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Original Studies |
Divisions of Endocrinology, Departments of Medicine (R.S., X.F.) and Pediatrics (L.P., M.A.L.), and the Ilyssa Center for Molecular and Cellular Endocrinology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21287; Department of Pediatrics, Vanderbilt University (J.A.P., K.L.F.), Nashville, Tennessee 37232; Department of Pediatric Endocrinology, Hospital Universitario Virgen de las Nieves (R.E.-M., I.M.d.L.), 18014 Granada, Spain; and Department of Pediatrics, King Faisal Specialist Hospital (A.A.-A.), 11211 Riyadh, Saudi Arabia
Address all correspondence and requests for reprints to: Roberto Salvatori, M.D., Division of Endocrinology, Johns Hopkins University School of Medicine, 1830 East Monument Street #333, Baltimore, Maryland 21287. E-mail: salvator{at}jhmi.edu
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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A variety of mutations in the gene encoding GH (GH1) have been described in patients with IGHD, including deletions, frame shifts, splice site defects, and nonsense mutations (6), and can cause IGHD type IA, IB, or II (7). In 1 large study of 151 patients with IGHD from 83 families, including 51 families with more than 1 affected member, the prevalence of GH1 mutations in subjects with IGHD IA was 66.7% (8). However, GH1 mutations were found in only 1.7% of cases with the most common form of IGHD, type IB (8). Thus, IGHD IB could result from genetic defects that impair GHRH synthesis, secretion, or action. Although the GHRH gene has been excluded as a candidate gene by linkage analysis (9), mutations in the gene for the GHRH receptor (GHRHR) have been recently identified (10, 11, 12, 13, 14). The GHRHR is a member of a large family of heptahelical transmembrane receptors that couple to G proteins upon receptor activation. The significant sequence homology between the GHRHR and the receptors for secretin, vasoactive intestinal peptide, pituitary adenyl cyclase-activating peptide, and glucagon-like peptide has allowed the categorization of these receptors into a new subfamily (B) of the G protein-coupled receptor superfamily (15). Binding of GHRH to GHRHRs expressed on the surface of somatotroph cells activates Gs and leads to a consequent increase in cAMP synthesis that induces cellular proliferation and GH secretion. In the little mouse, a naturally occurring murine model for human IGHD type IB, a missense mutation (D60G) in the extracellular domain of the GHRHR inhibits binding to GHRH (16, 17). Mice homozygous for this mutation have complete GH deficiency and proportionate dwarfism. Similar mutations have been identified in humans with IGHD IB. Affected subjects from 3 distantly related families from the Indian subcontinent are homozygous for a nonsense mutation that introduces a premature terminator codon (E72X) in the extracellular domain of the GHRHR and predicts synthesis of a truncated, nonfunctional receptor (10, 11, 12, 13). In addition, our group recently described an extended kindred from Brazil in which IGHD IB was caused by a mutation in a donor splice site of this gene (14). Despite these initial reports, the prevalence of mutations in the GHRHR gene in families with IGHD IB is unknown. Here we describe novel missense mutations in the GHRHR gene in 3 previously unreported families with autosomal recessive IGHD.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Twenty-eight families with at least two members with autosomal recessive IGHD IB were analyzed by linkage analysis. In three families siblings with IGHD were concordant for alleles at the D7S632 and D7S526 loci that map 0 cR from the GHRHR locus (18). These three families plus two additional families in which linkage analysis was not performed were then screened for mutations in the GHRHR gene. Three families were from North America, one from Spain, and one was originally from Pakistan, residing in Saudi Arabia.
All of the affected patients had GH deficiency and severe,
proportionate short stature (less than third percentile for age) and
had no evidence of other endocrine defects. There were no reports of
neonatal hypoglycemia. Imaging studies of the sellar region (skull
x-ray, computed tomography, or magnetic resonance imaging) showed no
evidence of sellar or parasellar masses. All subjects had GH deficiency
based on a subnormal response to a variety of stimuli (insulin-induced
hypoglycemia, GHRH, or arginine). All of the index cases were
prepubertal at the time of the GH evaluation, with the exception of the
two patients from family B (see below), who were evaluated at ages 16
and 17 yr, when they had advanced pubertal development (Tanner stages 3
and 4, respectively). The pedigrees of the three families in which
GHRHR mutations were found are shown in Fig. 1
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Both affected subjects in family B (BII-1 and BII-2) had marked short stature (-5.2 and -4.5 SD from normal, respectively). Subject BII-1 had a bone age of 13 yr at a chronological age of 17 and 6/12 yr, and subject BII-2 had a bone age of 14 yr at a chronological age of 16 and 2/12 yr. They both had subnormal serum GH responses to insulin-induced hypoglycemia (peak serum GH concentrations of 3.8 and 2.3 µg/L, respectively) despite glucose nadirs of 1.94 and 1.6 mmol/L and cortisol peaks of 772.5 and 775.3 nmol/L, respectively. Moreover, there were no serum GH spikes during sleep. By contrast, they both showed a GH response to a physical exercise test, with peak GH levels of 14.3 and 13.9 µg/L, respectively. This differential response to exercise is unexplained, but probably reflects in part the imprecision of the commercial polyclonal RIA used in 1977 to measure serum GH.
Both affected subjects from family C came to medical attention for evaluation of severe short stature. Patient CII-2 presented at age 11 2/12 yr with a stature of 98 cm (-6.8 SD), and patient CII-3 presented at age 2 10/12 yr with a stature of 74 cm (-5.2 SD). Review of their medical records indicated that IGHD had been documented during the initial hormonal evaluation, but the actual GH values were not recorded in the charts. However, at their last examination before being lost to follow-up both patients showed a dramatic response to GH therapy; at age 16 yr patient CII-2 had a stature that was only -2 SD below average, and at age 7 6/12 yr patient CII-3 had a normal stature (50th percentile), indicating that GH deficiency had been the basis of the severe growth failure in these subjects.
Patients from family A responded to exogenous GH therapy with a robust increase in growth velocity. The patients from family B were treated only for 1 yr due to advanced pubertal stage at the time of diagnosis.
All subjects or their parental guardians gave informed consent.
Amplification of the GHRH-R gene and mutation detection
Genomic DNA was extracted from peripheral blood leukocytes by standard techniques. The 13 exons and the corresponding intron-exon boundaries of the GHRHR gene were individually amplified via PCR. Exons 2 and 3 were amplified individually using the following primers: exon 2 antisense, 5'-ATCAGAGAAGCCACCACCTGC-3'; and exon 3 sense, 5'-TGCACCTGGGCTGAGTCTCTG-3'. All other exons and their exon-intron boundaries were amplified using primers and conditions described previously (14). All oligonucleotide primers correspond to intronic sequences that are located 9–41 bases from intron-exon junctions. The exon 1 sense primer annealed to the promoter region, and the exon 13 antisense primer annealed to the untranslated region of exon 13. The primers used to amplify the promoter region were: sense primer, 5'-ATTGACCAAGTGGCCTGTGGC-3'; and antisense primer, 5'-CAGCCTCAGTAAGCCTTGGCT-3', according to the published sequence (20). They amplify the proximal 285 bases (from -327 to -42) of the promoter region, and the amplification product overlaps the exon 1 amplicon.
One member of each primer pair was synthesized with a 40-bp-long GC-rich 5'-extension to increase the sensitivity of mutation detection during denaturing gel electrophoresis (DGGE). All amplified fragments were less than 320 bp long.
PCR products were first analyzed by electrophoresis through 8% acrylamide gels and then subjected to mutation analysis by DGGE (21, 22, 23). DGGE was performed at constant voltage (80 V) for 14–16 h at 60 C using a 7.5% acrylamide gel containing a linear gradient of 30–90% of the denaturants urea and formamide (100% denaturant = 8 mol/L urea and 40% formamide). Each abnormally migrating band was isolated and sequenced directly. Sequencing was performed using the Thermo-Sequenase Cycle Sequencing Kit (Amersham Pharmacia Biotech, Arlington Heights, IL). The prevalence of each newly discovered change in the coding region was determined by DGGE analysis of a commercial panel of genomic DNAs from 44 normal subjects (88 chromosomes) obtained from the DNA Polymorphism Discovery Resource, which includes DNA from anonymous unrelated individuals of diverse ethnicity (24). After the identification of a mutation in an index patient, all other available members of the family were genotyped via direct sequencing of the appropriate gene region(s).
Transient expression of GHRHR and cAMP assay
To determine whether the missense mutations alter receptor function, we used site-directed mutagenesis (25) to introduce each amino acid change into a wild-type GHRHR complementary DNA (cDNA) that contained a Hemophilis influenza hemagglutinin (HA) epitope tag in the intracellular C-terminal domain (26). This HA-tagged cDNA has the same functional activity as the native receptor (26). Mutagenesis was confirmed by direct sequencing of the cDNA clones. Wild-type and mutant GHRHR cDNAs were cloned in the pcDNA1.0 Amp expression plasmid (Invitrogen, Carlsbad, CA) and were transiently expressed in Chinese hamster ovary (CHO) cells. Briefly, CHO cells at 70–80% confluence were transfected with 8 µg plasmid DNA using Lipofectamine (Life Technologies, Inc., Gaithersburg, MD). Twenty-four hours after transfection, cells were harvested by gentle trypsinization and seeded in 24-well plates at 2 x 105 cells/well. The cells were cultured for an additional 24 h. The culture medium was then replaced with serum-free medium containing 0.5 mmol/L isobutylmethylxanthine and various concentrations of (His1,Nle27)-GHRH-(1–32) (Peninsula Laboratories, Inc., Belmont, CA) or forskolin (10-5 mol/L). After 15-min incubation at 37 C, total cAMP was extracted by addition of HCl to a final concentration of 0.1 N and a cycle of freeze-thawing. Cellular cAMP in the acid extracts was measured by RIA as previously described (27). Results were normalized to the cAMP response to forskolin and were expressed as picomoles of cAMP produced per well. Data are the mean ± SE of three independent experiments, each performed in triplicate. Results were analyzed by ANOVAs run on experimental means.
Cellular expression of GHRHRs
CHO cells were cultured on glass coverslips to 70–80% confluence and transfected as described above with wild-type or mutant cDNAs encoding GHRHRs in which a HA epitope had been introduced (26). Forty-eight hours after transfection the cells were washed twice with PBS and fixed with 0.4% acetic acid in methanol for 15 min at -20 C. Cells were washed three times in PBS containing 0.1% Triton X-100 and then incubated overnight at 4 C with 2 µg/ml mouse anti-HA monoclonal antibody 12CA5 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). After washing, cells were incubated for 30 min at 4 C with 2 µg/ml fluorescein isothiocyanate-conjugated goat antimouse antibody (Santa Cruz Biotechnology, Inc.). Cells were washed with phosphate-buffered saline and examined for fluorescence by confocal laser scanning microscopy (LSM 410, Carl Zeiss, Inc., Oberkochen, Germany) using a x40 objective. Samples were scanned for equivalent times with the same contrast and brightness settings.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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We found nucleotide changes in the GHRHR gene (28) that resulted in amino acid substitutions in two families (B and C) in which affected siblings were concordant for microsatellite markers 0 cR from the GHRHR locus and in one family (A) in which linkage analysis had not been performed.
The index case in family A (see pedigree in Fig. 1
) was homozygous for
a T
A transversion in codon 144 that replaces leucine (CTC) with
histidine (CAC) (L144H) in the first transmembrane domain of the
receptor (Fig. 2). The normal-statured
parents are heterozygous for this mutation, and the normal-statured
sibling is homozygous for the wild-type allele. This family is from
Spain, and the parents denied consanguinity.
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) is a compound
heterozygote and carries the L144H mutation described above (data not
shown) as well as a novel TG transversion in codon 242 (Figs. 3 and 4) that replaces phenylalanine
(TTC) with cysteine (TGC) (F242C) at the junction between the second
intracellular loop and the fourth transmembrane domain. The mother
(BI-2) is heterozygous for the F242C mutation, and the father (BI-1) is
heterozygous for the L144H mutation. A normal-statured maternal
half-brother (BII-3) is homozygous for the wild-type allele. This
family is from the northeastern United States, with no evidence of
Spanish ancestry. Measurement of serum concentrations of insulin-like
growth factor I are not available, because these subjects were first
evaluated clinically in 1978, when this test was not routinely
performed. Their GH studies are described in Subjects and
Methods.
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A transversion in
codon 222, which replaces alanine (GCA) with glutamic acid (GAA)
(A144E) in the third transmembrane domain (Fig. 4). The normal-statured
parents are heterozygous for the mutation, and the normal-statured
sibling is homozygous for the wild-type allele. This family is from
Pakistan, and the parents are second cousins. The locations of the
above mutations in the mature GHRHR protein are shown in Fig. 5.
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Functional studies
We introduced each missense mutation into the HA-epitope tagged
wild-type GHRH-R cDNA by in vitro mutagenesis and
transiently expressed it in CHO cells, which do not express endogenous
GHRHRs. The results are shown in Fig. 6.
Cells transfected with Lipofectamine alone (Mock) did not show a
significant cAMP response to GHRH. GHRH-stimulated cAMP production was
significantly less in cells expressing the mutant receptors than in
cells that were expressing wild-type GHRHR (WT) at
10-9–10-7
mol/L GHRH (Fig. 6), demonstrating that these amino acid changes
impaired the ability of the receptor to transmit GHRH signaling.
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Because the ability of the GHRHR to activate adenylyl cyclase
depends upon the number of receptors expressed on the cell surface, we
assessed cellular expression of wild-type and mutant receptors by
immunofluorescent staining. As shown in Fig. 7, cells that had been transfected with
no plasmid (Mock) showed no fluorescence (panel 1),
indicating that CHO cells have no background binding of the reagents
used to detect HA-tagged receptors. Cells transfected with wild-type
(panel 2) and with the 3 mutant receptors (panels
3–5) cDNAs showed comparable surface and intracellular
distribution of fluorescence; indicating equivalent cellular expression
of GHRHRs.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Although adenylyl cyclase appears to be the primary regulator of GH secretion, recent work suggests that other signal transduction pathways may play a complementary role. Synthetic peptides and analogs that bind to a novel G protein-coupled receptor expressed in the hypothalamus and the pituitary increase GH secretion via activation of phospholipase C (30). The naturally occurring ligand for this receptor has recently been identified from the stomach (31), but its importance in the physiology of the regulation of GH secretion is presently unknown.
The importance of GHRH to the proliferation of somatotroph cells and GH secretion has been recently highlighted by the discovery of unique inactivating mutations in the GHRHR gene (one nonsense and one splicing mutation) in affected members of IGHD IB kindreds (10, 11, 12, 13, 14). In each kindred affected individuals have severe GH deficiency and dwarfism, and homozygosity for mutant alleles is explained by common ancestral descent (14, 32). These initial results notwithstanding, the prevalence of GHRHR mutations in familial IGHD type IB remains unknown. In 1995, Cau et al. (33) analyzed a portion of the GHRHR gene in 65 unrelated subjects with presumed IGHD type IB, although the percentage of patients with familial occurrence of the disease was not reported. These workers did not find gross alterations, but their study was limited due to its ability to analyze only the sequence of the extracellular domain of the GHRHR.
In this work we screened the GHRHR gene in five families in which IGHD type IB was present in at least two family members, greatly increasing the likelihood of finding genetic abnormalities. In four of the five families the GH1 gene was normal (data not shown), and in three families linkage analysis showed segregation of IGHD phenotype with microsatellite markers close to the GHRHR gene located at 7p14-p15 (e.g. 2.6 cM from microsatellite D7S510) (13, 34). We identified three novel GHRHR gene missense mutations in two of the families with positive linkage and in one family in which linkage had not been performed. Because DGGE failed to identify a mutation in affected members of one family in which IGHD IB was linked to GHRHR markers, we also analyzed the GHRHR gene of the index case by direct sequencing, but no mutation was found. It is conceivable that this family may have a mutation in the promoter region upstream of the 327 bp that we analyzed. However, the proximal -319 bases of the GHRHR promoter region are sufficient to confer maximal activity in vitro (20), making it unlikely that mutations upstream of -319 cause a significant reduction in gene expression. It is also possible that a second gene in the same area is responsible for IGHD in this family.
As we started with 30 families with autosomal recessive IGHD IB, we conclude that the prevalence of GHRHR mutations in IGHD IB is 10% (3 in 30).
Affected patients from family B are compound heterozygotes for two different mutations. This is the first report of compound heterozygosity for mutations in the GHRHR gene. This family is from the northeastern United States. The L144H mutation carried by the index case’s father (BI-1) is identical to the mutation found in patients from family A, who live in Spain. Several lines of evidence support the idea that these two mutations arose independently. First, there is no history to indicate that the father’s family is of Spanish ancestry. Second, linkage studies showed that affected subjects from these two families do not share haplotypes near the GHRHR gene and therefore have inherited different chromosomes. Third, the L144H mutations occur in a CpG dinucleotide, a sequence that exhibits increased mutagenicity due to the spontaneous deamination of a methyl cytosine (35, 36).
All subjects with GHRHR mutations shared a similar phenotype that included severe GH deficiency, marked growth failure, and no evidence of additional pituitary hormone deficiency. Interestingly, the two affected subjects from family B had frankly subnormal GH responses to insulin-induced hypoglycemia and absent nighttime GH peaks, but both had significant GH increases after physical exercise. Despite this response, we believe it likely that these patients have GH deficiency. We noted a similar differential response to exercise (peak GH after exercise test, 10–15 µg/L) in as many as 12% of proven GH-deficient patients who were studied during the time that the patients in kindred B were evaluated (37). As these studies were performed during the 1970s with an early commercial polyclonal GH RIA, it is possible that the actual GH peaks would be lower were they measured using the more accurate monoclonal immunoradiometric or immunochemiluminometric assays available today (38). On the other hand, perhaps normal GHRHR action is not required for exercise-induced GH secretion. In this regard it is noteworthy that affected members of family A, who are homozygous for the L144H mutation, also showed higher GH serum peaks after propranolol and exercise than after GHRH, suggesting that physical exercise-induced GH secretion is less dependent on the presence of an intact GHRH receptor than other stimuli. Although the mechanism by which physical exercise stimulated GH release is not known (39), it has been speculated that it may act at least in part by reducing somatostatin inhibitory tone on somatotroph cells (40). This might explain why exercise may work as a GH secretagogue in subjects with mutations in the GHRHR. The greater GH response to exercise in affected subjects from family B, who are compound heterozygotes for the L144H and the F242C mutations, might indicate that the F242C receptor retains some activity. This is consistent with the clinical information, suggesting that patients from family B may be less severely GH deficient than other patients who have severe loss of function mutations.
The fact that none of these mutations was found in 88 chromosomes from unrelated normal subjects indicated that these base changes are not polymorphisms, nor are the mutant alleles present commonly in the general population.
The amino acids involved are located in the first (L144H) and third (A222E) transmembrane domains and at the junction of the second intracellular loop and the third transmembrane domain (F242C). These three amino acid residues are conserved in all of the mammalian GHRHR cDNAs (rat, mouse, pig, bovine, and human) cloned to date, pointing to their importance in receptor function (41). The L144 residue is also highly conserved in other family B human G protein-coupled receptors, such as the vasoactive intestinal peptide, secretin, glucagon-like peptide, and pituitary adenyl cyclase-activating peptide receptors. Transient expression of GHRHR cDNA containing these mutations showed that the expressed receptors failed to stimulate an increase in cAMP in response to GHRH, proving that they cause the IGHD phenotype in these families. The mechanism by which these 3 mutations interfere with receptor function remains to be determined. Immunofluorescence studies showed that all 3 mutants were expressed at similar levels as the wild-type receptor. Although the extracellular N-terminal domain of the GHRHR is necessary for GHRH binding, chimeric studies have shown that the transmembrane helexes and the intervening loops are crucial to confer binding specificity (26). Therefore, these mutations could disrupt GHRH binding. Alternatively, they could interfere with coupling with G proteins. It is unlikely that they would interfere with protein glycosylation, as the only glycosylation site is located in the extracellular domain (15). Further in vitro studies will answer this question and possibly give important information on which specific functions are associated with different regions of the receptor.
In conclusion, we have identified three new GHRHR missense mutations that cause IGHD IB in families with different ethnic backgrounds. Our data indicate that mutations in this gene cause a significant percentage of IGHD type IB.
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Acknowledgments |
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Footnotes |
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Received June 15, 2000.
Revised August 25, 2000.
Revised September 26, 2000.
Accepted October 3, 2000.
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