Clinical utility - Information about the usefulness of tests.

The clinical utility of a diagnostic test refers to its usefulness in improving patient outcomes, informing clinical decision-making, and optimizing healthcare resources. A diagnostic test with high clinical utility provides accurate, reliable, and actionable information that can guide appropriate treatment decisions, monitor treatment response, and identify potential adverse events or complications. Ultimately, the clinical utility of a diagnostic test depends on how well it can improve patient outcomes by guiding appropriate treatment decisions, improving clinical outcomes, and optimizing healthcare resource utilization. Healthcare providers need to weigh the benefits and drawbacks of using a particular diagnostic test in their clinical practice to determine its clinical utility.

Methods for determining clinical utility.

Measuring the clinical utility of a diagnostic test involves evaluating its impact on patient outcomes, clinical decision-making, and healthcare resource utilization. Determining clinical utility requires accessing patient medical history and outcomes data. These studies involve enrolling patients undergoing diagnostic tests and tracking their clinical outcomes. Researchers can determine the test's clinical utility by comparing the outcomes of patients who receive the diagnostic test to those who do not. These outcomes include benefits and harm. The highest level of evidence to support clinical utility determinations may be obtained from clinical trials. However, clinical laboratories are often not involved in clinical trials, and laboratory specialists may not be experienced in conducting such trials. Many established laboratory tests have never had clinical utility determined. Prospective studies assessing a diagnostic test's impact on clinical outcomes may require long-term patient monitoring, which is problematic. This paper presents methods that may be used to assess clinical utility.

SuPreme Study: a protocol to study the neuroprotective potential of sulfate among very/extremely preterm infants.

INTRODUCTION: Antenatal maternal magnesium sulfate (MgSO4) administration is a proven efficacious neuroprotective treatment reducing the risk of cerebral palsy (CP) among infants born preterm. Identification of the neuroprotective component with target plasma concentrations could lead to neonatal treatment with greater efficacy and accessibility. METHODS AND ANALYSIS: This is a prospective observational cohort study, in three tertiary Australian centres. Participants are preterm infants, irrespective of antenatal MgSO4 exposure, born in 2013-2020 at 24+0 to 31+6 weeks gestation, and followed up to 2 years corrected age (CA) (to September 2023). 1595 participants are required (allowing for 17% deaths/loss to follow-up) to detect a clinically significant reduction (30% relative risk reduction) in CP when sulfate concentration at 7 days of age is 1 SD above the mean.A blood sample is collected on day 7 of age for plasma sulfate and magnesium measurement. In a subset of participants multiple blood and urine samples are collected for pharmacokinetic studies, between days 1-28, and in a further subset mother/infant blood is screened for genetic variants of sulfate transporter genes.The primary outcome is CP. Surviving infants are assessed for high risk of CP at 12-14 weeks CA according to Prechtl's Method to assess General Movements. Follow-up at 2 years CA includes assessments for CP, cognitive, language and motor development, and social/behavioural difficulties.Multivariate analyses will examine the association between day 7 plasma sulfate/magnesium concentrations with adverse neurodevelopmental outcomes. A population pharmacokinetic model for sulfate in the preterm infant will be created using non-linear mixed-effects modelling. ETHICS AND DISSEMINATION: The study has been approved by Mater Misericordiae Ltd Human Research Ethics Committee (HREC/14/MHS/188). Results will be disseminated in peer-reviewed journal publications, and provided to the funding bodies. Using consumer input, a summary will be prepared for participants and consumer groups.

Hyperlactatemia in diabetic ketoacidosis is common and can be prolonged: lactate time-series from 25 intensive care admissions.

Hyperlactatemia is a documented complication of diabetic ketoacidosis (DKA). Lactate responses during DKA treatment have not been studied and were the focus of this investigation. Blood gas and electrolyte data from 25 DKA admissions to ICU were sequenced over 24 h from the first Emergency Department sample. Hyperlactatemia (> 2 mmol/L) was present in 22 of 25 DKA presentations [mean concentration = 3.2 mmol/L]. In 18 time-series (72%), all concentrations normalized in ≤ 2.6 h (aggregate decay t1/2 = 2.29 h). In the remaining 7 (28%), hyperlactatemia persisted > 12 h. These were females (P = 0.04) with relative anemia (hemoglobin concentrations 131 v 155 g/L; P = 0.004) and lower nadir glucose concentrations (5.2 v 8.0 mmol/L, P = 0.003). Their aggregate glucose decay curve commenced higher (42 mmol/L v 29 mmol/L), descending towards a lower asymptote (8 mmol/L v 11 mmol/L). Tonicity decay showed similar disparities. There was equivalent resolution of metabolic acidosis and similar lengths of stay in both groups. Hyperlactatemia is common in DKA. Resolution is often rapid, but high lactates can persist. Females with high glucose concentrations corrected aggressively are more at risk. Limiting initial hyperglycemia correction to ≥ 11 mmol/L may benefit.

A direct comparison of patient-based real-time quality control techniques: The importance of the analyte distribution.

BACKGROUND: Patient-based real-time quality control (PBRTQC) techniques have been described in clinical chemistry for over 50 years. PBRTQC has a number of advantages over traditional quality control including commutability, cost and the opportunity for real-time monitoring. However, there are few systematic investigations assessing how different PBRTQC techniques perform head-to-head. METHODS: In this study, we compare moving averages with and without truncation and moving medians. For analytes with skewed distributions such as alanine aminotransferase and creatinine, we also investigate the effect of Box-Cox transformation of the data. We assess the ability of each technique to detect simulated analytical bias in real patient data for multiple analytes and to retrospectively detect a real analytical shift in a creatinine and urea assay. RESULTS: For analytes with symmetrical distributions, we show that error detection is similar for a moving average with and without four standard deviation truncation limits and for a moving median. In contrast to analytes with symmetrically distributed results, moving averages perform poorly for right skewed distributions such as alanine aminotransferase and creatinine and function only with a tight upper truncation limit. Box-Cox transformation of the data both improves the performance of moving averages and allows all data points to be used. This was also confirmed for retrospective detection of a real analytical shift in creatinine and urea. CONCLUSIONS: Our study highlights the importance of careful assessment of the distribution of patient results for each analyte in a PBRTQC program with the optimal approaches dependent on whether the patient result distribution is symmetrical or skewed.

Intact transferrin and total plasma glycoprofiling for diagnosis and therapy monitoring in phosphoglucomutase-I deficiency.

Phosphoglucomutase 1 (PGM1) deficiency results in a mixed phenotype of a Glycogen Storage Disorder and a Congenital Disorder of Glycosylation (CDG). Screening for abnormal glycosylation has identified more than 40 patients, manifesting with a broad clinical and biochemical spectrum which complicates diagnosis. Together with the availability of D-galactose as dietary therapy, there is an urgent need for specific glycomarkers for early diagnosis and treatment monitoring. We performed glycomics profiling by high-resolution QTOF mass spectrometry in a series of 19 PGM1-CDG patients, covering a broad range of biochemical and clinical severity. Bioinformatics and statistical analysis were used to select glycomarkers for diagnostics and define glycan-indexes for treatment monitoring. Using 3 transferrin glycobiomarkers, all PGM1-CDG patients were diagnosed with 100% specificity and sensitivity. Total plasma glycoprofiling showed an increase in high mannose glycans and fucosylation, while global galactosylation and sialylation were severely decreased. For treatment monitoring, we defined 3 glycan-indexes, reflecting normal glycosylation, a lack of complete glycans (LOCGI) and of galactose residues (LOGI). These indexes showed improved glycosylation upon D-galactose treatment with a fast and near-normalization of the galactose index (LOGI) in 6 out of 8 patients and a slower normalization of the LOCGI in all patients. Total plasma glycoprofiling showed improvement of the global high mannose glycans, fucosylation, sialylation, and galactosylation status on D-galactose treatment. Our study indicates specific glycomarkers for diagnosis of mildly and severely affected PGM1-CDG patients, and to monitor the glycan-specific effects of D-galactose therapy.

Oral D-galactose supplementation in PGM1-CDG.

PurposePhosphoglucomutase-1 deficiency is a subtype of congenital disorders of glycosylation (PGM1-CDG). Previous casereports in PGM1-CDG patients receiving oral D-galactose (D-gal) showed clinical improvement. So far no systematic in vitro and clinical studies have assessed safety and benefits of D-gal supplementation. In a prospective pilot study, we evaluated the effects of oral D-gal in nine patients.MethodsD-gal supplementation was increased to 1.5 g/kg/day (maximum 50 g/day) in three increments over 18 weeks. Laboratory studies were performed before and during treatment to monitor safety and effect on serum transferrin-glycosylation, coagulation, and liver and endocrine function. Additionally, the effect of D-gal on cellular glycosylation was characterized in vitro.ResultsEight patients were compliant with D-gal supplementation. No adverse effects were reported. Abnormal baseline results (alanine transaminase, aspartate transaminase, activated partial thromboplastin time) improved or normalized already using 1 g/kg/day D-gal. Antithrombin-III levels and transferrin-glycosylation showed significant improvement, and increase in galactosylation and whole glycan content. In vitro studies before treatment showed N-glycan hyposialylation, altered O-linked glycans, abnormal lipid-linked oligosaccharide profile, and abnormal nucleotide sugars in patient fibroblasts. Most cellular abnormalities improved or normalized following D-gal treatment. D-gal increased both UDP-Glc and UDP-Gal levels and improved lipid-linked oligosaccharide fractions in concert with improved glycosylation in PGM1-CDG.ConclusionOral D-gal supplementation is a safe and effective treatment for PGM1-CDG in this pilot study. Transferrin glycosylation and ATIII levels were useful trial end points. Larger, longer-duration trials are ongoing.

Defining the Phenotype and Assessing Severity in Phosphoglucomutase-1 Deficiency.

OBJECTIVE: To define phenotypic groups and identify predictors of disease severity in patients with phosphoglucomutase-1 deficiency (PGM1-CDG). STUDY DESIGN: We evaluated 27 patients with PGM1-CDG who were divided into 3 phenotypic groups, and group assignment was validated by a scoring system, the Tulane PGM1-CDG Rating Scale (TPCRS). This scale evaluates measurable clinical features of PGM1-CDG. We examined the relationship between genotype, enzyme activity, and TPCRS score by using regression analysis. Associations between the most common clinical features and disease severity were evaluated by principal component analysis. RESULTS: We found a statistically significant stratification of the TPCRS scores among the phenotypic groups (P < .001). Regression analysis showed that there is no significant correlation between genotype, enzyme activity, and TPCRS score. Principal component analysis identified 5 variables that contributed to 54% variance in the cohort and are predictive of disease severity: congenital malformation, cardiac involvement, endocrine deficiency, myopathy, and growth. CONCLUSIONS: We established a scoring algorithm to reliably evaluate disease severity in patients with PGM1-CDG on the basis of their clinical history and presentation. We also identified 5 clinical features that are predictors of disease severity; 2 of these features can be evaluated by physical examination, without the need for specific diagnostic testing and thus allow for rapid assessment and initiation of therapy.

Reference intervals for plasma sulfate and urinary sulfate excretion in pregnancy.

BACKGROUND: Sulfate is important for fetal growth and development. During pregnancy, the fetus relies on sulfate from the maternal circulation. We report reference intervals for maternal plasma sulfate levels and fractional excretion index (FEI) for sulfate in pregnancy, as well as sulfate levels in cord blood from term pregnancies. METHODS: Plasma and urine were collected from 103 pregnant women of 10-20 weeks gestation and 106 pregnant women of 30-37 weeks gestation. Venous cord plasma was collected from 80 healthy term babies. Sulfate levels were measured by ion chromatography. Plasma and urinary creatinine levels were used to calculate FEI sulfate in pregnant women. Analyses provide reference intervals, and explored the relationship between maternal sulfate data with several prenatal factors. RESULTS: Median maternal plasma sulfate levels were 452 µmol/L and 502 µmol/L at 10-20 and 30-37 weeks gestation, respectively, and inversely correlated with FEI sulfate median values of 0.15 and 0.11. Overall reference intervals were 305-710 and 335-701 µmol/L (2.5th; 97.5th percentile; for 10-20 and 30-37 weeks gestation, respectively) for maternal plasma sulfate, and 0.06-0.31 and 0.05-0.28 for maternal FEI sulfate. Term venous cord plasma sulfate median levels were significantly (p = 0.038) higher in female babies (375 µmol/L) when compared to male babies (342 µmol/L), with an overall reference interval of 175-603 µmol/L. CONCLUSIONS: We provide the first reference intervals for maternal plasma sulfate levels and FEI sulfate, as well as cord plasma sulfate levels. These findings provide reference data for further studies of sulfate levels in both mother and child.

Sulphate in pregnancy.

Sulphate is an obligate nutrient for healthy growth and development. Sulphate conjugation (sulphonation) of proteoglycans maintains the structure and function of tissues. Sulphonation also regulates the bioactivity of steroids, thyroid hormone, bile acids, catecholamines and cholecystokinin, and detoxifies certain xenobiotics and pharmacological drugs. In adults and children, sulphate is obtained from the diet and from the intracellular metabolism of sulphur-containing amino acids. Dietary sulphate intake can vary greatly and is dependent on the type of food consumed and source of drinking water. Once ingested, sulphate is absorbed into circulation where its level is maintained at approximately 300 µmol/L, making sulphate the fourth most abundant anion in plasma. In pregnant women, circulating sulphate concentrations increase by twofold with levels peaking in late gestation. This increased sulphataemia, which is mediated by up-regulation of sulphate reabsorption in the maternal kidneys, provides a reservoir of sulphate to meet the gestational needs of the developing foetus. The foetus has negligible capacity to generate sulphate and thereby, is completely reliant on sulphate supply from the maternal circulation. Maternal hyposulphataemia leads to foetal sulphate deficiency and late gestational foetal death in mice. In humans, reduced sulphonation capacity has been linked to skeletal dysplasias, ranging from the mildest form, multiple epiphyseal dysplasia, to achondrogenesis Type IB, which results in severe skeletal underdevelopment and death in utero or shortly after birth. Despite being essential for numerous cellular and metabolic functions, the nutrient sulphate is largely unappreciated in clinical settings. This article will review the physiological roles and regulation of sulphate during pregnancy, with a particular focus on animal models of disturbed sulphate homeostasis and links to human pathophysiology.

Analyzing the metabolome.

Metabolites, the chemical entities that are transformed during metabolism, provide a functional readout of cellular biochemistry that offers the best prediction of the phenotype and the nature of a disease. Mass spectrometry now allows thousands of metabolites to be quantitated. The targeted or untargeted data from metabolic profiling can be combined with either supervised or unsupervised approaches to improve interpretation. These sophisticated statistical techniques are computationally intensive. This chapter reviews techniques applicable to metabolomics approaches to disease.

A randomized, controlled, crossover trial of fish oil treatment for impulsive aggression in children and adolescents with disruptive behavior disorders.

OBJECTIVE: Epidemiological research links aggression to low serum concentrations of omega-3 fatty acids, such as those found in fish oil. However, no studies have specifically examined whether fish oil supplementation can reduce the frequency and severity of impulsive aggression in children with disruptive behavior disorders. METHODS: Children presenting with impulsive aggression and meeting research criteria for diagnosis of disruptive behavior disorders were randomized to receive either: 1) Fish oil capsules (4 g daily) for 6 weeks followed by placebo (identical-looking capsules) for 6 weeks; or 2) placebo for 6 weeks, followed by fish oil for 6 weeks, in a double-blind, crossover design. Primary outcomes were the Children's Aggression Scale and the Modified Overt Aggression Scale. Secondary outcomes included emotional and behavioral functioning (Strengths and Difficulties Questionnaire [SDQ]), hyperactivity symptoms (Attention-Deficit/Hyperactivity Disorder [ADHD] Rating Scale), family functioning (Family Assessment Device), and cognitive functioning (Stop Signal Task, Trail-Making Task, and Eriksen Flanker Task). Serum concentrations of omega-3 and omega-6 fatty acids were measured at baseline, and at 6 and 12 weeks. RESULTS: Twenty-one children participated (81% male; mean age 10.3±2.2 years; range 7-14). Fish oil treatment increased serum concentrations of eicosapentanoic acid (F=14.76, p<0.05) and total omega-3s (F=20.56, p<0.05), but did not influence primary ratings of aggression. In fact, a trend suggested that fish oil worsened a secondary measure of aggression (SDQ Conduct Subscale, F=4.34, p=0.06). Fish oil treatment was associated with an improvement in one rating of hyperactivity (SDQ Hyperactivity Subscale, F=2.22, p<0.05), but did not influence any other outcome measures. CONCLUSIONS: These findings suggest that fish oil treatment does not improve aggression in children with disruptive behavior disorders.

Inosine triphosphate pyrophosphohydrolase (ITPA) polymorphic sequence variants in adult hematological malignancy patients and possible association with mitochondrial DNA defects.

BACKGROUND: Inosine triphosphate pyrophosphohydrolase (ITPase) is a 'house-cleaning' enzyme that degrades non-canonical ('rogue') nucleotides. Complete deficiency is fatal in knockout mice, but a mutant polymorphism resulting in low enzyme activity with an accumulation of ITP and other non-canonical nucleotides, appears benign in humans. We hypothesised that reduced ITPase activity may cause acquired mitochondrial DNA (mtDNA) defects. Furthermore, we investigated whether accumulating mtDNA defects may then be a risk factor for cell transformation, in adult haematological malignancy (AHM). METHODS: DNA was extracted from peripheral blood and bone marrow samples. Microarray-based sequencing of mtDNA was performed on 13 AHM patients confirmed as carrying the ITPA 94C>A mutation causing low ITPase activity, and 4 AHM patients with wildtype ITPA. The frequencies of ITPA 94C>A and IVS2+21A>C polymorphisms were studied from 85 available AHM patients. RESULTS: ITPA 94C>A was associated with a significant increase in total heteroplasmic/homoplasmic mtDNA mutations (p<0.009) compared with wildtype ITPA, following exclusion of haplogroup variants. This suggested that low ITPase activity may induce mitochondrial abnormalities. Compared to the normal population, frequencies for the 94C>A and IVS2+21A>C mutant alleles among the AHM patients were higher for myelodyplastic syndrome (MDS) - but below significance; were approximately equivalent for chronic lymphoblastic leukemia; and were lower for acute myeloid leukemia. CONCLUSIONS: This study invokes a new paradigm for the evolution of MDS, where nucleotide imbalances produced by defects in 'house-cleaning' genes may induce mitochondrial dysfunction, compromising cell integrity. It supports recent studies which point towards an important role for ITPase in cellular surveillance of rogue nucleotides.

Plasma and urinary sulfate determination in a cohort with autism.

Sulfate is important for mammalian development but is not routinely measured in clinical settings. The renal NaS1 sulfate transporter maintains circulating sulfate levels and is linked to renal sulfate wasting in mice. Some autistic individuals exhibit renal sulfate wasting, but the etiology is yet unknown. We measured plasma and urinary sulfate levels, calculated the fractional excretion index (FEI) of sulfate, and screened for two loss-of-function NaS1 sequence variants (R12X and N174S) in 23 autistic individuals. The FEI sulfate values ranged from 0.13 to 0.50. NaS1 variants were detected in 18 of the 23 individuals (11 heterozygous N174S, four homozygous N174S, two heterozygous R12X, and one individual carried both R12X and N174S). Those individuals with neither sequence variant had FEI sulfate ≤ 0.34, whereas FEI sulfate ≥ 0.35 was found in about 60 % (11 of 18) of individuals that had R12X and/or N174S. This study links renal sulfate wasting with loss-of-function NaS1 sequence variants in humans.

Insights into N-calls of mitochondrial DNA sequencing using MitoChip v2.0.

BACKGROUND: Developments in DNA resequencing microarrays include mitochondrial DNA (mtDNA) sequencing and mutation detection. Failure by the microarray to identify a base, compared to the reference sequence, is designated an 'N-call.' This study re-examined the N-call distribution of mtDNA samples sequenced by the Affymetrix MitoChip v.2.0, based on the hypothesis that N-calls may represent insertions or deletions (indels) in mtDNA. FINDINGS: We analysed 16 patient mtDNA samples using MitoChip. N-calls by the proprietary GSEQ software were significantly reduced when either of the freeware on-line algorithms ResqMi or sPROFILER was utilized. With sPROFILER, this decrease in N-calls had no effect on the homoplasmic or heteroplasmic mutation levels compared to GSEQ software, but ResqMi produced a significant change in mutation load, as well as producing longer N-cell stretches. For these reasons, further analysis using ResqMi was not attempted. Conventional DNA sequencing of the longer N-calls stretches from sPROFILER revealed 7 insertions and 12 point mutations. Moreover, analysis of single-base N-calls of one mtDNA sample found 3 other point mutations. CONCLUSIONS: Our study is the first to analyse N-calls produced from GSEQ software for the MitoChipv2.0. By narrowing the focus to longer stretches of N-calls revealed by sPROFILER, conventional sequencing was able to identify unique insertions and point mutations. Shorter N-calls also harboured point mutations, but the absence of deletions among N-calls suggests that probe confirmation affects binding and thus N-calling. This study supports the contention that the GSEQ is more capable of assigning bases when used in conjunction with sPROFILER.

Pyridoxine supply in human development.

Vitamin B(6) has an important role in the function of the human nervous system. Experimental data are not generally available on the role in human development, but significant conclusions may be made from studies of the effect of disorders of B(6) vitamer metabolism. Vitamin B(6) comprises seven compounds - pyridoxal, pyridoxine, pyridoxamine and their respective 5' phosphates. The common active form in human tissue is the 5'-phosphate form of pyridoxal (PLP) most of which is found in muscle bound to phosphorylase. Like many vitamins, B(6) can function both as a co-enzyme and as a chaperone. Pyridoxal-5'-phosphate is the metabolically active form and is involved in 100 enzymatic reactions including carbohydrate, amino acid, and fatty acid metabolism. There is evidence that in some situations B(6) vitamers can function as antioxidants. The fetus is dependent on the placenta for supply of vitamin B(6) and the demand correlates with amino acid metabolism. Few reports are available on the role of B(6) in embryogenesis. Studies of human disorders where B(6) metabolism is blocked show a major role in neurotransmitter function with secondary cerebral and cerebellar hypoplasia. Pyridoxine potentiates vitamin A teratogenicity and an excess leads to peripheral nerve cell degeneration. The key role of vitamin B(6) in the developing human is in metabolism, especially of the neurotransmitters.

Expanded newborn screening: outcome in screened and unscreened patients at age 6 years.

OBJECTIVE: Tandem mass spectrometry is widely applied to routine newborn screening but there are no long-term studies of outcome. We studied the clinical outcome at six years of age in Australia. METHODS: In a cohort study, we analyzed the outcome at 6 years for patients detected by screening or by clinical diagnosis among >2 million infants born from 1994 to 1998 (1,017,800, all unscreened) and 1998 to 2002 (461,500 screened, 533,400 unscreened) recording intellectual and physical condition, school placement, other medical problems, growth, treatment, diet, and hospital admissions. Results were analyzed separately for medium-chain acyl-CoA dehydrogenase deficiency (MCADD) and other disorders, and grouped patients as those who presented clinically or died in the first 5 days of life; patients presented later or diagnosed by screening, and those with substantially benign disorders. RESULTS: Inborn errors, excluding phenylketonuria, were diagnosed in 116 of 1,551,200 unscreened infants (7.5/100,000 births) and 70 of 461,500 screened infants (15.2/100,000 births). Excluding MCADD, 21 unscreened patients with metabolic disorders diagnosed after 5 days of life died or had a significant intellectual or physical handicap (1.35/100,000 population) compared with 2 of the screened cohort (0.43/100,000; odds ratio: 3.1 [95% CI: 0.73-13.32]). Considering the likely morbidity or mortality among the expected number of never-diagnosed unscreened patients, there would be a significant difference. Growth distribution was normal in all cohorts. CONCLUSION: Screening by tandem mass spectrometry provides a better outcome for patients at 6 years of age, with fewer deaths and fewer clinically significant disabilities.

The association between ketoacidosis and 25(OH)-vitamin D levels at presentation in children with type 1 diabetes mellitus.

BACKGROUND: There is considerable evidence supporting the role of vitamin D deficiency in the pathogenesis of type 1 diabetes mellitus (T1DM). Vitamin D deficiency is also associated with impairment of insulin synthesis and secretion. There have been no formal studies looking at the relationship between 25(OH)-vitamin D(3) and the severity of diabetic ketoacidosis (DKA) in children at presentation with T1DM. OBJECTIVE: To determine the relationship between measured 25(OH)-vitamin D(3) levels and the degree of acidosis in children at diagnosis with T1DM. SUBJECTS: Children presenting with new-onset T1DM at a tertiary children's hospital. METHODS: 25(OH)-vitamin D(3) and bicarbonate levels were measured in children at presentation with newly diagnosed T1DM. Those with suboptimal 25(OH)-vitamin D(3) levels (<50 nmol/L) had repeat measurements performed without interim vitamin D supplementation. RESULTS: Fourteen of the 64 children had low 25(OH)-vitamin D(3) levels at presentation, and 12 of these had low bicarbonate levels (<18 mmol/L) (p = 0.001). Bicarbonate explained 20% of the variation in vitamin D level at presentation (partial r(2) = 0.20, p < 0.001) and ethnic background a further 10% (partial r(2) = 0.10, p = 0.002). The levels of 25(OH)-vitamin D(3) increased in 10 of the 11 children with resolution of the acidosis. CONCLUSIONS: Acid-base status should be considered when interpreting 25(OH)-vitamin D(3) levels in patients with recently diagnosed T1DM. Acidosis may alter vitamin D metabolism, or alternatively, low vitamin D may contribute to a child's risk of presenting with DKA.

Molecular characterization of Prader-Willi syndrome by real-time PCR.

Prader-Willi syndrome (PWS) is a contiguous gene syndrome caused by the loss of function of genes situated within the 15q11-q13 region. The loss of function arises as a result of paternally derived mutations complemented by maternal imprinting. The molecular events underlying the disorder include interstitial deletions (70%), uniparental disomy (UPD) (25%), imprinting center defects (<5%), and rarely chromosomal translocations (<1%). The standard diagnosis of PWS is based on clinical observations and genetic investigations involving DNA methylation studies and fluorescence in situ hybridization (FISH) analysis. The absence of a paternal methylation pattern within 15q11 is sufficient for a diagnosis of PWS, and FISH analyses are used for the additional categorization of patients as either deletion or nondeletion. The main limitation of these investigations is that they neither determine the size of the molecular deletions nor permit detection of individuals with microdeletions in the PWS imprinting center that regulates imprinting in this region. We have designed and implemented a real-time PCR assay using genomic DNA and SYBR green I intercalating dye to determine the size of the chromosomal deletions in patients with PWS. This has been successfully performed on genomic DNA isolated from both peripheral blood leukocytes and buccal epithelial cells. Through this assay, the two common deletion classes in PWS were observed, and all results were 100% concordant with previous FISH assays performed on the same patients.