0021-972x/92/7502-0437$03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright 8 1992 by The Endocrine Society
Vol. 75, No. 2 Printed in U.S.A.
Isolated Growth Hormone (GH) Deficiency Type IA Associated with a 45-Kilobase Gene Deletion within Human GH Gene Cluster* AYSEHAN AKINCI?, SAD1 VIDINLISAN,
CHRISTINA PRIMUS
AND
Department of Pediatrics, Inselspital Pediatrics, Dr. Sami Ulus Children’s Hospital (S. V.), Ankara, Turkey
KANAKA, E. MULLIS
ANDRfiE
EBLR,
NEJAT
AKAR,
(A.A., C.K., A.E., P.E.M.), Bern, Switzerland; and the Department Hospital (A.A.), Ankara University (N.A.); and Social Security
ABSTRACT
the
of
hGH-2 genes, were deleted. The end points of the deletion lay within two regions of highly homologous DNA sequence situated 5’ to the hGH-1 gene and 5’ to the hCS-B gene. The retention of only the hCSB gene was associated with normal weight and length at birth and normal postpartum lactation in the mother heterozygous for the deletion. The parents, who are consanguineous, both presented with a DNA restriction pattern consistent with heterozygosity for this deletion. (J Clin Endocrirwl Metab 75: 437-441,1992)
A Turkish family of seven individuals (two parents and five offspring) is described in which three children presented with isolated GH deficiency type IA, as defined by Illig et al. The gene deletion responsible for the isolated GH deficiency was characterized by Southern blotting and hybridization analysis of genomic DNA using a 32P-labeled hGH cDNA clone as a probe. In the affected patients, a total of approximately 45 kilobases of DNA, encompassing the human (h) GH1, human chorionic somatomammotropin-L (hCS-L), hCS-A, and
A
VARIETY of genetic and acquired interruptions in the hypothalamo-pituitary-peripheral axis may cause short stature associatedwith abnormalities of GH secretion. Phillips III (1) classified four familial types of isolated GH deficiency (IGHD) differing in their mode of inheritance, degreeof GH secretion after stimulation tests, or responsivenessto human recombinant GH (r-hGH) treatment. Type IA IGHD, first described by Illig et al. (2, 3), has an autosomal recessivemode of inheritance and presents with a homozygousdeletion of DNA containing the hGH-1 structural gene. Subjects with IGHD type IA are occasionally characterized by short stature at birth and hypoglycemia, but severegrowth retardation by the age of 6 months is a consistent finding. They have a strong initial anabolic responseto r-hGH treatment, frequently followed by the development of anti-hGH antibodies associated with immune intolerance to r-hGH replacement (2-6). The hGH gene locus expanded during evolution into a cluster of five highly sequence-conservedgenes [5’ hGH-1, human chorionic somatomammotropin-L (hCS-L), hCS-A, hGH-2, and hCS-B 3’1, which spans approximately 66.5 kilobases(kb) on the long arm of chromosome 17q22-24 (7). Although there is heterogeneity in the size of the deletion reported within this gene cluster, most (70-80%) of those
characterized are of 6.7 kb, and the remainder (20-30%) are of 7.6 kb (6). In both cases,the hGH-1 locus is deleted. Both of the deletions are believed to result from unequal recombinational events within the hGH gene cluster, leaving the remaining genesof the cluster intact (6). However, Goossens et al. (8) reported a deletion of 40-kb DNA due to two separate deletions flanking the hCS-L gene in two affected siblings presenting with the clinical features of IGHD type IA. While hGH-1 gene deletion causesdisease,deletion of placental genesmay only produce an abnormal biochemical phenotype (9, 10). The present paper describesa Turkish family with three affected boys presenting with a 45-kb gene deletion involving the hGH-1, hCS-L, hCS-A, and hGH-2 genes. Subjects Patients We studied five offspring. cousins in good The father (III-l) is 160 cm (-1.2
Patient
Received September 12, 1991. Address all correspondence and requests for reprints to: Primus E. Mullis, M.D., Department of Pediatrics, Inselspital, CH-3010 Bern, Switzerland. * This work was supported by the Swiss National Science Foundation (Grant 32-28580.90), Kabi-Pharmacia Sweden (to A.A.), and KabiPharmacia Switzerland (to C.K.). t Present address: Department of Pediatrics, Dr. Sami Ulus Children’s Hospital, Ankara, Turkey.
and Methods
a Turkish family of seven individuals, two parents and The pedigree is shown in Fig. 1. The parents are first health and with height and weight within normal limits. is 168 cm [-1 SD score (SDS)], and the mother (111-2) SDS).
IV-l
The first child, a boy, was born in 1975 at term after a normal pregnancy. The delivery and post- and perinatal course were uncomplicated. His birth weight was 2900 g (-1.2 SDS); there is no record on length at birth. Growth retardation became evident during the first year of life, and the boy was referred to the hospital at 2.5 yr of age for growth retardation, failure to thrive, and episodic hypoglycemia. Psychomotor development was normal. His height was 62 cm (-6.6 SDS), and his weight was 5.7 kg (-4.1 SDS). Renal, intestinal, or metabolic
437
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 November 2015. at 08:09 For personal use only. No other uses without permission. . All rights reserved.
AKINCI
438
reasons for failure to thrive were excluded. A computed tomographic scan of the head showed no abnormalities. GH was not detectable using a Tandem immunoradiometric kit (Hybritech) before and after stimulation by insulin, L-dopa, and GH-releasing hormone (11). All other pituitary functions were normal. After establishing the diagnosis of IGHD, there was no human GH (hGH) available in Turkey, and therefore, no replacement therapy could be given. However, treatment was started when he was 12 yr old. At that time his height was 82 cm (-7.3 SDS), and his weight was 12.3 kg (-3.8 SDS). His pretreatment height velocity was 2 cm/yr. He was given 12 W/m2 r-hGH (Somatonorm, Kabi-Pharmacia, Stockholm, Sweden), SC, in three injections a week. With an increase in his height velocity to 8 cm/yr, he responded well to the treatment. Unfortunately, he developed anti-hGH antibodies (64% maximum binding of [‘251]hGH), blunting the positive effect to the rhGH replacement. His height velocity dropped to 1 cm/yr, and the treatment was stopped when he was 13.5 yr old. His height was 91 cm (-8.6 SDS), and his weight was 14 kg (-4.0 SDS).
Subjects IV-2 and IV-3
weight, 6.5 kg (-4.7 SDS)], he was admitted to the hospital to confirm the diagnosis of IGHD as in his two other brothers. Three vears later, rhGH replacement was started. Pretreatment height velo&y increased from 3.1 to 6 cm/yr. He was given 12 lU/m Somatonorm (KabiPharmacia). Six months later, height velocity decreased to 2 cm/yr, and r-hGH treatment was stopped. A short period after the cessation of rhGH treatment, in 1989, the patient died at the age of 9 yr suffering from hypoglycemia and convulsions.
Methods DNA isolation. Genomic DNA was isolated from peripheral leucocytes of affected subjects and relatives, as previously described (13, 14). The concentration of each sample was determined by measuring the optical density of the purified DNA at 260 and 280 nm.
Restriction endonuclease diRestion and Southern
blotting analysis. Samples of DNA (5 rg) were digested to completion with the restriction enzymes BumHI, Hindlll, EcoRl, Hincll, Pstl, Pvull, Msal, Bell, Balll, or Ball. After ” electrophoresis in 0.5-1.2 (wt/vol) agarose gels, the DNA fragments were transferred onto nylon membranes (Hybond-N, Amersham lnternational, Aylesbury, Buckinghamshire, United Kingdom), and hybridized with the 32P-labeled hGH probe, as previously described (13, 14). The filters were then washed at 65 C and autoradiographed at -70 C using intensifying screens (13, 14).
IV-4
In 1978, the second boy was born. His weight was 4000 g (1.8 SDS) after a normal term pregnancy. At the age of 8 months, the mother, after having given birth to two unaffected daughters, suspected the same disorder as presented in his brother, and he was referred to the out-patient clinic at Dr. Sami Ulus Children’s Hospital in Ankara, Turkey. His length was 58 cm (-3.8 SDS), and his weight was 4.8 kg (-3.7 SDS). At the age of 2 yr, he was tested, and no GH was measurable after insulin, L-dopa, and GH-releasing hormone stimulation tests. Six years later, when he was 8 yr old [length, 79 cm (-8.0 SDS); weight, 9.5 kg (-4.2 SDS)], r-hGH treatment was given in a dose of 12 W/m’, divided into three injections/week. During the first 6 months of treatment, he responded well, with an increase in his height velocity from 2.2 to 14.0 cm/yr. After that period, his height velocity dropped again coincidentally with the production of anti-hGH antibodies (67% maximum binding of [iz51]hGH) that blunted the growth effect. The height velocity in the second year of treatment was 1 cm/yr, and r-hGH treatment was discontinued. At 10 yr of age, he was 88 cm tall (-8.2 SDS), and his weight was 11 kg (-4.0 SDS)
Patient
JCE & M. 1992 Vol75.No2
Y
In 1976 and 1977, two girls were born at term after normal pregnancies They developed without any problems and were normal for age and sex (12).
Patient
ET AL.
IV-5
The third boy was born Unfortunately, his notes are been 3000 g (-1 SDS). At became obvious, and at the
at term in 1980 after a missing, but his weight the age of 12 months, age of 3 8/12 yr [length,
normal pregnancy. is reported to have growth retardation 67 cm (-6.6 SDS);
I
II
III
FIG. 1. Pedigree of the children with IGHD type 1A. Squares represent males, and circles females. The affected individuals are indicated by solid symbols. The boy IV-5 died at 9 yr of age suffering from hypoglycemia. The parents (111-l and 111-2) and the girls (IV-2 and IV-3) were heterozygous for the deletion.
Results Autoradiography of Southern-blotted genomic DNA from the affected boy (IV-l) digested with BamHI and hybridized with the hGH cDNA probe showed only a single fragment of 6.6 kb (Fig. 2). This fragment (Fig. 3) carries the hCS-B gene and extends from nucleotides 50234-56802 of the hGH gene cluster (7). The other five normal BamHI fragments, of 8.4 kb (hCS-L), 5.3 kb (hCS-A), 3.8 kb (hGH-l), 3.0 kb (hGH-2), and 1.1 kb (hGH-2), were missing(Fig. 2). A similar result was obtained with DNA from the other affected boy (IV-4). The pattern of hybridization after BamHI digestion of genomic DNA from heterozygotes and normal homozygotes did not show any differences (Fig. 2). These data indicate that the deletion may map up-stream of nucleotide 50234 and removes all of the genesof the hGH gene cluster except the hCS-B gene (Fig. 3). To confirm the presenceor absenceof particular genesof the hGH gene cluster and to define the end points of the deletion, further restriction analyses of genomic DNA from the family and normal controls were performed. HincII digestsof the patients’ DNA contained only a 7.3-kb fragment (Fig. 2). Since the 6.6-kb BamHI fragment between positions 50,234 and 56,802 seemsto be intact (Figs. 2 and 3), the 3’end of the 7.3-kb HincII fragment must correspond to the HincII site at position 55,461. Furthermore, the next Hi&I site up-stream, at position 49,935, must be deleted (Fig. 3), as must all of the HincII sites between this site and the 5’end of the hGH-1 gene. The 5’-end of the 7.3-kb HincII fragment could, therefore, correspond to the HincII site at position 3,079 (Fig. 3) (7). The pattern of hybridization after HincII digestion of heterozygote DNA shown in Fig. 2 reveals the additional fragment of 7.3 kb derived from the deleted gene.The 7.7-, 6.6- and 5.4/5.5-kb fragments carry the hGH2 gene, the hGH-1 gene, and the hCS-L, hCS-A, and hCSB genes, respectively (Fig. 4) (7). After EcoRI digestion of patient’s DNA, a 2.9-kb fragment
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 November 2015. at 08:09 For personal use only. No other uses without permission. . All rights reserved.
IGHD
DUE TO A 45-kb GENE DELETION
n
Eacl -
FIG. 2. Autoradiograph of Southernblotted DNA from an affected boy (IV1; solid symbol), from the heterozygous father (III-l; hatched symbol), and from a normal male control (open symbol) after restriction endonuclease digestion with BamHI, HincII, and EcoRI and hybridization with the hGH cDNA clone probe. Fragment sizes in kilobases are indicated at the margins.
-
6.6
-
5.3
-
3.8
E
#I -
52692
&I-7300-# x------.6568-r 50234
7.7
: -
7.3 6.6
/ -
5.5 5.4
55461’
3.0 -
I
+*goo-
3079
/_
-
Hincll -
t
8.4
:. \
BzH
4671
439
I 56802
FIG. 3. Approximate location of the breakpoints for the deletion of DNA encompassing the hGH-1 (N), hCS-L (L), hCS-A (A), and hGH2 (V) genes are indicated. The restriction sites and the sizes of the BamHI (B; x)-, EcoRI (E; l )-, and HincII (H; #)-derived fragments are shown.
2.6
The restriction patterns obtained by digesting the DNA with BamHI, Hz&II, and EcoRI support the idea of a deletion of approximately 45 kb within the hGH gene cluster, from approximately position 4,900 to approximately position 50,000 (Fig. 3). The HincII/BclI double digest of genomic DNA from patients and parents confirmed this hypothesis, as shown in Figs. 4 and 5. As the pedigree shows (Fig. l), the gene deletion has an autosomal recessivemode of inheritance, and it is purely by chance that all affected individuals were males. Discussion
The clinical characteristicsof patients with IGHD type IA, asdescribed by Illig et al. (2), demonstrate short body length, early growth retardation, typical facies, and a strong anabolic response to an initial course of r-hGH treatment, which is rapidly followed by the production of anti-hGH antibodies,
A Hindll bp
FIG. 4. Restriction map of the hGH gene cluster. N, hGH-1 gene; L, hCS-L, A, hCS-A, V, hGH-2; B, hCS-B. Al, HincII restriction sites (0) and the sizes of the HincII-derived fragments containing the different genes are shown. A2, BclI restriction sites (*) and the sizes of the HincII/BclI-derived fragments are indicated. B, The deletion of DNA encompassing 45 kb described is shown. Cl, H&II (0) and C2, BclI (*) restriction sites after the deletion of the 45-kb DNA are shown, and the expected 7.3-kb band after HincII/BclI digestion is presented.
was seen (Fig. 2). The 3’-end of the 2.9-kb EcoRI fragment must correspond to the EcoRI site at position 52,692. The 5’end of this fragment could correspond to the EcoRI site at position 4,671 (Fig. 3) (7). The pattern of hybridization in heterozygotes and normal controls showed the expected three EcoRI-derived fragments of 9.4, 2.9, and 2.6 kb, carrying the hCS-L, hCS-A and hCS-B, and hGH-1 and hGH2 genes,respectively (Fig. 2) (7).
H&II @I
/
-
23130
-
9416
-
6557
-
4361
Lb 7.3 6.5
-
FIG. 5. Autoradiograph of Southern-blotted DNA from individuals III1, III-P, IV-l, and IV-4 after digestion with HincII/BclI and hybridization with the hGH cDNA clone probe. Fragment sizes in kilobases are shown on the left. X Hi&III marker fragments are shown on the right.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 November 2015. at 08:09 For personal use only. No other uses without permission. . All rights reserved.
440
AKINCI
leading to growth arrest (1). Restriction endonuclease analyses in families with IGHD type IA have shown that the affected patients present with a deletion of DNA encompassing the hGH-1 gene that encodes for hGH. The majority of deletions characterized are of 6.7 kb, and a smaller group is 7.6 kb in size (6). Vnencak-Jones et al. (6) localized the possible end points of the 6.7-kb deletions within two 1250-basepair (bp) regions that flank the hGH-1 gene and have a high sequence homology. The end points of the 7.6-kb deletions are located within a 29-bp perfect repeat sequences flanking the hGH-1 gene (6). Different families presenting with the same size gene deletion have different alleles of restriction fragment length polymorphisms within the hGH gene locus. This suggests that most deletions result from independent unequal recombination events (15). In addition, a double deletion in which the hGH-1 gene and the hCS-A, hGH-2, and hCS-B genes are lost (8) and deletions of the hCS-A gene alone and of the hCS-A, hGH-2, and hCS-B genes have been previously reported (9, 10, 16). Restriction endonuclease analysis in our Turkish family demonstrated a 45-kb gene deletion encompassing the hGH1, hCS-L, hCS-A, and hGH-2 genes. Based on restriction endonuclease mapping, we believe that the breakpoints are within approximately 300 bp 5’ to the hGH-1 gene (between .. a position down-stream from the EcoRI restriction site at position 4,671 and the beginning of the hGH-1 gene at position 5,163) and approximately 300 bp 5’ to the hCS-B gene (between the HincII restriction site at position 49,935 and the beginning of the hCS-B gene at position 50,234). These two regions are highly sequence conserved (7) and suggest an independent unequal recombination event causing the deletion. Four genes (hCS-L, hCS-A, hGH-2, and hCS-B) belonging to the hGH gene cluster are known to be expressed in the placenta (7,9), but the hCS-L protein has not been found in either the circulation or the placenta, whereas the hCS-A and hCS-B genes code for hCS (human placental lactogen), which is massively produced in fetal placental tissues (17). The hGH-2 gene encodes placental GH (pGH), which seems to be a potent pituitary GH analog (18, 19), and immunocytochemical localization indicates that both maternal and fetal cells in the placenta may produce the protein (20). It has been hypothesized that hCS and pGH are important for the third trimester increase and maintenance of insulin-like growth factor-I levels seen in normal pregnancy and may account for the disappearance of maternal circulating pituitary GH in the second half of pregnancy, which results from inhibition of somatotroph secretion by increasing concentrations of IGF-I (21). In the family that we studied, three and two copies of the normal diploid complement of the hCS genes (hCS-A and hCS-B) are present in the mother and the affected patients, respectively. A direct relationship between the fetal hCS gene dosage and the maternal hCS concentration has been found (9), and the reported cases of complete or partial hCS deficiency have so far been associated with normal fetal and postnatal growth and no breastfeeding problems in the moth-
ET AL.
JCE & M. 1992 Vol75.No2
ers (9, 10, 16, 22). In contrast, Orlando et al. (23) reported that 4.5% of the alleles in a cohort of Spanish patients with IGHD presented with a deleted hCS-A gene. In our three patients, however, retention of the hCS-B gene alone was compatible with normal size and weight at birth. This can be interpreted in several ways. First, hormones other than hCS and pGH could be more important factors in fetal growth. Second, although Barrera-Saldana (17, 24) reported that there are roughly equivalent levels of hCS-A mRNA and hCS-B mRNA in normal human term placenta, the expression of hCS-B may exceed the expression of hCS-A in some patients, suggesting that some of the hormones of the hCS and hGH families must be able to substitute for those missing. In addition, it is intriguing to speculate that some deletions might bring the 5’-promoter region of the deleted gene to a position preceding the transcription initiation site of another gene, which may then substitute for the missing one (25). To summarize, we describe a Turkish family with a 45-kb gene deletion within the hGH gene cluster, encompassing the hGH-1, hCS-L, hCS-A, and hGH-2 genes. There was normal fetal growth, associated with normal birth weight and length, in three affected boys and normal postpartum lactation in the mother heterozygous for the deletion. Acknowledgment The authors want to thank Dr. P. M. Brickell (Department of Biochemistry and Molecular Biology, University College and Middlesex School of Medicine, London, United Kingdom) for helpful discussions.
References 1. Phillips III JA. 1983 The growth hormone (hGH) genes and human disease. In: Caskey CT, White R (eds) Banbury report 14. Recombinant DNA: application to human disease. Cold Spring _ - Harbor: Cold Spring Harbor Laboratory; 305-15. 2. Illie R, Prader A, Ferrandez Zachmann M. 1971 Hereditarv m-enati growth hormone deficiency with increased tendency to gro&wth hormone antibody formation (“A-type” of isolated growth hormone deficiency). Acta Pediatr Stand. 60:607. 3. Phillips III JA, Hjelle BL, Seeburg PH, Zachmann M. 1981 Molecular basis for familial isolated growth hormone deficiency. Proc Nat1 Acad Sci USA. 78:6372-5. 4. Laron Z, Kelijman M, Pertzelan A, Keret R, Shoffner IM, Parks JS. 1985 Human growth hormone gene deletion without antibody formation or growth arrest during treatment-a new disease entity? Isr J Med Sci. 21:999-1006. 5. Braga S, Phillips III JA, Joss E, Schwarz H, Zuppinger K. 1986 Familial growth hormone deficiency resulting from a 7.6 kb deletion within the growth hormone gene cluster. Am J Med Genet. 25:44352. 6. Vnencak-Jones CL, Phillips III JA, Chen EY, Seeburg PH. 1988 Molecular basis of human growth hormone gene deletions. Proc Nat1 Acad Sci USA. 85:5615-9. 7. Chen EY, Liao YC, Smith DH, Barrera-Saldana HA, Gelinas RE, Seeburg PH. 1989 The human growth hormone locus: nucleotide sequence, biology, and evolution. Genomics. 4:479-97. 8. Goossens M, Brauner R, Czernichow P, Duquesnoy P, Rappaport R. 1986 Isolated growth hormone (GH) deficiency type IA associated with a double deletion in the human GH gene cluster. J Clin Endocrinol Metab. 62:712-6. 9. Parks JS, Nielson PV, Sexton LA, Jorgenson EH. 1985 An effect of gene dosage on production of human chorionic somatomammo-
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 November 2015. at 08:09 For personal use only. No other uses without permission. . All rights reserved.
IGHD
DUE
TO A 45-kb
tropin. J Clin Endocrinol Metab. 60:994-7. 10. Simon P. Decoster C. Brocas H, Schwers I, Vassart G. 1986 Absence ‘of human chorionic somatomammob-opin during pregnancy associated with two types of gene deletion. Hum Genet. 74:235-a. 11. Blethan SL, Chaslow FI. 1983 Use of a two-site immunoradiometric assav for growth hormone (GH) in indentifying children with GH dependenrgrowth failure. J.Clin Endocrinol-Metab. 57:1031-5. H, Oleun P. 1978 Turk Cocuklarinda biiviime ve 12. Nevzi 0. Gun& gel&me normlari 1, Tartyve boy degerleri. 1st Tip Fak Met. il(ek74). 13. Mullis P, Pate1 M, Brickell PM, Brook CGD. 1990 Isolated growth hormone deficiency: analysis of the growth hormone (GH) releasing hormone gene and the GH gene cluster. J Clin Endocrinol Metab. 70:187-91. PE, Pate1 MS, Brickell PM, Brook CGD. 1991 Constitution14 Mullis ally short stature: analysis of the insulin-like growth factor-I gene and the human growth hormone gene cluster. Pediatr Res. 29:4125. III JA, Ferrandez A, Frisch H, Illig R, Zuppinger K. 1986 15 Phillips Defects of GH genes. In: Raiti S, ed. Clinical syndromes in human growth hormone. New York: Plenum Press; 211-26. J, Parks JS, Herd HE, Nielson PV. 1982 A gene deletion is 16 Wurzel responsible for absence of human chorionic somatomammotropin. DNA. 1:251-7. 17 -, Barrera-Saldana HA, Seeburg PH, Saunders GF. 1983 Two structurally different genes produce the same human placental lactogen hormone. J Biol Chem. 258:3787-93. 18. Frankenne F, Closset J, Gomez F, Scippo ML, Smal J, Hennen G.
GENE
19.
20.
21.
22.
23.
24.
25.
DELETION
1988 The physiology of growth hormone (GHs) in pregnant women and partial characterization of the placental GH variant. J Clin Endocrinol Metab. 66:1171-80. Frankenne F, Scippo ML, Van Beeumen J, Igout A, Hennen G. 1990 Identification of placental human growth hormone as the growth hormone-V gene expression product. J Clin Endocrinol Metab. 71:15-8. Jara CS, Salud AT, Bryant-Greenwood GD, Pirens G, Hennen G, Frankenne F. 1989 Immunocytochemical localization of the human growth hormone variant in the human placenta. J Clin Endocrinol Metab. 69:1069-72. Wilson DM, Benett A, Adamson GD, et al. 1982 Somatomedins in pregnancy: a cross-sectional study on insulin-like growth factors I and II and somatomedin peptide content in normal pregnancies. J Clin Endocrinol Metab. 55:858-61. Hennen G, Frankenne F. 1987 Influence des hormones proteiques placentaires sur la physiologie maternelle. Ann Endocrinol (Paris). 48:278-88. Orlando PI, Phillips III JA, Ferrandez AN, Arnal TM, Woodard MJ, BuondM. 1987 Frequency and types of deletions-in the growth hormone (GH) gene clusters of Spanish subjects with GH deficiency. Am J Hum Genet. 41:A105. Fitzuatrick SL. Premont R, Barrera-Saldana HA, Saunders GF. 1985 Expression of two human placental lactogen genes in placentas from several individuals [Abstract]. Proc of the 65th Annual Meet of The Endocrine Sot. 63 1. Nickel BE, Kardami E, Cattini PA. 1990 Differential expression of human placental growth hormone variant and chorionic somatomammotropin in culture. Biochem J. 267:653-8.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 November 2015. at 08:09 For personal use only. No other uses without permission. . All rights reserved.
Vol. 75, No. 2 Printed in U.S.A.
Isolated Growth Hormone (GH) Deficiency Type IA Associated with a 45-Kilobase Gene Deletion within Human GH Gene Cluster* AYSEHAN AKINCI?, SAD1 VIDINLISAN,
CHRISTINA PRIMUS
AND
Department of Pediatrics, Inselspital Pediatrics, Dr. Sami Ulus Children’s Hospital (S. V.), Ankara, Turkey
KANAKA, E. MULLIS
ANDRfiE
EBLR,
NEJAT
AKAR,
(A.A., C.K., A.E., P.E.M.), Bern, Switzerland; and the Department Hospital (A.A.), Ankara University (N.A.); and Social Security
ABSTRACT
the
of
hGH-2 genes, were deleted. The end points of the deletion lay within two regions of highly homologous DNA sequence situated 5’ to the hGH-1 gene and 5’ to the hCS-B gene. The retention of only the hCSB gene was associated with normal weight and length at birth and normal postpartum lactation in the mother heterozygous for the deletion. The parents, who are consanguineous, both presented with a DNA restriction pattern consistent with heterozygosity for this deletion. (J Clin Endocrirwl Metab 75: 437-441,1992)
A Turkish family of seven individuals (two parents and five offspring) is described in which three children presented with isolated GH deficiency type IA, as defined by Illig et al. The gene deletion responsible for the isolated GH deficiency was characterized by Southern blotting and hybridization analysis of genomic DNA using a 32P-labeled hGH cDNA clone as a probe. In the affected patients, a total of approximately 45 kilobases of DNA, encompassing the human (h) GH1, human chorionic somatomammotropin-L (hCS-L), hCS-A, and
A
VARIETY of genetic and acquired interruptions in the hypothalamo-pituitary-peripheral axis may cause short stature associatedwith abnormalities of GH secretion. Phillips III (1) classified four familial types of isolated GH deficiency (IGHD) differing in their mode of inheritance, degreeof GH secretion after stimulation tests, or responsivenessto human recombinant GH (r-hGH) treatment. Type IA IGHD, first described by Illig et al. (2, 3), has an autosomal recessivemode of inheritance and presents with a homozygousdeletion of DNA containing the hGH-1 structural gene. Subjects with IGHD type IA are occasionally characterized by short stature at birth and hypoglycemia, but severegrowth retardation by the age of 6 months is a consistent finding. They have a strong initial anabolic responseto r-hGH treatment, frequently followed by the development of anti-hGH antibodies associated with immune intolerance to r-hGH replacement (2-6). The hGH gene locus expanded during evolution into a cluster of five highly sequence-conservedgenes [5’ hGH-1, human chorionic somatomammotropin-L (hCS-L), hCS-A, hGH-2, and hCS-B 3’1, which spans approximately 66.5 kilobases(kb) on the long arm of chromosome 17q22-24 (7). Although there is heterogeneity in the size of the deletion reported within this gene cluster, most (70-80%) of those
characterized are of 6.7 kb, and the remainder (20-30%) are of 7.6 kb (6). In both cases,the hGH-1 locus is deleted. Both of the deletions are believed to result from unequal recombinational events within the hGH gene cluster, leaving the remaining genesof the cluster intact (6). However, Goossens et al. (8) reported a deletion of 40-kb DNA due to two separate deletions flanking the hCS-L gene in two affected siblings presenting with the clinical features of IGHD type IA. While hGH-1 gene deletion causesdisease,deletion of placental genesmay only produce an abnormal biochemical phenotype (9, 10). The present paper describesa Turkish family with three affected boys presenting with a 45-kb gene deletion involving the hGH-1, hCS-L, hCS-A, and hGH-2 genes. Subjects Patients We studied five offspring. cousins in good The father (III-l) is 160 cm (-1.2
Patient
Received September 12, 1991. Address all correspondence and requests for reprints to: Primus E. Mullis, M.D., Department of Pediatrics, Inselspital, CH-3010 Bern, Switzerland. * This work was supported by the Swiss National Science Foundation (Grant 32-28580.90), Kabi-Pharmacia Sweden (to A.A.), and KabiPharmacia Switzerland (to C.K.). t Present address: Department of Pediatrics, Dr. Sami Ulus Children’s Hospital, Ankara, Turkey.
and Methods
a Turkish family of seven individuals, two parents and The pedigree is shown in Fig. 1. The parents are first health and with height and weight within normal limits. is 168 cm [-1 SD score (SDS)], and the mother (111-2) SDS).
IV-l
The first child, a boy, was born in 1975 at term after a normal pregnancy. The delivery and post- and perinatal course were uncomplicated. His birth weight was 2900 g (-1.2 SDS); there is no record on length at birth. Growth retardation became evident during the first year of life, and the boy was referred to the hospital at 2.5 yr of age for growth retardation, failure to thrive, and episodic hypoglycemia. Psychomotor development was normal. His height was 62 cm (-6.6 SDS), and his weight was 5.7 kg (-4.1 SDS). Renal, intestinal, or metabolic
437
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 November 2015. at 08:09 For personal use only. No other uses without permission. . All rights reserved.
AKINCI
438
reasons for failure to thrive were excluded. A computed tomographic scan of the head showed no abnormalities. GH was not detectable using a Tandem immunoradiometric kit (Hybritech) before and after stimulation by insulin, L-dopa, and GH-releasing hormone (11). All other pituitary functions were normal. After establishing the diagnosis of IGHD, there was no human GH (hGH) available in Turkey, and therefore, no replacement therapy could be given. However, treatment was started when he was 12 yr old. At that time his height was 82 cm (-7.3 SDS), and his weight was 12.3 kg (-3.8 SDS). His pretreatment height velocity was 2 cm/yr. He was given 12 W/m2 r-hGH (Somatonorm, Kabi-Pharmacia, Stockholm, Sweden), SC, in three injections a week. With an increase in his height velocity to 8 cm/yr, he responded well to the treatment. Unfortunately, he developed anti-hGH antibodies (64% maximum binding of [‘251]hGH), blunting the positive effect to the rhGH replacement. His height velocity dropped to 1 cm/yr, and the treatment was stopped when he was 13.5 yr old. His height was 91 cm (-8.6 SDS), and his weight was 14 kg (-4.0 SDS).
Subjects IV-2 and IV-3
weight, 6.5 kg (-4.7 SDS)], he was admitted to the hospital to confirm the diagnosis of IGHD as in his two other brothers. Three vears later, rhGH replacement was started. Pretreatment height velo&y increased from 3.1 to 6 cm/yr. He was given 12 lU/m Somatonorm (KabiPharmacia). Six months later, height velocity decreased to 2 cm/yr, and r-hGH treatment was stopped. A short period after the cessation of rhGH treatment, in 1989, the patient died at the age of 9 yr suffering from hypoglycemia and convulsions.
Methods DNA isolation. Genomic DNA was isolated from peripheral leucocytes of affected subjects and relatives, as previously described (13, 14). The concentration of each sample was determined by measuring the optical density of the purified DNA at 260 and 280 nm.
Restriction endonuclease diRestion and Southern
blotting analysis. Samples of DNA (5 rg) were digested to completion with the restriction enzymes BumHI, Hindlll, EcoRl, Hincll, Pstl, Pvull, Msal, Bell, Balll, or Ball. After ” electrophoresis in 0.5-1.2 (wt/vol) agarose gels, the DNA fragments were transferred onto nylon membranes (Hybond-N, Amersham lnternational, Aylesbury, Buckinghamshire, United Kingdom), and hybridized with the 32P-labeled hGH probe, as previously described (13, 14). The filters were then washed at 65 C and autoradiographed at -70 C using intensifying screens (13, 14).
IV-4
In 1978, the second boy was born. His weight was 4000 g (1.8 SDS) after a normal term pregnancy. At the age of 8 months, the mother, after having given birth to two unaffected daughters, suspected the same disorder as presented in his brother, and he was referred to the out-patient clinic at Dr. Sami Ulus Children’s Hospital in Ankara, Turkey. His length was 58 cm (-3.8 SDS), and his weight was 4.8 kg (-3.7 SDS). At the age of 2 yr, he was tested, and no GH was measurable after insulin, L-dopa, and GH-releasing hormone stimulation tests. Six years later, when he was 8 yr old [length, 79 cm (-8.0 SDS); weight, 9.5 kg (-4.2 SDS)], r-hGH treatment was given in a dose of 12 W/m’, divided into three injections/week. During the first 6 months of treatment, he responded well, with an increase in his height velocity from 2.2 to 14.0 cm/yr. After that period, his height velocity dropped again coincidentally with the production of anti-hGH antibodies (67% maximum binding of [iz51]hGH) that blunted the growth effect. The height velocity in the second year of treatment was 1 cm/yr, and r-hGH treatment was discontinued. At 10 yr of age, he was 88 cm tall (-8.2 SDS), and his weight was 11 kg (-4.0 SDS)
Patient
JCE & M. 1992 Vol75.No2
Y
In 1976 and 1977, two girls were born at term after normal pregnancies They developed without any problems and were normal for age and sex (12).
Patient
ET AL.
IV-5
The third boy was born Unfortunately, his notes are been 3000 g (-1 SDS). At became obvious, and at the
at term in 1980 after a missing, but his weight the age of 12 months, age of 3 8/12 yr [length,
normal pregnancy. is reported to have growth retardation 67 cm (-6.6 SDS);
I
II
III
FIG. 1. Pedigree of the children with IGHD type 1A. Squares represent males, and circles females. The affected individuals are indicated by solid symbols. The boy IV-5 died at 9 yr of age suffering from hypoglycemia. The parents (111-l and 111-2) and the girls (IV-2 and IV-3) were heterozygous for the deletion.
Results Autoradiography of Southern-blotted genomic DNA from the affected boy (IV-l) digested with BamHI and hybridized with the hGH cDNA probe showed only a single fragment of 6.6 kb (Fig. 2). This fragment (Fig. 3) carries the hCS-B gene and extends from nucleotides 50234-56802 of the hGH gene cluster (7). The other five normal BamHI fragments, of 8.4 kb (hCS-L), 5.3 kb (hCS-A), 3.8 kb (hGH-l), 3.0 kb (hGH-2), and 1.1 kb (hGH-2), were missing(Fig. 2). A similar result was obtained with DNA from the other affected boy (IV-4). The pattern of hybridization after BamHI digestion of genomic DNA from heterozygotes and normal homozygotes did not show any differences (Fig. 2). These data indicate that the deletion may map up-stream of nucleotide 50234 and removes all of the genesof the hGH gene cluster except the hCS-B gene (Fig. 3). To confirm the presenceor absenceof particular genesof the hGH gene cluster and to define the end points of the deletion, further restriction analyses of genomic DNA from the family and normal controls were performed. HincII digestsof the patients’ DNA contained only a 7.3-kb fragment (Fig. 2). Since the 6.6-kb BamHI fragment between positions 50,234 and 56,802 seemsto be intact (Figs. 2 and 3), the 3’end of the 7.3-kb HincII fragment must correspond to the HincII site at position 55,461. Furthermore, the next Hi&I site up-stream, at position 49,935, must be deleted (Fig. 3), as must all of the HincII sites between this site and the 5’end of the hGH-1 gene. The 5’-end of the 7.3-kb HincII fragment could, therefore, correspond to the HincII site at position 3,079 (Fig. 3) (7). The pattern of hybridization after HincII digestion of heterozygote DNA shown in Fig. 2 reveals the additional fragment of 7.3 kb derived from the deleted gene.The 7.7-, 6.6- and 5.4/5.5-kb fragments carry the hGH2 gene, the hGH-1 gene, and the hCS-L, hCS-A, and hCSB genes, respectively (Fig. 4) (7). After EcoRI digestion of patient’s DNA, a 2.9-kb fragment
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 November 2015. at 08:09 For personal use only. No other uses without permission. . All rights reserved.
IGHD
DUE TO A 45-kb GENE DELETION
n
Eacl -
FIG. 2. Autoradiograph of Southernblotted DNA from an affected boy (IV1; solid symbol), from the heterozygous father (III-l; hatched symbol), and from a normal male control (open symbol) after restriction endonuclease digestion with BamHI, HincII, and EcoRI and hybridization with the hGH cDNA clone probe. Fragment sizes in kilobases are indicated at the margins.
-
6.6
-
5.3
-
3.8
E
#I -
52692
&I-7300-# x------.6568-r 50234
7.7
: -
7.3 6.6
/ -
5.5 5.4
55461’
3.0 -
I
+*goo-
3079
/_
-
Hincll -
t
8.4
:. \
BzH
4671
439
I 56802
FIG. 3. Approximate location of the breakpoints for the deletion of DNA encompassing the hGH-1 (N), hCS-L (L), hCS-A (A), and hGH2 (V) genes are indicated. The restriction sites and the sizes of the BamHI (B; x)-, EcoRI (E; l )-, and HincII (H; #)-derived fragments are shown.
2.6
The restriction patterns obtained by digesting the DNA with BamHI, Hz&II, and EcoRI support the idea of a deletion of approximately 45 kb within the hGH gene cluster, from approximately position 4,900 to approximately position 50,000 (Fig. 3). The HincII/BclI double digest of genomic DNA from patients and parents confirmed this hypothesis, as shown in Figs. 4 and 5. As the pedigree shows (Fig. l), the gene deletion has an autosomal recessivemode of inheritance, and it is purely by chance that all affected individuals were males. Discussion
The clinical characteristicsof patients with IGHD type IA, asdescribed by Illig et al. (2), demonstrate short body length, early growth retardation, typical facies, and a strong anabolic response to an initial course of r-hGH treatment, which is rapidly followed by the production of anti-hGH antibodies,
A Hindll bp
FIG. 4. Restriction map of the hGH gene cluster. N, hGH-1 gene; L, hCS-L, A, hCS-A, V, hGH-2; B, hCS-B. Al, HincII restriction sites (0) and the sizes of the HincII-derived fragments containing the different genes are shown. A2, BclI restriction sites (*) and the sizes of the HincII/BclI-derived fragments are indicated. B, The deletion of DNA encompassing 45 kb described is shown. Cl, H&II (0) and C2, BclI (*) restriction sites after the deletion of the 45-kb DNA are shown, and the expected 7.3-kb band after HincII/BclI digestion is presented.
was seen (Fig. 2). The 3’-end of the 2.9-kb EcoRI fragment must correspond to the EcoRI site at position 52,692. The 5’end of this fragment could correspond to the EcoRI site at position 4,671 (Fig. 3) (7). The pattern of hybridization in heterozygotes and normal controls showed the expected three EcoRI-derived fragments of 9.4, 2.9, and 2.6 kb, carrying the hCS-L, hCS-A and hCS-B, and hGH-1 and hGH2 genes,respectively (Fig. 2) (7).
H&II @I
/
-
23130
-
9416
-
6557
-
4361
Lb 7.3 6.5
-
FIG. 5. Autoradiograph of Southern-blotted DNA from individuals III1, III-P, IV-l, and IV-4 after digestion with HincII/BclI and hybridization with the hGH cDNA clone probe. Fragment sizes in kilobases are shown on the left. X Hi&III marker fragments are shown on the right.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 November 2015. at 08:09 For personal use only. No other uses without permission. . All rights reserved.
440
AKINCI
leading to growth arrest (1). Restriction endonuclease analyses in families with IGHD type IA have shown that the affected patients present with a deletion of DNA encompassing the hGH-1 gene that encodes for hGH. The majority of deletions characterized are of 6.7 kb, and a smaller group is 7.6 kb in size (6). Vnencak-Jones et al. (6) localized the possible end points of the 6.7-kb deletions within two 1250-basepair (bp) regions that flank the hGH-1 gene and have a high sequence homology. The end points of the 7.6-kb deletions are located within a 29-bp perfect repeat sequences flanking the hGH-1 gene (6). Different families presenting with the same size gene deletion have different alleles of restriction fragment length polymorphisms within the hGH gene locus. This suggests that most deletions result from independent unequal recombination events (15). In addition, a double deletion in which the hGH-1 gene and the hCS-A, hGH-2, and hCS-B genes are lost (8) and deletions of the hCS-A gene alone and of the hCS-A, hGH-2, and hCS-B genes have been previously reported (9, 10, 16). Restriction endonuclease analysis in our Turkish family demonstrated a 45-kb gene deletion encompassing the hGH1, hCS-L, hCS-A, and hGH-2 genes. Based on restriction endonuclease mapping, we believe that the breakpoints are within approximately 300 bp 5’ to the hGH-1 gene (between .. a position down-stream from the EcoRI restriction site at position 4,671 and the beginning of the hGH-1 gene at position 5,163) and approximately 300 bp 5’ to the hCS-B gene (between the HincII restriction site at position 49,935 and the beginning of the hCS-B gene at position 50,234). These two regions are highly sequence conserved (7) and suggest an independent unequal recombination event causing the deletion. Four genes (hCS-L, hCS-A, hGH-2, and hCS-B) belonging to the hGH gene cluster are known to be expressed in the placenta (7,9), but the hCS-L protein has not been found in either the circulation or the placenta, whereas the hCS-A and hCS-B genes code for hCS (human placental lactogen), which is massively produced in fetal placental tissues (17). The hGH-2 gene encodes placental GH (pGH), which seems to be a potent pituitary GH analog (18, 19), and immunocytochemical localization indicates that both maternal and fetal cells in the placenta may produce the protein (20). It has been hypothesized that hCS and pGH are important for the third trimester increase and maintenance of insulin-like growth factor-I levels seen in normal pregnancy and may account for the disappearance of maternal circulating pituitary GH in the second half of pregnancy, which results from inhibition of somatotroph secretion by increasing concentrations of IGF-I (21). In the family that we studied, three and two copies of the normal diploid complement of the hCS genes (hCS-A and hCS-B) are present in the mother and the affected patients, respectively. A direct relationship between the fetal hCS gene dosage and the maternal hCS concentration has been found (9), and the reported cases of complete or partial hCS deficiency have so far been associated with normal fetal and postnatal growth and no breastfeeding problems in the moth-
ET AL.
JCE & M. 1992 Vol75.No2
ers (9, 10, 16, 22). In contrast, Orlando et al. (23) reported that 4.5% of the alleles in a cohort of Spanish patients with IGHD presented with a deleted hCS-A gene. In our three patients, however, retention of the hCS-B gene alone was compatible with normal size and weight at birth. This can be interpreted in several ways. First, hormones other than hCS and pGH could be more important factors in fetal growth. Second, although Barrera-Saldana (17, 24) reported that there are roughly equivalent levels of hCS-A mRNA and hCS-B mRNA in normal human term placenta, the expression of hCS-B may exceed the expression of hCS-A in some patients, suggesting that some of the hormones of the hCS and hGH families must be able to substitute for those missing. In addition, it is intriguing to speculate that some deletions might bring the 5’-promoter region of the deleted gene to a position preceding the transcription initiation site of another gene, which may then substitute for the missing one (25). To summarize, we describe a Turkish family with a 45-kb gene deletion within the hGH gene cluster, encompassing the hGH-1, hCS-L, hCS-A, and hGH-2 genes. There was normal fetal growth, associated with normal birth weight and length, in three affected boys and normal postpartum lactation in the mother heterozygous for the deletion. Acknowledgment The authors want to thank Dr. P. M. Brickell (Department of Biochemistry and Molecular Biology, University College and Middlesex School of Medicine, London, United Kingdom) for helpful discussions.
References 1. Phillips III JA. 1983 The growth hormone (hGH) genes and human disease. In: Caskey CT, White R (eds) Banbury report 14. Recombinant DNA: application to human disease. Cold Spring _ - Harbor: Cold Spring Harbor Laboratory; 305-15. 2. Illie R, Prader A, Ferrandez Zachmann M. 1971 Hereditarv m-enati growth hormone deficiency with increased tendency to gro&wth hormone antibody formation (“A-type” of isolated growth hormone deficiency). Acta Pediatr Stand. 60:607. 3. Phillips III JA, Hjelle BL, Seeburg PH, Zachmann M. 1981 Molecular basis for familial isolated growth hormone deficiency. Proc Nat1 Acad Sci USA. 78:6372-5. 4. Laron Z, Kelijman M, Pertzelan A, Keret R, Shoffner IM, Parks JS. 1985 Human growth hormone gene deletion without antibody formation or growth arrest during treatment-a new disease entity? Isr J Med Sci. 21:999-1006. 5. Braga S, Phillips III JA, Joss E, Schwarz H, Zuppinger K. 1986 Familial growth hormone deficiency resulting from a 7.6 kb deletion within the growth hormone gene cluster. Am J Med Genet. 25:44352. 6. Vnencak-Jones CL, Phillips III JA, Chen EY, Seeburg PH. 1988 Molecular basis of human growth hormone gene deletions. Proc Nat1 Acad Sci USA. 85:5615-9. 7. Chen EY, Liao YC, Smith DH, Barrera-Saldana HA, Gelinas RE, Seeburg PH. 1989 The human growth hormone locus: nucleotide sequence, biology, and evolution. Genomics. 4:479-97. 8. Goossens M, Brauner R, Czernichow P, Duquesnoy P, Rappaport R. 1986 Isolated growth hormone (GH) deficiency type IA associated with a double deletion in the human GH gene cluster. J Clin Endocrinol Metab. 62:712-6. 9. Parks JS, Nielson PV, Sexton LA, Jorgenson EH. 1985 An effect of gene dosage on production of human chorionic somatomammo-
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 November 2015. at 08:09 For personal use only. No other uses without permission. . All rights reserved.
IGHD
DUE
TO A 45-kb
tropin. J Clin Endocrinol Metab. 60:994-7. 10. Simon P. Decoster C. Brocas H, Schwers I, Vassart G. 1986 Absence ‘of human chorionic somatomammob-opin during pregnancy associated with two types of gene deletion. Hum Genet. 74:235-a. 11. Blethan SL, Chaslow FI. 1983 Use of a two-site immunoradiometric assav for growth hormone (GH) in indentifying children with GH dependenrgrowth failure. J.Clin Endocrinol-Metab. 57:1031-5. H, Oleun P. 1978 Turk Cocuklarinda biiviime ve 12. Nevzi 0. Gun& gel&me normlari 1, Tartyve boy degerleri. 1st Tip Fak Met. il(ek74). 13. Mullis P, Pate1 M, Brickell PM, Brook CGD. 1990 Isolated growth hormone deficiency: analysis of the growth hormone (GH) releasing hormone gene and the GH gene cluster. J Clin Endocrinol Metab. 70:187-91. PE, Pate1 MS, Brickell PM, Brook CGD. 1991 Constitution14 Mullis ally short stature: analysis of the insulin-like growth factor-I gene and the human growth hormone gene cluster. Pediatr Res. 29:4125. III JA, Ferrandez A, Frisch H, Illig R, Zuppinger K. 1986 15 Phillips Defects of GH genes. In: Raiti S, ed. Clinical syndromes in human growth hormone. New York: Plenum Press; 211-26. J, Parks JS, Herd HE, Nielson PV. 1982 A gene deletion is 16 Wurzel responsible for absence of human chorionic somatomammotropin. DNA. 1:251-7. 17 -, Barrera-Saldana HA, Seeburg PH, Saunders GF. 1983 Two structurally different genes produce the same human placental lactogen hormone. J Biol Chem. 258:3787-93. 18. Frankenne F, Closset J, Gomez F, Scippo ML, Smal J, Hennen G.
GENE
19.
20.
21.
22.
23.
24.
25.
DELETION
1988 The physiology of growth hormone (GHs) in pregnant women and partial characterization of the placental GH variant. J Clin Endocrinol Metab. 66:1171-80. Frankenne F, Scippo ML, Van Beeumen J, Igout A, Hennen G. 1990 Identification of placental human growth hormone as the growth hormone-V gene expression product. J Clin Endocrinol Metab. 71:15-8. Jara CS, Salud AT, Bryant-Greenwood GD, Pirens G, Hennen G, Frankenne F. 1989 Immunocytochemical localization of the human growth hormone variant in the human placenta. J Clin Endocrinol Metab. 69:1069-72. Wilson DM, Benett A, Adamson GD, et al. 1982 Somatomedins in pregnancy: a cross-sectional study on insulin-like growth factors I and II and somatomedin peptide content in normal pregnancies. J Clin Endocrinol Metab. 55:858-61. Hennen G, Frankenne F. 1987 Influence des hormones proteiques placentaires sur la physiologie maternelle. Ann Endocrinol (Paris). 48:278-88. Orlando PI, Phillips III JA, Ferrandez AN, Arnal TM, Woodard MJ, BuondM. 1987 Frequency and types of deletions-in the growth hormone (GH) gene clusters of Spanish subjects with GH deficiency. Am J Hum Genet. 41:A105. Fitzuatrick SL. Premont R, Barrera-Saldana HA, Saunders GF. 1985 Expression of two human placental lactogen genes in placentas from several individuals [Abstract]. Proc of the 65th Annual Meet of The Endocrine Sot. 63 1. Nickel BE, Kardami E, Cattini PA. 1990 Differential expression of human placental growth hormone variant and chorionic somatomammotropin in culture. Biochem J. 267:653-8.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 November 2015. at 08:09 For personal use only. No other uses without permission. . All rights reserved.
