James B. Gibson

Detection of Clinically Relevant Exonic Copy-Number Changes by Array CGH

Array comparative genomic hybridization (aCGH) is a powerful tool for the molecular elucidation and diagnosis of disorders resulting from genomic copy‐number variation (CNV). However, intragenic deletions or duplications—those including genomic intervals of a size smaller than a gene—have remained beyond the detection limit of most clinical aCGH analyses. Increasing array probe number improves genomic resolution, although higher cost may limit implementation, and enhanced detection of benign CNV can confound clinical interpretation. We designed an array with exonic coverage of selected disease and candidate genes and used it clinically to identify losses or gains throughout the genome involving at least one exon and as small as several hundred base pairs in size. In some patients, the detected copy‐number change occurs within a gene known to be causative of the observed clinical phenotype, demonstrating the ability of this array to detect clinically relevant CNVs with subkilobase resolution. In summary, we demonstrate the utility of a custom‐designed, exon‐targeted oligonucleotide array to detect intragenic copy‐number changes in patients with various clinical phenotypes. Hum Mutat 31:1–17, 2010.

Endogenous synthesis of galactose in normal men and patients with hereditary galactosaemia

Despite restricted ingestion of lactose, patients with galactose-1-phosphate uridyltransferase deficiency have raised concentrations of galactose metabolites in blood and urine. Endogenous production of galactose may underlie this phenomenon. Using isotopically labelled galactose in a continuous intravenous infusion, we employed the steady-state flux method to calculate endogenous galactose production rate in three normal men and three patients with classic galactosaemia. We found that galactosaemic patients and normal subjects synthesise gram quantities of galactose per day. The rate of synthesis ranged from 0.53-1.05 mg/kg per h. Endogenous production of galactose may be an important factor in the pathogenesis of the complications of the brain and ovary, and could explain the persistent elevation of galactose metabolites in patients despite dietary restriction of galactose.

Int22h-1/int22h-2-mediated Xq28 rearrangements: intellectual disability associated with duplications and in utero male lethality with deletions

Background X linked intellectual disability (XLID) is common, with an estimated prevalence of 1/1000. The expanded use of array comparative genomic hybridisation (CGH) has led to the identification of several XLID-associated copy-number variants. Methods Array CGH analysis was performed using chromosomal microarray with ∼105 000 oligonucleotides covering the entire genome. Confirmatory fluorescence in situ hybridisation analyses were subsequently performed. Chromosome X-inactivation (XCI) was assessed using methylation-sensitive restriction enzyme digestion followed by PCR amplification. Results A novel ∼0.5 Mb duplication in Xq28 was identified in four cognitively impaired males who share behavioural abnormalities (hyperactivity and aggressiveness) and characteristic facial features (high forehead, upper eyelid fullness, broad nasal bridge and thick lower lip). These duplications were inherited from mothers with skewed XCI and are mediated by nonallelic homologous recombination between the low-copy repeat regions int22h-1 and int22h-2, which, in addition to int22h-3, are also responsible for inversions disrupting the factor VIII gene in haemophilia A. In addition, we have identified a reciprocal deletion in a girl and her mother, both of whom exhibit normal cognition and completely skewed XCI. The mother also had two spontaneous abortions. Conclusions The phenotypic similarities among subjects with int22h-1/int22h-2-mediated Xq28 duplications suggest that such duplications are responsible for a novel XLID syndrome. The reciprocal deletion may not be associated with a clinical phenotype in carrier females due to skewed XCI, but may be lethal for males in utero. Advancements in array CGH technology have enabled the identification of such small, clinically relevant copy-number variants.

The Role of Molecular Testing and Enzyme Analysis in the Management of Hypomorphic Citrullinemia

Expanded newborn screening detects patients with modest elevations in citrulline; however it is currently unclear how to treat these patients and how to counsel their parents. In order to begin to address these issues, we compared the clinical, biochemical, and molecular features of 10 patients with mildly elevated citrulline levels. Three patients presented with clinical illness whereas seven came to attention as a result of expanded newborn screening. One patient presented during pregnancy and responded promptly to IV sodium phenylacetate/sodium benzoate and arginine therapy with no long‐term adverse effects on mother or fetus. Two children presented with neurocognitive dysfunction, one of these responded dramatically to dietary protein reduction. ASS enzyme activity was not deficient in all patients with biallelic mutations suggesting this test cannot exclude the ASS1 locus in patients with mildly elevated plasma citrulline. Conversely, all symptomatic patients who were tested had deficient activity. We describe four unreported mutations (p.Y291S, p.R272H, p.F72L, and p.L88I), as well as the common p.W179R mutation. In silico algorithms were inconsistent in predicting the pathogenicity of mutations. The cognitive benefit in one patient of protein restriction and the lack of adverse outcome in seven others restricted from birth, suggest a role for protein restriction and continued monitoring to prevent neurocognitive dysfunction.

Comparison of erythrocyte uridine sugar nucleotide levels in normals, classic galactosemics, and patients with other metabolic disorders

By limiting galactosylation mechanisms, a cellular deficiency of the uridine sugar nucleotide, UDPgalactose, has been implicated as a cause of the long-term complications seen in patients with classic galactosemia despite dietary treatment. As a result, great interest has been generated in the accurate assessment of UDPgalactose, as well as UDPglucose, from which UDPgalactose may be derived by the function of a ubiquitous, active UDPgalactose-4-epimerase. Since several series of values for the concentration of these compounds in red blood cells (RBCs) of galactosemics have been flawed by the use of methods subsequently shown to be unsuitable, we have quantified erythrocyte UDPgalactose and UDPglucose levels by an accurate high-performance liquid chromatography (HPLC) assay in 116 normals, 76 galactosemics, and 39 patients with other metabolic disorders. These large groups have permitted the evaluation of age, diet, and genotype as influential factors in the steady-state RBC levels of the sugar nucleotides. The data show that age is an important determinant of RBC levels, with children younger than 10 years having higher values than individuals older than 10 years. Mean UDPgalactose levels in galactosemic children younger than 10 years and those older than 10 years were 30% and 18% lower, respectively, than levels in comparable normals. Although the mean differences were highly significant, there was considerable overlap of individual values. There was no difference in UDPglucose levels between galactosemics and normals.(ABSTRACT TRUNCATED AT 250 WORDS)

De Novo Disruption of the Proteasome Regulatory Subunit PSMD12 Causes a Syndromic Neurodevelopmental Disorder

Degradation of proteins by the ubiquitin-proteasome system (UPS) is an essential biological process in the development of eukaryotic organisms. Dysregulation of this mechanism leads to numerous human neurodegenerative or neurodevelopmental disorders. Through a multi-center collaboration, we identified six de novo genomic deletions and four de novo point mutations involving PSMD12, encoding the non-ATPase subunit PSMD12 (aka RPN5) of the 19S regulator of 26S proteasome complex, in unrelated individuals with intellectual disability, congenital malformations, ophthalmologic anomalies, feeding difficulties, deafness, and subtle dysmorphic facial features. We observed reduced PSMD12 levels and an accumulation of ubiquitinated proteins without any impairment of proteasome catalytic activity. Our PSMD12 loss-of-function zebrafish CRISPR/Cas9 model exhibited microcephaly, decreased convolution of the renal tubules, and abnormal craniofacial morphology. Our data support the biological importance of PSMD12 as a scaffolding subunit in proteasome function during development and neurogenesis in particular; they enable the definition of a neurodevelopmental disorder due to PSMD12 variants, expanding the phenotypic spectrum of UPS-dependent disorders.

Recurrent ACADVL molecular findings in individuals with a positive newborn screen for very long chain acyl-coA dehydrogenase (VLCAD) deficiency in the United States

Very long chain acyl-coA dehydrogenase deficiency (VLCADD) is an autosomal recessive inborn error of fatty acid oxidation detected by newborn screening (NBS). Follow-up molecular analyses are often required to clarify VLCADD-suggestive NBS results, but to date the outcome of these studies are not well described for the general screen-positive population. In the following study, we report the molecular findings for 693 unrelated patients that sequentially received Sanger sequence analysis of ACADVL as a result of a positive NBS for VLCADD. Highlighting the variable molecular underpinnings of this disorder, we identified 94 different pathogenic ACADVL variants (40 novel), as well as 134 variants of unknown clinical significance (VUSs). Evidence for the pathogenicity of a subset of recurrent VUSs was provided using multiple in silico analyses. Surprisingly, the most frequent finding in our cohort was carrier status, 57% all individuals had a single pathogenic variant or VUS. This result was further supported by follow-up array and/or acylcarnitine analysis that failed to provide evidence of a second pathogenic allele. Notably, exon-targeted array analysis of 131 individuals screen positive for VLCADD failed to identify copy number changes in ACADVL thus suggesting this test has a low yield in the setting of NBS follow-up. While no genotype was common, the c.848T>C (p.V283A) pathogenic variant was clearly the most frequent; at least one copy was found in ~10% of all individuals with a positive NBS. Clinical and biochemical data for seven unrelated patients homozygous for the p.V283A allele suggests that it results in a mild phenotype that responds well to standard treatment, but hypoglycemia can occur. Collectively, our data illustrate the molecular heterogeneity of VLCADD and provide novel insight into the outcomes of NBS for this disorder.

Pitfalls in Measuring Cerebrospinal Fluid Glycine Levels in Infants With Encephalopathy

In encephalopathic infants, cerebrospinal fluid hyperglycinemia and elevated cerebrospinal fluid to plasma glycine ratio are considered pathognomonic of nonketotic hyperglycinemia. To evaluate the significance of cerebrospinal fluid hyperglycinemia and elevated cerebrospinal fluid to plasma glycine ratio in acutely encephalopathic infants, a retrospective chart review of all cases of isolated elevation of cerebrospinal fluid glycine levels at Arkansas Children’s Hospital from January 1995 to December 2000 was performed. Twenty-two patients (14 males) were included. The most common diagnosis was hypoxic ischemic encephalopathy (n = 8). Nine patients had elevated cerebrospinal fluid to plasma glycine ratio, which was transient in 7 patients. This study shows that elevated cerebrospinal fluid to plasma glycine ratio can be encountered in a variety of clinical conditions. The significance of this observation in light of the poor prognosis of nonketotic hyperglycinemia and the possible role of glycine in the mechanism of ischemic neuronal injury is addressed.

Uridine diphosphate hexoses in leukocytes and fibroblasts of classic galactosemics and patients with other metabolic diseases.

ABSTRACT: To examine uridine diphosphate hexose (UDPhexose) content of cells that have more complete metabolic patterns than erythrocytes, which have been commonly used in the study of galactosemia, the concentrations of uridine diphosphate galactose (UDPgalactose) and uridine diphosphate glucose (UDPglucose) were determined in white blood cells (WBC) and fibroblasts cultured from skin biopsies. Leukocyte UDPgalactose and UDPglucose values were determined in 60 normal individuals, 14 classic gamactosemics, and 18 patients with other metabolic diseases on protein-restricted and low-lactose diets. There was no difference in the average concentration of these compounds between any of these groups. There was no relationship between age and WBC UDPhexose content or correlation of WBC and erythrocyte UDPhexose levels in the same blood specimens. WBC from galactosemic individuals differ from their red blood cells because the former do not show the low average UDPgalactose levels and abnormal UDPglucose to UDPgalactose ratio previously reported for erythrocytes from galactose-1-phosphate uridyltransferase-deficient individuals. Fibroblast cell lines from 10 normal and 10 galactosemic individuals, cultured and grown to confluence in glucose medium, also showed no difference in nucleotide sugar concentrations. Thus far, of the cell types easily available, red blood cells appear to be unique in showing an abnormality in nucleotide sugar metabolism. The fact that galactosemic fibroblasts demonstrate no abnormality in the concentration of these compounds suggests that the defective galactosylation that has been observed in galactosemic fibroblasts is not due to unavailability of UDPgalactose.

Uridine diphosphoglucose content of human erythrocytes: assessment by conversion to uridine diphosphoglucuronate.

To settle the ongoing controversy regarding differential uridine diphosphoglucose (UDPG) and uridine diphosphogalactose (UDPGal) content of erythrocytes, which may be important in evaluating the metabolic abnormality in patients with galactosemia, we derived a combined enzymatic-high-performance liquid chromatography (HPLC) assay. Uridine diphosphoglucuronate (UDPGA), the unique product of UDPG dehydrogenase activity, was separated and quantified by HPLC in extracts of human erythrocytes. The quantity of UDPGA produced in cell filtrates incubated with the enzyme corresponds to the amount of UDPG directly determined by HPLC. The amount of UDPGA produced was independent of the enzyme purity or activity used. On the other hand, the amounts of UDPG estimated by fluorometric measurement of the production of reduced nicotinamide adenine dinucleotide varied with the enzyme purity and activity. The combined enzymatic-HPLC method confirms the direct determinations of UDPG content of normal erythrocytes. The results indicate that, under appropriate conditions, the fluorometric-based assay will give accurate estimates of UDPG, but the direct HPLC method yields consistent and correct UDPG and UDPGal determinations.