Cloning of the human genomic amiloride-sensitive Na+H+ antiporter gene, identification of genetic polymorphisms, and localization on the genetic map of chromosome 1p

doi:10.1016/0888-7543(90)90530-8

Genomics

Volume 7, Issue 1, May 1990, Pages 131-135

https://doi.org/10.1016/0888-7543(90)90530-8 Get rights and content

Abstract

We have cloned and mapped a 90-kb region spanning the human genomic amiloride-sensitive $Na^{+} H^{+}$ antiporter gene on overlapping cosmid clones. This cloned region extends 27 kb upstream of the 5′ end of the longest antiporter cDNA and reveals a primary transcription unit of at least 60 kb. Using these genomic clones we have identified polymorphisms in the antiporter gene in human genomic DNA cleaved with enzymes TaqI and MspI; both are two-allele systems. We have localized both polymorphisms to the same segment within an intron of the antiporter transcription unit. Observed heterozygosity for both markers is 47% in 175 unrelated individuals, with the two polymorphic systems showing complete linkage disequilibrium. We have determined genotypes at the antiporter locus in 667 individuals in 59 reference families. Linkage analysis using 28 other markers on human chromosome 1 precisely locates the antiporter gene (locus APNH) on the genetic map of 1p. The antiporter gene is closely flanked by two highly informative loci, lying 3 cM proximal to the rhesus blood group locus (Rh) and 4 cM distal to the anonymous DNA marker CMM8 (locus D1S79). These antiporter polymorphisms and informative flanking markers will prove useful in genetic linkage studies employing the antiporter gene.

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We have examined the sequence of the cDNA encoding the sodium/hydrogen exchanger isoform 1 (NHE1), from 23 bases upstream of the start codon to 28 bases downstream of the stop codon. Template was prepared from (1) peripheral blood mononuclear cells (PBMC) isolated from 10 healthy unrelated Caucasian volunteers; (2) PBMCs isolated from 6 leukemic patients (acute lymphoblastic leukemia [ALL], n = 3; chronic lymphocytic leukemia [CLL], n = 1; chronic myelogenous leukemia [CML], n = 2); and (3) samples of 4 leukemic cell lines (ALL: CEM, MOLT4; AML: KG1a; CML: K562). NHE1 cDNA in normal PBMCs showed silent polymorphism of nucleotides 112 (N1: T, frequency 0.70; C, frequency 0.30; prevalence of heterozygosity 0.42); 2248 (N2: G, frequency 0.90; A, frequency 0.10; heterozygosity 0.18); and 2493 (N3: G, frequency 0.90; A, frequency 0.10; heterozygosity 0.18). Deduced primary structure of NHE1 protein in all normal volunteers was identical to that previously published for NHE1 from renal and cardiac tissue. Similar to normal PBMCs, NHE1 cDNA from leukemic cells showed polymorphism of nucleotides N1, N2, and N3, but failed to demonstrate leukemia-specific sequence differences. We conclude that the coding region of NHE1 cDNA shows a greater level of polymorphism than is currently recognized, but that sequence mutation of NHE1 is not a key event in the pathogenesis of leukemia.
Evidence for effective suppression of recombination the chromosome 17q21 segment spanning RNU2-BRCA1
1999, American Journal of Human Genetics
Characterization of associations between polymorphic sites located throughout the ∼200–400-kb variable-length region spanning RNU2–BRCA1 reveals nearly complete linkage disequilibrium. This segment spans the RNU2 array, which includes 6–30 tandem copies of the U2 snRNA gene, and an adjacent region containing NBR1, the LBRCA1 pseudogene, NBR2, and BRCA1 in a tandemly duplicated structure. A series of biallelic polymorphisms define two common haplotypes that do not vary significantly, in structure or frequency, between populations of primarily European (n=275) or Asian (n=34) ancestry. Lower-frequency variants occurring at distantly located sites within this region also show very strong associations. The rarer haplotype classes appear to be distinguished by mutational alteration and are not recombination products of the two major classes. The two major haplotypes also exhibit significantly different allele-length distributions for local simple tandem-repeat markers. The conservation of extensive distinct chromosomal haplotypes during a long period of human population expansion and divergence indicates that selective forces or specific chromosomal mechanisms result in effective recombination suppression. The extreme degree of long-range linkage disequilibrium at this locus may be exceeded only by that reported for the human MHC locus, where allele-specific functional interactions are believed to be significant. These findings have implications for the estimation of the time of origin of BRCA1 mutations having a founder effect, the interpretation of the significance of rare allelic variants, and the study of the origins of modern populations.
Genomic organization glucocorticoid transcriptional activation of the rat Na+/H+ exchanger Nhe3 gene
1996, Journal of Biological Chemistry
The activity of the apical membrane Na⁺/H⁺ exchanger NHE3 isoform of renal or intestinal epithelial cells is chronically regulated by a wide variety of stimuli, including acidosis, cAMP, glucocorticoids, and thyroid hormone. To understand the molecular mechanisms responsible for long term regulation of this cation transporter, we have isolated and determined the structure of this gene from a rat genomic library. The Nhe3 gene spans >40 kilobases and contains 17 exons that are flanked by typical splice donor and acceptor sequences at the exon-intron boundaries. The transcription initiation site was mapped by S1 nuclease protection analyses of mRNA from rat kidney and intestine. Multiple start sites were clustered between nucleotides −100 and −96 relative to the translation initiation codon. An atypical TATA-box and CCAAT-box are centered 30 and 147 nucleotides, respectively, upstream of the predominant transcription initiation site. Sequence analysis of approximately 1.4 kilobases of the 5′-flanking promoter region also revealed the presence of other putative cis-acting elements recognized by various transcription factors (e.g. AP-1, AP-2, C/EBP, NF-I, OCT-1/OTF-1, PEA3, Sp1, glucocorticoid, and thyroid hormone receptors), some of which may participate in the chronic regulation of this gene. The glucocorticoid responsiveness of the Nhe3 gene was assessed by fusing its 5′ regulatory region to the firefly luciferase reporter gene and then by measuring the expression of the chimeric gene in transiently transfected renal epithelial OK and LLC-PK₁ cells. Glucocorticoid treatment significantly increased the luciferase activity of the chimeric gene in both cell lines, thereby indicating that glucocorticoid regulation of Nhe3 is mediated primarily by a transcriptional mechanism.
High-resolution fluorescence mapping of 46 dna markers to the short arm of human chromosome 1
1993, Genomics
We describe a high-resolution cytogenetic map for 46 DNA markers previously assigned to the short arm of human chromosome 1. Using fluorescence in situ hybridization on simultaneously R-banded prometaphase chromosomes, a refined map position was found for 45 probes. For 6 of these probes, additional hybridization sites were observed and for another 7 probes, conflicting results were found with regard to previous localizations. For some probes with overlapping map positions, probe order could be determined by dual-color hybridization on elongated chromosomes. The present high-resolution map can be used to refine the previously published composite map and also provides additional landmarks for the construction of a contig map of the short arm of chromosome 1.
Physical and Genetic Mapping of a Human Apical Epithelial Na</H< Exchanger (NHE3) Isoform to Chromosome 5p15.3
1993, Genomics
A gene family of Na⁺/H⁺ exchanger isoforms has been identified. Characterization of rabbit NHE3 suggests that it is the apical epithelial Na⁺/H⁺ exchanger isoform responsible for transepithelial, electroneutral Na⁺ absorption in intestinal and renal epithelial cells. We have previously isolated from a human kidney cortex library a partial human NHE3 cDNA, clone HKC-3. Using HKC-3 to probe human/rodent somatic cell hybrid mapping panels, the human NHE3 gene was physically mapped to the distal portion of chromosome 5p15.3. Southern analysis of EcoRI-digested human genomic DNAs of CEPH pedigrees probed with HKC-3 detected three polymorphic sites containing a total of nine alleles that segregate in a Mendelian fashion. The observed heterozygosity for the NHE3 locus in unrelated individuals was 71%. Linkage analysis between NHE3 and other markers known to map at 5p15 confirmed the localization of NHE3 to chromosome 5p15.3, making NHE3 the most telomeric gene identified on the short arm of chromosome 5.
Localization of the mouse Na+/H+ exchanger gene on distal chromosome 4
1993, Genomics
A cDNA probe of the human Na⁺/H⁺ exchanger (formerly, antiporter) was used to probe Southern blots of DNA obtained from various inbred mouse strains. On the basis of fragment length polymorphisms generated by each of two restriction enzymes, three different alleles could be identified. The distribution of alleles of the Nhe-1 gene was determined in the BXD recombinant inbred strain series. Comparison with previously reported strain distribution patterns shows that the gene encoding the mouse Na⁺/H⁺ exchanger maps to distal mouse Chromosome 4, between Lck and Akp-2.

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Genomics

Abstract

References (12)

Cosmid vectors for genomic walking and rapid restriction mapping

Techniques for radiolabeling DNA to high specific activity

Anal. Biochem

Twenty-eight loci form a continuous linkage map of markers for human chromosome 1

Genomics

Molecular cloning, primary structure, and expression of the human growth factor-activatable $Na^{+} H^{+}$ antiporter

Cell

Unequal crossing over between homologous chromosomes is not the major mechanism involved in the generation of new alleles at VNTR loci

Genomics

Genetic difference in lithium-sodium exchange and regulation of the sodium-hydrogen exchanger in essential hypertension

J. Cardiovasc. Pharacol

Cited by (34)

Silent polymorphisms within the coding region of human sodium/hydrogen exchanger isoform-1 cDNA in peripheral blood mononuclear cells of leukemia patients: A comparison with healthy controls

Evidence for effective suppression of recombination the chromosome 17q21 segment spanning RNU2-BRCA1

Genomic organization glucocorticoid transcriptional activation of the rat Na<sup>+</sup>/H<sup>+</sup> exchanger Nhe3 gene

High-resolution fluorescence mapping of 46 dna markers to the short arm of human chromosome 1

Physical and Genetic Mapping of a Human Apical Epithelial Na<sup><</sup>/H<sup><</sup> Exchanger (NHE3) Isoform to Chromosome 5p15.3

Localization of the mouse Na<sup>+</sup>/H<sup>+</sup> exchanger gene on distal chromosome 4

Short communicationCloning of the human genomic amiloride-sensitive Na+H+ antiporter gene, identification of genetic polymorphisms, and localization on the genetic map of chromosome 1p

Abstract

Anal. Biochem

Genomics

Cell

Genomics

Genetic difference in lithium-sodium exchange and regulation of the sodium-hydrogen exchanger in essential hypertension

J. Cardiovasc. Pharacol

Short communication
Cloning of the human genomic amiloride-sensitive $Na^{+} H^{+}$ antiporter gene, identification of genetic polymorphisms, and localization on the genetic map of chromosome 1p