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J W Belmont

Publications and source records attributed to J W Belmont.

At least 19 recordsLinked to original sources
PubMed2003

Omphalocele in trisomy 3q: further delineation of phenotype.

We report a case of a patient with omphalocele, dysmorphic features, and mild developmental delay associated with a chromosomal aberration. Chromosome studies showed that the propositus carries a maternally derived unbalanced translocation der(4)t(3;4)(q27.3;q32.3), resulting in trisomy for region 3q27.3-->qter and monosomy for 4q32.3-->qter. Because the association between dup3q and omphalocele has been reported in several cases, we analyzed the data on 93 previously reported patients with partial trisomy of the long arm of chromosome 3 and compared the clinical features between the cases. The imbalance of chromosome 3 in the patient was further defined by fluorescence in situ hybridization (FISH) studies using bacterial artificial chromosome (BAC) clones. BAC clone RP11-171N2 was identified as a breakpoint-spanning clone in the patient and his mother. Based on our comparative analysis, we have delineated that the smallest region of overlap (SRO) associated with omphalocele is from BAC 171N2 to 3qter. We hypothesize that the SRO contains a gene(s) important in normal abdominal wall development and is of potential interest for further investigation.

Chromosomes, Human, Pair 3↗
PubMed2001

Supravalvular aortic stenosis: genetic and molecular dissection of a complex mutation in the elastin gene.

We have identified two elastin gene (ELN) mutations located in cis in two related families with supravalvular aortic stenosis (SVAS). These mutations included an in-frame duplication in exon 18 (1034-1057dup) and a single base substitution in exon 26 (1829G-->A) predicted to result in the amino acid substitution R610Q. Haplotype analysis in one of the families identified an individual with a recombination between exon 18 and 26 of the elastin gene. This individual was unaffected and carried the exon 18 insertion mutation but not 1829G-->A. Skin fibroblasts were established from this recombinant normal individual and from an affected individual carrying both of the mutations. Reverse transcription/polymerase chain reaction (RT-PCR) analysis indicated that the expression of the mutant allele was reduced to 12%-27% of the normal allele in the affected but not in the unaffected individual. RNA-blot hybridization and immunoprecipitation experiments revealed reduced steady-state elastin mRNA levels and tropoelastin synthesis in the affected individual. RT-PCR analysis of the mRNA rescued by cycloheximide treatment indicated that mutation 1829G-->A created a cryptic donor splice site within exon 26, resulting in the deletion of four nucleotides at the 3'-end of exon 26 and a frameshift in the mRNA. This frameshift mutation generated a premature termination codon in the domain encoded by exon 28, clearly resulting in nonsense-mediated decay (NMD) of this frameshift RNA product. Despite considerable variability in the molecular nature of mutations responsible for SVAS, the unifying mechanism appears to be the generation of null alleles by NMD leading to elastin haploinsufficiency.

Alleles↗
PubMed2001

Cell cycle-dependent expression and nucleolar localization of hCAP-H.

Condensin is a conserved 13S heteropentamer composed of two nonidentical structural maintenance of chromosome (SMC) family proteins, in Xenopus XCAP-C and XCAP-E, and three regulatory subunits, XCAP-D2, XCAP-G, and XCAP-H. Both biochemical and genetic analyses have demonstrated an essential role for the 13S condensin complex in mitotic chromosome condensation. Further, a potential requirement for condensin in completion of chromatid arm separation in early anaphase is demonstrated by the mutational phenotypes of the Drosophila homologues of XCAP-H, barren and XCAP-C, DmSMC4. In this study we have investigated the expression and subcellular distribution of hCAP-H, the human homolog of XCAP-H, in order to better understand its cellular functions. Transcription of hCAP-H was restricted to proliferating cells with highest expression during the G(2) phase of the cell cycle. In contrast, cellular hCAP-H protein levels were constant throughout the cell cycle. hCAP-H was found to be associated with mitotic chromosomes exhibiting a nonuniform but symmetric distribution along sister chromatids. The symmetry of hCAP-H association with sister chromatids suggests that there are sequence-dependent domains of condensin aggregation. During interphase hCAP-H, -C, and -E, have distinct punctate nucleolar localization, suggesting that condensin may associate with and modulate the conformation and function of rDNA. hCAP-H association with condensed chromatin was not observed in the early phase of chromosome condensation when histone H3 phosphorylation has already taken place. This finding is consistent with the hypothesis that histone H3 phosphorylation precedes condensin-mediated condensation.

Amino Acid Sequence↗
PubMed2000

Long-distance DD-PCR and cDNA microarrays.

Analysis of differential gene expression is a classic tool in experimental biology. Broadly applicable new methods to identify and quantitative differential mRNA profiles, such as long distance differential display PCR and cDNA microarrays, promise to greatly accelerate understanding of mechanisms of development, differentiation, and disease.

Animals↗
PubMed2000

Zinc-induced anemia and neutropenia in an adolescent.

We report an adolescent who developed anemia, leukopenia, and neutropenia after prolonged use of over-the-counter zinc for treatment of acne. Hypocupremia and sideroblastic anemia may result from long-term or excessive exposure to zinc.

Acne Vulgaris↗
PubMed1999

Gene trap screening using negative selection: identification of two tandem, differentially expressed loci with potential hematopoietic function.

A fusion gene between Escherichia coli lacZ and herpes simplex virus thymidine kinase (HSV-tk) was constructed and used in a gene trap screen for hematopoietic loci in mouse embryonic stem (ES) cells. This gene, galtek, allowed both convenient histochemical detection of expression as well as ablation of expressing cells under 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodouracil (FIAU) selection. Individual ES cell clones bearing gene trap insertions were differentiated in the presence of FIAU and scored for erythropoietic activity at day 9 of differentiation. Screening of a total of 235 independent gene trap lines identified one clone, F3, which consistently demonstrated FIAU-sensitive erythropoiesis during in vitro differentiation. Cloning of endogenous transcribed sequences from the F3 insertion site identified two distinct transcription units, F3-1 and F3-2, encoding mRNAs of approximately 1.3 kb and 3.35 kb, respectively. The transcripts were unrelated and did not exhibit similarity to known sequences. Both loci demonstrated similar relative levels of expression in the heart, testis, kidney, and lung as assessed by Northern blot hybridization. Whole-mount in situ hybridization detected F3-2 expression at multiple sites in embryonic day (E) 10.5 embryos, including the genital ridges, the aortic endothelium, and endothelium-associated cell clusters within the aortic lumen. Expression of F3-2 in the aortic endothelium and endothelium-associated clusters overlapped that of gata-2, a gene required for hematopoietic development. The FIAU sensitivity of hematopoiesis in F3 embryoid bodies may result from expression of galtek during the formation of early hematopoietic cells, directed by regulatory signals from one or both of these endogenous loci.

Amino Acid Sequence↗
PubMed1998

Identification and cloning of differentially expressed genes by long-distance differential display.

Differential mRNA display (DD-PCR) amplifies short cDNAs (average size 100-350 bp), representing mainly the 3' untranslated regions (3' UTR) of transcripts. Sequencing of these cDNAs is predominantly uninformative for prediction of function and selection of clones for further analysis. Differential display of longer amplicons (0.5-2.0 kb) could enable isolation of cDNAs that encompass both 3' UTR and at least part of the 3' end of the coding region. The coding sequence information could facilitate selection of candidate clones for further analysis without the necessity of screening cDNA libraries. By combining DD-PCR protocols with long-distance PCR and using hot-start PCR with rTth DNA polymerase we have successfully amplified and comparatively displayed cDNAs ranging in size from 150 bp to 2 kb. Long-distance DD-PCR (LDD-PCR) has generated highly reproducible primer-specific patterns of cDNA fragments, as well as reproducible duplicate fingerprints, obtained from different RNA and cDNA samples. Sequencing and expression analyses of LDD-PCR clones have shown that LDD-PCR (a) enables nonredundant clone sampling, (b) generates many clones that encompass part of the coding region, and (c) samples both abundant and rare transcripts, approximately 60% of which are differentially expressed as confirmed by Northern analysis. Coupled with high-throughput cDNA sequencing and multiplex hybridization of cDNA microarrays for confirmation of differential expression, LDD-PCR could prove to be useful for simultaneous scanning of transcripts from multiple cDNA samples and faster selection of differentially expressed transcripts of interest.

Animals↗
PubMed1998

DNA deletion confined to the iduronate-2-sulfatase promoter abolishes IDS gene expression.

Deficiency of the enzyme iduronate-2-sulfatase (IDS) results in Hunter syndrome, an X-linked recessive lysosomal storage disorder. In this study, analysis of a patient with features of moderate to severe Hunter syndrome identified a 178-bp deletion upstream of IDS exon 1 spanning a predicted promoter element. Sequencing of all nine IDS exons from this patient failed to identify any additional mutations within the coding regions or in intron-exon boundaries. The 178-bp deletion is flanked by two 13-bp direct repeats and potential DNA topoisomerase II recognition sites. These findings point toward nonhomologous recombination as a possible mechanism for this mutation. Expression studies on this patient do not detect any IDS transcripts, indicating that the deletion spans sequences essential for IDS expression. Complete lack of expression of IDS is consistent with the moderate to severe phenotype observed in this patient.

Base Sequence↗
PubMed1998

Recent progress in the molecular genetics of congenital heart defects.

Congenital heart defects (CHD) constitute the single most common anatomic class of birth defects and are a major cause of infant mortality. Correlation of normal and pathological embryology/anatomy has led to the formulation of mechanistic models, but there is limited understanding of the genetic basis for the inferred embryological processes. Most evidence points to extensive etiologic heterogeneity and a re-evaluation of simple multifactorial models is required. The recent identification of several genes responsible for congenital heart defects in the context of more complex clinical disorders provides significant entry points for the genetic analysis of human heart development. The association of aneusomies (particularly microdeletion syndromes) with specific cardiac lesions provides further strong support for mechanistic classification. Studies in the mouse are laying the groundwork for a comprehensive genetic model of cardiac organogenesis. Nevertheless, the basis for the large majority of CHD, especially isolated defects, remains obscure. Dissection of the genetic components of CHD is one of the greatest challenges in medical genetics for the coming decades.

Animals↗
PubMed1998

Reassessment of biochemically determined Hunter syndrome carrier status by DNA testing.

Deficiency of iduronate-2-sulphatase (IDS) results in the X linked recessive lysosomal storage disorder Hunter syndrome. Determination of carrier status in families affected by this disorder has been performed using a variety of enzymatic tests. None of these tests has proved to be 100% effective at identifying carriers. The aim of this study was to perform carrier testing in a family affected by the disorder, where testing was complicated by the fact that no surviving affected subjects were available for study. Direct dye primer sequencing of PCR products was used to identify mixed bases in an obligate carrier. Two mixed bases were observed within exon VIII. The first base change (T-->A) at nucleotide position 1150 results in a missense mutation (H342Q), while the second base change (G-->T) at nucleotide position 1151 results in a nonsense mutation (G343X). Four additional female family members were screened for the same mutation. Using this approach it is possible to provide unambiguous information about a subject's carrier status and, unlike biochemical testing, this approach will be equally effective when applied to families with the mild form of this disorder.

DNA↗
PubMed1998

Comparative sequence analysis of a gene-rich cluster at human chromosome 12p13 and its syntenic region in mouse chromosome 6.

The Human Genome Project has created a formidable challenge: the extraction of biological information from extensive amounts of raw sequence. With the increasing availability of genomic sequence from other species, one approach to extracting coding and regulatory element information is through cross-species sequence comparison. To assess the strengths and weaknesses of this methodology for large-scale sequence analysis, 227 kb of mouse sequence syntenic to a gene-rich cluster on human chromosome 12p13 was obtained. Primarily through percent identity plots (PIPs) of SIM comparative sequence alignments, the sequence of coding regions, putative alternative exons, conserved noncoding regions, and correlation in repetitive element insertions were easily determined. The analysis demonstrated that the number, order, and orientation of all 17 genes are conserved between the two species, whereas two human pseudogenes are absent in mouse. In addition, apart from MIRs, no direct correlation of distribution or position of the majority of repetitive elements between the two species is seen. Finally, in examining the synonymous and nonsynonymous substitution rates in the conserved genes, a large variation in nonsynonymous rates is observed indicating that the genes in this region are diverging at different rates. This study indicates the utility and strength of large-scale cross-species sequence comparisons in the extraction of biological information from raw sequence, especially when combined with other computational tools such as GRAIL and BLAST.

Amino Acid Sequence↗
PubMed1997

Protection of primary human T cells from HIV infection by Trev: a transdominant fusion gene.

Gene therapy is one of several approaches that are being tested in the search for an effective anti-human immunodeficiency virus (HIV) treatment. In this strategy, a "protective" gene would be introduced into target cells, rendering them relatively resistance to the virus-induced cytopathicity. Tat and Rev are viral proteins essential for HIV gene expression. Tat increases viral gene transcription and Rev is responsible for the nuclear export of mRNA encoding structural viral proteins. A fusion protein (Trev) was constructed, joining Tat and Rev transdominant mutant gene sequences. Previously, we showed that Trev inhibits both Tat and Rev activities in Jurkat T cells. To determine whether Trev could inhibit HIV replication in primary cells, we transferred the trev gene to peripheral blood lymphocytes and challenged them with different HIV strains. Levels of HIV p24 antigen (Ag) were reduced 4- to 15-fold in cultures of Trev-CD4+ T cells infected with two HIV primary clinical isolates and were not detectable in cultures infected with HIV strains NL4-3 and SF2. In contrast, cultures of nontransduced CD4+ T cells infected with the same viruses had levels of HIV p24 Ag up to 10 ng/ml. Trev-transduced CD4+ T cells demonstrated increased survival following HIV challenge for the length of the experiments (30 days). We did not observe rapid emergence of Trev-resistant HIV in our cultures. Following HIV challenge, cell-associated Trev protein was increased, supporting the hypothesis that cells surviving Trev expression provided a cell survival advantage. This work showed that Trev was able to inhibit HIV replication in primary CD4+ T cells, and, therefore the trev gene could be a candidate for gene therapy against HIV.

CD4-Positive T-Lymphocytes↗
PubMed1997

Large-scale comparative sequence analysis of the human and murine Bruton's tyrosine kinase loci reveals conserved regulatory domains.

Large-scale genomic DNA sequencing of orthologous and paralogous loci in different species should contribute to a basic understanding of the evolution of both the protein-coding regions and noncoding regulatory elements. We compared 93 kb of human sequence to 89 kb of mouse sequence in the Bruton's tyrosine kinase (BTK) region. In addition to showing the conservation of both position and orientation of the five functionally unrelated genes in the region (BTK, alpha-D-galactosidase A, L44L, FTP-3, and FCI-12), the comparison revealed conservation of clusters of noncoding sequence flanking the first exon of each gene. Furthermore, in the sequence comparison at the BTK locus, the conservation of clusters of noncoding sequence extends throughout the locus; the noncoding sequence is more highly conserved in the BTK locus in comparison to the flanking loci. This suggests a correlation with the complex developmental regulation of expression of btk. To determine whether a highly conserved 3.5-kb segment flanking the first exon of BTK contains transcriptional regulatory signals, we tested various portions of the segment for promoter and expression activity in several appropriate cell lines. The results demonstrate the contribution of the conserved region flanking the first exon to the cell lineage-specific expression pattern of btk. These data show the usefulness of large scale sequence comparisons to focus investigation on regions of noncoding sequence that play essential roles in complex gene regulation.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed1996

A single strand conformation polymorphism study of CD40 ligand. Efficient mutation analysis and carrier detection for X-linked hyper IgM syndrome.

Mutations in the gene for CD40 ligand are responsible for the X-linked form of hyper IgM syndrome. However, no clinical or laboratory findings that reliably distinguish X-linked disease from other forms of hyper IgM syndrome have been reported, nor are there tests available that can be used to confidently provide carrier detection. To identify efficiently mutations in the gene for CD40 ligand, eight pairs of PCR primers that could be used to screen genomic DNA by single strand conformation polymorphism (SSCP) were designed. 11 different mutations were found in DNA from all 13 patients whose activated T cells failed to bind a recombinant CD40 construct. The exact nature of four of these mutations, a deletion and three splice defects, could not be determined by cDNA sequencing. In addition, SSCP analysis permitted rapid carrier detection in two families in whom the source of the mutation was most likely a male with gonadal chimerism who passed the disorder on to some but not all of his daughters. These studies document the utility of SSCP analysis for both mutation detection and carrier detection in X-linked hyper IgM syndrome.

Base Sequence↗