical effectiveness has been hard to obtain because of the rarity of individual disorders, often precluding randomized controlled trials, the increase in diagnosis of individual disorders by screening, compared with clinical diagnosis, variable definitions of what constitutes a case, uncertainty about completeness of ascertainment, and differences in treatment in different geographical areas or at different times. Multiplex testing has introduced some new problems. There have been recent attempts to standardize screening in several countries, which have taken different approaches. Public pressure has driven the introduction of screening for inborn errors in some areas. Since it seems inevitable that screening may often be implemented ahead of hard evidence of benefit, ongoing evaluation of clinical effectiveness is a necessary part of any screening programme.

Objective: Vascular disease is associated with increased plasma asymmetric dimethylarginine (ADMA) and homocysteine, and both are increased in renal failure. In cystathionine beta-synthase deficiency (CBS) there is severe hyperhomocysteinaemia, precocious vascular disease, and endothelial dysfunction. We investigated whether ADMA levels are elevated in CBS patients with and without renal impairment, and whether lowering plasma homocysteine also lowers ADMA., Methods: We measured plasma homocysteine, arginine, asymmetric and symmetric dimethylarginines, nitrate + nitrite, creatinine and cystatin C in 23 CBS-deficient patients and 24 age-matched controls., Results: In the patients, nitrate + nitrite and the ratio L: -arginine/ADMA were markedly reduced (21.6 +/- 6.1 vs 57.7 +/- 7.5 micromol/L and 132.9 +/- 24.7 vs 181.9 +/- 56.1, respectively, p < 0.001 for both), reflecting endothelial dysfunction. Plasma ADMA for the group was moderately increased (0.55 +/- 0.08 vs 0.49 +/- 0.07 micromol/L, p = 0.018), but this was due to significantly higher levels than controls in only those 7 of the 23 patients who had elevated cystatin C levels (0.59 +/- 0.08 vs 0.49 +/- 0.07 mg/L, p = 0.007). Posttreatment total homocysteine in patients varied widely (15-285, median 92 micromol/L), but was not correlated with ADMA or other measured variables. In three newly-diagnosed patients, marked reduction of total homocysteine during treatment produced minimal changes in ADMA., Conclusions: ADMA levels were significantly increased only in the CBS-deficient patients with elevated cystatin C levels, and not in those with normal renal function. The reported relationship between hyperhomocysteinaemia and ADMA may not be direct, but could be secondary to reduced renal function.

Context: Many developed countries are reexperiencing iodine deficiency. One World Health Organization index of iodine deficiency in populations is the percentage of neonates with TSH levels greater than 5 mIU/liter 72 h after delivery. Measured TSH levels vary with methodology and are influenced by external factors including iodine exposure at time of delivery., Objective: We wished to determine whether babies delivered by cesarean section have higher levels of TSH than babies delivered vaginally because this factor could influence determination of iodine deficiency., Design and Setting: This was a cohort study of mothers delivering at a teaching hospital in 2002-2003 and their babies., Patients and Methods: Women delivering a live infant were eligible for the study. Demographic data, mode of delivery, and subsequent routine newborn screening TSH levels were recorded., Results: Of 2031 infants, 31.2% were delivered by cesarean section; 1864 babies were born after 37 wk, with blood samples collected by d 5 (>91% on d 3). Significant predictors of TSH concentration were the day TSH was measured and type of delivery. The distribution curve of TSH values was right shifted in infants born by cesarean section. The percentage of neonates born by cesarean with at TSH value greater than 5 was 7.1%. The figure for those born vaginally was 4.3%., Conclusions: Babies delivered by cesarean section are significantly more likely to have TSH levels greater than 5 mIU/liter on d 3 than those delivered vaginally. With the rise in the rate of births by cesarean, this could be an important factor in assessing population iodine deficiency using neonatal TSH levels.

Over 400 rare, biochemically diverse genetic metabolic disorders (inborn errors of metabolism) have been described and the list is growing by the month. Although recent advances in the diagnosis and treatment of these disorders have substantially improved the prognosis for many of them, including those presenting in the neonatal period, a proportion of affected individuals die before the diagnosis can be confirmed and, in some cases, before the diagnosis is even considered. This review will provide an outline of the range of clinical presentations seen in neonates with genetic metabolic disorders and provide a practical approach for rapid biochemical screening for these disorders. In addition, suggested guidelines are given for the collection of relevant samples in the perimortem period, the aim being to maximize the chance of identifying any underlying genetic metabolic disorder., (Copyright 2004 Elsevier Ltd.)

An efficient newborn screening test for detecting cystic fibrosis has been available for over 20 years but is only now coming into widespread use. Blood immunoreactive trypsin is elevated in babies with cystic fibrosis and its measurement in dried blood spots is the primary screening tool. Poor discrimination in the first week requires a re-sampling step. The identification of the cystic fibrosis transmembrane conductance regulator gene and the discovery of a common mutation has allowed a combination of the primary screening test with a DNA test using the same sample. Differing genetic backgrounds have led to the development of population-specific protocols. A false-negative rate of around 5% is usual. Specificity is high. In all protocols involving a DNA test, confirmation of the diagnosis by sweat test is necessary when only one mutation is identified, identification of some carriers therefore being unavoidable. Careful counselling is needed for the families of these carriers.

Medical ethics is an integral part of medical practice. The general principles are well known: autonomy (the right to choose), beneficence (do good), non-maleficence (do no harm), and justice (be fair and equitable). In newborn screening these principles must be especially carefully applied, as the intervention, screening, has not been sought by the patient, but is a form of preventive medicine. In proposing a screening programme questions to be asked are: should we do it? (is there enough benefit, not too much harm?); can we do it? (do we have the technology and skill to find the cases sought); can we afford it? The first question, with its ethical implications, is often ignored. New issues have arisen with new technology, but underlying ethical themes are the same. Tandem mass spectrometry can be used to detect about 30 very rare disorders in a single test. Proving the benefit of this (and other screening tests) is difficult because randomised controlled trials seem impractical, because of power considerations, long follow-up time, and because there is already a perceived benefit. Best possible evidence of a lower order must be sought. In future, DNA microarray technology is likely to become sufficiently inexpensive to apply to newborn screening. It is difficult to predict all the future possibilities of DNA technology in this fast-moving field. Major ethical problems are likely with the ability to detect adult-onset disorders or susceptibility to these in babies. Under what circumstances would this be ethical? We need to start debating these issues.

The conversion of 3-methylglutaconyl-CoA to 3-hydroxy-3-methylglutaryl-CoA is the only step in leucine catametabolism yet to be characterized at enzyme and DNA levels. The deficiency of the putative mitochondrial enzyme 3-methylglutaconyl-CoA hydratase associates with the rare organic aciduria 3-methylglutaconic aciduria type I (MGA1), but neither the enzyme nor its gene have been described in any organism. Here we report that human 3-methylglutaconyl-CoA hydratase is identical with a previously described RNA-binding protein (designated AUH) possessing enoyl-CoA hydratase activity. Molecular analyses in five patients from four independent families revealed homozygosity or compound heterozygosity for mutations in the AUH gene; most mutations are predicted to completely abolish protein function. Mutations identified include c.80delG, R197X, IVS8-1G>A, A240V, and c.613&#95;614insA. Clinical severity of MGA1 in published patients has been quite variable. Included in the present study is an additional patient with MGA1 who was detected by neonatal screening and has remained asymptomatic up to his present age of 2 years. The boy is homozygous for an N-terminal frameshift mutation in the AUH gene. Complete absence of 3-methylglutaconyl-CoA hydratase/AUH appears to be compatible with normal development in some cases. Further work is required to identify external or genetic factors associated with development of clinical problems in patients with MGA1., (Copyright 2003 Wiley-Liss, Inc.)

Treatment of metabolic disease aims to restore homeostasis, where possible. This can be achieved in a number of ways. For disorders of intermediary metabolism, treatment involves a thorough understanding of the disorder and the pathogenesis of the deleterious effects The various approaches indicated may involve substrate restriction, replacement of deficient products, removal of toxic metabolites or stimulation of residual enzymes. Newer therapies include enzyme replacement and gene therapy. Often, the cornerstone of treatment is dietary. Substrate restriction includes not only a diet low in the substrate indicated by the disorder, but also strict calorie support in times of illness to avoid catabolism. Useful levels of substrate restriction may require the use of supplements of "medical foods", for example amino acid mixtures. Provision of the deficient products is important in disorders affecting energy metabolism. To understand the problems involved in nutritional treatment it is helpful to consider examples of different types of disorders. In Maple syrup urine disease (MSUD), treatment with a very strict low-protein diet, supplemented by a branched-chain-free amino acid mixture is successful, but each intercurrent illness is hazardous, regimens for sick days vital, and strict lifelong treatment is needed. Treatment for phenylketonuria is similar in restricting a substrate but there is no tendency for systemic illness if the phenylalanine levels are too high. Disorders of the urea cycle are difficult dietary challenges because while a very low-protein diet is required, no specific amino acid needs to be avoided and there is a fine line between adequate protein intake and chronic catabolism. Fatty acid oxidation disorders affect energy production and can be detected by newborn screening using tandem mass spectrometry. For long-chain fatty acid disorders, long chain fats must largely be avoided and medium-chain fats must be substituted while strictly avoiding catabolism. Glycogen storage disorders require strict attention to providing carbohydrate, at all times including throughout the night. Many patients with inborn errors do not need any specific dietary therapy, (eg those with storage or neurodegenerative disorders), although all children benefit from an optimal diet, and sick children need this especially.

Computerization within newborn screening programs is a developing issue. To date two basic approaches to data storage have been used: (1) a storage system for babies diagnosed with a disorder, (2) a comprehensive system with long-term details for all patient samples, tests performed, test results and interpretations. It usually provides efficient real-time reports for various clinical and quality control requirements and easy access to an inborn errors registry. Within the last decade there have been two new technologies adapted to routine use in newborn screening laboratories: (1) tandem mass spectrometry for selected amino acids and acyl carnitine, and (2) DNA mutational analysis of PCR products. Both technologies present data storage challenges. Both are capable of providing large files of information for a sample. Consideration must be given to how these data are stored, whether all results including a graphical representation or DNA sequence data are kept or whether only final results for specific analytes are stored. Many new analytical technologies can only be incorporated into routine programs as a result of advances in hardware and software allowing better access to, and storage of, data.

Newborn screening is a medical intervention. For every program, there should be evidence of its effect and effectiveness. The four questions to be addressed, very broadly, are: What is the effectiveness for case-finding (sensitivity, specificity, and positive predictive value)? What are the benefits of early detection versus clinical detection? What harm results from the program? Are the costs reasonably balanced in relation to benefits? Ideally, there would be randomized controlled trials (RCTs) of screening for each disorder. In practice, power calculations reveal that for very rare disorders this is not feasible. The numbers of screened and unscreened babies required would be huge, and trials would last for decades. There have only been RCTs of newborn screening for cystic fibrosis (birth prevalence 25-40 per 100,000 in Caucasians). No such trials were ever attempted for hypothyroidism, with a similar birth prevalence, and it may not now be ethical to mount one. Instead, lower orders of evidence must be used. Double-blind randomized controlled trials should be planned for not-so-rare disorders where possible. Where it is not feasible, careful planning and collection of data, plus the use of both historical controls and contemporaneous controls from other regions may have to suffice. To introduce programs with no plans for full evaluation is not best practice. Evaluation of outcomes of all kinds, not simply of case-finding, must be mandatory. Data for case-finding should be collected actively, with systematic searching for missed cases. Data about benefits need to be collected in well-planned long-term studies, although short-term benefits are also valuable. Good studies of harm, mainly from false positive results, are urgently needed. The problem of costs and benefits is difficult, and a "reasonable balance" rather than positive cost/benefit ratio seems desirable.

The incorporation of tandem mass spectrometry (MSMS) into an existing newborn screening program is an evolving process. Limited worldwide experience has ensured that all stages of reliability testing need to be followed. These include a literature review to establish methodology and analytes/disorders for testing and a pilot screening project including assaying archival samples from subjects with proven target disorders. Algorithms used for analyte concentrations and the relationships of various analytes to one another for resample criteria need to be continually reassessed to maximise screening specificity, sensitivity and positive predictive value. Since 1st of April 1998, the NSW Newborn Screening Program has screened 320, 848 babies using electrospray MSMS for selected amino acids and acyl camitines. Screening for amino acids has led to requests for 415 repeat samples with 94 babies referred for further testing. Of these 73 had a disorder of amino acid metabolism, including 43 with persistent hyperphenylalaninemia (36 of whom had PKU, 2 had a pterin pathway defect, 5 HPAA). Screening for acyl carnitines has led to requests for 245 repeat samples with 63 babies referred for further investigation. Of these 44 had a diagnosed disorder, including 15 with medium chain acyl CoA dehydrogenase deficiency. Five babies with confirmed disorders detectable with MS/MS had negative test results. The cost of screening using MSMS was only $A0.50 more than the method for screening for PKU and homocystinuria alone (ie the bacterial inhibition assays) and has allowed detection of an additional 74 babies at least 48 of whom have a diagnosis for which early treatment seems clearly beneficial. MSMS has shown a sensitivity of 95.9% and specificity of 99.8% in our laboratory with a positive predictive value of 18%.

Cystathionine beta-synthase (CBS) deficiency is the most common cause of homocystinuria. It is inherited as an autosomal recessive trait and common clinical features are: dislocation of the optic lens, osteoporosis, mental retardation, and thromboembolism. We determined the molecular basis of CBS deficiency in 36 Australian patients from 28 unrelated families, using direct sequencing of the entire coding region of the CBS gene. The G307S and I278T mutations were the most common mutations. They were present in 19% and 18% of independent alleles, respectively. In total, seven novel and 20 known mutations were detected. Of those, the two novel missense mutations (C109R and G347S), as well as two known missense mutations (L101P and N228K), were expressed in E. Coli. All mutant proteins completely lacked catalytic activity. Furthermore, we studied the correlation between genotype and the biochemical response to pyridoxine treatment in the patients of whom 13 were pyridoxine responsive, 21 were non-responsive, and two were partially responsive. The G307S mutation always resulted in a severe non-responsive phenotype, whereas I278T resulted in a milder B6 responsive phenotype. From our results, we were also able to establish three other mild mutations: P49L, R369C, and V371M., (Copyright 2002 Wiley-Liss, Inc.)