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Biomedical subjects

P G Gallagher

Publications and source records attributed to P G Gallagher.

At least 19 recordsLinked to original sources
PubMed1997

Structure and organization of the human ankyrin-1 gene. Basis for complexity of pre-mRNA processing.

Ankyrin-1 (ANK-1) is an erythrocyte membrane protein that is defective in many patients with hereditary spherocytosis, a common hemolytic anemia. In the red cell, ankyrin-1 provides the primary linkage between the membrane skeleton and the plasma membrane. To gain additional insight into the structure and function of this protein and to provide the necessary tools for further genetic studies of hereditary spherocytosis patients, we cloned the human ANK-1 chromosomal gene. Characterization of the ANK-1 gene genomic structure revealed that the erythroid transcript is composed of 42 exons distributed over approximately 160 kilobase pairs of DNA. Comparison of the genomic structure with the protein domains reveals a near-absolute correlation between the tandem repeats encoding the membrane-binding domain of ankyrin with the location of the intron/exon boundaries in the corresponding part of the gene. Erythroid stage-specific, complex patterns of alternative splicing were identified in the region encoding the regulatory domain of ankyrin-1. Novel brain-specific transcripts were also identified in this region, as well as in the "hinge" region between the membrane-binding and spectrin-binding domains. Utilization of alternative polyadenylation signals was found to be the basis for the previously described, stage-specific 9.0- and 7.2-kilobase pair transcripts of the ANK-1 gene.

Alternative Splicing↗
PubMed1997

Spectrin St Claude, a splicing mutation of the human alpha-spectrin gene associated with severe poikilocytic anemia.

An alpha-spectrin variant with increased susceptibility to tryptic digestion, alpha(II/47), was previously observed in a child with severe, recessively inherited, poikilocytic anemia. The molecular basis of this variant, spectrin St Claude, has now been identified as a splicing mutation of the alpha-spectrin gene due to a T --> G mutation in the 3' acceptor splice site of exon 20. This polypyrimidine tract mutation creates a new acceptor splice site, AT --> AG, and leads to the production of two novel mRNAs. One mRNA contains a 12 intronic nucleotide insertion upstream of exon 20. This insertion introduces a termination codon into the reading frame and is predicted to encode a truncated protein (108 kD) that lacks the nucleation site and thus cannot be assembled in the membrane. In the other mRNA, there is in-frame skipping of exon 20, predicting a truncated (277 kD) alpha-spectrin chain. The homozygous propositus has only truncated 277 kD alpha-spectrin chains in his erythrocyte membranes. His heterozygous parents are clinically and biochemically normal. This allele was identified in 3% of asymptomatic individuals from Benin, Africa.

Adult↗
PubMed1997

Mutation of a highly conserved residue of betaI spectrin associated with fatal and near-fatal neonatal hemolytic anemia.

We studied an infant with severe nonimmune hemolytic anemia and hydrops fetalis at birth. His neonatal course was marked by ongoing hemolysis of undetermined etiology requiring repeated erythrocyte transfusions. He has remained transfusion-dependent for more than 2 yr. A previous sibling born with hemolytic anemia and hydrops fetalis died on the second day of life. Peripheral blood smears from the parents revealed rare elliptocytes. Examination of their erythrocyte membranes revealed abnormal mechanical stability as well as structural and functional abnormalities in spectrin. Genetic studies revealed that the proband and his deceased sister were homozygous for a mutation of betaIsigma1 spectrin, L2025R, in a region of spectrin that is critical for normal function. The importance of leucine in this position of the proposed triple helical model of spectrin repeats is highlighted by its evolutionary conservation in all beta spectrins from Drosophila to humans. Molecular modeling demonstrated the disruption of hydrophobic interactions in the interior of the triple helix critical for spectrin function caused by the replacement of the hydrophobic, uncharged leucine by a hydrophilic, positively charged arginine. This mutation must also be expressed in the betaIsigma2 spectrin found in muscle, yet pathologic and immunohistochemical examination of skeletal muscle from the deceased sibling was unremarkable.

Anemia, Hemolytic, Congenital↗
PubMed1997

Amino-acid substitution in alpha-spectrin commonly coinherited with nondominant hereditary spherocytosis.

Nondominant hereditary spherocytosis (ndHS) is a disorder characterized in some patients by severe hemolytic anemia and marked deficiency of erythrocyte spectrin. This report describes the identification of a variant spectrin chain, alpha-spectrin Bughill or alpha(BH), that is associated with this disorder in a number of patients. Tryptic maps of spectrin from affected individuals revealed an acidic shift in isoelectric point of the alphaII domain peptides at 46 kD and 35 kD. A point mutation at codon 970 of the alpha-spectrin gene (GCT-->GAT), that changes the encoded amino acid from an alanine to an aspartic acid, was identified in genomic DNA of affected patients. The alpha(BH) variant was present in 8 patients with ndHS from five different kindreds but was absent in 4 patients from two other kindreds. The 8 ndHS patients with the alpha(BH) variant appeared to be homozygous for the alpha(BH) variant by analysis of peptide maps of limited tryptic digests of erythrocyte spectrin. However, following genomic DNA analysis, only 2 of these patients were true homozygotes, whereas 6 were found to be doubly heterozygous for the alpha(BH) allele and a second, presumably abnormal, alpha-spectrin gene. These results suggest that, in these 6 patients, the second alpha-spectrin allele is in fact associated with one or more genetic defect(s), causing decreased accumulation of alpha-spectrin. The pattern of transmission of the alpha(BH) allele in certain families suggests that the alpha(BH) amino-acid substitution is not itself responsible for ndHS but is more likely a polymorphic variant that, in some but not all cases, is in linkage disequilibrium with another uncharacterized alpha-spectrin gene defect that itself is a cause of ndHS.

Alleles↗
PubMed1997

Genetic basis of the polymorphisms of the alphaI domain of spectrin.

Defects of alpha spectrin have been identified in many cases of hereditary elliptocytosis (HE) and hereditary pyropoikilocytosis (HPP). To aid in the genetic analysis of families with these disorders, the locations of three alpha-spectrin gene polymorphisms were mapped, the genetic basis of these polymorphisms identified, and PCR-based assays designed for their identification. The frequencies of these polymorphisms were determined in two populations and in patients with alphaI/50a HE and HPP. These studies identified two distinct haplotypes and provided evidence that two HE/HPP mutations associated with the alphaI/50a protein phenotype, L207P and L260P, arose on separate chromosomal backgrounds.

Alleles↗
PubMed1997

Hyponatremia and weight changes in an ultradistance triathlon.

OBJECTIVE: To describe the weight changes and the incidence of hyponatremia during an ultradistance triathlon in the athletes who attend medical care after the race. DESIGN: Descriptive research. SETTING: The 1996 New Zealand Ironman Triathlon in which each athlete swam 3.8 km, cycled 180 km, and ran 42 km. PARTICIPANTS: Ninety-five athletes attending for medical care after the race were studied. One hundred sixty-nine athletes who did not attend for medical care were also weighed before and after the race. MAIN OUTCOME MEASURES: Weights were measured at race registration and on finishing the race. Whole-blood sodium concentration was measured in those athletes with clinical evidence of fluid or electrolyte disturbances. RESULTS: Weights were significantly decreased at the end of the race in the athletes seeking medical care (n = 48, mean % delta wt = -2.5%, p < 0.001) and also in the athletes who did not seek medical care (n = 169, mean % delta wt = -2.9%, p < 0.001). Seventeen percent of race starters sought medical attention. Dehydration accounted for 26% of primary diagnoses and hyponatremia for 9%. One athlete with hyponatremia (Na 130 mEq/L) is described who drank 16 L over the course of the race, with a weight gain of 2.5 kg. This is consistent with the hypothesis of fluid overload as the cause of his hyponatremia. Hyponatremia accounted for four of five admissions to the hospital after the race. An inverse relationship between postrace sodium concentrations and percentage change in body weight was observed (r = -0.63). CONCLUSIONS: Hyponatremia is an important risk to the health of athletes competing in an ultradistance triathlon, with fluid overload the likely aetiology.

Adult↗
PubMed1997

Molecular basis of erythrocyte membrane disorders.

Disorders of the erythrocyte membrane, such as hereditary spherocytosis, hereditary elliptocytosis, and hereditary pyropoikilocytosis, are characterized by heterogeneity in their clinical and laboratory manifestations. Recent work has shown that these disorders also exhibit marked genetic heterogeneity. Multiple genetic loci have been implicated, and various genetic defects, including gene deletions, point mutations, and defects in mRNA processing, have been described. The spherocytosis syndromes, with a few rare exceptions, are due to "private" mutations, ie, each individual kindred has a unique mutation. In contrast, the elliptocytosis/pyropoikilocytosis syndromes, although also quite heterogeneous, have been associated with distinct mutations in persons of similar genetic backgrounds, suggesting a "founder effect" for these mutations.

Animals↗
PubMed1996

Genomic organization and 5'-flanking DNA sequence of the murine stomatin gene (Epb72).

Stomatin is a poorly understood integral membrane protein that is absent from the erythrocyte membranes of many patients with hereditary stomatocytosis. This report describes the cloning of the murine stomatin chromosomal gene, determination of its genomic structure, and characterization of the 5'-flanking genomic DNA sequences. The stomatin gene is encoded by seven exons spread over approximately 25 kb of genomic DNA. There is no concordance between the exon structure of the stomatin gene and the locations of three domains predicted on the basis of protein structure. Inspection of the 5'-flanking DNA sequences reveals features of a TATA-less housekeeping gene promoter and consensus sequences for a number of potential DNA-binding proteins.

Amino Acid Sequence↗
PubMed1996

A nonsense mutation in the erythrocyte band 3 gene associated with decreased mRNA accumulation in a kindred with dominant hereditary spherocytosis.

We studied a French kindred with typical hereditary spherocytosis (HS). Studies of erythrocytes and erythrocyte membranes from HS individuals revealed abnormal erythrocyte membrane mechanical stability as well as 15-20% deficiency of band 3, the anion transporter. Anion transport studies of red cells from two affected individuals revealed decreased sulfate flux. Nucleotide sequence of cDNA encoding the distal third of the cytoplasmic domain and the entire transmembrane domain of band 3 obtained by RT-PCR of reticulocyte RNA of an affected family member was normal. Sequence analysis of genomic DNA from an HS individual identified a nonsense mutation of the band 3 gene, Q330X, near the end of the band 3 cytoplasmic domain. This mutation was present in genomic DNA of all HS family members and absent in DNA of unaffected family members. Using an RT-PCR-based assay, a marked quantitative decrease in accumulation of the mutant band 3 RNA was detected. Thus the codon 330 nonsense mutation is responsible for the decreased accumulation of mutant band 3 RNA and the deficiency of band 3 protein in this kindred. These results have important implications for the role of band 3 defects in the membrane pathobiology of HS as well as for the techniques used in detection of HS mutations.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1996

Ankyrin-1 mutations are a major cause of dominant and recessive hereditary spherocytosis.

Hereditary spherocytosis (HS) is the most common inherited haemolytic anaemia in Northern Europeans. The primary molecular defects reside in the red blood cell (RBC) membrane, particularly in proteins that link the membrane skeleton to the overlying lipid bilayer and its integral membrane constituents. Ankyrin-1 is the predominant linker molecule. It attaches spectrin, the major skeletal protein, to the cytoplasmic domain of band 3, the RBC anion exchanger. Two-thirds of patients with HS have combined spectrin and ankyrin-1 deficiency; deficiency of band 3 occurs in about 15 to 20% (ref.1). These data suggest that ankyrin-1 or band 3 defects may be common in HS. To test this we screened all 42 coding exons plus the 5' untranslated/promoter region of ankyrin-1 and the 19 coding exons of band 3 in 46 HS families. Twelve ankyrin-1 mutations and five band 3 mutations were identified. Missense mutations and a mutation in the putative ankyrin-1 promoter were common in recessive HS. In contrast, ankyrin-1 and band 3 frameshift and nonsense null mutations prevailed in dominant HS. Increased accumulation of the normal protein product partially compensated for the ankyrin-1 or band 3 defects in some of these null mutations. Our findings indicate that ankyrin-1 mutations are a major cause of dominant and recessive HS (approximately 35 to 65%), that band 3 mutations are less common (approximately 15 to 25%), and that the severity of HS is modified by factors other than the primary gene defect.

Ankyrins↗
PubMed1996

Epidemiological studies of spectrin mutations related to hereditary elliptocytosis and spectrin polymorphisms in Benin.

We studied an African population in Benin and discovered an unexpectedly high frequency (1.6%) of hereditary elliptocytosis (HE) among the 1447 subjects studied. In approximately two-thirds of HE individuals we identified molecular defects, primarily those in erythrocyte alpha-spectrin (dupL154, L260P and L207P mutations), as well as a novel mutation of erythrocyte beta-spectrin (beta-W2061R mutation). We also identified the genetic basis of a previously identified protein polymorphism of the alpha III domain of spectrin (R1331I mutation). The genetic background of HE in the African population was studied using a number of polymorphisms of the alpha-spectrin gene, including the alpha III domain polymorphism. These studies suggest that the HE mutations appear to have originated from separate genetic backgrounds in this population.

Benin↗
PubMed1996

The lethal hemolytic mutation in beta I sigma 2 spectrin Providence yields a null phenotype in neonatal skeletal muscle.

Point mutations in beta I sigma 1 spectrin that impair the self-association of spectrin alpha beta heterodimers cause mild to severe hemolytic disease and erythrocyte shape abnormalities. Most such mutations act in a dominant negative fashion. One mutation that is particularly devastating is found in beta spectrin Providence. The Providence mutation replaces serine2019 with proline. Heterozygotes display microcytic and fragile erythrocytes; homozygotes die in the neonatal period. It has recently been determined that an alternative transcript of the same beta I sigma 1 spectrin gene expressed in erythroid lineage cells is the major spectrin in skeletal and cardiac muscle and in some neurons. Because the site of the Providence mutation is common to both beta I sigma 1 and beta I sigma 2 spectrin, defective protein must also be expressed in these tissues. Yet the impact of this or any other beta I spectrin mutation outside of the red cell is unexplored. To address this question with respect to skeletal muscle, we have examined the effects of the Providence mutation in cultured muscle cells, after adoptive gene transfer to adult mice, and in two infants homozygous for spectrin Providence. Transfection of the FLAG epitope tagged wild-type beta I sigma 2 or Providence beta I sigma 2 cDNA constructs into C2C12 myoblasts demonstrated by sedimentation velocity analysis that spectrin beta I sigma 2 Providence formed alpha II/-beta I sigma 2 heterodimers in muscle cells but not heterotetramers. Correspondingly, wild-type beta I sigma 2 spectrin formed both alpha II/beta I sigma 1 dimers and heterotetramers, although the proportion of dimers was surprisingly high, which suggested some limitation on self-association in the muscle environment. After adoptive gene transfer into adult mouse skeletal muscle in vivo, both the wild-type and mutant beta I sigma 2 spectrins assembled into a subsarcolemmal complex in a pattern indistinguishable from the native spectrin skeleton. Skeletal muscle taken at autopsy from two infants homozygous for spectrin Providence was normal histologically, as was the intracellular distribution of beta I sigma 2 spectrin as measured by immunoperoxidase staining. These patients also revealed no clinical evidence of myopathy or muscle wasting. It is unknown if they would have experienced dystrophic or myopathic changes if they had lived longer, although we believe that this is unlikely based on the absence of clinical myopathies in patients with other (albeit less severe) beta I spectrin self-association defects. Collectively, these observations indicate that the spectrin mutations that impact tetramer and oligomer formation, even those with a severe hemolytic phenotype, do not impact skeletal muscle function primarily because skeletal muscle does not use the oligomerizing feature of the spectrin skeleton to the same degree as erythrocytes.

Amino Acid Sequence↗
PubMed1996

Molecular basis and haplotyping of the alphaII domain polymorphisms of spectrin: application to the study of hereditary elliptocytosis and pyropoikilocytosis.

Hereditary elliptocytosis (HE) and hereditary pyropoikilocytosis (HPP) are inherited disorders of erythrocyte shape that are frequently associated with abnormalities in alpha-spectrin, one of the principal structural proteins of the erythrocyte membrane skeleton. Five polymorphisms of the alpha-spectrin gene, located in a 6-kb interval of genomic DNA, were identified and analyzed in normal and mutant alpha-spectrin alleles. Three of these polymorphisms are due to single nucleotide substitutions in the alpha-spectrin gene coding region that lead to changes in the amino acid sequence. In combination, these three polymorphisms are responsible for the different peptide phenotypes of the alphaII domain previously observed following limited tryptic digestion of spectrin protein. The most common haplotype, type 1, was found predominantly in Caucasians and was the only haplotype identified in Asians. Haplotypes 2, 3, and 4 were identified predominantly in individuals of African ancestry and were commonly found in patients with HE or HPP. Analysis of coinheritance of alphaII domain polymorphisms with alpha-spectrin gene mutations causing HE or HPP in African-American patients with HE and HPP suggests that, with one exception, a given HE/HPP mutation is present in an alpha-spectrin gene of only one haplotype, indicating a founder effect. The other two polymorphisms located in this region of the alpha-spectrin gene do not change the amino acid sequence of the encoded alpha-spectrin chain and are not in linkage disequilibrium with three of the four alphaII domain haplotypes. A model is proposed for the evolutionary origin of the different haplotypes.

Amino Acid Sequence↗
PubMed1995

Structure, organization, and expression of the human band 7.2b gene, a candidate gene for hereditary hydrocytosis.

Band 7.2b is an integral membrane phosphoprotein absent from the erythrocyte membranes of patients with hereditary hydrocytosis, a hemolytic anemia inherited in an autosomal dominant fashion and characterized by stomatocytic red blood cells with abnormal permeability to Na+ and K+. The precise role of band 7.2b is unknown, but it may interact with other proteins of the junctional complex of the membrane skeleton. To gain additional insight into the structure and function of this protein and to provide the necessary tools for further genetic studies of hydrocytosis patients, we determined the sequence of the full-length human band 7.2b cDNA, characterized the genomic structure of the band 7.2b gene, studied its pattern of expression in different tissues, and characterized the promoter of the gene. The composite band 7.2b gene cDNA was 3047 base pairs in length. Northern blot analysis revealed a wide tissue distribution of expression of the band 7.2b gene, with utilization of alternative polyadenylation signals generating transcripts of 2.2 and 3.1 kilobases. Cloning of the band 7.2b chromosomal gene revealed that it is composed of seven exons distributed over 40 kilobases of DNA. The band 7.2b gene promoter was identified as a TATA-less, (G+C)-rich promoter with a typical InR recognition sequence and a single transcription initiation site. It directed high level expression of a reporter gene in both erythroid and nonerythroid cells. An imperfect simple sequence repeat polymorphism was identified in the 5'-flanking DNA, and an assay was developed for its analysis by PCR.

3T3 Cells↗
PubMed1995

cDNA structure, tissue-specific expression, and chromosomal localization of the murine band 7.2b gene.

Band 7.2b is an integral phosphoprotein absent from the erythrocyte membranes of patients with hydrocytosis, an autosomal, dominantly inherited, hemolytic anemia characterized by stomatocytic red blood cells with abnormal permeability to Na+ and K+. The role of this protein in the erythrocyte membrane is not well understood. To gain additional insight into the structure and function of this protein, we have cloned the murine band 7.2b cDNA and studied its tissue-specific expression. 2,873 bp of cDNA with an open reading frame of 852 bp were isolated. This fragment encodes a protein of 284 amino acids with a predicted molecular weight of 31 kD. The band 7.2b gene had a wide pattern of expression, with high levels of mRNA in heart, liver, skeletal muscle, and testis and low levels in lung, brain, and spleen. Using fluorescent in situ hybridization, the murine band 7.2b gene was mapped to chromosome 2, at the border of the distal region of 2B and proximal region of C1, syntenic to 9q33-q34, the location of the human homologue. Models of the predicted protein structure showed a short NH2-terminal head, a strongly hydrophobic 28-amino acid stretch presumably encoding a single membrane-spanning domain, and a large domain composed of beta sheet and alpha helix. Database searching showed no significant homology of other known proteins to murine or human band 7.2b.

Amino Acid Sequence↗
PubMed1995

Hematologic disorders and nonimmune hydrops fetalis.

Hematologic disorders are implicated in approximately 10% to 27% of cases of nonimmune hydrops fetalis. In almost all of these disorders, anemia leading to heart failure, edema, ascites, and anasarca is the final common denominator. The etiology of the anemia in these cases can be conveniently divided into two categories: (1) excessive erythrocyte loss by hemolysis or hemorrhage, and (2) erythrocyte underproduction. The former include intrinsic erythrocyte abnormalities such as alpha-thalassemia and glucose-6-phosphate dehydrogenase deficiency, and conditions with excessive fetal blood loss such as fetomaternal hemorrhage and twin-twin transfusion. The latter include bone marrow replacement syndromes and conditions associated with failure of erythrocyte production. The presentation, diagnosis, and management of hematologic disorders associated with nonimmune hydrops fetalis are reviewed.

Erythrocyte Count↗