C ytogenet Cell G en et 5 7 :3 9 -4 0 (1991)

© 1991 S. Karger AG. Basel 0301-0171/91 /0571-0039$2.75/0

T h e g en e for h u m an grow th h o r m o n e -r e le a s in g factor (G H R F ) m a p s to or near ch ro m o so m e 2 0 p l2 V .V .N .G . R a o , C. L ö ffle r, S . S c h n i t t g e r , I. H a n s m a n n

Abstract. Growth hormone-releasing factor (GHRF), a hy­ pothalamic releasing factor also named somatocrinin, in­ fluences the secretion and synthesis of growth hormone. Human GHRF is encoded by a single gene which was assigned to chro-

mosome 20 by dot-blot analysis of DNA from dual laser sorted chromosomes. Using a radioactive cDNA probe, we localized the GHRF gene to chromosome 20pl2 or near band 20pl2.

Two hypothalamic releasing factors, growth hormone-releas­ ing factor (GHRF) and somatostatin, participate in regulating the complex process of growth hormone (GH) synthesis and secretion in the anterior pituitary. While GHRF or somatocrinin (Gubler et al„ 1983) functions as a specific stimulator of GH secretion, somatostatin may act as an inhibitory counter­ part (Guillemin et al„ 1982). GHRF, isolated from human pan­ creatic tumors causing acromegaly (Guillemin et al„ 1982; Rivier et al., 1982), is a 44-amino acid peptide reported to be amidated at its COOH terminus. GHRF is synthesized as a 107- and 108-amino acid precursor protein, preprosomatocrinin, with a molecular weight of approximately 13,000 (Gubler et al., 1983; Mayo et al., 1983). The human GHRF is encoded by a single gene on chromosome 20 containing five exons separated by four introns, and spans approximately 10 kb of genomic DNA. The five exons encode the functionally dis­ crete domains of the precursor molecule, with exon 3 encoding most of the mature GHRF peptide. An T/w-repeat was identified upstream from exon 2 (Mayo et al., 1985). Since GHRF is involved in the synthesis and regulation of growth hormone secretion, it was assumed that mutations in this gene result in diseases with growth hormone deficiency such as the familial isolated growth hormone deficiency (IGHD) and pituitary dwarfism (Philips, 1983). Regional localization of the human GHRF gene to chromosome 20 might be of utility in studies involving diseases with the growth hormone deficiency. Hence, we have performed in situ hybridization with a radioactively-labeied cDNA probe for the GHRF gene, to confirm its chromosomal assignment and to map it regionally on chromo­ some 20.

T h e 560-bp cD N A probe (G R F-8 in G u b ler et al., 1983) was radioactivcly labeled w ith 3H -d A T P . 3H -d C T P . an d 3H -d T T P by the ran d o m p rim in g m ethod to a specific activity o f 3.5 x 107 cpm . P rom elaphase chrom osom es w ere o b tain ed from the lym phocytes o f a karyotypically norm al m ale using the m ethod o f R ybak et al. (1982). In situ hy b rid izatio n w as carried o u t using the m ethod o f C h an d ler and Y unis (1978). as m odified by B eerm ann et al. (1988). T he slides, after hybridization, w ere w ashed stringently w ith differen t co n cen tratio n s o f SSC and 5% T C A an d w ere th en dipped in A m ersham LM1 photographic em ulsion and exposed for 6 d at 4 ° C. S ubsequently, they were developed an d G -b a n d e d using the trypsin-G iem sa m ethod.

Supported by the Deutsche Forschungsgemeinschaft and a fellowship to G.R. from DAAD. Received 18 February 1991; accepted 18 February 1991. Request reprints from Prof. Dr. 1. Hansmann. Institut fur Humangenetik, Universität Göttingen, Gosslerstrasse 12d, 3400 Göttingen (Germany).

A total of 227 silver grains were observed from the 148 meta­ phases analyzed (1.5 grains/metaphase). These silver grains were distributed more or less randomly on all chromosomes with no noticeable cluster except for chromosome 20 (Fig. 1). A large cluster of grains, i.e., 34 of the 227 grains (15%) were local­ ized on chromosome 20 (P < 0.001), thus confirming the assignment of the GHRF gene to this chromosome (Fig. 2). Con­ sidering the DNA content/relative length of chromosome 20, a total of 5.2 (2.3%) grains were expected on this chromosome. A large peak of grains (13/34,38.2%) was observed at band 20pl2, while two smaller peaks of 4 grains each (4/34, 11.8%) were observed at bands 20p 11 and 20p 13. Thus, based on our results we assign the GHRF gene locus to or near band 20pl 2. Shohat et al. (1989) measured GHRF gene dosage in DNA from a patient with a suspected terminal deletion of 20p [46,XX,del(20)(pl 1.23)]. The patient had a growth hormone deficiency associated with a neurosecretory defect in GHRF secretion, in addition to other clinical features like peculiar facies, congenital heart defects, and severe failure to thrive. Quantitative Southern blotting revealed a normal dose of two copies of GHRF in the patient’s genome. The authors, there­ fore, excluded the GHRF gene locus from the region 20pl 1.23-»pter. The breakpoint of the structural anomaly found in the patient’s chromosome 20 was assigned by cytological means to band pi 1.23. This observation does not contradict our assignment of the GHRF locus to or near band p 12. Further-

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Fig. 1. H istogram o f grain d istrib u tio n in 148 m etaphases hybridized in situ w ith a p ro b e fo r G H R F .

Fig. 2. Idiogram o f chrom osom e 20 a t th e 500-band stage, show ing grain d istrib u tio n .

more molecular analysis of DNA from this patient with various 20p DNA probes defined the deletion to be interstitial rather than terminal (Schnittger et al., unpublished). Moreover, it can not be excluded that the regular gene order on 20p was already altered prior to breakage, or that the structural anomaly resulted from a more complex event of breakage and reunion. In situ hybridization, on the other hand, does not permit precise assignment of a locus in question, to an individual chromosome

band, such as 20p 12. Considering the results from these studies, we assume that the GHRF gene is located either between the centromere and the proximal breakpoint or above the distal breakpoint, but either way close to band pi 2.

Guillemin R, Brazeau P, Böhlen P, Esch F. Ling N, Wehrenberg WB: Growth hormone-releasing factor from a human pancreatic tumor that caused acro­ megaly. Science 218:585-587 (1982). Mayo KE, Cerclli GM, Lebo RV, Bruce BD, Rosenfeld MG, Evans RM: Gencencodinghuman growth hor­ mone-releasing factor precursor: structure, se­ quence, and chromosomal assignment. Proc natl Acad Sei, USA 82:63-67 (1985). MayoKE, Vale W, Rivier J. Rosenfeld MG, Evans RM: Expression-cloning and sequence of a cDNA encod­ ing human growth hormone-releasing factor. Na­ ture 306:86-88 ( 1983). Philips JA III: The growth hormone (hGH) gene and human disease, in Caskey CT. White RL (eds): Recombinant DNA: Applications to Human Dis­

ease, pp 305-315 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 1983). RivierJ.Spiess J.T horner M. Vale W: Characterization of a growth hormone-releasing factor from a human pancreatic islet tumour. Nature 300:276-278 (1982). Rybak J, Tharapel A. Robinett S. Garcia M, Maniken C. Freeman M: A simple reproducible method for prometaphase chromosome analysis. Hum Genet 60:328-333(1982). Shohat M, Herman V. Schrcck R. Pulst S-M. Neufeld N, Melmed S. Korenberg JR: Growth hormone neuro­ secretory disorder due to deletion of 20pl 1.23-pter but with normal growth hormone releasing factor genes (GHRF). Cytogenet Cell Genet 51:1078 (1989).

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Beermann F, Hummler E, Franke U. Hansmann I: Allo­ cation of a transgenic c-myc integration site to mouse chromosome 8B3-C1. Cytogenet Cell Genet 49:311-312(1988). Chandler ME. Yunis JJ: A high resolution in situ hybridization technique for the direct visualization of labeled G-banded early metaphase and prophase chromosomes. Cytogenet Cell Genet 22:352-356 (1978). Gubler U, Monahan JJ, Lomedico PT. Bhatt RS. Collier KJ, Hoffman BJ. Böhlen P. Esch F, Ling N, Zeytin F. Brazcau P, Poonian MS, Gage LP: Cloning and sequence analysis of cDNA for the precursor of human growth hormone-releasing factor, somaiocrinin. Proc natl Acad Sei, USA 80:4311-4314 (1983).

T h e au th o rs gratefully acknow ledge D r. U eli G ubler, D ept, o f M olecular G enetics, R oche R esearch C enter, H offm an-L aR oche. Inc. N utley. New Je r­ sey 07110. for kindly prov id in g the cD N A probe. T h an k s are d u e to M r. D ietm a r Schlote for th e co m p u ter graphics.