Characterization of the Human Growth Hormone Receptor Gene And

Proc. Natl. Acad. Sci. USA Vol. 86, pp. 8083-8087, October 1989 Medical Sciences Characterization of the human growth hormone receptor gene and demonstration of a partial gene deletion in two patients with Laron-type dwarfism (exon structure/DNA sequence/gene defects) PAUL J. GODOWSKI*, DAVID W. LEUNG*, LILLIAN R. MEACHAMt, JOHN P. GALGANIt, RENATE HELLMISS*, RUTH KERET§, PETER S. ROTWEIN¶, JOHN S. PARKSt, ZVI LARON§, AND WILLIAM I. WOOD* *Departmenlts of Developmental Biology and Molecular Biology, Genentech, Inc., 460 Point San Bruno Boulevard, South San Francisco, CA 94080; tDepartment of Pediatrics, Emory University School of Medicine, 2030 Ridgewood Drive, Northeast, Atlanta, GA 30322; Departments of tPediatrics and 1lnternal Medicine and Genetics, Washington University School of Medicine, 660 South Euclid Avenue, Saint Louis, MO 63110; and Institute of Pediatric and Adolescent Endocrinology, Tel Aviv University, Beilinson Medical Center, 49100 Petah Tikva, Israel Communicated by William H. Daughaday, July 24, 1989 (receivedfor review June 8, 1989)

ABSTRACT Laron-type dwarfism is an autosomal reces- individuals with LTD have been shown to lack GH binding in sive genetic disorder that is characterized by high levels of liver biopsy samples (17), to lack GH binding activity in their growth hormone and low levels of insulin-like growth factor I serum (18-20), or to lack an IGF-I response in transformed in the circulation. Several lines of evidence suggest that this T lymphoblasts (21). These studies suggest that LTD results disease is caused by a defect in the growth hormone receptor. from a defect in the GH receptor. In theory, LTD could result In order to analyze the receptor gene in patients with Laron- from defects in the gene that encodes the GH receptor itself type dwarfism and with other growth disorders, we have first as well as in other genes required for its expression or determined the gene structure in normal individuals. There are function. nine exons that encode the receptor and several additional In order to define the role of the GH receptor in human exons in the 5' untranslated region. The coding exons span at growth disorders, we present here the characterization ofthe least 87 kilobase pairs of chromosome 5. Characterization of gene for the human GH receptor. 11 We use these data to the growth hormone receptor gene from nine patients with demonstrate the deletion of a large portion of the receptor Laron-type dwarfism shows that two individuals have a dele- gene in two LTD patients. A portion of this work has been tion of a large portion of the extracellular, hormone binding presented previously.** domain of the receptor gene. Interestingly, this deletion in- cludes nonconsecutive exons, suggesting that an unusual rearrangement may have occurred. Thus, we provide direct evi- HUMAN SUBJECTS dence that Laron-type dwarfism can result from a defect in the Patient 147 is a male with LTD born to related parents of structural gene for the growth hormone receptor. Jewish Iraqi origin. Birth length (49 cm) and weight (3.0 kg) were normal. He showed slow physical and mental develop- Growth hormone (GH) is secreted from the pituitary and has ment and had clinical features ofGH deficiency but with high direct and indirect actions on various tissues, causing effects GH levels (>40 ng/ml) and low levels of IGF-I. At age 35 his on growth and metabolism (1, 2). Though high-affinity bind- height was 128.3 cm (7.3 SD below expected) and his weight ing sites for GH have been identified in a number of tissues was 41 kg. No serum GH binding protein activity was (1, 3), the highest concentration of GH receptors is found in detected. No 251I-labeled GH binding was detected in liver the liver (4) where GH induces the expression and secretion microsomal membranes from this patient after liver biopsy of insulin-like growth factor I (IGF-I) (3). The GH receptor [case 2 (17)]. recently has been purified and characterized from rabbit liver Patient D1 is a girl with LTD born to related parents of (5), and clones for the rabbit and human GH receptor have Jewish Iraqi origin. Birth length (45 cm) and weight (2.85 kg) been isolated from liver cDNA libraries (6). These clones were normal. At the age of 6 months she showed typical encode a protein of 620 amino acids with a single, centrally features of GH deficiency but with high plasma levels of GH located transmembrane domain. Comparison of the primary (20-100 ng/ml). After 5 days of GH therapy, there was no sequences of the GH receptor and of the related prolactin increase in the plasma IGF-I level. At age 8 years, 10 months, receptor (7) suggests that they may be members of a new her length was 104 cm (4.2 SD below expected) and her family of membrane-bound receptors. weight was 22.5 kg. No serum GH binding protein activity A high-affinity GH binding protein has been demonstrated was detected (20). in the plasma ofa number ofmammals, including man (8-10). Patient 1 is a boy with LTD born to unrelated parents in the Characterization of this binding protein (11, 12), including United States. He lacks serum GH binding protein activity direct amino acid sequence data (5, 6), shows that the GH [see patient 1 (18) for a full clinical description]. DNA was binding protein is the extracellular, hormone binding domain obtained from fibroblasts. of the GH receptor. The role of this protein in the regulation The other six LTD patients, who had no alteration in their of growth is not known. genomic DNA pattern, include three of oriental Jewish Laron-type dwarfism (LTD) is a rare, autosomal recessive background, two from the United States, and one from disorder that is characterized by high circulating levels of biologically active GH accompanied by low levels of IGF-I Abbreviations: GH, growth hormone; IGF-I, insulin-like growth (13-16) and a failure to respond to GH therapy (15). Some factor I; LTD, Laron-type dwarfism. "The sequence reported in this paper has been deposited in the GenBank data base (accession no. M26401). The publication costs of this article were defrayed in part by page charge **Meacham, L. R., Parks, J. S., McKean, M. C., Keret, R. & payment. This article must therefore be hereby marked "advertisement" Laron, Z., 71st Annual Meeting of the Endocrine Society, Seattle, in accordance with 18 U.S.C. §1734 solely to indicate this fact. June 21-24, 1989, abstr. 1651.

8083 Downloaded by guest on September 28, 2021 8084 Medical Sciences: Godowski et al. Proc. Natl. Acad. Sci. USA 86 (1989) Poland (fibroblasts provided by Maria Malinowska andThom- observed in genomic blots of the receptor (see below) were asz Romer, Child Health Center, Warsaw, Poland). The three due to a family ofclosely related genes or to the several exons oriental Jewish patients [described previously as patients 8, of a single-copy receptor gene. Six clones were isolated from 9, and 10 (14)] are from one clan and have clinical features of human genomic libraries by screening with the cloned human GH deficiency but with high plasma levels of GH, low levels GH receptor cDNA (Fig. 1). These clones were mapped, and ofIGF-I, and undetectable GH serum binding protein activity the hybridizing regions were sequenced in order to determine (20). All three showed no response to GH therapy. The two precisely the extent of each exon (Fig. 2). With the exception U.S. patients lack serum binding protein activity and showed of a single base difference in exon 10 (Fig. 2, legend), the no response to GH therapy [described previously as patients sequence of the coding region determined for the genomic 2 and 3 (18) or as patients 6 and 8, respectively (22)]. clones matched that determined previously from cDNA clones (6). METHODS The coding and 3' untranslated regions of the receptor are encoded by 9 exons, numbered 2-10 (Fig. 1). Exons 2-9 range Genomic clones for the GH receptor were isolated from A in size from 66 to 179 bp; exon 10, which encodes nearly all libraries either described previously, A4X (23) (AGG.9, -19, of the cytoplasmic domain as well as the 3' untranslated and -20), or kindly provided by John McLean (Genentech) region, is about 3400 bp. Exons 2 and 8 are nearly coincident (AGG.33, -47, and -48). The libraries were screened with with the putative secretion signal sequence and the trans- 32P-labeled (24) cDNA probes from the human GH receptor membrane domain, respectively. The extracellular, GH bind- (6). The hybridization was performed at 420C in 50% form- ing region of the receptor is encoded by exons 3-7. Most of amide/0.75 M NaCl/0.075 M trisodium citrate, and the filters the 5' untranslated region appears to be present on a series of were washed at 420C in 0.03 M NaCl/3 mM trisodium alternatively spliced exons that have not yet been localized citrate/0.1% SDS. DNA sequencing was performed by the (see below). The gene for the GH receptor has previously dideoxy chain-termination method (25) using genomic frag- been localized to chromosome 5p13.1-p12 (27) and spans at ments subcloned into plasmid vectors (26). least 87 kbp. Gaps of unknown length occur between the Genomic DNA was isolated from normal or LTD blood or genomic clones in two places. Thus, the 2/3 and 6/7 introns from fibroblasts. Six micrograms of DNA was digested with are at least 14 and 24 kbp; the 3/4 intron is probably 27 kbp HindIII or EcoRI, electrophoresed on a 0.7% agarose gel, (Fig. 1). We have used the genomic map and hybridization transferred to nitrocellulose, hybridized as above, and data (see below) to assign exons 2-10 to the bands observed washed at 500C as above (24). Probes for the genomic in genomic blots probed for the receptor (Fig. 3). hybridization were labeled with 32p (24). The full-length Analysis of LTD Patient DNA. The GH receptor gene was probe was a 1928-base-pair (bp) Xho I to SnaBI fragment (bp analyzed in DNA samples from patients with LTD. A cDNA -11 to +1917) of the human GH receptor cDNA (6). To fragment encompassing the complete protein coding region of generate the exon-specific probes, pairs of 60-mer oligonu- the receptor was used to probe genomic blots of DNA from cleotides were synthesized; each pair contains a 10-bp com- control and nine LTD individuals. No obvious alterations in plementary region at the 3' end. Complementary oligonucle- the receptor gene were observed in six LTD DNA samples otides were annealed and extended with the large fragment of (data not shown). Patient 1 was shown to be heterozygous for DNA polymerase I in the presence of 32P-labeled nucleotides a 200-bp insertion in the 3' untranslated region (data not (24). The marker lanes contained 32P-labeled A HindIII DNA shown). Since it is unclear whether this insertion would affect (24). the expression of the GH receptor gene, we have not analyzed it further. It is possible that this insertion is due to the expansion of the Alu repeat that is in the 3' untranslated RESULTS region of the gene (Fig. 1). Characterization of the GH Receptor Gene. In order to The restriction pattern of HindIII-digested DNA from two analyze human gene defects in the GH receptor, we first patients, 147 and D1, appeared abnormal, with the absence of characterized the structure of the normal gene. This analysis the 16-kbp band assigned to exons 4 and 5 and the 3.5-kbp also would allow us to determine whether the multiple bands band for exon 6 (Fig. 3). In addition, the EcoRI band assigned A 0 1 2 3 4 kbp - -181 200 400 600620 Amino Acids 11 1 1 11 234 5 6 7 89 10 FeYnncqrAvl , FIG. 1. (A) Schematic representation of the 5' Cytoplasmic 3' K A GH receptor mRNA and protein. Shown are L!xtracella _j scales of nucleotides and amino acids, the loca- Signal Transmembrane Alu Repeat tion of the exon boundaries, and the major features of the protein and mRNA. (B) Map of B the GH hormone receptor gene. Shown are a 0 ,, 20 40 60 , 80 100 scale of nucleotides, location of the exons, re- kbp I A/ I I /' I I I striction map for four enzymes, and the location Exons 2 e, 3 4 5 6 , 8 910 of six genomic clones. Two gaps in the map are --- 7 Gene X 14Z 1 1 I -r 7,/>f I_ shown at their minimum length. The length of

I - ...... I the 3/4 intron is established only by the coinci- Hindill dence of genomic Sst I fragments in the region EcoRi III 1-I-III I I I 11111 and thus could be greater. The BamHI sites Sstl indicated by an asterisk may represent more BamHI I I II I I than one site separated by an unknown distance. * * The order of the bracketed EcoRI sites is un- XGG.'"33 XGG.48 XGG.20 known. The restriction map is unknown in the XGG.9 XGG.47 XGG.19 shaded regions. Downloaded by guest on September 28, 2021 l Medical Sciences: Godowski et al. Proc. Natl. Acad. Sci. USA 86 (1989) 8085

* EXON 2 1 TTTCATGATAAT GGTCTGCTTTTAATT GCTGGGCTTTACCTT ACCCTTTTTGTGATT GCAGGTCCTACAGGT ATGGATCTCTGGCAG CTGCTGTTGACCTTG GCACTGGCAGGATCA AGTGATGCTTTTTCT GGAAGTGAGGGTGAG -18 M D L W Q LL L TL A L A G S S D A F S G S E A

148 TTCTGCTTTTCCATT TCCACCCTCAGTGTT TTGAAACAACACTaA ACTGTATTC . 14 kbp or more . .. * ~~~~~~~~~~~~~~~~~~EXON3* 1 GATGGACTAGATGGT TTTGCCTTCCTCTTT CTGTTTCAGCCACAG CAGCTATCCTTAGCA GAGCACCCTGGAGTC TGCAAAGTGTTAATC CAGGCCTAAAGACAA GTAAGAATTTCAGTC CTTTTTCTTCCTTCG AATGATATTTTCCAT 7 TA A I L S R A P W S L Q S V N P G L K T N

151 GTTTTAGTGTAATTA AGCTACTATCCT 27 kbp . . pstI EXON 4 1 AGGATCACATATGAC TCACCTGATTTCATG CCTTGCCTTTTCTTT TTATTCTGCAGATTC TTCTAAGGAGCCTAA ATTCACCAAGTGCCG TTCACCTGAGCGAGA GACTTTTTCATGCCA CTGGACAGATGAGGT TCATCATGGTACAAA 29 S S K E P K F T K C R S P E R E TF S C H W T D E V H H G T K

* ~~~~~~~~~~~~~ncoI 151 GAACCTAGGACCCAT ACAGCTGTTCTATAC CAGAAGGTGCCACCA TCATGCCTTTCTGAT TTTCCTCTCCATGGA TGTACCTACTAAAGT ACACTA 6 kbp 60 N L G P I Q L F Y T R R * ~~~~~~~~~EXON5 1 ACTTAAGCTACAACA TGATTTTTGGAACAA TTAATCTTTTTTTAA CCCTTCATTTTAGGA ACACTCAAGAATGGA CTCAAGAATGGAAAG AATGCCCTGATTATG TTTCTGCTGGGGAAA ACAGCTGTTACTTTA ATTCATCGTTTACCT 72 N T Q E W T Q E W K E C P D Y V S A G E N S C Y F N S SF T S

151 CCATCTGGATACCTT ATTGTATCAAGCTAA CTAGCAATGGTGGTA CAGTGGATGAAAAGT GTTTCTCTGTTGATG AAATAGGTAAATCAC AGGTTTTTGTTTCAT TTGACATAGTTTTAG ACTAAATAAATGGGG AAGC 5 kbp 103 I N I P Y C I K L T S N G G T V DEK C F S V D E I V EXON 6 ecoRV 1 CCATTAATATTAAAT TGTGTCTGTCTGTGT ACTAATGCTCTGTTG AATTGCACAGTGCAA CCAGATCCACCCATT GCCCTCAACTGGACT TTACTGAACGTCAGT TTAACTGGGATTCAT GCACATATCCAAGTG AGATGGGAAGCACCA 130 Q P D P P I A L N W T L L NV S L T G I H A D I Q V R W E A P

151 CGCAATGCAGATATT CAGAAAGGATGGATG GTTCTGGAGTATGAA CTTCAATACAAAGAA GTAAATGAAACTAAA TGGAAAATGGTAAGA TGTTGCTACACCTTA CACTTTGACTTTTCT TTCTATT . 24 kbp or more 161 R N A D I Q K G I K V L E Y E L Q Y K E V N E TK WN M * EEXON 7 1 ATACCTGTAGTGTTC ATTGGCATTGAGTTG TTGACTCTTTGGCCA ATATGGCGTTTATAT TTTTGTCTTGAAAGA TGGACCCTATATTGA CAACATCAGTTCCAG TGTACTCATTGAAAG TGGATAAGGAATATG AAGTGCGTGTGAGAT 189 M D P I L T T S V P V YSL KV D K E Y E V R V R S

151 CCAAACAACGAAACT CTGGAAATTATGGCG AGTTCAGTGAGGTGC TCTATCTAACACTTC CTCAGATCACCCAAT TTACATGTCAACAAG GTAAAAGAAATAAAA GATTAAAATAGTAGC TAACCTGGCTTTTGT CAATATAACAGTTGA 215 K Q R N S G N Y G E F S E V L Y V T L P 0 M S Q F T C E E D

ecoRV 301 TTCACCCCTGCACTG GTAGTGTGTTGTCCA AATCAAAATATATTA ACATCAGATATCAGG AT 3 kbp ncoI EXON8 1 GAAACTGTGCTTCAA CTAGTCGTAATTCTG AAAGCGAAATATTCT TGTGTGTTTGCAGAT TTCTACTTTCCATGG CTCTTAATTATTATC TTTGGAATATTTGGG CTAACAGTGATGCTA TTTGTATTCTTATTT TCTAAACAGCAAAGG 245 F Y F P W L L I I I F G I F G L T V M L F V FL FS K Q Q R

*kpn I 151 TAGGATGTAGGMGG TAGTATTCTTTGGTA CCTTCTGTACCAGTT GTGTTAGACCTTGCC AT 4 kbp EXON 9 claI 1 GCTATAATTGAGAAT ATGTAGCTTTTAAGA TGTCAAAACCAAAAT TTTTATATGTTTTCA AGGATTAAAATGCTG ATTCTGCCCCCAGTT CCAGTTCCAAAGATT AAAGGAATCGATCCA GATCTCCTCAAGGTA ACTAATAATTTTATC 275 I K M L I L PP V P V P K I K G I D P D L L K

151 TAAAGTTGTAGCTAG TACTAATTAACACCT GAAGACTCCTGTCAT ATG .. 0.4 kbp EXON 10 ecoRI 1 GCTAATTCATTTAAT TATTATGAGTTTCTT TTCATAGATCTTCAT TTTCTTTCTATTTTC TAGGAAGGAAAATTA GAGGAGGTGAACACA ATCTTAGCCATTCAT GATAGCTATAAACCC GAATTCCACAGTGAT GACTCTTGGGTTGAA 298 E G K LEE V N T I L A I H D S Y K P E F H S D DS W V E

151 TTTATTGAGCTAGAT ATTGATGAGCCAGAT GAAAAGACTGAGGAA TCAGACACAGACAGA CTTCTAAGCAGTGAC CATGAGAAATCACAT AGTAACCTAGGGGTG AAGGATGGCGACTCT GGACGTACCAGCTGT TGTGAACCTGACATT 327 F I E L D I D E P D E K T E S D T D R L L S S D H E K S H S N L G V K D G D S G R T S C C E P D I

kpnI 301 CTGGAGACTGATTTC AATGCCAATGACATA CATGAGGGTACCTCA GAGGTTGCTCAGCCA CAGAGGTTAAAAGGG GAAGCAGATCTCTTA TGCCTTGACCAGAAG AATCAAAATAACTCA CCTTATCATGATGCT TGCCCTGCTACTCAG 377 L E T D F N A N D I H E G T S E V A Q P HR L K G E A D L L C L DI K N Q N S P Y H D A C P A T Q

451 CAGCCCAGTGTTATC CAAGCAGAGAAAAAC AAACCACAACCACTT CCTACTGAAGGAGCT GAGTCAACTCACCAA GCTGCCCATATTCAG CTAAGCAATCCAAGT TCACTGTCAAACATC GACTTTTATGCCCAG GTGAGCGACATTACA 427 Q P S V I Q A EK N K P Q P L P T E G A E S T H Q A A H I Q L S N P S S L S N I D F Y A Q V S D I T

sma I 601 CCAGCAGGTAGTGTG GTCCTTTCCCCGGGC CAAAAGAATAAGGCA GGGATGTCCCAATGT GACATGCACCCGGAA ATGGTCTCACTCTGC CAAGAAAACTTCCTT ATGGACAATGCCTAC TTCTGTGAGGCAGAT GCCAAAWAGTGCCTC 477 P A G S V V L S P G Q K N K A G M S Q C D M H P E MV S L C Q E N F L M D N A Y F C E A D A KK C L

hindIII 751 CCTGTGGCTCCTCAC ATCAAGGTTGAATCA CACATACAGCCAAGC TTAAACCAAGAGGAC ATTTACATCACCACA GAAAGCCTTACCACT GCTGCTGGGAGGCCT GGGACAGGAGAACAT GTTCCAGGTTCTGAG ATGCCTGTCCCAGAC 527 P V A P H I K V ES H I Q P S L N I E D I Y I T T E S L T T A A G R P G T G E H V P G S E M P V P D

901 TATACCTCCATTCAT ATAGTACAGTCCCCA CAGGGCCTCATACTC AATGCGACTGCCTTG CCCTTGCCTGACAAA GAGTTTCTCTCATCA TGTGGCTATGTGAGC ACAGACCAACTGAAC AAAATCATGCCTTAG CCTTTCTTTGGTTTC 577 Y T S I H I V Q S P Q G L I L N A T A L P L P D K E F L SS C G Y V S T D Q L NK I M P C

1051 CCAAGAGCTACGTAT TTAATAGCAAAGAAT TGACTGGGGCAATAA CGTTTAAGCCAAAAC AATGTTTAAACCTTT TTTGGGGGAGTGACA GGATGGGGTATGGAT TCTAAAATGCCTTTT CCCAAAATGTTGAAA TATGATGTTAAAAAA

1201 ATAAGAAGAATGCTT AATCAGATAGATATT CCTATTGTGCAATGT AAATATTTTAAAGAA TTGTGTCAGACTGTT TAGTAGCAGTGATTG TCTTAATATTGTGGG TGTTAATTTTTGATA CTAAGCATTGAATGA CTATGTTTTTAATGT

1351 ATAGTAAATCACGCT TTTTGAAAAAGCGAA AAAATCAGGTGGCTT TTGCGGTTCAGGAAA ATTGAATGCAAACCA TAGCACAGGCTAATT TTTTGTTGTTTCTTA AATAAGAAACTTTTT TATTTAAAAAACTAA AAACTAGAGGTGAGA ostI 1501 AATTTAAACTATAAG CAAGAAGGCAAAAAT AGTTTGGATATGTAA AACATTTATTTTGAC ATAAAGTTGATAAAG ATATTTTTTAATAAT TTAGACTTCAAGCAT GGCTATTTTATATTA CACTACACACTGTGT ACTGCAGTTGGTATG

1651 ACCCCTCTAAGGAGT GTAGCAACTACAGTC TAAAGCTGGTTTAAT GTTTTGGCCAATGCA CCTAAAGAAAAACAA ACTCGTTTTTTACAA AGCCCTTTTATACCT CCCCAGACTCCTTCA ACAATTCTAAAATGA TTGTAGTAATCTGCA

1801 TTATTGGAATATAAT TGTTTTATCTGAATT TTTAAACAAGTATTT GTTAATTTAGAAAAC TTTAAAGCGTTTGCA CAGATC about 1530 bp unsequenced . .

3411 CCTTCAAAGTTTAAT AAATTTATTTTCTTG GATTCCTGATAGTGT GCTTCTGTTATCAAA CACCAACATAAAAAT GATCTAAACCA FIG. 2. DNA sequence ofthe GH receptor genomic clones. Exons 2-10 are overlined, and the encoded protein sequence is shown. Asterisks indicate the extent of the exon-specific oligonucleotide probes. Nucleotides that differ from the cDNA sequence (6) are underlined. to exon 4 is absent. New bands not found in these and other pattern, but clearly much ofthe receptor gene is present. This control DNAs are observed for patient 147 (HindIII, 4.4 and result suggests that either the two patients contain a partial 1.8 kbp; EcoRI, 4.8 and 1.3 kbp) (Fig. 3). The two large-sized deletion in the GH receptor gene or, alternatively, they have HindIII bands for patient 147 (ofabout 20 and 11 kbp) appear an unusual polymorphism. to be a polymorphic pattern found in other control DNAs of In order to reduce the complexity of the analysis and to Israeli origin (data not shown). The integrity ofthe remaining examine individual exons, replicate blots containing DNA bands is difficult to judge because of the complex restriction from the two LTD patients were hybridized with synthetic Downloaded by guest on September 28, 2021 8086 Medical Sciences: Godowski et al. Proc. Natl. Acad. Sci. USA 86 (1989) Hind/I _ EcoRi with full-length or other exon-specific probes. Interestingly, 1 2 3 4 M Ml 2 3 4 an abnormal size band was found with the exon 4 probe in digests from both patients (Fig. 4). These results show that a exon kbp exon substantial portion of the GH receptor gene is missing from both alleles of these two indicate that more 23.123.1 patients yet than 4.5_ogo a simple deletion has occurred. Blots hybridized with an exon 7, go A 9.4 .4e 3+1 0 -. show that 8,9,10 2 6.6 4. 4 10-specific probe for both patients two EcoRI 2 bands of 11 and 16 are found not 4.4 kbp (data shown). The *. Ae29,10 larger "'- 6 4. band obscures the lack of the exon 5/6 EcoRI band for the 10-*.,-w 2.3 8 two patients (Fig. 3). 4s-P .2. 10 DISCUSSION

0.56 We have determined the exon structure of the human GH receptor gene, and, using this information, we have examined the receptor gene in nine LTD patients. The data show that two patients have a deletion in both receptor alleles that removes a large part of the coding region for the GH binding FIG. 3. Genomic blot hybridized with the full-lengtth GH receptor domain. With such a large deletion, we consider it very cDNA probe. Blots contained DNA from two nornnal individuals unlikely that the receptor would retain its normal function. (adult male, United States) (lanes 1 and 4) or DNA froim LTD patient Thus, these data provide direct evidence that LTD can result 147 (lanes 2) or patient D1 (lanes 3). The sizes of the marker DNAs from defects in the structural gene for the GH receptor. These (lanes M) are shown between the panels. Assignmeints of the GH findings also provide convincing genetic data showing that receptor exons to the HindIII or EcoRI bands are she)wn on the left the gene identified by GH binding (1, 5) and cloned as a and right of the panels, respectively. putative GH receptor (6) is, in fact, required for proper growth. probes specific for exons 2-7. A normal patteirn of hybrid- No obvious alterations were observed in the restriction ization was observed with the exon 2 and 7 prc)bes (Fig. 4). pattern of seven other LTD individuals, clearly demonstrat- DNAs from the two LTD patients failed to hybriidize with the ing that LTD is caused by a heterogeneity of gene defects. exon 3, 5, and 6 probes. The failure to detect hhybridization Based on the wide collection of gene defects found in other was not due to the lack of DNA in the LTD laLnes or to an inherited diseases (28-31), we expect that point mutations in artifact of the transfer since similar results vvere seen in the GH receptor gene also may account for many LTD cases. multiple experiments and since these same filtezrs produced The detection of these defects will require a more detailed normal hybridization signals when stripped and:rehybridized analysis than studies employing genomic blots as described here. In addition, mutations in other genes, such as those Exon 2 Exon 3 Exon 4 required for the expression ofthe receptor or for transduction Hind/i EcoRi of the GH also result in a similar or identical Hind/li EcoRi Hindi. I// EdoRl signal, might M 1 2 3 41 2 3 4M M 1 2 3 4 1 2 3 4 M 3 4 1 2 3 4 M phenotype. 1 One surprising finding is that noncontiguous exons have been deleted in the two LTD patients. Such a mutation could

IN. VW result from two independent deletion events or from a .. ~~~* complex deletion and rearrangement. A more detailed anal- 4..4 aw - 4w ysis of the genome structure of these A*_ ... ,- two patients may suggest which mutational event is more likely. Both patients :w are from families with consanguinity and, at the level of resolution determined here, are homozygous for the mutation. Although both patients are from the same ethnic background, their hybridization patterns with the full-length probe are not identical (Fig. 3), showing that their GH receptor genes do have polymorphic differences.

Exon 5 Exon 6 Exon 7 Assignment ofthe GH receptorexons to the multiple bands found on genomic blots (Fig. 3) shows that the complex Hind//i EcoRl Hind/l/ EcoRi Hindt/I/ EcoRi pattern observed results from the multiple exons of a single- M 2 1 34 12 3 4M M 1 2 3 4 1 2 3 4 M Ml1 2 copy gene rather than from a gene family. However, the many bands could obscure other cross-hybridizating genes,

qw and our data do not exclude the existence of other genes a* - -m 4w 4. 4.. closely related to the GH receptor. Under our hybridization 40 4. 4. 4 conditions, we do not detect the distantly related prolactin dw 4. qw 4w receptor gene (7). Ad a a a g When cDNA clones for the human and rabbit GH receptor were isolated, nearly all of these clones diverged 12 bp 5' of the initiating methionine codon (6). We speculated that these clones resulted from multiple splicing 5' of the coding region (6) and perhaps from transcription initiated from different promoters. The current sequence data show that the point of FIG. 4. Genomic blots hybridized with exon-sp4ecific probes. 5' divergence corresponds to the beginning of exon 2. Thus, Blots containing DNA from two normal individuals (laines 1 and 4) or there is a diverse splicing pattern in the 5' untranslated region LTD patient 147 (lanes 2) or patient D1 (lanes 3) were h3ybridized with of the GH receptor gene. Localization of these 5' exons and exon-specific probes as indicated. Marker DNA (lan(es M) is as in characterization of the promoter(s) and their possible tissue- Fig. 3. specific regulation will await further studies. Downloaded by guest on September 28, 2021 Medical Sciences: Godowski et al. Proc. Natl. Acad. Sci. USA 86 (1989) 8087 Human and rabbit GH receptor cDNA clones also have Kelly, P. A. (1988) Cell 53, 69-77. been isolated that diverge within the coding region (6). Most 8. Ymer, S. I. & Herington, A. C. (1985) Mol. Cell. Endocrinol. coincide with the 41, 153-161. (six in all) of these points of divergence 9. Herington, A. C., Ymer, S. & Steverson, J. (1986) J. Clin. exon boundaries determined here (data not shown). These Invest. 77, 1817-1823. clones represent a series ofunspliced or differentially spliced 10. Baumann, G., Stolar, M. W., Ambrun, K., Barsano, C. P. & mRNAs, including two clones, ghr.244 and ghr.438, that have DeVries, B. C. (1986) J. Clin. Endocrinol. Methods 62, 134- exon 3 or 4 missing, respectively. The biological significance 141. of these observations awaits further analysis. 11. Herington, A. C., Ymer, S., Roupas, P. & Steverson, J. (1986) receptor mRNAs found Biochim. Biophys. Acta 881, 236-240. Recently, the two prominent GH 12. Barnard, R. & Waters, M. J. (1986) Biochem. J. 237, 885-892. in mouse liver have been cloned (32). These mRNAs encode 13. Laron, Z., Pertzelan, A. & Mannheimer, S. (1966) Isr. J. Med. two forms of the mouse receptor, a high molecular weight, Sci. 2, 152-155. membrane-bound form and a low molecular weight form that 14. Laron, Z., Pertzelan, A. & Karp, M. (1968) Isr. J. Med. Sci. 4, diverges prior to the transmembrane domain and appears 883-894. likely to encode the secreted GH binding protein (33). The 15. Laron, Z. (1984) in Advances in Internal Medicine and Pedi- point ofdivergence ofthese two clones matches the exon 7/8 atrics, eds. Frick, H. P., Harnack, G. A., Kochsiek, K., Mar- boundary found here, supporting the proposal that alterna- tini, G. A. & Prader, A. (Springer, New York), pp. 117-150. receptor mRNA generates the two 16. McKusick, V. A. (1988) Mendelian Inheritance in Man (Johns tive splicing of the GH Hopkins Univ. Press, Baltimore), 8th Ed., pp. 1141-1142. receptor species (32). 17. Eshet, R., Laron, Z., Petzelan, A. & Dintzman, M. (1984) Isr. cDNA clones encoding the prolactin receptor also have J. Med. Sci. 20, 8-11. been isolated from rat, rabbit, and mouse libraries (7, 34, 35). 18. Daughaday, W. H. & Trivedi, B. (1987) Proc. Natl. Acad. Sci. Some of these clones encode long and short forms of the USA 84, 4636-4640. prolactin receptor. The points of divergence of these clones 19. Baumann, G., Shaw, M. A. & Winter, R. J. (1987) J. Clin. match the 9/10 exonjunction found here for the GH receptor. Endocrinol. Methods 65, 814-816. Thus, it would appear that the prolactin and GH receptor will 20. Laron, Z., Klinger, B., Erster, B. & Silbergeld, A. (1989) Acta have at least some similarity in their exon structures. This Endocrinol., in press. the two 21. Geffner, M. E., Golde, D. W., Lippe, B. M., Kaplan, S. A., coupled with an overall amino acid identity of Bersch, N. & Li, C. H. (1987) J. Clin. Endocrinol. Methods 64, receptors of about 25% shows that the two receptors have 1042-1046. evolved by duplication and divergence ofa common ancestral 22. Heath-Monnig, E., Wohltmann, H. J., Mills-Dunlap, B. & gene. Daughaday, W. H. (1987) J. Clin. Endocrinol. Metab. 64, 501-507. We thank Ellen Heath-Mannig for providing fibroblasts for the 23. Wood, W. I., Capon, D. J., Simonsen, C. C., Eaton, D. L., three U.S. LTD patients, Jeroen Knops for the initial analysis of Gitschier, J., Keyt, B., Seeburg, P. H., Smith, D. H., Holl- patient 1 DNA, and the Genentech organic synthesis group for ingshead, P., Wion, K. L., Delworth, E., Tuddenham, oligonucleotide probes. Z.L. is incumbent of the Irene and Nicholas E. G. D., Vehar, G. A. & Lawn, R. M. (1984) Nature (London) Marsh Chair for Pediatric Endocrinology and Diabetes at Tel Aviv 312, 330-337. University. L.R.M. was supported by a J. N. Goddard Research 24. Maniatis, T., Frisch, E. F. & Sambrook, J. (1982) Molecular Fellowship and by a National Institutes of Health Fellowship (5 T32 Cloning:A Laboratory Manual (Cold Spring Harbor Lab., Cold DKO 7298-09); J.P.G. was supported by a National Institutes of Spring Harbor, NY). Health Training Grant (DK 07120). This work was supported by 25. Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Natl. grants from the March of Dimes (Clinical Research Grant 6-454, Acad. Sci. USA 74, 5463-5467. J.S.P. and Z.L.), from the National Institutes of Health (R01 26. Vieira, J. & Messing, J. (1987) Methods Enzymol. 153, 3-11. HD24960, J.S.P.; and P01 HD20805, P.S.R.), and from the Emory 27. Barton, D. E., Foellmer, B. E., Wood, W. I. & Francke, U. Egleston Children's Research Center (J.S.P.) and by Genentech, Inc. (1989) Cytogenet. Cell Genet., in press. 28. Kazazian, H. H. & Boehm, C. D. (1988) Blood 72, 1107-1116. 1. Hughes, J. P. & Friesen, H. G. (1985) Annu. Rev. Physiol. 47, 29. Gitschier, J., Wood, W. I., Tuddenham, E. G. D., Shuman, 469-482. M. A., Goralka, T. M., Chen, E. Y. & Lawn, R. M. (1985) 2. Rechler, M. M., Nissley, S. P. & Roth, J. (1987) N. Engl. J. Nature (London) 315, 427-430. Med. 316, 941-943. 30. Phillips, J. A., Hjelle, B. L., Seeburg, P. H. & Zachmann, M. 3. Isaksson, 0. G. P., Eden, S. & Jamsson, J.-O. (1985) Annu. (1981) Proc. Natl. Acad. Sci. USA 78, 6372-6375. Rev. Physiol. 47, 483-499. 31. Laron, Z., Kelijman, M., Erster, B., Keret, R., Shoffner, J. M. 4. Posner, B. I., Kelly, P. A., Shiu, R. P. C. & Friesen, H. G. & Parks, J. S. (1985) Isr. J. Med. Sci. 21, 999-1006. (1974) Endocrinology 95, 521-531. 32. Smith, W. C., Kuniyoshi, J. & Talamantes, F. (1989) Mol. 5. Spencer, S. A., Hammonds, R. G., Henzel, W. J., Rodriguez, Endocrinol. 3, 984-990. H., Waters, M. J. & Wood, W. I. (1988) J. Biol. Chem. 263, 33. Smith, W. C., Linzer, D. I. H. & Talamantes, F. (1988) Proc. 7862-7867. Natl. Acad. Sci. USA 85, 9576-9579. 6. Leung, D. W., Spencer, S. A., Cachaines, G., Hammonds, 34. Edery, M., Jolicoeur, C., Levi-Meyrueis, C., Dusanter-Foust, R. G., Collins, C., Henzel, W. J., Barnard, R., Waters, M. J. I., Petridou, B., Boutin, J.-M., Lesueur, L., Kelly, P. A. & & Wood, W. I. (1987) Nature (London) 330, 537-543. Djiane, J. (1989) Proc. Natl. Acad. Sci. USA 86, 2112-2116. 7. Boutin, J.-M., Jolicoeur, C., Okamura, H., Gagnon, J., Edery, 35. Davis, J. A. & Linzer, D. I. H. (1989) Mol. Endocrinol. 3, M., Shirota, M., Banville, Dusanter-Fourt, I., Djiane, J. & 674-680. Downloaded by guest on September 28, 2021