Enhanced interpretation of newborn screening results without analyte cutoff values.

PURPOSE: To improve quality of newborn screening by tandem mass spectrometry with a novel approach made possible by the collaboration of 154 laboratories in 49 countries. METHODS: A database of 767,464 results from 12,721 cases affected with 60 conditions was used to build multivariate pattern recognition software that generates tools integrating multiple clinically significant results into a single score. This score is determined by the overlap between normal and disease ranges, penetration within the disease range, differences between conditions, and weighted correction factors. RESULTS: Ninety tools target either a single condition or the differential diagnosis between multiple conditions. Scores are expressed as the percentile rank among all cases with the same condition and are compared to interpretation guidelines. Retrospective evaluation of past cases suggests that these tools could have avoided at least half of 279 false-positive outcomes caused by carrier status for fatty-acid oxidation disorders and could have prevented 88% of known false-negative events. CONCLUSION: Application of this computational approach to raw data is independent from single analyte cutoff values. In Minnesota, the tools have been a major contributing factor to the sustained achievement of a false-positive rate below 0.1% and a positive predictive value above 60%.

Increased GABAergic output in the ventromedial hypothalamus contributes to impaired hypoglycemic counterregulation in diabetic rats.

OBJECTIVE: Impaired glucose counterregulation during hypoglycemia is well documented in patients with type 1 diabetes; however, the molecular mechanisms underlying this defect remain uncertain. We reported that the inhibitory neurotransmitter γ-aminobutyric acid (GABA), in a crucial glucose-sensing region within the brain, the ventromedial hypothalamus (VMH), plays an important role in modulating the magnitude of the glucagon and epinephrine responses to hypoglycemia and investigated whether VMH GABAergic tone is altered in diabetes and therefore might contribute to defective counterregulatory responses. RESEARCH DESIGN AND METHODS: We used immunoblots to measure GAD(65) protein (a rate-limiting enzyme in GABA synthesis) and microdialysis to measure extracellular GABA levels in the VMH of two diabetic rat models, the diabetic BB rat and the streptozotocin (STZ)-induced diabetic rat, and compared them with nondiabetic controls. RESULTS: Both diabetic rat models exhibited an ~50% increase in GAD(65) protein as well as a twofold increase in VMH GABA levels compared with controls under baseline conditions. Moreover, during hypoglycemia, VMH GABA levels did not change in the diabetic animals, whereas they significantly declined in nondiabetic animals. As expected, glucagon responses were absent and epinephrine responses were attenuated in diabetic rats compared with their nondiabetic control counterparts. The defective counterregulatory response in STZ-diabetic animals was restored to normal with either local blockade of GABA(A) receptors or knockdown of GAD(65) in the VMH. CONCLUSIONS: These data suggest that increased VMH GABAergic inhibition is an important contributor to the absent glucagon response to hypoglycemia and the development of counterregulatory failure in type 1 diabetes.

Glucose prevents the fall in ventromedial hypothalamic GABA that is required for full activation of glucose counterregulatory responses during hypoglycemia.

Local delivery of glucose into a critical glucose-sensing region within the brain, the ventromedial hypothalamus (VMH), can suppress glucose counterregulatory responses to systemic hypoglycemia. Here, we investigated whether this suppression was accomplished through changes in GABA output in the VMH. Sprague-Dawley rats had catheters and guide cannulas implanted. Eight to ten days later, microdialysis-microinjection probes were inserted into the VMH, and they were dialyzed with varying concentrations of glucose from 0 to 100 mM. Two groups of rats were microdialyzed with 100 mM glucose and microinjected with either the K(ATP) channel opener diazoxide or a GABA(A) receptor antagonist. These animals were then subjected to a hyperinsulinemic-hypoglycemic glucose clamp. As expected, perfusion of glucose into the VMH suppressed the counterregulatory responses. Extracellular VMH GABA levels positively correlated with the concentration of glucose in the perfusate. In turn, extracellular GABA concentrations in the VMH were inversely related to the degree of counterregulatory hormone release. Of note, microinjection of either diazoxide or the GABA(A) receptor antagonist reversed the suppressive effects of VMH glucose delivery on counterregulatory responses. Some GABAergic neurons in the VMH respond to changes in local glucose concentration. Glucose in the VMH dose-dependently stimulates GABA release, and this in turn dose-dependently suppresses the glucagon and epinephrine responses to hypoglycemia. These data suggest that during hypoglycemia a decrease in glucose concentration within the VMH may provide an important signal that rapidly inactivates VMH GABAergic neurons, reducing inhibitory GABAergic tone, which in turn enhances the counterregulatory responses to hypoglycemia.

Establishing a consortium for the study of rare diseases: The Urea Cycle Disorders Consortium.

The Urea Cycle Disorders Consortium (UCDC) was created as part of a larger network established by the National Institutes of Health to study rare diseases. This paper reviews the UCDC's accomplishments over the first 6years, including how the Consortium was developed and organized, clinical research studies initiated, and the importance of creating partnerships with patient advocacy groups, philanthropic foundations and biotech and pharmaceutical companies.

Clinical and genomic characterization of distal duplications and deletions of chromosome 4q: study of two cases and review of the literature.

Variable clinical presentations of patients with chromosomally detected deletions in the distal long arm (q) of chromosome 4 have been reported. The lack of molecular characterization of the deletion sizes and deleted genes hinders further genotype-phenotype correlation. Using a validated oligonucleotide array comparative genomic hybridization (oaCGH) analysis, we examined two patients with apparent chromosomal deletions in the distal 4q region. In the first, oaCGH identified a 2.441 megabase (Mb) duplication and a 12.651 Mb deletion at 4q34.1 in a pregnant female who transmitted this aberration to her son. This mother has only learning disabilities while her son had both renal and cardiac anomalies in the newborn period. Unrecognized paternal genetic factors may contribute to the variable expression. The second patient is a 17-year-old female with a history of Pierre Robin sequence, cardiac abnormalities and learning disabilities. She was diagnosed prenatally with a de novo 4q deletion, and oaCGH defined a 16.435 Mb deletion of 4q34.1-4q35.2. Phenotypic comparison and subtractive genomic mapping between these two cases suggested a 4 Mb region possibly harboring a candidate gene for Pierre Robin sequence. Our cases and review of reported cases with genomic findings indicated the presence of familial variants with variable expressivity as well as de novo or inherited pathogenic simple deletion, duplication and complex deletion and duplication in the distal 4q region.

Safety and efficacy of 22 weeks of treatment with sapropterin dihydrochloride in patients with phenylketonuria.

Phenylketonuria (PKU) is an inherited metabolic disease characterized by phenylalanine (Phe) accumulation, which can lead to neurocognitive and neuromotor impairment. Sapropterin dihydrochloride, an FDA-approved synthetic formulation of tetrahydrobiopterin (6R-BH4, herein referred to as sapropterin) is effective in reducing plasma Phe concentrations in patients with hyperphenylalaninemia due to tetrahydrobiopterin (BH4)-responsive PKU, offering potential for improved metabolic control. Eighty patients, > or =8 years old, who had participated in a 6-week, randomized, placebo-controlled study of sapropterin, were enrolled in this 22-week, multicenter, open-label extension study comprising a 6-week forced dose-titration phase (5, 20, and 10 mg/kg/day of study drug consecutively for 2 weeks each), a 4-week dose-analysis phase (10 mg/kg/day), and a 12-week fixed-dose phase (patients received doses of 5, 10, or 20 mg/kg/day based on their plasma Phe concentrations during the dose titration). Dose-dependent reductions in plasma Phe concentrations were observed in the forced dose-titration phase. Mean (SD) plasma Phe concentration decreased from 844.0 (398.0) micromol/L (week 0) to 645.2 (393.4) micromol/L (week 10); the mean was maintained at this level during the study's final 12 weeks (652.2 [382.5] micromol/L at week 22). Sixty-eight (85%) patients had at least one adverse event (AE). All AEs, except one, were mild or moderate in severity. Neither the severe AE nor any of the three serious AEs was considered related to sapropterin. No AE led to treatment discontinuation. Sapropterin is effective in reducing plasma Phe concentrations in a dose-dependent manner and is well tolerated at doses of 5-20 mg/kg/day over 22 weeks in BH4-responsive patients with PKU.

Increased GABAergic tone in the ventromedial hypothalamus contributes to suppression of counterregulatory responses after antecedent hypoglycemia.

OBJECTIVE: We have previously demonstrated that modulation of gamma-aminobutyric acid (GABA) inhibitory tone in the ventromedial hypothalamus (VMH), an important glucose-sensing region in the brain, modulates the magnitude of glucagon and sympathoadrenal responses to hypoglycemia. In the current study, we examined whether increased VMH GABAergic tone may contribute to suppression of counterregulatory responses after recurrent hypoglycemia. RESEARCH DESIGN AND METHODS: To test this hypothesis, we quantified expression of the GABA synthetic enzyme, glutamic acid decarboxylase (GAD), in the VMH of control and recurrently hypoglycemic rats. Subsequently, we used microdialysis and microinjection techniques to assess changes in VMH GABA levels and the effects of GABA(A) receptor blockade on counterregulatory responses to a standardized hypoglycemic stimulus. RESULTS: Quantitative RT-PCR and immunoblots in recurrently hypoglycemic animals revealed that GAD(65) mRNA and protein were increased 33 and 580%, respectively. Basal VMH GABA concentrations were more than threefold higher in recurrently hypoglycemic animals. Furthermore, whereas VMH GABA levels decreased in both control and recurrently hypoglycemic animals with the onset of hypoglycemia, the fall was not significant in recurrently hypoglycemic rats. During hypoglycemia, recurrently hypoglycemic rats exhibited a 49-63% reduction in glucagon and epinephrine release. These changes were reversed by delivery of a GABA(A) receptor antagonist to the VMH. CONCLUSIONS: Our data suggest that recurrent hypoglycemia increases GABAergic inhibitory tone in the VMH and that this, in turn, suppresses glucagon and sympathoadrenal responses to subsequent bouts of acute hypoglycemia. Thus, hypoglycemia-associated autonomic failure may be due in part to a relative excess of the inhibitory neurotransmitter, GABA, within the VMH.

A novel syndrome of cerebral cavernous malformation and Greig cephalopolysyndactyly. Laboratory investigation.

OBJECT: Greig cephalopolysyndactyly syndrome (GCPS) is one of a spectrum of overlapping clinical syndromes resulting from mutations in the gene GLI3 on chromosome 7p. Cerebral cavernous malformation (CCM) is caused by mutations in three distinct genes, including Malcavernin (CCM2), which also maps to chromosome 7p and is located 2.8 Mbp from GLI3. The authors describe a new syndrome that combines the vascular lesions characteristic of CCM with the hallmarks of GCPS, including polydactyly, hypertelorism, and developmental delay. METHODS: The authors used high-resolution array-based comparative genome hybridization (CGH) analysis to characterize the 3 million-bp deletion on chromosome 7 that accounts for this novel clinical presentation. A 4-year-old girl presented with polydactyly, hypertelorism, and developmental delay and was also found to have multiple CCMs after suffering a seizure. RESULTS. Genetic analysis using array-based CGH revealed a deletion affecting multiple genes in the 7p14-13 locus, the interval that includes both CCM2 and GLI3. Quantitative real-time polymerase chain reaction (RT-PCR) on genomic DNA confirmed this genomic lesion. CONCLUSIONS: A novel syndrome, combining features of CCM and GCPS, can be added to the group of entities that result from deleterious genetic variants involving GLI3, including GCPS, acrocallosal syndrome, Pallister-Hall syndrome, and contiguous gene syndrome. The deletion responsible for this new entity can be easily detected using either array-based chromosomal analysis or quantitative RT-PCR.

Microarray-based CGH detects chromosomal mosaicism not revealed by conventional cytogenetics.

Somatic chromosomal mosaicism is a well-established cause for birth defects, mental retardation, and, in some instances, specific genetic syndromes. We have developed a clinically validated, targeted BAC clone array as a platform for comparative genomic hybridization (aCGH) to enable detection of a wide range of pathologic copy number changes in DNA. It is designed to provide high sensitivity to detect well-characterized submicroscopic micro-deletion and duplication disorders while at the same time minimizing detection of variation of uncertain clinical significance. In the course of studying 2,585 samples submitted to our clinical laboratory, chromosomal mosaicism was detected in 12 patient samples; 10 of these cases were reported to have had a normal blood chromosome analysis. This enhanced ability of aCGH to detect mosaicism missed by routine chromosome analysis may be due to some combination of testing multiple cell lineages and/or failure of cytogenetically abnormal T lymphocytes to respond to mitogens. This suggests that aCGH may detect somatic chromosomal mosaicism that would be missed by conventional cytogenetics.

Newborn screening and the pediatric practitioner.

Since the beginning of newborn screening for metabolic and other disorders in 1964, advances in the understanding of the disorders identified and development of new methods of testing newborn screening blood spots have contributed to improved health in children. Pediatricians and others involved in the health care of infants must be able to participate in the assessment and confirmatory testing of infants who have an abnormal test result and in the care of infants identified with a disorder. Expansion in the technology and number of disorders identified has complicated this process. As more and a greater variety of disorders are tested for and identified, a crucial collaborative role has emerged for the newborn screening programs and their public health professionals, the tertiary care specialists in the disorders and the primary care clinicians who comprise the medical home of the infants identified. This collaboration needs to provide prompt results of the newborn screening tests, expeditious and expert confirmatory testing and an effective care plan for the affected infant to realize the benefits of treatment for children with otherwise devastating disorders.

Effect of expanded newborn screening for biochemical genetic disorders on child outcomes and parental stress.

CONTEXT: Tandem mass spectrometry now allows newborn screening for more than 20 biochemical genetic disorders. Questions about the effectiveness and risks of expanded newborn screening for biochemical genetic disorders need to be answered prior to its widespread acceptance as a state-mandated program. OBJECTIVES: To compare newborn identification by expanded screening with clinical identification of biochemical genetic disorders and to assess the impact on families of a false-positive screening result compared with a normal result in the expanded newborn screening program. DESIGN: Prospective study involving an inception cohort of newly diagnosed children. SETTING: Massachusetts, Maine, and a private laboratory in Pennsylvania with expanded newborn screening; other New England states with limited screening. PARTICIPANTS: Families of 50 affected children identified through expanded newborn screening (82% of eligible cases); 33 affected children identified clinically (97% of eligible cases); 94 screened children with false-positive results (75% of eligible cases); and 81 screened children with normal results (63% of eligible cases). MAIN OUTCOME MEASURES: Child's health and development and the Parental Stress Index. RESULTS: Within the first 6 months of life, 28% of children identified by newborn screening compared with 55% of clinically identified children required hospitalization (P =.02). One child identified by newborn screening compared with 8 (42%) identified clinically performed in the range of mental retardation (P<.001). Mothers in the screened group reported lower overall stress on the Parental Stress Index than mothers in the clinically identified group (z = 3.38, P<.001). Children with false-positive results compared with children with normal results were twice as likely to experience hospitalization (21% [n = 20] vs 10% [n = 8], respectively; P =.06). Mothers of children in the false-positive group compared with mothers of children with normal screening results attained higher scores on the Parental Stress Index (z = 4.25, P<.001) and the Parent-Child Dysfunction subscale (z = 5.30, P<.001). CONCLUSIONS: Expanded newborn screening may lead to improved health outcomes for affected children and lower stress for their parents. However, false-positive screening results may place families at risk for increased stress and parent-child dysfunction.