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Biomedical subjects

Edward R B McCabe

Publications and source records attributed to Edward R B McCabe.

At least 19 recordsLinked to original sources
PubMed2007

DAX1: Increasing complexity in the roles of this novel nuclear receptor.

DAX1 (NR0B1) is a nuclear receptor with a characteristic C-terminal ligand binding domain, but an atypical DNA binding domain. Mutations in the DAX1 gene cause adrenal hypoplasia congenita (AHC) establishing its biological importance. Recent studies highlight the complexities of DAX1 regulation and function. There is considerable phenotypic variability in AHC suggesting the existence of DAX1 modifier genes and environmental influences on DAX1 function. The findings of an alternatively spliced DAX1A, more common than DAX1 in all tissues except testis, of DAX1 homodimers, and of DAX1 heterodimers with a number of transcription factor partners including DAX1A and SHP point to an expanded transcription regulatory network under DAX1 control. Model organisms (mice and zebrafish) are being used to identify other DAX1 functions and modifier genes to understand the pathogenesis of AHC and the lack of genotype-phenotype correlation.

Adrenal Gland Diseases↗
PubMed2007

Deletion mapping in Xp21 for patients with complex glycerol kinase deficiency using SNP mapping arrays.

Infantile or complex glycerol kinase deficiency (cGKD) is a contiguous gene deletion syndrome caused by a loss of GK (MIM# 300474), along with its neighboring genes, Duchenne muscular dystrophy (DMD; MIM# 300377) and/or Nuclear Receptor Subfamily 0, Group B, Member 1 (NR0B1; MIM# 300473). Patients with cGKD present with glyceroluria and hyperglycerolemia in association with DMD and/or adrenal hypoplasia congenita (AHC). The purpose of these investigations was to determine whether the Affymetrix GeneChip Mapping Array (SNP chip) could be utilized to detect and map breakpoints in patients with cGKD. Genomic DNAs from several primary lymphoblastoid cell lines from patients with cGKD were analyzed on the Affymetrix platform. The Affymetrix SNP chip is a high-density oligonucleotide array that allows a standardized, parallel interrogation of thousands of SNPs across the entire genome (except for the Y chromosome). Analysis of the array features' hybridization intensities enabled clear delineation of the patient deletions with a high degree of confidence. Many of these patient deletions had been mapped by PCR and their breakpoints confirmed by sequencing. This study demonstrates the utility of the Affymetrix Mapping GeneChips for molecular cytogenetic analysis, beyond the SNP genotyping for which the arrays were initially designed. With one out of 160 live births (approximately 25,000 U.S. neonates annually) reported to have cytogenetic disorders, we envision a significant need for such a standardized platform to carry out rapid, high-throughput, genomic analyses for molecular cytogenetics applications.

Cells, Cultured↗
PubMed2006

Dosage-sensitive sex reversal adrenal hypoplasia congenita critical region on the X chromosome, gene 1 (DAX1) (NR0B1) and small heterodimer partner (SHP) (NR0B2) form homodimers individually, as well as DAX1-SHP heterodimers.

Dosage-sensitive sex reversal adrenal hypoplasia congenita critical region on the X chromosome, gene 1 (DAX1) (NR0B1), and small heterodimer partner (SHP) (NR0B2) are atypical nuclear receptor superfamily members that function primarily as corepressors through heterodimeric interactions with other nuclear receptors. Mutations in DAX1 cause adrenal hypoplasia congenita, and mutations in SHP lead to mild obesity and insulin resistance, but the mechanisms are unclear. We investigated the existence and subcellular localization of DAX1 and SHP homodimers and the dynamics of homodimerization. We demonstrated DAX1 homodimerization in the nucleus and cytoplasm, and dissociation of DAX1 homodimers upon heterodimerization with steroidogenic factor 1 (SF1) or ligand-activated estrogen receptor-alpha (ERalpha). DAX1 homodimerization involved an interaction between its amino and carboxy termini involving its LXXLL motifs and activation function (AF)-2 domain. We observed SHP homodimerization in the nucleus of mammalian cells and showed dissociation of SHP homodimers upon heterodimerization with ligand-activated ERalpha. We observed DAX1-SHP heterodimerization in the nucleus of mammalian cells and demonstrated the involvement of the LXXLL motifs and AF-2 domain of DAX1 in this interaction. We further demonstrate heterodimerization of DAX1 with its alternatively spliced isoform, DAX1A. This is the first evidence of homodimerization of individual members of the unusual NR0B nuclear receptor family and heterodimerization between its members. Our results suggest that DAX1 forms antiparallel homodimers through the LXXLL motifs and AF-2 domain. These homodimers may function as holding reservoirs in the absence of heterodimeric partners. The formation of DAX1 and SHP homodimers and DAX1-SHP and DAX1-DAX1A heterodimers suggests the possibility of novel functions independent of their coregulator roles, suggesting additional complexity in the molecular mechanisms of DAX1 and SHP action.

Amino Acid Motifs↗
PubMed2006

Conserved family of glycerol kinase loci in Drosophila melanogaster.

Glycerol kinase (GK) is an enzyme that catalyzes the formation of glycerol 3-phosphate from ATP and glycerol, the rate-limiting step in glycerol utilization. We analyzed the genome of the model organism Drosophila melanogaster and identified five GK orthologs, including two loci with sequence homology to the mammalian Xp21 GK protein. Using a combination of sequence analysis and evolutionary comparisons of orthologs between species, we characterized functional domains in the protein required for GK activity. Our findings include additional conserved domains that suggest novel nuclear and mitochondrial functions for glycerol kinase in apoptosis and transcriptional regulation. Investigation of GK function in Drosophila will inform us about the role of this enzyme in development and will provide us with a tool to examine genetic modifiers of human metabolic disorders.

Amino Acid Sequence↗
PubMed2006

IMAGe association and congenital adrenal hypoplasia: no disease-causing mutations found in the ACD gene.

The spontaneous mouse mutant adrenocortical dysplasia (acd) is characterized by defects in the adrenals, kidneys, and gonads of adult mutant mice and by caudal dysgenesis and vertebral segmentation defects in acd embryos. This association of defects mirrors those identified in patients with known or suspected abnormalities in adrenocortical development, including adrenal hypoplasia congenita and IMAGe association. The identification of the Acd gene in mice has prompted the study of its human homolog ACD, which has recently been shown to be a regulator of telomere length. Sequencing of ACD in 15 patients revealed no coding mutations, but three novel SNPs were identified.

Adrenal Insufficiency↗
PubMed2006

Novel role for the orphan nuclear receptor Dax1 in embryogenesis, different from steroidogenesis.

Cytomegalic adrenal hypoplasia congenita (AHC) is an X-linked disease caused by mutations in DAX1-encoding gene NR0B1, previously thought to function primarily in steroidogenesis. We sought to determine the expression pattern for Dax1 along with known network partners in early embryogenesis and to determine a steroidogenic capacity for the embryo prior to the establishment of the urogenital ridge at embryonic day 9 (E9). Here, we report that murine Dax1 is a unique marker in early embryonic development, distinguishing the extraembryonic (proximal) endoderm from the remainder of the developing embryo. We showed that Wilms tumor 1, steroidogenic factor 1, and estrogen receptor beta were expressed throughout the embryo, but the progesterone, estrogen alpha and androgen receptors, cytochrome P450 (Cyp11a1) and Nur77 were not observed in any of the embryonic layers. Lack of Cyp11A1 expression at this stage confirmed an absence of inherent steroidogenic capacity for the early embryo. The role of Nr0b1 in embryonic stem (ES) cells was investigated using siRNA knockdown, resulting in differentiation toward endoderm-like fate. Nr0b1 conditional knockout in ES cells led to differentiation, confirming our knockdown results. Our investigations suggest that Nr0b1 functions in a novel role in the maintenance of a relatively undifferentiated state. Our results further suggest that the failure of conventional murine Nr0b1 knockout attempts may be due to disregulated differentiation.

Animals↗
PubMed2006

A familial missense mutation in the hinge region of DAX1 associated with late-onset AHC in a prepubertal female.

Mutations in the DAX1 (Dosage-sensitive sex reversal-Adrenal hypoplasia congenita (AHC) critical region on the X chromosome gene 1; NR0B1) cause X-linked AHC, a disease characterized by primary adrenal failure in infancy and hypogonadotropic hypogonadism. All known missense mutations impair DAX1 repression of steroidogenic factor 1 (SF1) transactivation and have been localized to the putative ligand binding domain. Here, an asymptomatic father and his late-onset AHC daughter were both shown to share a novel DAX1 mutation (C200W), the first missense mutation identified in the hinge region of DAX1. Using immunohistochemistry we demonstrate that the sub-cellular localization of the C200W mutant DAX1 protein is shifted from the nucleus to the cytoplasm. The disturbed localization of the C200W mutant in transfected cells correlates with impaired transcriptional repression activity. The import defect is relatively mild, retaining 80% of wild-type activity, which may explain the unusually mild phenotype. Import of DAX1 into the nucleus involves a direct interaction with SF1. In vitro assays demonstrate that the C200W mutant retains the ability to functionally interact with SF1, which suggests that SF1-independent interactions of DAX1 could be responsible for the import defect.

Active Transport, Cell Nucleus↗
PubMed2006

Use of electrochemical DNA biosensors for rapid molecular identification of uropathogens in clinical urine specimens.

We describe the first species-specific detection of bacterial pathogens in human clinical fluid samples using a microfabricated electrochemical sensor array. Each of the 16 sensors in the array consisted of three single-layer gold electrodes-working, reference, and auxiliary. Each of the working electrodes contained one representative from a library of capture probes, each specific for a clinically relevant bacterial urinary pathogen. The library included probes for Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa, Enterocococcus spp., and the Klebsiella-Enterobacter group. A bacterial 16S rRNA target derived from single-step bacterial lysis was hybridized both to the biotin-modified capture probe on the sensor surface and to a second, fluorescein-modified detector probe. Detection of the target-probe hybrids was achieved through binding of a horseradish peroxidase (HRP)-conjugated anti-fluorescein antibody to the detector probe. Amperometric measurement of the catalyzed HRP reaction was obtained at a fixed potential of -200 mV between the working and reference electrodes. Species-specific detection of as few as 2,600 uropathogenic bacteria in culture, inoculated urine, and clinical urine samples was achieved within 45 min from the beginning of sample processing. In a feasibility study of this amperometric detection system using blinded clinical urine specimens, the sensor array had 100% sensitivity for direct detection of gram-negative bacteria without nucleic acid purification or amplification. Identification was demonstrated for 98% of gram-negative bacteria for which species-specific probes were available. When combined with a microfluidics-based sample preparation module, the integrated system could serve as a point-of-care device for rapid diagnosis of urinary tract infections.

Biosensing Techniques↗
PubMed2006

Asymptomatic isolated human glycerol kinase deficiency associated with splice-site mutations and nonsense-mediated decay of mutant RNA.

Isolated glycerol kinase deficiency (GKD) is an X-linked inborn error of metabolism that is either symptomatic or asymptomatic. GKD is due to deletions of, or mutations within, the GK gene, and there is no genotype-phenotype correlation. We identified three patients with asymptomatic GKD, determined that they had GK splice-site mutations, and studied the stability of their GK mRNA to understand the molecular mechanism of the GKD. All three patient mutations caused a frameshift and introduction of a premature stop codon. A fourth patient had an Alu insertion in intron 4 that led to alternative splicing. To study the effect of splice-site mutations on RNA species, we performed reverse transcriptase PCR and found only normal-sized products for all patients. Incubation with anisomycin to block nonsense-mediated decay (NMD), revealed two RNA species for each individual. Sequence analysis revealed that the larger bands represented the wild-type GK RNA and smaller bands represented mutant misspliced RNA, suggesting that the abnormal RNA species were targeted by NMD. Normal RNA species observed in each patient are likely responsible for their mild phenotypes. We speculate that influences on RNA processing and protein stability represent modifiers of the GKD phenotype.

Aged↗
PubMed2005

Targeted disruption of glycerol kinase gene in mice: expression analysis in liver shows alterations in network partners related to glycerol kinase activity.

Glycerol kinase deficiency (GKD) is an X-linked inborn error of metabolism with metabolic and neurological crises. Liver shows the highest level of glycerol kinase (GK) activity in humans and mice. Absence of genotype-phenotype correlations in patients with GKD indicates the involvement of modifier genes, including other network partners. To understand the molecular pathogenesis of GKD, we performed microarray analysis on liver mRNA from neonatal glycerol kinase (Gyk) knockout (KO) and wild-type (WT) mice. Unsupervised learning revealed that the overall gene expression profile of the KO mice was different from that of WT. Real-time PCR confirmed the differences for selected genes. Functional gene enrichment analysis was used to find 56 increased and 37 decreased gene functional categories. PathwayAssist analysis identified changes in gene expression levels of genes involved in organic acid metabolism indicating that GK was part of the same metabolic network which correlates well with the patients with GKD having metabolic acidemia during their episodic crises. Network component analysis (NCA) showed that transcription factors sterol regulatory element-binding protein (SREBP)-1c, carbohydrate response element-binding protein (ChREBP), hepatocyte nuclear factor-4 alpha (HNF-4alpha) and peroxisome proliferative-activated receptor-alpha (PPARalpha) had increased activity in the Gyk KO mice compared with WT mice, whereas SREBP-2 was less active in the Gyk KO mice. These studies show that Gyk deletion causes alterations in expression of genes in several regulatory networks and is the first time NCA has been used to expand on microarray data from a mouse KO model of a human disease.

Animals↗
PubMed2005

Two-tiered universal newborn screening strategy for severe combined immunodeficiency.

Outcomes for infants with severe combined immunodeficiency (SCID) would be improved by universal newborn screening, but there are not yet screening tests of sufficient accuracy for the disorder. In a pilot study, we assessed the ability of a two-tiered strategy to improve accuracy. Dried blood samples from patients were assessed with two tests for lymphopenia: interleukin-7, a T-cell growth cytokine, and TRECs, a byproduct of T-cell receptor recombination. IL-7 screening has a specificity of 96.1% and TRECs have a specificity of 92.3%. Combining these tests in a two-tiered strategy increases specificity to 100% (97-100% CI). Sensitivity was 85% for IL-7 screening and 100% for TREC screening. A two-tiered strategy may be of sufficient accuracy to enable universal SCID screening, and should be assessed in a prospective trial.

DNA, Circular↗
PubMed2005

Human and murine glycerol kinase: influence of exon 18 alternative splicing on function.

Glycerol kinase (GK) is a key enzyme in glycerol metabolism with two alternatively spliced forms-one with an 87bp insertion corresponding to exon 18 (GK+EX18), and one lacking exon 18 (GK-EX18). We report the expression of GK+/-EX18 in various tissues and cell lines, as well as their enzymatic characteristics and subcellular localization. RT-PCR revealed differential expression in tissues and cell lines. Northern blot analysis revealed that both forms of the murine ortholog, Gyk, were highly expressed in murine heart and increased during embryonic development. K(m) values for glycerol for GK+/-EX18 were not significantly different, although GK-EX18 had a higher V(max) for glycerol. GK-EX18 had a lower K(m) and V(max) for ATP than GK+EX18. Immunofluorescence experiments showed that GK+EX18 co-localized to the mitochondria and the perinuclear region while GK-EX18 had a diffuse expression pattern. These data suggest specific and divergent roles for GK+EX18 and GK-EX18 in cellular metabolism and development.

Alternative Splicing↗
PubMed2005

Single-gene disorders: what role could moonlighting enzymes play?

Single-gene disorders with "simple" Mendelian inheritance do not always imply that there will be an easy prediction of the phenotype from the genotype, which has been shown for a number of metabolic disorders. We propose that moonlighting enzymes (i.e., metabolic enzymes with additional functional activities) could contribute to the complexity of such disorders. The lack of knowledge about the additional functional activities of proteins could result in a lack of correlation between genotype and phenotype. In this review, we highlight some notable and recent examples of moonlighting enzymes and their possible contributions to human disease. Because knowledge and cataloging of the moonlighting activities of proteins are essential for the study of cellular function and human physiology, we also review recently reported and recommended methods for the discovery of moonlighting activities.

Animals↗
PubMed2005

Molecular genetic confirmatory testing from newborn screening samples for the common African-American, Asian Indian, Southeast Asian, and Chinese beta-thalassemia mutations.

beta-Thalassemia is a serious health problem in the United States, especially in California, due to increased Asian immigration. Neonatal screening by using high-performance liquid chromatography (HPLC) or isoelectric focusing (IEF) may lead to confusion due to interactions of various hemoglobinopathies with beta-thalassemia. Our purpose was to develop single-tube multiplexed PCR assays using original neonatal screening specimens to identify the mutations responsible for beta-thalassemia in order to expedite diagnostic confirmation. Primers were designed for two to six common ethnic-specific mutations using the amplification refractory mutation system (ARMS). This multiplex ARMS approach was standardized using DNA samples with known mutations for beta-thalassemia in those of Asian (Southeast Asian, Chinese, and Asian Indian) and African-American descent. Specimens from African-American neonates were tested for two mutations (-88 and -29); Asian Indians for five mutations (IVSI-1, IVSI-5, codons (Cd) 41/42, Cd 8/9, and 619-bp deletion); Chinese, Taiwanese, and Southeast Asians for seven mutations (Cd 41/42, Cd 17, -28, IVSII-654, Cd 71/72, IVSI-5, and IVSI-1). We identified each of these beta-thalassemia mutations in multiplexed ARMS from positive control samples. We tested 25 anonymized dried blood specimens from neonates who had been diagnosed with beta-thalassemia and who also belonged to these ethnic groups. We detected a mutation specific to the neonate's ethnic group using the ARMS approach in nearly all specimens, and the results were confirmed by sequencing. Multiplexed ARMS for ethnic-specific beta-thalassemia mutations from the original newborn screening dried blood specimens is a rapid and efficient approach for diagnostic confirmation.

Asian People↗
PubMed2005

Multiplex-PCR assay for the deletions causing hereditary persistence of fetal hemoglobin.

INTRODUCTION: Hereditary persistence of fetal hemoglobin (HPFH) is a benign condition caused by the failure of normal switching from the fetal to the adult beta-globin gene, resulting in continuous production of fetal hemoglobin beyond the perinatal period. To date, eight deletions of variable size and position have been reported for HPFH. Southern hybridization and PCR are the most common methods used to detect each deletion. AIM: Our aim was to develop a multiplex-PCR assay to detect these deletions in a single tube in order to facilitate rapid and accurate molecular diagnosis. METHODS AND RESULTS: This report is the first application of multiplex-gap-PCR to detect all HPFH deletions simultaneously to expedite diagnosis. The deletion breakpoints were precisely identified for each deletion and primers were designed in the unique regions across the breakpoints of HPFH-1 (Black), HPFH-2 (Ghanaian), HPFH-3 (Asian Indian), HPFH-4 (Italian), HPFH-5 (Italian), HPFH-6 (Vietnamese), HPFH-7 (Kenyan), and SEA-HPFH (Southeast Asian). As many as 16 primers were used in a single amplification reaction by adjusting the relative primer concentrations. The multiplex-PCR approach was standardized on known positive control samples. We identified unique deletion-specific products for each deletion. The results were confirmed by sequence analysis. CONCLUSIONS: We conclude that our multiplex-gap PCR strategy provides the most rapid and accurate diagnosis for the deletions in the beta-globin gene cluster causing HPFH.

Fetal Hemoglobin↗