Showing papers by "Stylianos E. Antonarakis published in 1991"

Hypoxia-inducible nuclear factors bind to an enhancer element located 3' to the human erythropoietin gene.

Abstract: Human erythropoietin gene expression in liver and kidney is inducible by anemia or hypoxia. DNase I-hypersensitive sites were identified 3' to the human erythropoietin gene in liver nuclei. A 256-base-pair region of 3' flanking sequence was shown by DNase I protection and electrophoretic mobility-shift assays to bind four or more different nuclear factors, at least two of which are induced by anemia in both liver and kidney, and the region functioned as a hypoxia-inducible enhancer in transient expression assays. These results provide insight into the molecular basis for the regulation of gene expression by a fundamental physiologic stimulus, hypoxia.

Cloning of the gamma-aminobutyric acid (GABA) rho 1 cDNA: a GABA receptor subunit highly expressed in the retina.

Abstract: Type A gamma-aminobutyric acid (GABAA) receptors are a family of ligand-gated chloride channels that are the major inhibitory neurotransmitter receptors in the nervous system. Molecular cloning has revealed diversity in the subunits that compose this heterooligomeric receptor, but each previously elucidated subunit displays amino acid similarity in conserved structural elements. We have used these highly conserved regions to identify additional members of this family by using the polymerase chain reaction (PCR). One PCR product was used to isolate a full-length cDNA from a human retina cDNA library. The mature protein predicted from this cDNA sequence in 458 amino acids long and displays between 30 and 38% amino acid similarity to the previously identified GABAA subunits. This gene is expressed primarily in the retina but transcripts are also detected in the brain, lung, and thymus. Injection of Xenopus oocytes with RNA transcribed in vitro produces a GABA-responsive chloride conductance and expression of the cDNA in COS cells yields GABA-displaceable muscimol binding. These features are consistent with our identification of a GABA subunit, GABA rho 1, with prominent retinal expression that increases the diversity and tissue specificity of this ligand-gated ion-channel receptor family.

Parental Origin of the Extra Chromosome in Trisomy 21 as Indicated by Analysis of DNA Polymorphisms

Abstract: Background. Over the past 20 years, the parental origin of the extra chromosome in children with trisomy 21 has been investigated with cytogenetic methods of identifying morphologic variations in chromosome 21. These studies have concluded that the origin of the extra chromosome 21 was maternal in approximately 80 percent of cases and paternal in about 20 percent. Methods. We studied 200 families, each with a single child with trisomy 21, using DNA polymorphisms as markers to determine the parental origin of the nondisjunction causing the extra chromosome 21. These polymorphisms spanned a region of about 120 centimorgans on the long arm of chromosome 21, from the D21S13 locus (the most centromeric) to the COL6A1 gene (the most telomeric). Results. The parental origin of nondisjunction could be determined for all but 7 of the 200 children. It was maternal in 184 children (proportion [±SE], 95.3±1.5 percent) and paternal in 9 (4.7±1.5 percent). In a subgroup of 31 families, we compared the results ...

Cell-type-specific and hypoxia-inducible expression of the human erythropoietin gene in transgenic mice.

Abstract: Synthesis of erythropoietin, the primary humoral regulator of erythropoiesis, in liver and kidney is inducible by anemia or hypoxia. Analysis of human erythropoietin gene expression in transgenic mice revealed that sequences located 6-14 kilobases 5' to the gene direct expression to the kidney, whereas sequences within the immediate 3'-flanking region control hepatocyte-specific expression. Human erythropoietin transcription initiation sites were differentially utilized in liver and kidney. Inducible transgene expression was precisely targeted to peritubular interstitial cells in the renal cortex that synthesize endogenous mouse erythropoietin. These studies demonstrate that multiple erythropoietin gene regulatory elements control cell-type-specific expression and inducibility by a fundamental physiologic stimulus, hypoxia.

Haemophilia A: database of ncleotide substituttions, deletions, insertions and rearrangements of the factor VIII gene

Abstract: Mutations at the factor VIII gene locus causing Haemophilia A have now been identified in many patients from many ethnic groups. Earlier studies used biased methods which detected repetitive mutations at a few CG dinucleotides. More recently rapid gene scanning methods have uncovered an extreme diversity of mutations. Over 80 different point mutations, 6 insertions, 7 small deletions, and 60 large deletions have been characterised. Repetitive mutation has been proved for at least 16 CpG sites. All nonsense mutations cause severe disease. Most missense mutations appear to cause instability of the protein, but some are associated with production of dysfunctional factor VIII molecules, thereby localising functionally critical regions of the cofactor. Variable phenotype has been observed in association with three of the latter class of genotype. This catalogue of gene lesions in Haemophilia A will be updated annually.

Protocols to establish genotype-phenotype correlations in Down syndrome.

Abstract: Charles J. Epstein,* Julie R. Korenberg,$ Goran Anneren,§ Stylianos E. Antonarakis,II Segolene Ayme, # Eric Courchesne, * * Lois B. Epstein,*t Anne Fowler, TT Yoram Groner,$ Jean L. Huret,§§ Thomas L. Kemper,III Ira T. Lott, * * Bertram H. Lubin,ttT Ellen Magenis,144 John M. Opitz,§§§ David Patterson, 11111 Jean H. Priest, ### Siegfried M. Pueschel,**** Stanley 1. Rapoport,tttt Pierre-Marie Sinet,§§§§ Rudolph E. Tanzi,## and Felix de la Cruzt14$

Molecular characterization of severe hemophilia A suggests that about half the mutations are not within the coding regions and splice junctions of the factor VIII gene.

Abstract: Hemophilia A is an X chromosome-linked disorder resulting from deficiency of factor VIII, an important protein in blood coagulation. A large number of disease-producing mutations have been reported in the factor VIII gene. However, a comprehensive analysis of the mutations has been difficult because of the large gene size, its many scattered exons, and the high frequency of de novo mutations. Recently, we have shown that nearly all mutations resulting in mild-to-moderate hemophilia A can be detected by PCR and denaturing gradient gel electrophoresis (DGGE). In this study, we attempted to discover the mutations causing severe hemophilia A by analyzing 47 unselected patients, 30 of whom had severe hemophilia and 17 of whom had mild-to-moderate disease. Using DGGE as a screening method, we analyzed 99% of the coding region, 94% of the splice junctions, the promoter region, and the polyadenylylation site of the gene. We found the mutation in 16 of 17 (94%) patients with mild-to-moderate disease but in only 16 of 30 (53%) patients with severe hemophilia A. Since DGGE after computer analysis appears to detect all mutations in a given fragment, the lower-than-expected yield of mutations in patients with severe disease is likely not due to failure of the detection method; it is probably due to the presence of mutations in DNA sequences outside the regions studied. Such sequences may include locus-controlling regions, other sequences within introns or outside the gene that are important for its expression, or another gene involved in factor VIII expression that is very closely linked to the factor VIII gene.

Molecular characterization of mild-to-moderate hemophilia A : detection of the mutation in 25 of 29 patients by denaturing gradient gel electrophoresis

Abstract: To date it has been difficult to characterize completely a genetic disorder, such as hemophilia A, in which the involved gene is large and unrelated affected individuals have different mutations, most of which are point mutations. Toward this end, we analyzed the DNA of 29 patients with mild-to-moderate hemophilia A in which the causative mutation is likely to be a missense mutation. Using computer analysis, we determined the melting properties of factor VIII gene sequences to design primer sets for PCR amplification and subsequent denaturing gradient gel electrophoresis (DGGE). A total of 45 primer sets was chosen to amplify 99% of the coding region of the gene and 41 of 50 splice junctions. To facilitate detection of point mutations, we mixed DNA from two male patients, and both homoduplexes and heteroduplexes were analyzed. With these 45 primer sets, 26 DNAs containing previously identified point mutations in the factor VIII gene were studied, and all 26 mutations were easily distinguishable from normal. After analyzing the 29 patients with unknown mutations, we identified the disease-producing mutation in 25 (86%). Two polymorphisms and two rare normal variants were also found. Therefore, DGGE after computer analysis is a powerful method for nearly complete characterization of disease-producing mutations and polymorphisms in large genes such as that for factor VIII.

DNA polymorphism haplotypes of the human lipoprotein lipase gene: possible association with high density lipoprotein levels.

Abstract: Lipoprotein lipase (LPL) plays a central role in the metabolism of lipoproteins by hydrolyzing the core triglycerides of circulating very low density lipoproteins and chylomicrons. The enzyme is encoded by a gene about 30kb in size located on the short arm of human chromosome 8. We have determined the locations of the four common DNA polymorphisms along the gene, including a polymorphism that occurred only among an American black population examined. These restriction site polymorphisms were used for haplotype analysis of Mediterranean and US black families. Estimation of the extent of nonrandom association between these polymorphisms indicated considerable linkage disequilibrium between these sites. No correlation was observed between the level of linkage disequilibrium and the physical distance of the polymorphic sites. The polymorphism information content of the haplotypes ranged from 0.65 to 0.74, thereby constituting a relatively useful genetic marker on chromosome 8. We tested for possible associations between the polymorphisms and circulating lipoprotein phenotypes in a population of 139 Caucasians undergoing coronary arteriography and 50 of their spouses. Some possibly significant associations between LPL gene polymorphisms and levels of high density lipoprotein cholesterol (P = 0.015) and total plasma cholesterol (P = 0.025) were observed. In contrast to a previous report, we found no significant associations with the levels of plasma triglycerides.

Use of short sequence repeat DNA polymorphisms after PCR amplification to detect the parental origin of the additional chromosome 21 in Down syndrome.

Abstract: The origin of nondisjunction in trisomy 21 has so far been studied using cytogenetic heteromorphisms and DNA polymorphisms using Southern blot analysis. Short sequence repeats have recently been described as an abundant class of DNA polymorphisms in the human genome, which can be typed using the polymerase chain reaction (PCR) amplification. We describe the usage of such markers on chromosome 21 in the study of parental origin of the additional chromosome 21 in 87 cases of Down syndrome. The polymorphisms studied were (a) two (GT)n repeats and a poly(A) tract of an Alu sequence within the HMG14 gene and (b) a (GT)n repeat of locus D21S156. The parental origin was determined in 68 cases by studying the segregation of polymorphic alleles in the nuclear families (either by scoring three different alleles in the proband or by dosage comparison of two different alleles in the proband). Our results demonstrate the usefulness of highly informative PCR markers for the study of nondisjunction in Down syndrome.

Down syndrome due to de novo Robertsonian translocation t(14q;21q): DNA polymorphism analysis suggests that the origin of the extra 21q is maternal.

Abstract: Down syndrome is rarely due to a de novo Robertsonian translocation t(14q;21q). DNA polymorphisms in eight families with Down syndrome due to de novo t(14q;21q) demonstrated maternal origin of the extra chromosome 21q in all cases. In seven nonmosaic cases the DNA markers showed crossing-over between two maternal chromosomes 21, and in one mosaic case no crossing-over was observed (this case was probably due to an early postzygotic nondisjunction). In the majority of cases (five of six informative families) the proximal marker D21S120 was reduced to homozygosity in the offspring with trisomy 21. The data can be best explained by chromatid translocation in meiosis I and by normal crossover and segregation in meiosis I and meiosis II.

The COL6A1 and COL6A2 genes exist as a gene cluster and detect highly informative DNA polymorphisms in the telomeric region of human chromosome 21q

Abstract: The genes that encode the alpha 1 (VI) and alpha 2 (VI) collagen chains, designated COL6A1 and COL6A2, map to human chromosomal band 21q22.3. Using pulsed-field gel electrophoresis and somatic cell hybrids, we found that COL6A1 and COL6A2 form a gene cluster on the most distal part of chromosome 21. Furthermore, we detected several DNA polymorphisms (both restriction site and VNTRs) associated with these loci. These polymorphisms make the COL6A1 and COL6A2 genes among the most informative markers on human chromosome 21.

The vanishing twin: an explanation for discordance between chorionic villus karyotype and fetal phenotype.

Abstract: One ‘erroneous’ diagnosis occurred in 200 first-trimester chorionic villus samples (CVS) analysed. In direct preparations following 24 h incubation as well as in long-term cultures, a 46.XX karyotype was observed in the villi (28 and 25 cells, respectively). At 20 weeks of gestation, labour was induced because of fetal death in utero. An autopsy performed on the fetus revealed a male phenotype. Placenta and fetal tissues were not submitted for cytogenetic studies. The discordant CVS karyotype (46,XX), in view of the male fetal phenotype, prompted further cytogenetic and molecular studies. Chromosome marker studies on the parents' blood and chorionic villi confirmed both maternal and paternal inheritance of chromosomes in the CVS. DNA studies on formalin-fixed skin using a Y-specific probe, DYZ1, confirmed the presence of a Y chromosome in the fetus. The most likely cause of the discrepant CVS karyotype is the presence of an undetected degenerating dizygotic twin.

A 48,XXY,+21 Down syndrome patient with additional paternal X and maternal 21

Abstract: The origin of meiotic nondisjunction of the extra chromosomes X and 21 was studied in a patient with the karyotype 48,XXY,+21 using DNA polymorphisms. The extra chromosome X was the result of paternal first meiotic nondisjunction of X and Y. The extra chromosome 21 was derived from the mother. The meiotic error in the mother most probably occurred in meiosis II. Thus, this is a combination caused by the chance occurrence of two independent events.

Cystic fibrosis mutation cluster

Abstract: Four mutations have been found clustered in exon 11 of the CFTR (cystic fibrosis transmembrane conductance regulator) gene. These mutations occur within a set of amino acids highly conserved among ATP-dependent transport proteins. Humans can be tested to determine whether they carry one of these mutations using a number of methods and/or probes taught herein. Specifically the mutations include: Asn 549 , Asp 551 , Stop 553 , and Thr 559 .

Linkage mapping of D21S171 to the distal long arm of human chromosome 21 using a polymorphic (AC)n dinucleotide repeat.

Abstract: An (AC)n repeat within the anonymous DNA sequence D21S171 was shown to be highly polymorphic in members of the 40 Centre d'Etude du Polymorphisme Humain (CEPH) families Ten different alleles at this marker locus were detected by electrophoresis on polyacrylamide gels of DNA amplified by the polymerase chain reaction (PCR) using primers flanking the (AC)n repeat The observed heterozygosity was 66% PCR amplification of DNA from somatic cell hybrids mapped D21S171 to human chromosome 21, and linkage analysis localized this marker close to the loci CD18, PFKL, D21S113 and D21S112 in chromosomal band 21q223 In CEPH family 12 a de novo allele has been observed in a maternally derived chromosome

A molecular genetic approach to amyotrophic lateral sclerosis

Abstract: Disorders of the motor neurons may affect both the upper and lower neurons, primarily the lower motor neurons as in the spinal muscular atrophies are primarily the upper motor neurons as in the familial spastic paraplegias. Amyotrophic lateral sclerosis is a degenerative disorder of the motor neuron that results in paralysis and wasting of voluntary muscles. Large motor neurons in the cerebral cortex, brain stem and spinal cord degenerate or are lost. Hyaline inclusions may be seen in the cytoplasm of surviving motor neurons. Acute axonal degeneration of peripheral motor fibers occurs at all levels, including the distal axon. Subclinical involvement of the spinecerebellar tracts, posterior column and Clarke's column as well as loss of large neurons in the dorsal root ganglia and neurons of oculomotor nuclei has been reported. The average duration of life onset of symptoms of amyotrophic lateral sclerosis is three years and ninety per cent of patients died within 5 years. The basic mechanism of disease in amyotrophic lateral sclerosis remains unknown. There is no known treatment that will prevent, reverse or otherwise alter the course of the disease. Autosomal dominant and autosomal recessive forms of amyotrophic lateral sclerosis are genetic models of amyotrophic lateral sclerosis which may provide insight into the disease mechanism of sporadic amyotrophic lateral sclerosis, five to ten percent of adult cases of amyotrophic lateral sclerosis with early onset of symptoms and a more benign course. It is conceivable that both genetic and sporadic forms of amyotrophic lateral sclerosis result from failure of the same or similar neuronal mechanism triggered by defective genes and by an environment agent in sporadic amyotrophic lateral sclerosis.

ic andhypoxia-inducibl e expression ofthehuman erythropoietin geneintransgenic mice (enhancer elements/gene regulation/in situ hybridization/polycythemia)

Abstract: Synthesis oferythropoietin, theprimary hu- moralregulator oferythropoiesis, inliver andkidney is inducible byanemia orhypoxia. Analysis ofhumanerythro- poietin geneexpression intransgenic micerevealed thatse- quences located 6-14kilobases 5'tothegenedirect expression tothekidney, whereas sequences within theimmediate 3'- flanking region control hepatocyte-specific expression. Human erythropoietin transcription initiation sites weredifferentially utilized inliver andkidney. Inducible transgene expression was precisely targeted toperitubular interstitial cells intherenal cortex that synthesize endogenous mouseerythropoietin. These studies demonstrate that multiple erythropoietin generegula- toryelements control cell-type-specific expression andinduc- ibility byafundamental physiologic stimulus, hypoxia.