This Article Abstract Full Text (PDF) Submit a related Letter to the Editor Purchase Article View Shopping Cart Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Rosenbloom, A. L. Articles by Francke, U. Search for Related Content PubMed PubMed Citation Articles by Rosenbloom, A. L. Articles by Francke, U.

Stature in Ecuadorians Heterozygous for Growth Hormone Receptor Gene E180 Splice Mutation Does Not Differ From That of Homozygous Normal Relatives¹

Department of Pediatrics, University of Florida College of Medicine, Children’s Medical Services Center (A.R.), 1701 SW 16th Avenue, Gainesville, Florida 32608; Institute of Endocrinology Metabolism and Reproduction (J.G-A.), Quito, Ecuador; and Department of Genetics and Howard Hughes Medical Institute, Stanford University Medical Center (M.S.B., U.F.), Stanford, California 94305-5323

Address all correspondence and requests for reprints to: Arlan L. Rosenbloom, Department of Pediatrics, University of Florida College of Medicine, Children’s Medical Services Center (A.R.), 1701 SW 16th Avenue, Gainesville, Florida 32608. E-mail: rosenal{at}peds.ufl.edu

	Abstract

Top Abstract Introduction Methods Results Discussion Note Added in Proof References

 
Heterozygosity for certain mutations of the GH receptor (GHR) gene has been proposed as the cause of partial resistance to GH, and there has been a recent demonstration of a dominant-negative effect of such a mutation in a mother and child. To examine the effect of heterozygosity in a large genetically homogeneous population with GHR deficiency, in which a substantial number of heterozygous (carrier) subjects and homozygous normal individuals can be compared, we studied a population in Ecuador in which 70 individuals with GHR deficiency were homozygous for the E180 splice mutation. We found that 58 heterozygous relatives of probands were not significantly shorter than 37 homozygous normal relatives [SD score (SDS) for height -1.85 ± 1.04 (SD) vs. -1.55 ± 0.96, P > 0.10]. When only those families with both homozygous normals and carriers were compared, the 33 heterozygous and 29 normal relatives did not differ significantly in height SDS (-1.98 ± 1.07 vs. -1.77 ± 0.91, P > 0.3).

If heterozygosity for the E180 splice mutation were to influence stature, heights of heterozygous parents of probands would be expected to correlate with those of probands and of carriers who are their offspring and not with heights of their homozygous normal children. Parental height SDS did not correlate with height SDS of affected offspring (r = 0.24). For unaffected siblings as a group or analyzed separately as normals or carriers, there was a strong correlation between parental and offspring SDS for height (P < 0.01 for all comparisons). Thus, the effect of homozygosity for the GHR mutation was so profound as to abolish parental influence on height, and there was no difference in the influence of parental stature between carrier and noncarrier offspring. These findings demonstrate no meaningful effect on stature of heterozygosity for the E180 splice mutation of the GHR, which is a functional null mutation and, in the homozygous state, results in profound short stature from severe insulin-like growth factor-I deficiency.

	Introduction

Top Abstract Introduction Methods Results Discussion Note Added in Proof References

 
OVER 30 distinct mutations have been described since characterization of the GH receptor (GHR) nearly a decade ago. These include a deletion and missense, nonsense, frameshift, and splice site mutations that, in the homozygous state, produce profound effects on stature and physical features typical of severe GH/insulin-like growth factor-I (IGF-I) deficiency (1). The recent finding of heterozygosity for GHR mutations associated with partial resistance to GH in a group of short statured patients partially responsive to GH and with low GHBP levels, raised the question of whether certain mutations might affect growth in the heterozygous state (2). It has been recently demonstrated that alternatively spliced isoforms of the GHR, expressed in transfected cells, modulate the function of the normal full-length receptor (3), and that a single heterozygous mutation involving the cytoplasmic domain of the GHR appears to have a dominant-negative effect in a mother and daughter with short stature (4).

There has been little information about the influence on stature of heterozygosity of those GHR mutations that cause severe short stature and other clinical manifestations of GHR deficiency (GHRD) in the homozygous state. Stature was thought to be below normal in unaffected siblings and presumed heterozygous parents of Israeli patients (5, 6), but the reference standard for these observations was not the immigrant middle eastern population of which they were a part (7). To appropriately address this issue requires an adequate number of individuals proven to be homozygous normal or heterozygous for the GHR defect causing GHRD in probands. The use of parents as presumed heterozygotes is problematic, because they would have to be compared with their siblings, who may or may not be heterozygotes, as well.

The Ecuadorian population with GHRD is the only large genetically homogeneous population that has been reported with GHRD, currently numbering 70 probands, all but one homozygous for the E180 splice mutation (8). This made it possible to apply contemporary molecular genetic techniques to identify the carrier state. In 1994, we reported an insignificantly shorter mean stature among 41 individuals who were carriers, compared with 24 homozygous normal siblings (9). We have now expanded this study to a larger number of relatives, and analyses indicate that heterozygosity for the E180 splice mutation does not affect stature.

	Methods

Top Abstract Introduction Methods Results Discussion Note Added in Proof References

 
Subjects

Parents or other relatives of 62 probands were measured for height and studied for carrier status. Subjects and, if minors, their parents, agreed to have height measurements with the understanding that these were for study purposes and comparisons with affected relatives. Blood spots were obtained for genetic analysis after explanation of the mechanism and inheritance of GHRD in this population and agreement by the subject or parent with our policy for reporting results. This policy, developed to avoid labeling or other social problems, is to report only to the individual tested in a counseling session with J.G.-A.; in the case of minors, results are retained at the Institute for Endocrinology Metabolism and Reproduction in Quito, Ecuador and are available when the person reaches adulthood or marries. This approach was discussed extensively within the community and universally approved.

There were 13 affected sibling pairs and one family with 3 siblings affected; in these cases the average SD score (SDS) for height of the affected siblings was used for the comparisons. Genetic and statural data were available for 41 parent pairs and 6 single parents and 95 other relatives. The latter included 74 first-degree relatives of probands (71 siblings and 3 offspring) and 21 second-degree relatives of probands (6 cousins, 8 aunts or uncles, 4 grandparents, and 3 nieces or nephews). Non-first-degree relatives were added to expand the number of normal relatives for comparison; this group provided 12 of the 37 normal relatives and 9 of the 58 heterozygous relatives. All subjects were between the ages of 5 and 50 yr.

Height measurements

Stature was measured in centimeters, either with a fixed stadiometer (Harpenden, Holtain Ltd., Crosswell, Crymych, Dyled, UK) at the Institute in Quito, or in the subjects’ homes with a Raven Minimetre (Raven Equipment Ltd., Unit #4, Ford Farm Industrial Complex, Raintree Road, Dunnow Essex CM6 1HU, UK) (10). Measurements were done three times, and the average recorded. These height measurements were converted to SDS using U.S. population data (11). For affected children, the last recorded height before starting replacement therapy with recombinant IGF-I was used (12).

Genetic analysis

All probands had been proven to be homozygous for the E180 splice mutation of the GHR gene responsible for GHRD in this population by restriction analysis of a PCR product from exon 6 with the enzyme MnlI (13). This method was also used to determine whether the unaffected relatives were heterozygous carriers of this mutation or homozygous normal.

Data analysis

The t test was used to compare mean SDS for height for homozygous normal and heterozygous relatives. To explore the possible influence of heterozygosity for the E180 splice mutation on stature, correlations for height SDS were calculated between parents and their unaffected offspring, separately as carriers and normals, and combined. Within each family, the mean SDS was used if more than one proband, parent, or sibling of either type was studied.

	Results

Top Abstract Introduction Methods Results Discussion Note Added in Proof References

 
There was no significant difference between mean SDS in homozygous normal vs. heterozygous relatives (Table 1). Sibships of probands with both normal and heterozygous (carrier) siblings were separately analyzed to determine whether there was any bias introduced by inclusion of families having only heterozygous and no homozygous normal siblings. The mean SDS for normals and heterozygotes in the subset of families with both kinds of siblings also did not differ significantly (Table 1).

As we noted in the early description of a segment of the Ecuadorian population with GHRD, there was no correlation between mean parental height SDS and proband height SDS (14) (Table 2). Correlations between stature of unaffected siblings and parents were strong, and not different when parents were compared with all their unaffected offspring, or separately to their offspring who were homozygous normal or carriers (Table 2).

	Discussion

Top Abstract Introduction Methods Results Discussion Note Added in Proof References

As we have previously noted, the effect of homozygosity for this mutation is so profound that there is not a significant influence of parental heights on proband heights, nor do the proband heights correlate with unaffected sibling heights (14). This lack of correlation for stature between the heterozygous carrier parents and affected offspring argues against an effect on stature of the carrier state. This contrasts with conditions such as Turner syndrome in which, despite substantial growth retardation, the probands’ heights are still strongly influenced by parental endowment and correlate with those of normal siblings (15). If heterozygosity for the GHR mutation in this population were to be phenotypically influential, one would also expect that the correlation between stature of offspring who do not have GHRD and that of their heterozygous parents would be influenced by whether the offspring are homozygous normal or carriers. In contrast to the lack of significant correlation of parental with proband height SDS, there was a highly significant correlation between parental and unaffected offspring SDS, whether the offspring were carriers or not.

The distinct mutations that have been identified in the GHR gene include a number resulting in GHRD in the homozygous or compound heterozygous state, several purported to result in partial GH insensitivity in the heterozygous state (2), and one proven to do so in a dominant-negative fashion (4). In addition, a number of polymorphisms have been described that do not appear to influence the GH/IGF-I axis (16, 17). The findings reported here indicate that there is no effect on stature of heterozygosity for the E180 splice mutation that causes GHRD in the homozygous state. This mutation creates a new splice site within exon 6 that is exclusively used and causes a predicted deletion of eight amino acids. The predicted mutant receptor molecule has the potential to act in a dominant-negative fashion by heterodimerization with the products of the normal allele. The lack of clinical evidence for this occurring supports our previous hypothesis that the eight-amino acid deletion causes protein misfolding and degradation (18). Therefore, it is likely that the E180 splice mutation is a functional null mutation. In contrast, missense mutations and structural mutations that lead to a stable mutant protein might have effects on growth in heterozygotes.

	Note Added in Proof

Top Abstract Introduction Methods Results Discussion Note Added in Proof References

 
Since submission of this report, another family has been reported with a heterozygous mutation resulting in moderate growth failure through a presumed dominant negative effect on GH signaling (Iida K, Takahashi Y, Kaji H, et al. 1998 Growth hormone (GH) insensitivity syndrome with high serum GH-binding protein levels caused by a heterozygous splice site mutation of the GH receptor gene producing a lack of intracellular domain. J Clin Endocrinol Metab. 83:531–537).

	Acknowledgments

 
We are grateful to Linda Allen of the Department of Health Policy and Epidemiology of the University of Florida College of Medicine for assistance with data analysis, Drs. Victor Martinez and Oswaldo Vasconez of the Institute for Endocrinology Metabolism and Reproduction in Quito for assistance with data collection, and Margaret Stanley for manuscript preparation.

	Footnotes

 
¹ This work was supported by NIH Grant DK-45830 and the Howard Hughes Medical Institute.

² Current address: Department of Internal Medicine, University of Iowa College of Medicine, Iowa City, Iowa 52242.

Received January 15, 1998.

Revised February 23, 1998.

Accepted April 10, 1998.

	References

Top Abstract Introduction Methods Results Discussion Note Added in Proof References

 

1. Rosenbloom AL, Rosenfeld RG, Guevara-Aguirre J. 1997 Growth hormone insensitivity. Ped Clin North Am. 44:423–442.[CrossRef][Medline]
2. Goddard AD, Covello R, Luoh S-M, et al. 1995 Mutations of the growth hormone receptor in children with idiopathic short stature. N Engl J Med. 333:1093–1098.[Abstract/Free Full Text]
3. Ross RJM, Esposito N, Shen XY, et al. 1997 A short isoform of the human growth hormone receptor functions as a dominant negative inhibitor of the full length receptor and generates large amounts of binding protein. Mol Endocrinol. 11:265–273.[Abstract/Free Full Text]
4. Ayling RM, Ross R, Towner P, et al. 1997 A dominant-negative mutation of the growth hormone receptor causes familial short stature. Nature Genet. 16:13–14.[CrossRef][Medline]
5. Pertzelan A, Adam A, Laron Z. 1968 Genetic aspects of pituitary dwarfism due to absence or biological inactivity of growth hormone. Isr J Med Sci. 4:895–900.[Medline]
6. Laron Z. 1993 Laron syndrome: from description to therapy. Endocrinologist. 3:21–28.
7. Weingarten MA. 1992 Changing health and changing culture: the Yemenite Jews in Israel. Westport, CT: Praeger.
8. Berg MA, Guevara-Aguirre JG, Rosenbloom AL, Rosenfeld RG, Francke U. 1992 Mutation creating a new donor splice site in the growth hormone receptor genes of 37 Ecuadorean patients with Laron syndrome. Hum Mut. 1:24–34.[CrossRef][Medline]
9. Rosenbloom AL, Guevara-Aguirre J, Rosenfeld RG, Fielder PJ. 1994 Is there heterozygote expression for growth hormone receptor deficiency? Acta Paediatr Suppl. 399:125–127.[Medline]
10. Diamond FB Jr, Guevara-Aguirre J, Rosenbloom AL. 1994 Accurate determination of height using an inexpensive measuring device. Clin Pediatr. 33:172–174.
11. National Center for Health Statistics. 1997 NCHS growth curves for children birth to 18 years: United States. In: Vital and health statistics. Series 11, number 165. (DHEW Publication No. (PHS) 78–1650). Washington, DC: Government Printing Office.
12. Guevara-Aguirre J, Rosenbloom AL, Vasconez O, Martinez V, Gargosky SE, Rosenfeld RG. 1997 Two year treatment of growth hormone receptor deficiency (GHRD) with recombinant insulin-like growth factor-I in 22 children: comparison of two dosage levels and to GH treated GH deficiency. J Clin Endocrinol Metab. 82:629–633.[Abstract/Free Full Text]
13. Berg MA, Peoples R, Perez-Jurado L, et al. 1994 Receptor mutations and haplotypes in growth hormone receptor deficiency: a global survey and identification of the Ecuadorian E-180 splice mutation in an oriental Jewish patient. Acta Paediatr Suppl. 399:112–114.[Medline]
14. Guevara-Aguirre J, Rosenbloom AL, Vaccarello MA, Fielder PJ, Diamond FB, Rosenfeld RG. 1991 Growth hormone receptor deficiency (Laron syndrome): clinical and genetic characteristics. Acta Paediatr Scand [Suppl] 377:96–103.
15. Lemli L, Smith DW. 1963 The XO syndrome: a study of the differentiated phenotype in 25 patients. J Pediatr. 63:577–588.[CrossRef][Medline]
16. Kou K, Lajara R, Rotwein P. 1993 Amino acid substitutions in the intracellular part of the growth hormone receptor in a patient with the Laron syndrome. J Clin Endocrinol Metab. 76:54–59.[Abstract]
17. Chujo S, Kaji H, Takahashi Y, Okimura Y, Abe H, Chihara K. 1996 No correlation of growth hormone receptor gene mutation P561T with body height. Eur J Endocrinol. 134:560–562.[Abstract]
18. Francke U, Berg MA. 1993 Genetic heterogeneity in Laron syndrome. Acta Paediatr Suppl. 391:3–7.

This article has been cited by other articles:

				R. M. C. Pereira, M. H. Aguiar-Oliveira, A. Sagazio, C. R. P. Oliveira, F. T. Oliveira, V. C. Campos, C. T. Farias, T. A. R. Vicente, M. B. Gois Jr, J. L. M. Oliveira, et al. Heterozygosity for a Mutation in the Growth Hormone-Releasing Hormone Receptor Gene Does Not Influence Adult Stature, But Affects Body Composition J. Clin. Endocrinol. Metab., June 1, 2007; 92(6): 2353 - 2357. [Abstract] [Full Text] [PDF]

				J. E. Rowland, L. M. Kerr, M. White, P. G. Noakes, and M. J. Waters Heterozygote Effects in Mice with Partial Truncations in the Growth Hormone Receptor Cytoplasmic Domain: Assessment of Growth Parameters and Phenotype Endocrinology, December 1, 2005; 146(12): 5278 - 5286. [Abstract] [Full Text] [PDF]

				Z. Laron Laron Syndrome (Primary Growth Hormone Resistance or Insensitivity): The Personal Experience 1958-2003 J. Clin. Endocrinol. Metab., March 1, 2004; 89(3): 1031 - 1044. [Abstract] [Full Text] [PDF]

				C. K. Buckway, K. A. Selva, K. L. Pratt, E. Tjoeng, J. Guevara-Aguirre, and R. G. Rosenfeld Insulin-Like Growth Factor Binding Protein-3 Generation as a Measure of GH Sensitivity J. Clin. Endocrinol. Metab., October 1, 2002; 87(10): 4754 - 4765. [Abstract] [Full Text] [PDF]

				P. Marzullo, C. Buckway, K. L. Pratt, A. Colao, J. Guevara-Aguirre, and R. G. Rosenfeld Leptin Concentrations in GH Deficiency: The Effect of GH Insensitivity J. Clin. Endocrinol. Metab., February 1, 2002; 87(2): 540 - 545. [Abstract] [Full Text] [PDF]

				C. K. Buckway, J. Guevara-Aguirre, K. L. Pratt, C. P. Burren, and R. G. Rosenfeld The IGF-I Generation Test Revisited: A Marker of GH Sensitivity J. Clin. Endocrinol. Metab., November 1, 2001; 86(11): 5176 - 5183. [Abstract] [Full Text] [PDF]

				M. Sjoberg, T. Salazar, C. Espinosa, A. Dagnino, A. Avila, M. Eggers, F. Cassorla, P. Carvallo, and M. V. Mericq Study of GH Sensitivity in Chilean Patients with Idiopathic Short Stature J. Clin. Endocrinol. Metab., September 1, 2001; 86(9): 4375 - 4381. [Abstract] [Full Text] [PDF]

				Growth Hormone Receptor Deficiency in Ecuador J. Clin. Endocrinol. Metab., December 1, 1999; 84(12): 4436 - 4443. [Full Text]

This Article Abstract Full Text (PDF) Submit a related Letter to the Editor Purchase Article View Shopping Cart Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Rosenbloom, A. L. Articles by Francke, U. Search for Related Content PubMed PubMed Citation Articles by Rosenbloom, A. L. Articles by Francke, U.