Margretta R. Seashore

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.

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, ≥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) µmol/L (week 0) to 645.2 (393.4) µmol/L (week 10); the mean was maintained at this level during the studys final 12 weeks (652.2 [382.5] µmol/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 Reponses After Antecedent Hypoglycemia

OBJECTIVE—We have previously demonstrated that modulation of γ-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 GABAA receptor blockade on counterregulatory responses to a standardized hypoglycemic stimulus. RESULTS—Quantitative RT-PCR and immunoblots in recurrently hypoglycemic animals revealed that GAD65 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 GABAA 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.

In Vivo Measurement of Phenylalanine in Human Brain by Proton Nuclear Magnetic Resonance Spectroscopy

Disorders of the CNS are the major causes of morbidity and mortality observed in untreated subjects with phenylketonuria (PKU). A method to measure cerebral concentrations of phenylalanine (Phe) in vivo would greatly enhance the ability to investigate both the pathophysiology and the efficacy of therapy of this aminoacidopathy. Twelve image-guided localized proton nuclear magnetic resonance spectroscopic studies were performed in seven subjects with PKU using pulse sequences optimized to detect the aromatic protons of Phe. Ten control studies were also performed using a 2.1-Tesla Bruker Biospec spectrometer. Plasma Phe was measured at the time of the spectroscopic examination in the PKU patients. A Phe signal was observed in all 12 studies performed on the group with PKU, and in five studies cerebral Phe concentrations were measured to be 480 to 780 μmol/g. Plasma Phe concentrations were 0.7 to 3.3 mM (10.8 to 54.8 mg/dL) in the subjects with PKU. Human cerebral Phe concentrations can be measured noninvasively using proton nuclear magnetic resonance spectroscopy. A simultaneous measure of Phe and several other cerebral metabolites is obtained with this innovative technology. Adaptations of this technique can be used to investigate PKU and other neurometabolic disorders with modifications of current clinical magnetic resonance imaging systems.

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 UCDCs 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.

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.

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.

High Incidence of Noonan Syndrome Features Including Short Stature and Pulmonic Stenosis in Patients carrying NF1 Missense Mutations Affecting p.Arg1809: Genotype–Phenotype Correlation

Neurofibromatosis type 1 (NF1) is one of the most frequent genetic disorders, affecting 1:3,000 worldwide. Identification of genotype–phenotype correlations is challenging because of the wide range clinical variability, the progressive nature of the disorder, and extreme diversity of the mutational spectrum. We report 136 individuals with a distinct phenotype carrying one of five different NF1 missense mutations affecting p.Arg1809. Patients presented with multiple café‐au‐lait macules (CALM) with or without freckling and Lisch nodules, but no externally visible plexiform neurofibromas or clear cutaneous neurofibromas were found. About 25% of the individuals had Noonan‐like features. Pulmonic stenosis and short stature were significantly more prevalent compared with classic cohorts (P < 0.0001). Developmental delays and/or learning disabilities were reported in over 50% of patients. Melanocytes cultured from a CALM in a segmental NF1‐patient showed two different somatic NF1 mutations, p.Arg1809Cys and a multi‐exon deletion, providing genetic evidence that p.Arg1809Cys is a loss‐of‐function mutation in the melanocytes and causes a pigmentary phenotype. Constitutional missense mutations at p.Arg1809 affect 1.23% of unrelated NF1 probands in the UAB cohort, therefore this specific NF1 genotype–phenotype correlation will affect counseling and management of a significant number of patients.

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 GAD65 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 GAD65 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 GABAA receptors or knockdown of GAD65 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.

Recurrent acute pancreatitis associated with propionic acidemia.

Propionic acidemia (PA) is a severe metabolic disorder of infants and children (1), resulting from a defect in the mitochondrial enzyme propionyl-coenzyme A carboxylase (PCC). PA is inherited in an autosomal recessive manner. Patients experience metabolic acidosis, ketosis, seizures, and mental retardation. Acute pancreatitis is a potentially life-threatening inflammatory disorder that has many known inciting factors, including metabolic derangements (2,3). However, although acute pancreatited with PA (4,5), recurrent acute