Localization of the human B-type natriuretic peptide precursor (NPPB) gene to chromosome 1p36

SHORT COMMUNICATION Localization of the Human B-Type Natriuretic Peptide Precursor (NPPB) Gene to Chromosome lp36 KAREN C. ARDEN,*'t '1 CARRIE S. VIARS,* SUZANNE WEISS,* STEFANIA ARGENTIN,t AND MONA NEMER~ *Ludwig Institute for Cancer Research and t Department of Medicine, University of California San Diego, 9500 Gilman Drive, La Jolla, California 92093-0660; and :#Laboratory of Cardiac Growth and Differentiation, Institut de Recherches Cliniques de Montreal, 110 Avenue des Pins Ouest, Montreal, Qu@bec, H2Wl R7 Canada Received October 12, 1994; accepted January 11, 1995

pathological cardiac development, suggesting that the two related peptides have distinct physiological or pathophysiological functions (3, 4, 10, 19, 20). The genes encoding NPPA and N P P B have been characterized in several species and show a strikingly well-conserved organization of three exons and two introns, with the second exon containing most of the coding sequences (1, 11, 15, 18). This, together with the similarities in amino acid sequence, peptide structure, and tissue distribution, suggests that NPPA and N P P B may have evolved from a common ancestral gene. Here we have determined the chromosomal localization of N P P B initially using Southern blot analysis of a series of h u m a n × hamster somatic cell hybrid cell lines containing the various combinations of h u m a n chromosomes. The hybrids designated with GM were obtained from the H u m a n Genetic Mutant Cell Repository (Camden, NJ) and those with A3 were from P. Pearson (Johns Hopkins University). Genomic DNA from the cell hybrids was digested with BamHI, separated by agarose gel electrophoresis, transferred to nylon membranes, and hybridized with a radiolabeled 522-bp genomic fragment, NP44 (Nemer, unpublished data) of the NPPB locus (Fig. 1). Under our conditions, no hamster or mouse crossreactive homologue was detected. Chromosomal localization was determined by scoring the presence or absence of human-specific bands on the autoradiograph

Cardiac m y o c y t e s s y n t h e s i z e a n d s e c r e t e a family o f p e p t i d e h o r m o n e s w i t h p o t e n t natriuretic, diuretic, and v a s o d i l a t o r y properties. T h e s e p e p t i d e s are der i v e d from p r e c u r s o r m o l e c u l e s that are e n c o d e d by t w o different genes, the atrial n a t r i u r e t i c p e p t i d e prec u r s o r A (NPPA) a n d the B-type natriuretic p e p t i d e or natriuretic p e p t i d e p r e c u r s o r B (NPPB). A h u m a n g e n o m i c c l o n e for the N P P B g e n e w a s u s e d to determ i n e the c h r o m o s o m a l l o c a t i o n o f the N P P B gene. A n a l y s i s of S o u t h e r n blot h y b r i d i z a t i o n to DNAs from v a r i o u s s o m a t i c cell h y b r i d s a n d f l u o r e s c e n c e in situ h y b r i d i z a t i o n a l l o w e d a s s i g n m e n t o f t h e N P P B locus to h u m a n c h r o m o s o m e lp36. This l o c a t i o n c o i n c i d e d w i t h that of the N P P A locus; pulsed-field gel electrop h o r e s i s p l a c e d N P P A a n d N P P B w i t h i n 50 kb of e a c h other. This close c h r o m o s o m a l linkage, t o g e t h e r w i t h the c o n s e r v e d p r i m a r y s e q u e n c e s a n d structural organ i z a t i o n of the t w o n a t r i u r e t i c p e p t i d e p r e c u r s o r genes, s u g g e s t s that the n a t r i u r e t i c p e p t i d e loci m a y h a v e e v o l v e d from a c o m m o n a n c e s t o r gene. © 1995 Academic Press, Inc.

Natriuretic peptides are a family of hormones that play important roles in the regulation of body fluid homeostasis. They include atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) (14). These peptides share structural and functional properties, bind to the same class of receptors, and elicit similar biological responses, including diuresis, natriuresis, and vasodilation. The three peptides are derived from the carboxy termini of distinct precursors (natriuretic peptide precursors A, B, and C) that are the product of three different genes. Whereas expression of the natriuretic peptide precursor C (NPPC) is most abundant in cells of the nervous system (7, 9), both A and B natriuretic peptide precursors (NPPA and NPPB) are synthesized predominantly in the heart (3, 10). Within the heart, expression of NPPA and N P P B is differentially regulated in atria and ventricles and during normal and

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FIG. 1.

Hybridization of the genomic h u m a n NPPB probe to from h u m a n x rodent somatic cell hybrids. The chromosomal localization was determined by scoring the presence or absence of the human-specific bands on the autoradiograph of the Southern blot. Only the presence or absence of chromosome 1 was concordant with hybridization signals.

BamHI-digested DNA

To whom correspondence should be addressed. Telephone: (619) 534-7807. Fax: (619) 534-7750. 385

GENOMICS 26, 385--389 (1995) 0888-7543/95 $6.00 Copyright © 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.

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FIG. 2. Southern hybridization of the genomic NPPB insert from NP44 B a m H I restriction enzyme digests of genomic DNA from hamster, mouse, human, and somatic cell hybrid cell lines. Lane 1, DNA from normal human male; lane 2, DNA from hybrid cell line A9/ 1492 containing l p t e r - p l 3 ; lane 3, DNA from hybrid cell line DCR1 containing p34-qter; lane 4, DNA from hybrid cell line NBE-N1 containing l p l 3 - q t e r ; lane 5, DNA from hybrid cell line A9/GM0201 containing lpter-p32; lane 6, DNA from hybrid cell line F5A containing 1p35-p32; lane 7, mouse DNA; lane 8, hamster DNA.

of the Southern blot shown in Fig. 1. Only the presence or absence of h u m a n chromosome 1 was concordant with NP44 hybridization, with at least two discordancies observed for every other chromosome. These data as well as the h u m a n chromosomal content of the somatic cell hybrids are summarized in Table 1. To confirm and refine the localization of NPPB on chromosome 1, additional hybrid cell lines each containing only a portion of chromosome 1 were used. One of these, A9/1492, contains most of the short arm of chromosome 1, from l p t e r to lp13. The cell line DCR1 retains the derivative 1 from a t(1; 17)(p34;q12) and therefore contains ip34 to lqter. NBE-NI contains the region of chromosome 1 from lp31 to lqter. A9/GM0201 contains only the portion of the short arm of chromosome I from l p t e r to lp32, and F5A contains a smaller region of the short arm from lp35 to lp32. Analysis of DNA prepared from these cell lines revealed hybridization of NP44 only to DNA from the two cell lines that retain the distal end of the short arm of chromosome 1, refining the localization of N P P B to lp36 (Fig. 2). This assignment was independently confirmed using two-color fluorescence in situ hybridization (FISH). A biotin-labeled a-satellite repeat probe (Oncor catalog No. P5008) that hybridizes to the centromeric regions of chromosomes 1 (DlZ7), 5 (D5Z2), and 19 (D19Z3), used to confirm chromosome identification, was detected by Texas red-labeled avidin. The entire NP44 plasmid containing the 522-kb genomic fragment of the N P P B locus was labeled by nick-translation using dig o x i g e n i n - l l - d U T P (Boehringer Mannheim) for FISH and detected using an FITC-conjugated antibody directed against digoxigenin. Normal h u m a n chromosome preparations were obtained from phytohemagglutinin-stimulated normal peripheral blood lymphocytes cultured for 72 h. Cytogenetic harvests and slide preparations were performed using standard methods. Hybridization to metaphase chromosomes was performed as described (12). Preparations were counterstained by mounting the slides in an antifade solution containing 1 #g/ml phenylenediamine, propidium iodide, and

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DAPI. Metaphase spreads were examined and photographed using a Zeiss Axiophot microscope and the appropriate filter combinations. Chromosome identification was performed by simultaneous hybridization with the a-satellite repeat probe as well as by counterstaining the chromosomes with DAPI, which produces a Q-banding pattern. In these experiments, 112 metaphase spreads were examined from normal lymphocytes, and signals were considered to be specific only when they were detected on each chromatid of a single chromosome. Specific signals were seen in 8% of the 112 metaphases examined. In each case the hybridization signals were located in the distal portion of chromosome 1, in band lp36 (Fig. 3). Pulsed-field gel electrophoresis (PFGE) was used to determine the physical distance between N P P B and NPPA. Agarose blocks containing high-molecularweight DNA were prepared from lymphoblastoid cells, and 1 #g of DNA was digested with the appropriate restriction enzymes according to standard techniques (16). Electrophoresis was carried out in a Bio-Rad CHEF DRII apparatus at 15°C for 26 h at a pulse time of 75 s followed by 10 h at a pulse time of 120 s in a 1% agarose gel at 150 V. Fragment sizes were determined by comparison with phage concatemers (New England Bio Labs) run in the outside lanes of each gel. After electrophoresis the gels were stained with ethidium bromide and exposed to UV light before being denatured and transferred. Consecutive hybridization of probes for N P P B (NP44) and NPPA ( J A l l 0 ) to human genomic DNA showed that both probes recognize several identical fragments including 300-kb SalI, 150kb SfiI, and 140-kb SmaI fragments. However, NPPB and NPPA hybridize to different XhoI fragments, demonstrating the presence of at least one XhoI site between the two loci (Fig. 4). In addition, both probes hybridized to 210-kb BssHII, 200-kb EagI, 340-kb NotI, 390-kb NruI, 100-kb SacII, and 50-kb PacI fragments (data not shown). The PacI digest resulted in the small-

FIG. 3. Localization of the NPPB gene by fluorescence in situ hybridization. Chromosome 1 can be identified by morphology and by hybridization of a labeled chromosome-specific a-satellite repeat probe, DIZ5. Specific hybridization of the NPPB cDNA clone to the distal lp36 region is indicated by an arrowhead.

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tal part of the short arm of chromosome 1 have also been reported in multiple endocrine neoplasia (8) and Merkel cell carcinoma (17). Whether the natriuretic peptides play a role in these latter diseases is unclear, but the present mapping of the NPPB locus may serve to focus efforts in this regard. Moreover, the present studies provide physical evidence in support of the idea t h a t NPPB and NPPA shared a common ancestry and necessitate further mapping at a more refined level.

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ACKNOWLEDGMENTS NP44

JA110

FIG. 4. Comparison of NPPB clone NP44 and NPPA clone JA110 by pulsed-field gel electrophoresis. Restriction fragment sizes were determined by comparison with phage concatemers run in the outside lanes of the gel. DNA from the same lymphoblastoid cell line was digested with SaII, SfiI, Sinai, and XhoI. The resulting pulsedfield blot was sequentially hybridized with NP44 and JAll0.

est fragment detected by both probes, indicating that NPPB and NPPA are within 50 kb of each other. The present study demonstrates t h a t the gene encoding NPPB is located on chromosome lp36 in the same chromosome band as NPPA (22). NPPB and NPPA are members of a gene family t h a t also includes NPPC (6), although the chromosome location of NPPC has not yet been established. It is possible t h a t these genes, as has been proposed for other families of genes, may have evolved from a common ancestor t h a t gave rise to at least two natriuretic peptide genes. Recently, the mouse NPPB was assigned to mouse chromosome 4 in close proximity to NPPA (18). The localization of NPPB on mouse chromosome 4 and on the short arm of h u m a n chromosome 1 adds yet another locus to the conserved syntenic group of homologous genes t h a t are located on h u m a n chromosome 1 and mouse chromosome 4. Interestingly, the natriuretic peptide receptors also show considerable sequence similarity to each other (6), raising the possibility t h a t they too are of common evolutionary origin. Differential expression of the two natriuretic peptide genes in cardiac and extracardiac tissue suggests t h a t while they may be located in close chromosomal proximity to one another, they are under independent regulatory control (3). Indeed, transcription of the ANP and BNP genes appears to be controlled by some common pathways and factors but also by additional regulatory mechanisms t h a t may be specific to each locus (1, 5). Furthermore, while expression of both NPPA and NPPB is upregulated in several cardiovascular diseases, including hypertension and cardiac hypertrophy, the NPPB locus appears to be hyperresponsive since changes in gene expression occur earlier and to greater extents (4, 10) such t h a t in m a n y h u m a n diseases, BNP becomes the predominant circulating natriuretic peptide (20). Finally, deletions of lp36.1-p36.2 are common findings in neuroblastoma (21), and alterations in the dis-

Part of this work was supported by the Medical Research Council of Canada. S.A. is a fellow and M.N. is a scholar of the Heart and Stroke Foundation of Canada. We thank Drs. Garrett Brodeur and Nicholas Dracopoli for providing the chromosome 1 regional somatic cell hybrid mapping panel. REFERENCES 1. Argentin, S., Nemer, M., Drouin, J., Scott, G. K., Kennedy, B. P., and Davies, P. L. (1985). The gene for rat atrial natriuretic factor. J. Biol. Chem. 260: 4568-4571. 2. Argentin, S., Ardati, A., Tremblay, S., Lihrmann, I., Robitaille, L., Drouin, J., and Nemer, M. (1994). Developmental stagespecific regulation of atrial natriuretic factor gene transcription in cardiac cells. Mol. Cell. Biol. 14: 777-790. 3. Dagnino, L., Drouin, J., and Nemer, M. (1991). Differential expression ofnatriuretic peptide genes in cardiac and extracardiac tissues. Mol. Endocrinol. 5: 1292-1300. 4. Dagnino, L., Lavigue, J.-P., and Nemer, M. (1992). Increased transcripts for B-type natriuretic peptide in spontaneously hypertensive rats. Hypertension 20: 690-700. 5. Gr6pin, C., Dagnino, L., Robitaille, L., Haberstroh, L., Antakly, T., and Nemer, M. (1994). A hormone coding gene identifies a pathway for cardiac but not skeletal muscle gene transcription. Mol. Cell. Biol. 14: 3115-3129. 6. Koller, K. J., and Goeddel, D. V. (1992). Molecular biology of the natriuretic peptides and their receptors [Review]. Circulation 86: 1081-1088. 7. Komatsu, Y., Nakao, K., Suga, S. I., Ogawa, Y., Mukoyama, M., Arai, H., Shirakami, G., Hosoda, K., Nakagawa, O., Hama, N., Kishimoto, I., and Imura, H. (1991). C-type natriuretic peptide (CNP) in rats and humans. Endocrinology 129: 1104-1106. 8. Mathew, C. G., Smith, B. A., Thorpe, K., Wong, Z., Royle, N. J., Jeffreys, A. J., and Ponder, B. A. (1987). Deletion of genes on chromosome 1 in endocrine neoplasia. Nature 328: 524-526. 9. Minamino, N., Aburaya, M., Kojima, M., Miyamoto, K., Kangawa, K., and Matsuo, H. (1993). Distribution of C-type natriuretic peptide and its messenger RNA in rat central nervous system and peripheral tissue. Biochem. Biophys. Res. Commun. 197: 326-335. 10. Mukoyama, M., Nakao, K., Hosoda, K., Suga, S. I., Saito, Y., Ogawa, Y., Shirakami, G., Jougasaki, M., Obata, K., Yasue, H., Kambayashi, Y., Inouye, K., and Imure, H. (1991). Brain natriuretic peptide as a novel cardiac hormone in humans. J. Clin. Invest. 87: 1402-1412. 11. Nemer, M., Chamberland, M., Sirois, D., Argentin, S., Drouin, J., Dixon, R. A., Zivin, R. A., and Condra, J. H. (1984). Gene structure of human cardiac hormone precursor, pro-natriodilatin. Nature 312: 654-656. 12. Pinkel, D., Straume, T., and Gray, J. W. (1986). Cytogenetic analysis using quantitative, high-sensitivity fluorescence hybridization. Proc. Natl. Acad. Sci. USA 83: 2934-2938. 13. Rigaud, G., Grange, T., and Pictet, R. (1987). The use of NaOH

SHORT COMMUNICATION as transfer solution of DNA onto nylon membrane decreases the hybridization efficiency. Nucleic Acids Res. 15: 857. 14. Rosenzweig, A., and Seidman, C. E. (1991). Atrial natriuretic factor and related peptide hormones. Annu. Rev. Biochem. 60: 229-255. 15. Seilhamer, J. J., Arfsten, A., Miller, J. A., Lundquist, P., Scarborough, R. M., Lewicki, J. A., and Porter, J. G. (1989). Human and canine gene homologs of porcine brain natriuretic peptide. Biochem. Biophys. Res. Commun. 165: 650-658. 16. Smith, C. L., and Cantor, C. (1990). Purification, specific fragmentation and separation of large DNA molecules. Methods Enzymol. 155: 449-467. 17. Sozzi, G., Bertoglio, M. G., Pilotti, S., Rilke, F., Pierotti, M. A., and Della Porta, G. (1988). Cytogenetic studies in primary and metastatic neuroendocrine Merkel cell carcinoma. Cancer Genet. Cytogenet. 30: 151-158. 18. Steinhelper, M. E. (1993). Structure, expression, and genomic

19.

20.

21.

22.

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mapping of the mouse natriuretic peptide type-B gene. Circ. Res. 72: 984-992. Takahashi, T., Allen, P. D., and Izumo, S. (1992). Expression of A-, B-, and C-type natriuretic peptide genes in failing and developing human ventricles: Correlation with expression of the Ca(2+)-ATPase gene. Circ. Res. 71: 9-17. Wei, C. M., Heublein, D. M., Perrella, M. A., Lerman, A., Rodeheifer, R. J., McGregor, C. G., Edwards, W. D., Schaff, H. V., and Burnett, J. C., Jr. (1993). Natriuretic peptide system in human heart failure. Circulation 88: 1004-1009. Weith, A., Martinsson, T., Cziepluch, C., Bruderlein, S., Amler, L. C., Berthold, F., and Schwab, M. (1989). Neuroblastoma consensus deletion maps to lp36.1-2. Genes Chrom. Cancer 1: 159-166. Yang-Feng, T. L., Floyd-Smith, G., Nemer, M., Drouin, J., and Francke, U. (1985). The pronatriodilatin gene is located on the distal short arm of human chromosome 1 and on mouse chromosome 4. Am. J. Hum. Genet. 37: 1117-1128.