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The Journal of Clinical Endocrinology & Metabolism Vol. 82, No. 2 444-451
Copyright © 1997 by The Endocrine Society

Clinical Studies

Clinical, Biochemical, and Molecular Investigations of a Genetic Isolate of Growth Hormone Insensitivity (Laron’s Syndrome)1

L. Baumbach, A. Schiavi, R. Bartlett, E. Perera, J. Day, M. R. Brown, S. Stein, M. Eidson, J. S. Parks and W. Cleveland

Departments of Pediatrics (L.B., A.S., E.P., M.E., W.C.) and Neurology (R.B., S.S.), University of Miami School of Medicine, Miami, Florida 33136; Research Atlantica, Inc. (J.D.), Coral Springs, Florida 33065; and the Department of Pediatrics, Emory University School of Medicine (M.R.B., J.S.P.), Atlanta, Georgia 30322

Address all correspondence and requests for reprints to: Lisa L. Baumbach, Ph.D., Room 6021, Mailman Center for Child Development, University of Miami School of Medicine, 1601 NW 12th Avenue, Miami, Florida 33136.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
We have characterized the GH receptor mutation that is responsible for extreme short stature and GH insensitivity in a Bahamian genetic isolate. Heights of affected individuals ranged from -4.0 to -6.3 SD. Like others with Laron’s syndrome, they had normal to high serum GH concentrations and low serum insulin-like growth factor I concentrations. Circulating levels of GH-binding protein activity were below limits of detection. Amplification of exons 2–7 and screening with single strand conformational polymorphism analysis located an abnormality in exon 7. Sequencing identified homozygosity for a C to T transition in the third position of codon 236. Reverse transcription and PCR amplification of complementary DNA from lymphocytes showed that this same sense mutation generated a new splice donor site 63 bp 5' to the normal exon 7 splice site. This novel site was used to the exclusion of the normal site in homozygotes. Both normal and variant messenger ribonucleic acid species were detected in heterozygotes. The predicted protein lacks 21 amino acids, including those defining the WS-like motif of the GH receptor extracellular domain. The high frequency of Laron’s syndrome in this isolated island population probably reflects the introduction of the G236 splice mutation by a settler early in the 300-yr history of English settlement.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
IN 1966, LARON et al. (1) described children with the phenotypic features of GH deficiency who differed from GH-deficient children in that they had normal to high concentrations of GH in serum and did not grow in response to GH treatment. They were later found to have low concentrations of insulin-like growth factor I (IGF-I), which also did not respond to GH treatment (2). Diminished binding of GH to membranes prepared from liver biopsy specimens suggested that the genetic defect in this autosomal recessive condition involved a receptor for GH (3).

Isolation of the GH receptor (GH-R) and cloning of GH-R complementary DNA (cDNA) identified it as a member of the cytokine receptor superfamily (4, 5). Members of this family share the common features of extracellular, transmembrane, and intracellular domains as well as a WS-like motif, which has been implicated in ligand binding and receptor activity (6). Limited proteolysis of GH-R in vivo leads to the release of a GH-binding protein (GHBP) representing the extracellular domain of GH-R (7). This protein has a binding affinity similar to that of GH-R, and under physiological conditions it binds approximately 50% of circulating GH (8, 9). GH-binding activity is undetectable or markedly reduced in the sera of most patients with Laron’s syndrome (LS) (10, 11).

Several types of mutations in the GH-R locus have been demonstrated in patients with LS (12, 13). Some appear to be limited to a particular region or ethnic group, and others are more widely distributed. Deletions of exons 3, 5, and 6 have only been observed in persons of Oriental Jewish ancestry (14, 15). Small, 1- or 2-bp deletions disrupt the reading frame and lead to recognition of premature stop codons (16). Single base substitutions generate translational stop codons (17), create missense mutations (18, 19), and alter messenger ribonucleic acid (mRNA) splicing. Abnormalities in splicing can result from disruption of a normal splice site (12, 20) or creation of a new, and preferred, donor splice site (21).

In 1990, Rosenbloom and colleagues described a remarkable concentration of patients with LS in Loja, Ecuador (22) involving a consanguineous population of Spanish Jewish ethnicity. Subsequently, a similar population was found in a neighboring province (23). All but 1 of 37 affected individuals were homozygous for a mutation at codon 180 (21). A substitution of G for A in the third base did not alter the sense of the E codon, but it did activate a donor splice site 24 bases upstream from the normal exon 6 donor splice site. Reverse transcription-PCR (RT-PCR) amplification of cDNA from lymphocytes showed that this site was used to the exclusion of the normal site. The predicted protein lacks 8 amino acids of the extracellular domain. The Ecuadorian cohort represents the largest known group of patients with LS in the world and the first genetic isolate to be reported.

Over the past 3 decades, several patients from an island in the Bahamas and others from Nassau and Florida have been referred to the University of Miami Department of Pediatrics for evaluation of extreme short stature. Clinical, biochemical, genetic, and molecular investigations have resulted in the identification of eight patients with LS from this Bahamian island. We report the results of these studies and the definition of the GH-R mutation in these Bahamians who represent the first cluster of patients with LS of Anglo-Saxon descent.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Subject studies and materials

Personal interviews and historical research were used to construct pedigrees outlining the genealogy of the island’s inhabitants. Subjects with short stature and their immediate families were examined. SD scores (Z-scores) for height were derived from the National Center for Health Statistics reference sample. Biochemical assays for serum GH and IGF-I levels were performed using RIA. Serum GHBP levels were determined by Genentech (South San Francisco, CA), using a ligand-mediated immunofunctional assay (24).

Twenty-one individuals were selected for further study based on clinical and biochemical criteria. They included seven suspected of having LS (Z-score, -4.0 to -6.3), four of their parents, and one grandparent; 4 with moderately short stature (Z-score, -2.5 to -2.9); and the mother of a child with short stature. Three control individuals were sampled. Either Epstein-Barr virus-transformed lymphocytes or skin fibroblasts were used for molecular studies. The involvement of human subjects in these studies followed the protocol and informed consent procedure approved by the University of Miami institutional review board.

Molecular methodology

DNA isolation and exon-specific PCR. Genomic DNA was isolated from peripheral blood and transformed lymphocytes by "salting out" (25). Exon-specific PCR was performed using the primers shown in Fig. 1Go. PCR reactions included 1 µmol/L of each primer, 2.5 U Taq polymerase, and 200–500 ng genomic DNA. Cycling conditions were 94 C for 5 min (1 cycle); 94 C for 30s, 55 C for 30s, and 72 C for 30 s (30 cycles); and 72 C for 7 min. Products were visualized on agarose gels.



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  		Figure 1. Primers used for exon-specific PCR.

 
Southern blot analysis. Southern blots of genomic DNA digests were transferred to nylon membrane (Hybond-N, Amersham) according to standard methodology (26). Restriction fragments representing exons 2–10 were detected with the cDNA insert from plasmid PBR.HGHR (14).

Single strand conformational polymorphism (SSCP) analysis. SSCP was performed to detect sequence divergence (27). Four microliters of the PCR reaction were mixed with 8 µL tracking dye and denatured at 95 C for 2 min, followed by immediate quenching on ice. Four microliters of this mixture were loaded on a 20 x 20-cm nondenaturing MDE gel (J. T. Baker, Phillipsberg, NJ) and electrophoresed at 4 watts for 6–7 h at constant temperature (25 C). Banding patterns were visualized using a silver-staining kit (Bio-Rad Laboratories, Richmond, CA).

Direct DNA cycle sequencing. PCR products were purified for sequencing by electroelution from low melting point agarose, followed by minicolumn purification, and sequenced using a double stranded DNA Cycle Sequencing System kit (BRL, Gaithersburg, MD). The primers used for sequencing (primers a, b, and c, Fig. 2Go) were end labeled with [32P]ATP. Dideoxy sequencing products were electrophoresed on an 8% denaturing polyacrylamide gel at 100 watts, dried on Whatman 3M paper (Clifton, NJ), under vacuum. Autoradiograms were analyzed after a 24-h exposure to Kodak XAR-5 film (Eastman Kodak, Rochester, NY).



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  		Figure 2. Partial sequence of GH-R cDNA (from DNASIS, Hitachi Software, Tokyo, Japan), with important PCR primers and exon information listed. Numbers correspond to numbering of nucleotides in cDNA sequence. a, Exon 6 PCR primer for DNA sequencing and forward primer for second round of RT-PCR; b, exon 6 sequencing primer; c, exon 7 reverse sequencing primer; d, reverse primer for second round RT-PCR; e, reverse primer for first round RT-PCR and reverse PCR primer for exon 8. A base pair with a square indicates the site of the LS mutation; an underlined sequence corresponds to the WS-like motif in the GH-R.

 
RNA isolation and RT-PCR of GH-R mRNA. Total RNA was isolated using the RNA NOW Liquid Matrix RNA isolation kit (Biogentex, Seabrook, MA 77586). GH-R mRNA-specific primers (Fig. 2Go) were used for RT-PCR amplification of GH-R cDNA with the following modifications of a previously described procedure (28). Approximately 4 µg RNA were mixed with 400 ng reverse primer (Fig. 2Go, primer e), heated, quenched on ice, and reverse transcribed with 400 U Superscript reverse transcriptase (Life Technologies, Grand Island, NY). Reactions were terminated and spin dialyzed in Amicon S-100 columns (Lexington, MA). Half (20 µL) of the final volume was used as the PCR template. PCR reactions were performed with first and second rounds [primers a and e, followed by heminested primers b (forward) and d (backward); Fig. 2Go] in the presence of 3 mmol/L MgCl2 and with the following cycling conditions: 94 C for 2 min (1 cycle); 94 C for 60 s, 55 C for 60 s, and 72 C for 60 s (30 cycles); and 72 C for 7 min.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Clinical information

Inhabitants of the island are thought to be of English origin, with some Scottish and Irish ancestry. The island founders were Puritan settlers who originally came to the Bahamas in 1645 (29, 30, 31). The current inhabitants have a limited number of surnames. Due to the long history of individuals marrying within the population, genetic drift has been quite limited, making this island a genetic isolate. For these reasons as well as a limited amount of direct consanguinity, LS is more prevalent in this, compared to the general, population.

Table 1Go summarizes the clinical and biochemical investigations of our study group. Several individuals have the phenotype described for LS (1). Features include marked growth failure, craniofacial disproportion with a relatively small face and a depressed nasal bridge, and truncal obesity.


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  		Table 1. Characteristics and serum GH, IGF-I, and GHBP levels of the subjects

 
Random serum GH measurements were elevated in the infants and children studied in this group, but low in two of the adult subjects (patients IC and PC). The low random serum GH values in adult Laron patients is a frequent, but unexplained, observation (22). Concentrations of IGF-I were low in all patients with LS studied, and GHBP levels were below the limits of detection.

Four other individuals with moderately short stature were also evaluated. Three (GB, MB, and PA) were related. They were not as short as the patients with LS, and their appearance was not typical for LS. GH, IGF-I, and GHBP protein levels were normal. They are considered to have moderately short stature unrelated to a GH receptor abnormality.

Four parents (MA, JA, JUP, and TP) of two LS patients were available for study. Two of the parents were siblings (MA and JUP). All had heights and serum IGF-I levels within the normal range. The mothers had normal GHBP levels, whereas the fathers had reduced levels. Serum IGF-I and GHBP levels were normal in two sets of grandparents and four population controls (two males and two females).

Identification of the mutation in the GH-R gene

An initial evaluation of the GH-R gene was performed by Southern blot to screen for exon deletions. No gross rearrangements involving GH-R exons in affected individuals were detected (data not shown). GH-R exon structure was further investigated using an exon-specific PCR amplification assay. Exon primer sequences are presented in Fig. 1Go. Results from PCR amplification of exons 2–7 are shown in Fig. 3Go. No obvious deletions of exons were detected. Each of the PCR primers is located approximately 20 bp from the beginning or end of an exon, and the average size of the PCR amplimer is 180 bp for the first seven exons.



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  		Figure 3. PCR amplification of GH-R exons 2–7 reveals no gross rearrangement of the GH-R gene. Genomic DNA was isolated from affected and control individuals as described in the molecular methodology section of Materials and Methods, and exons 2–7 of the GH-R gene were amplified in the standard PCR reaction using 500 ng genomic DNA. Twenty-two-microliter aliquots were electrophoresed in 1.5% agarose gels with appropriate mol wt markers. Only the expected mol wt species of approximately 170–180 bp were detected after 30 rounds of PCR. Lane a, Variant phenotype with moderate short stature; lanes b and c, LS patients; lane d, population control; controls (cont): A, nonpopulation control; -, no template DNA added; +, {lambda} DNA control.

 
These exon-specific PCR products were then screened for point mutations using SSCP analysis. An electrophoretic change, indicative of a conformational difference, was detected in exon 7 in two patients with LS (FA and SP), but not in control individuals and not in the individuals with moderately short stature (Fig. 4Go). These results suggested that alterations detected in the SSCP experiments did not represent an exon 7 polymorphism.



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  		Figure 4. SSCP analysis of GH-R exons 2–7 indicates a conformational shift in exon 7. Four microliters of a 50-µL PCR reaction were denatured at 90 C for 5 min before electrophoresis on an MDE acrylamide gel for 6.5 h at 4 watts and constant voltage. Visualization of DNA was accomplished using the Bio-Rad silver-staining kit. Results for exons 2, 3, and 7 appear from left to right; data for exons 4–6 are not shown. The order of patients from left to right is as follows: variant phenotype with moderate short stature (PA), two patients with LS (FA and SP), and a population control (HH).

 
To define the observed SSCP conformational shift, amplified exon 7 DNA from patients with LS, several sets of parents of these patients, and population controls (unrelated to patients with LS and/or their immediate families) was purified and sequenced as described using the reverse primer for exon 7 (Fig. 2Go, primer c). Homozygosity for a C to T transition was detected at the third base position of codon 236 (exon 7; position 766 of the GH-R cDNA) in all patients with LS studied (Fig. 5Go). The same alteration was detected in the heterozygous state in obligate carriers [TP, (Fig. 5Go) and others; data not shown], indicating that the mutation was identical by descent in this population. Control individuals and those with moderately short stature had the normal base pair at this position.



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  		Figure 5. DNA sequence analysis reveals a C->T transition at nucleotide position 766 in the GH-R cDNA. Approximately 6 ng of exon 7-specific PCR products from affected and nonaffected individuals were sequenced using an internal radiolabeled cycle sequencing primer (see Fig. 2Go for details). Each of the sequencing reactions was grouped by dideoxy terminator on the polyacrylamide gel as shown. Shown in the figure are portions of the DNA sequence surrounding the GH-R mutation in exon 7 for a population control (LP), two patients with LS (PC and SP), and an obligate carrier (TP). A C->T transition was detected in affected and heterozygous individuals and most likely accounts for the exon 7 SSCP abnormality shown in Fig. 4Go.

 
This mutation was not predicted to alter amino acid composition of the GH-R due to its occurrence in the third position of a codon. Further examination of the surrounding primary DNA sequence flanking the mutation was suggestive of a cryptic splice donor site (32). Comparison of this site and the native site 63 bp downstream at the 3'-end of exon 7 (Fig. 6aGo) to the published consensus sequences for splicing (33, 34) suggested that the newly created site would be preferred as the splice donor sequence for the intron between exons 7 and 8. Therefore, this mutation was predicted to alter normal mRNA splicing of the GH-R gene, resulting in a receptor missing 21 amino acids of the mature protein (Fig. 6bGo).



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  		Figure 6. a, Sequence comparison of the activated cryptic splice site to the natural GH-R exon 7 splice donor site and the consensus donor splice site sequence. The C->T transition (indicated with an asterisk) activates the cryptic splice donor site within exon 7. The cryptic site matches the splice consensus sequence better than the natural donor splice site at the 3'-end of exon 7, as indicated by the percentage that each base occupies in each respective position (33, 34). b, Predicted consequence of the activation of the cryptic splice site in LS patients. Through the activation of the cryptic splice site 63 bp upstream of the normal exon 7 3'-splice site (as shown in the figure), the GH-R mRNA in patients with LS is predicted to be truncated by 63 bp as shown, causing a 21-amino acid in-frame deletion in the GH-R.

 
GH-R mRNA was isolated from lymphoblastoid cell lines of affected, heterozygous, and control subjects to test this hypothesis. Using RT-PCR and sets of carefully designed PCR primers (Fig. 2Go, primers a, d, and e), a GH-R cDNA was reverse transcribed using primer e (Fig. 2Go). A 500-bp region of the cDNA, corresponding to the distal 3'-half of exon 6 through the proximal 5'-half of exon 8, was PCR amplified using primers a and d (Fig. 2Go). The results of these experiments are shown in Fig. 7Go. In the parent of a child with LS (lane JA), two GH-R mRNA species were detected: one of the correct size transcribed from the normal wild-type allele (WT, Fig. 7Go), and a shorter species (M, Fig. 7Go) transcribed from the allele carrying the codon 236 mutation. Only one GH-R mRNA species of shortened length, corresponding to transcription from two mutant GH-R alleles (mRNA species M, Fig. 7Go) is detected in the LS patient (lane FA). DNA sequencing of the shortened RNA species using primers a and c (Fig. 2Go) confirmed the splicing breakpoints predicted above (Fig. 8Go).



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  		Figure 7. RT-PCR of GH-R mRNA from lymphocytes confirms the predicted splice variant in affected and carrier individuals. Total RNA was isolated from transformed lymphocyte cultures, and 4 µg were used for RT using GH-R reverse primer e (see Fig. 2Go for further details) and Superscript (BRL). Products of the RT reaction are purified using Amicon S-100 filters, and 20 µL were used in a PCR amplification reaction using the outside PCR primers (a and e), followed by the nested, second round PCR primers, b and d, shown in Fig. 2Go. This methodology amplifies a cDNA species of approximately 500 bp corresponding to the 3'-portion of exon 6, all of exon 7, and the 5'-portion of exon 8. WT, Wild-type GH-R mRNA, detected at approximately 500 bp; M, mutant GH-R mRNA, 63 bp shorter than the wild type. JA, Heterozygous parent of FA (child with LS); Control, population control. RT-PCR products were electrophoretically separated and visualized on 2% agarose gels.

 


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  		Figure 8. Sequence comparison of the cDNA of an affected patient with LS and the population control. RT-PCR products shown in Fig. 7Go were agarose gel purified and sequenced as described in the text. The sequence shown indicates the predicted breakpoint in a LS patient compared to that in a population control. Shown is the actual cDNA sequence from bases 745–844 in the control and the missing bases in the mutant cDNA species from a patient with LS.

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
We have presented clinical, biochemical, and molecular evidence supporting the diagnosis of LS resulting from a mutation of the GH-R in a Bahamian genetic isolate of Anglo-Saxon origin. This isolate is indeed ethnically different from the Ecuadorian isolate described by Rosenbloom et al. (22), which is thought to have originated in Spain in a population of "conversos," Jews who converted to Catholicism before and during the Spanish Inquisition.

Several clinical observations of our patient population merit further comment. The random serum GH values obtained in our patients with LS are consistent with those reported in other studies (22). Increased GH levels in our children with LS, compared to those in the adult patients, are also consistent with previous observations, as is the marked diminution in serum IGF-I levels in our patient population and a moderate increase in these levels in older patients (22). Our patients with LS had serum GHBP levels below the level of detection.

The GH-R mutation identified in this population is unique. All current evidence points to the underlying mutation in the GH-R locus as being identical by descent. This observation is reflective of the genetic homogeneity of this population, as direct consanguinity has actually been quite limited. The mutation creates a preferred splice donor site in the middle of exon 7, which alters normal GH-R mRNA processing and is predicted to eliminate 21 amino acids from the extracellular domain. No cDNA species using the normal splice site at the end of exon 7 were produced from cells of affected individuals, suggesting that in homozygous affected individuals, the activated splice site is used exclusively in GH-R mRNA processing. These observations are analogous to those reported for the Ecuadorian patients with LS (21), which result from a similar splicing mutation in GH-R exon 6. In both of these cases, substitution of a single base pair within an exon not only creates a new splice site, but also creates an exclusively preferred site for splicing due to increased homology with conserved consensus splicing sequences (33, 34). Unlike the Ecuadorian mutation, which is predicted to cause the loss of eight amino acids in the extracellular domain, the codon 236 mutation occurs in the WS-like motif, a conserved region of structural homology shared among all members of the cytokine receptor family (6).

Both the Ecuadorian and the Bahamian mutations lead to the production of a GHBP that fails to bind GH. Woods et al. (20) recently described another mutation in GH-R that affects mRNA splicing. Two cousins belonging to a highly consanguineous pedigree originating in Pakistan were found to have a G to C substitution in codon 274. This mutation in the second base of the codon changes the normal arginine (AGG) to a threonine (ACG). Codon 274 is the last codon in exon 8. The mutation disrupts the 5' donor splice site for exon 8 and results in exclusion of exon 8 from the processed mRNA. There is omission of the transmembrane domain of the GH-R protein and a frame shift that generates a translational stop codon five codons downstream in exon 9. This results in the production of an altered protein lacking the transmembrane and intracellular domains of GH-R, but retaining full GH-binding activity. Overproduction of the mutant protein results in a phenotype of LS with increased GHBP activity in serum.

Several important issues are unresolved. The first is the immediate need for carrier screening within this population. In 1990, there were 1291 people living on the island (35). Eight patients with LS have been identified, indicating a prevalence of 1 in 163 and suggesting a carrier frequency of 1 in 7. The occasional occurrence of low GHBP levels in heterozygotes does not offer a viable alternative to molecular tests for carrier status. We are currently in negotiations with island officials regarding the institution of a carrier-screening program within this population.

The other issue is that of the biological effect of the codon 236 mutation on GH-R structure and function. We do not know whether the mutant protein is produced or whether it is normally processed and localized to the plasma membrane. The use of in vitro models to study effects on ligand binding, as has been described for other reported GH-R mutations (19, 36), is a viable experimental approach, as is the use of in vitro systems to assay possible effects on signal transduction (37). A complementary approach is the use of in situ and in vitro immunocytochemical techniques and cell fractionation (38) to determine the subcellular location of the GH-R in various tissues, thereby potentially detecting defects in receptor processing and trafficking. It is expected that a combination of these approaches will be necessary to fully address the potential biological effects of this mutation.


   Acknowledgments
 
The authors thank the subjects and their families who participated in these studies as well as the Bahamian government for their cooperation and support. The authors also thank Dr. Louis Underwood, Dr. Jan Gardner, and other members of the Underwood Laboratory for the endocrinological investigations of the patients. The authors acknowledge Dr. Cresio Alves for contributions to the clinical studies, and Ms. Susannah Tapley, Nicole Diez, and Cecelia Valdez for the molecular studies. We also extend our appreciation to Dr. Sandy Norman, Ms. Roxanne Gonzalez, Mr. Sergio Garcia, and Ms. Tamar Berman for their help in construction of the island genealogy. Plasmid PBR.HGHR, PCR primer information, and cDNA sequence information were provided by Dr. William Wood of Genentech, Inc. The authors also thank Drs. Roger Donahue, Mark Rabin, Paul Benke, Serge Amselem, Arlan Rosenbloom, Mary Anne Berg, Mike Waters, and Stephen Goodman. We acknowledge the editorial assistance of Dr. Magda Plewinska, Ms. Tammy Rossi, Ms. Barbara Johnson, and Ms. Rana Habash.


   Footnotes
 
1 This work was supported in part by awards from the Genentech Foundation for Growth and Development and from the University of Miami School of Medicine (to L.B.). This work was also supported in part by NIH R01-DK-46312 (to J.S.P.). Back

Received April 15, 1996.

Revised July 16, 1996.

Revised October 29, 1996.

Accepted November 5, 1996.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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