0021-972X/90/7001-0187$02.00/0 Journal of Clinical Endocrinology and Metabolism Copyright© 1990 by The Endocrine Society
Vol. 70, No. 1 Printed in U.S.A.
Isolated Growth Hormone Deficiency: Analysis of the Growth Hormone (GH)-Releasing Hormone Gene and the GH Gene Cluster P. MULLIS*, M. PATELf, P. M. BRICKELL, AND C. G. D. BROOK Endocrine Unit, Middlesex Hospital (P.M., C.G.D.B.), London WIN 8AA, and the Medical Molecular Biology Unit, Biochemistry Department, University College and Middlesex School of Medicine (M.P., P.M.B.) London W1T6DB, United Kingdom
found. The allelic frequencies for 5 common restriction fragment length polymorphisms were similar in patients and controls. The distribution and frequency of the distinct haplotypes in the hGH gene family revealed no differences between IGHD (n = 30 chromosomes) and controls (n = 48 chromosomes). No deletion or restriction fragment length polymorphisms could be found using a hGH-releasing hormone cDNA clone as a probe in patients or controls. This large volume of data gathered from a Caucasian population indicates that the great majority of patients with IGHD has no structural abnormalities of the hGH gene cluster, particularly no hGH-1 gene deletion. In addition, they have no gross deletions within the hGH-releasing hormone gene. (J Clin Endocrinol Metab 70: 187, 1990)
ABSTRACT. Isolated GH deficiency (IGHD) cannot be distinguished on the grounds of anti-human (h) GH antibodies and stunted growth response to exogenous hGH. DNA analysis was proposed to classify children with IGHD. Genomic DNA was extracted and studied by restriction endonuclease analysis after extraction from the circulating lymphocytes of 53 children with IGHD. These children included 5 pairs of siblings and 5 individuals from 10 families, whose parents (n = 20) and brothers and sisters (n = 5) were also analyzed. Twenty-five adults, including individuals from 3 families of normal height, were studied as controls. No deletion within the hGH gene cluster was identified using a [32P]hGH cDNA clone as a probe. A compound heterozygosity for a hGH-1 deletion or a mutation have not been
S
HORT stature associated with abnormalities of human (h) GH secretion can result from a variety of genetic and acquired interruptions in the hypothalamopituitary-peripheral tissue axis. Phillips III (1) classified three distinct familial types of isolated GH deficiency (IGHD) on the basis of inheritance and other hormone deficiencies: type 1A, autosomal recessive inherited, hGH-1 gene deletion, absent endogenous hGH secretion; type IB, autosomal recessive inherited, no hGH-1 gene deletion, diminished hGH secretion; type II , autosomal dominant inherited, no hGH-1 gene deletion, diminished hGH secretion; and type III, X-linked dominant inherited, no hGH-1 gene deletion, diminished hGH secretion. The hGH gene cluster consists of five very similar genes in the order 5' (GH-1, CSHP-1, CSH-1, GH-2, CSH-2) 3' encompassing a distance of around 50 kilobasepairs [1 kilobase (kb) = 1000 basepairs (bp)] on the long arm of chromosome 17 (2). Illig et al. (3) described the clinical features of IGHD type 1A. In contrast to children with IGHD type IB (non-hGH-1 deleted), the
most striking phenomenon in children with IGHD type 1A (hGH-1 deleted) is an initial anabolic response to exogenous hGH, followed by the development of antihGH antibodies in sufficient titer to cause arrest of response to hGH replacement (1). Laron et al. (4) described four patients who had hGH-1 gene deletions but did not develop anti-hGH antibodies and responded with normal growth to replacement therapy. This has led to the idea that IGHD type 1A cannot be distinguished on the grounds of anti-hGH antibody production, and it has been proposed that DNA analysis should be used to classify such children (5). We have studied a large number of children with IGHD to determine whether a significant number have deletions within the hGH gene cluster or other structural abnormalities detectable by linkage to restriction fragment length polymorphisms (RFLPs). We included in the analysis five families with affected sibling pairs and five families with one affected child and used the distribution and frequency of the haplotypes to perform a linkage analysis. As the frequency of certain RFLPs can differ between populations (6), all of the patients and their families and the controls were derived from the British Caucasian population. We also studied the same
Received May 11,1989. Address requests for reprints to: Prof. C. G. D. Brook, Middlesex Hospital, Mortimer Street, London, WIN 8AA United Kingdom. * Supported by the Swiss National Science Foundation. t Supported by the Cancer Research Campaign.
187
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MULLIS ET AL.
188
individuals for abnormalities of the GH-releasing hormone (GHRH) gene.
JCE & M • 1990 Vol 70 • No 1
glucose concentrations were measured using the YSI 23 AM glucose analyzer (Yellow Springs, OH). Preparation of genomic DNA
Subjects and Methods Patients Fifty-three children with IGHD attending the Growth Disorder Clinic at the Middlesex Hospital in London were studied, including 15 children from 10 families. Children of Mediterranean, Asian, African, and Semitic descent were excluded from the study. In 5 families there were 2 affected children, and in the remaining 5 families there was 1 affected and 1 unaffected child of normal height for age and sex (7). The parents of the 10 families with affected children studied were normal for height and sex (7) (Table 1). The criteria for IGHD included no demonstrable causes of IGHD, stature 3 SD score (SDS) or
less for chronological age and sex (7), delayed bone age (>2 yr) (8), a peak hGH level of 1.5 /ttg/L after standard pharmacological stimulation, height velocity SDS of 1 or less for chronological age and sex (7), and otherwise normal endocrinology. Standard auxological assessment was performed (Table 2) (9). No child had evidence of organic disease, psychosocial deprivation, or any eating disorder, and all had normal renal and hepatic function. The children showed a sustained response to hGH treatment over 1-6 yr (median, 2.2). Controls were 25 British Caucasian adults, including individuals from 3 families of normal stature and without any endocrine disease (Table 1). The families had 2 and 3 adult offspring, respectively. Demonstration of impaired GH secretion was undertaken using insulin-induced hypoglycemia (0.15 U/kg), and adequate hypoglycemia (blood glucose,
Vol. 70, No. 1 Printed in U.S.A.
Isolated Growth Hormone Deficiency: Analysis of the Growth Hormone (GH)-Releasing Hormone Gene and the GH Gene Cluster P. MULLIS*, M. PATELf, P. M. BRICKELL, AND C. G. D. BROOK Endocrine Unit, Middlesex Hospital (P.M., C.G.D.B.), London WIN 8AA, and the Medical Molecular Biology Unit, Biochemistry Department, University College and Middlesex School of Medicine (M.P., P.M.B.) London W1T6DB, United Kingdom
found. The allelic frequencies for 5 common restriction fragment length polymorphisms were similar in patients and controls. The distribution and frequency of the distinct haplotypes in the hGH gene family revealed no differences between IGHD (n = 30 chromosomes) and controls (n = 48 chromosomes). No deletion or restriction fragment length polymorphisms could be found using a hGH-releasing hormone cDNA clone as a probe in patients or controls. This large volume of data gathered from a Caucasian population indicates that the great majority of patients with IGHD has no structural abnormalities of the hGH gene cluster, particularly no hGH-1 gene deletion. In addition, they have no gross deletions within the hGH-releasing hormone gene. (J Clin Endocrinol Metab 70: 187, 1990)
ABSTRACT. Isolated GH deficiency (IGHD) cannot be distinguished on the grounds of anti-human (h) GH antibodies and stunted growth response to exogenous hGH. DNA analysis was proposed to classify children with IGHD. Genomic DNA was extracted and studied by restriction endonuclease analysis after extraction from the circulating lymphocytes of 53 children with IGHD. These children included 5 pairs of siblings and 5 individuals from 10 families, whose parents (n = 20) and brothers and sisters (n = 5) were also analyzed. Twenty-five adults, including individuals from 3 families of normal height, were studied as controls. No deletion within the hGH gene cluster was identified using a [32P]hGH cDNA clone as a probe. A compound heterozygosity for a hGH-1 deletion or a mutation have not been
S
HORT stature associated with abnormalities of human (h) GH secretion can result from a variety of genetic and acquired interruptions in the hypothalamopituitary-peripheral tissue axis. Phillips III (1) classified three distinct familial types of isolated GH deficiency (IGHD) on the basis of inheritance and other hormone deficiencies: type 1A, autosomal recessive inherited, hGH-1 gene deletion, absent endogenous hGH secretion; type IB, autosomal recessive inherited, no hGH-1 gene deletion, diminished hGH secretion; type II , autosomal dominant inherited, no hGH-1 gene deletion, diminished hGH secretion; and type III, X-linked dominant inherited, no hGH-1 gene deletion, diminished hGH secretion. The hGH gene cluster consists of five very similar genes in the order 5' (GH-1, CSHP-1, CSH-1, GH-2, CSH-2) 3' encompassing a distance of around 50 kilobasepairs [1 kilobase (kb) = 1000 basepairs (bp)] on the long arm of chromosome 17 (2). Illig et al. (3) described the clinical features of IGHD type 1A. In contrast to children with IGHD type IB (non-hGH-1 deleted), the
most striking phenomenon in children with IGHD type 1A (hGH-1 deleted) is an initial anabolic response to exogenous hGH, followed by the development of antihGH antibodies in sufficient titer to cause arrest of response to hGH replacement (1). Laron et al. (4) described four patients who had hGH-1 gene deletions but did not develop anti-hGH antibodies and responded with normal growth to replacement therapy. This has led to the idea that IGHD type 1A cannot be distinguished on the grounds of anti-hGH antibody production, and it has been proposed that DNA analysis should be used to classify such children (5). We have studied a large number of children with IGHD to determine whether a significant number have deletions within the hGH gene cluster or other structural abnormalities detectable by linkage to restriction fragment length polymorphisms (RFLPs). We included in the analysis five families with affected sibling pairs and five families with one affected child and used the distribution and frequency of the haplotypes to perform a linkage analysis. As the frequency of certain RFLPs can differ between populations (6), all of the patients and their families and the controls were derived from the British Caucasian population. We also studied the same
Received May 11,1989. Address requests for reprints to: Prof. C. G. D. Brook, Middlesex Hospital, Mortimer Street, London, WIN 8AA United Kingdom. * Supported by the Swiss National Science Foundation. t Supported by the Cancer Research Campaign.
187
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 18:27 For personal use only. No other uses without permission. . All rights reserved.
MULLIS ET AL.
188
individuals for abnormalities of the GH-releasing hormone (GHRH) gene.
JCE & M • 1990 Vol 70 • No 1
glucose concentrations were measured using the YSI 23 AM glucose analyzer (Yellow Springs, OH). Preparation of genomic DNA
Subjects and Methods Patients Fifty-three children with IGHD attending the Growth Disorder Clinic at the Middlesex Hospital in London were studied, including 15 children from 10 families. Children of Mediterranean, Asian, African, and Semitic descent were excluded from the study. In 5 families there were 2 affected children, and in the remaining 5 families there was 1 affected and 1 unaffected child of normal height for age and sex (7). The parents of the 10 families with affected children studied were normal for height and sex (7) (Table 1). The criteria for IGHD included no demonstrable causes of IGHD, stature 3 SD score (SDS) or
less for chronological age and sex (7), delayed bone age (>2 yr) (8), a peak hGH level of 1.5 /ttg/L after standard pharmacological stimulation, height velocity SDS of 1 or less for chronological age and sex (7), and otherwise normal endocrinology. Standard auxological assessment was performed (Table 2) (9). No child had evidence of organic disease, psychosocial deprivation, or any eating disorder, and all had normal renal and hepatic function. The children showed a sustained response to hGH treatment over 1-6 yr (median, 2.2). Controls were 25 British Caucasian adults, including individuals from 3 families of normal stature and without any endocrine disease (Table 1). The families had 2 and 3 adult offspring, respectively. Demonstration of impaired GH secretion was undertaken using insulin-induced hypoglycemia (0.15 U/kg), and adequate hypoglycemia (blood glucose,
