Francis Bowling

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.

Neonatal screening for cystic fibrosis.

Two groups of patients with cystic fibrosis were compared. The screened group, detected with an improved neonatal screening assay for immunoreactive trypsin, developed fewer chest infections requiring treatment and gained more weight than the unscreened group. Early diagnosis by screening seems to affect early morbidity.

The association between ketoacidosis and 25(OH)-vitamin D-3 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 D3 and the severity of diabetic ketoacidosis (DKA) in children at presentation with T1DM.

Utility of AVP gene testing in familial neurohypophyseal diabetes insipidus

Context  Familial neurohypophyseal diabetes insipidus (FNDI) is a rare disorder resulting from arginine vasopressin (AVP) gene mutations. A partial defect in AVP secretion occurs early in the course of FNDI and may not be detected by a water deprivation test (WDT). Testing for AVP gene mutations may confirm a diagnosis of FNDI when a WDT is inconclusive and may also predict individuals who will later develop FNDI.

Genotype-phenotype associations in patients with severe hyperinsulinism of infancy

In hyperinsulinism of infancy (HI), unregulated insulin secretion causes hypoglycemia. Pancreatectomy may be required in severe cases, most of which result from a defect in the β-cell KATP channel, encoded by ABCC8 and KCNJ11. Pancreatic histology may be classified as diffuse or focal disease (the latter associated with single paternal ABCC8 mutations), indicated by the presence of islet cell nuclear enlargement in areas of diffuse abnormality. We investigated genotype-phenotype associations in a heterogeneous Australian cohort. ABCC8 and KCNJ11 genes were sequenced and case histology was reviewed in 21 infants who had pancreatectomy. Ninety-eight control DNA samples were tested by single nucleotide polymorphism analysis. Eighteen ABCC8 mutations were identified, 10 novel. Eleven patients (4 compound heterozygote, 4 single mutation, 3 no mutation detected) had diffuse hyperinsulinism. Nine patients had focal hyperinsulinism (6 single paternal mutation, 2 single mutation of undetermined parental origin, 1 none found) with absence of islet cell nuclear enlargement outside the focal area, although centroacinar cell proliferation and/or nesidiodysplasia was present in 7 cases. Regeneration after near-total pancreatectomy was documented in 4 patients, with aggregates of endocrine tissue observed at subsequent operations in 3. Although the absence of enlarged islet cell nuclei is a useful discriminant of focal hyperinsulinism associated with a paternal ABCC8 mutation, further research is needed to understand the pathophysiology of other histological abnormalities in patients with HI, which may have implications for mechanisms of ductal and islet cell proliferation. Previous surgery should be taken into account when interpreting pancreatic histology.

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.

Maternal gonadal mosaicism causing ornithine transcarbamylase deficiency.

Ornithine transcarbamylase (OTC) deficiency (McKusick 311250), an X-linked inherited disorder, often presents in males with severe neonatal onset of hyperammonemia. Maternal gonadal mosaicism in OTC deficiency was postulated previously, but no cases have been reported. We report on a family in which two consecutive males were affected with OTC deficiency, which was proven biochemically with characteristic metabolites and absent enzyme activity in liver. OTC genotyping in both brothers showed a new mutation in exon 6 (Met206Arg: ATG-->AGG), which encodes part of the equatorial H6 alpha-helix. Biochemical investigations confirmed normal results in the mother and grandmother and the absence of OTC activity in the affected males. Genotyping of the mother and grandmother was performed on peripheral blood leukocytes and skin fibroblasts and showed no mutation in the somatic cells. The recurrence of OTC deficiency in offsprings of a woman with normal genotype strongly suggests gonadal mosaicism. Gonadal mosaicism needs to be considered when counseling couples in which the mother has had a previously affected child with OTC deficiency but apparently is not a carrier.

Adult-onset arginase deficiency.

(EC 3.5.3.1) is the last enzyme in the urea cycle. The enzyme is present in Arginase two forms. The cytosolic type I isoform, which is present in most tissues, predominates in the liver. Red cells contain type I arginase. The type II mitochondrial isoform predominates in kidney. The enzyme is thought to a†ect arginine availability for nitric oxide neurotransmitter synthesis and ornithine availability for both the urea cycle and the ornithine d-aminotransferase pathway. All cases of reported arginase deÐciency (McKusick 207800) have involved the liver type and several mutations have been characterized (Prasad et al 1997). The deÐciency presents in the neonatal period or early childhood, with a spastic paraparesis, seizures and psychomotor retardation. Cases have persistent hyperargininaemia, with episodic elevations of ammonium. Red cell and liver arginase activity are absent. While some cases of late diagnosis have been reported, all reported individuals have been a†ected from an early age. The present case was an 18-year-old woman who had been well as a child with normal growth and development. She presented for investigation of collapse with sudden onset of a spastic diplegia. No sensory deÐcit was evident. She had mild, tender hepatomegaly. She gave a history of being unwell with episodic nausea and vomiting over the previous 6 months, and had experienced some degree of lower limb weakness over the previous 2 weeks. She had commenced the oral contraceptive 1 year earlier and was on no other medications. Family history revealed consanguinity, with her parents being second cousins. Her father had died of a presumed cerebrovascular event at 47 years of age. Liver studies showed reduced synthetic function with coagulopathy, albumin 25 g/L, and raised transaminases. Metabolic investigation showed a normal ammonium of 23 kmol/L, elevated plasma arginine (546 kmol/L ; normal 20È130), homoarginine (25 kmol/L ; normal \3), glutamine (1050 kmol/L ; normal 400È850), but repeatedly normal urinary arginine (6 kmol/L ; normal \25) with mild elevations of urinary ornithine (74 kmol/L ; normal \50) and lysine (82 kmol/L ; normal \70). CSF showed increased arginine (50 kmol/L ; normal 15È18) and slightly increased homoarginine (3.1 mmol/L ; normal \3). Abetalipoproteinaemia, Wilson disease, porphyria, and vitamin deÐciencies were excluded. MRI of brain and spinal cord revealed no abnormality. The EEG

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.

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.