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Original Studies |
Departments of Medicine (S.M.J.d.B., M.A.L.), Pediatrics (C.-L.D., M.A.L.), and Psychiatry (M.C.L.), The Johns Hopkins University School of Medicine, Baltimore, Maryland 21287; and National Institute of Mental Health, National Institutes of Health (T.B.U.), Bethesda, Maryland 20892
Address all correspondence and requests for reprints to: Suzanne Jan de Beur, M.D., Division of Endocrinology and Metabolism, The Johns Hopkins University School of Medicine, 1830 East Monument Street, Suite 333, Baltimore, Maryland 21287.
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Abstract |
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Tissues that have been analyzed from patients with PHP 1a have a 50%
reduction in the expression and activity of Gs
(3, 4) due to heterozygous mutations in the gene encoding the -chain
of Gs (GNAS1) (2). In addition to PTH resistance,
patients with PHP 1a also show resistance to other hormones whose
receptors are coupled to Gs, an observation
consistent with the widespread deficiency of this protein (5, 6, 7).
Patients with type 1a also manifest an unusual constellation of
developmental and skeletal defects, collectively termed Albright’s
hereditary osteodystrophy (AHO) (8), whose relationship to
Gs deficiency remains unclear.
Patients with PHP type 1b have a genetically and biochemically distinct
disorder. Patients with PHP 1b lack features of AHO, have normal
expression of Gs protein in accessible
tissues, and manifest hormonal resistance that is limited to PTH target
tissues (9). Furthermore, PTH resistance may be limited to the kidney
with PTH responsiveness preserved in the bone, as evidenced by the
hyperparathyroid skeletal lesions observed in many of these patients
(10, 11).
Because Gs activity is normal in cells
analyzed from patients with PHP 1b, it is likely that a
defect(s) in other genes that are involved in PTH signal transduction
is responsible. To explore whether bioinactive PTH or defective PTH
receptors might account for PHP 1b, we performed linkage studies in
multigenerational PHP 1b kindreds. Using intragenic polymorphisms in
PTH and the PTH/PTH-related peptide (PTHrP) receptor (PTH type 1
receptor) genes, we identified patterns of inheritance that were
discordant with linkage of PHP 1b to the PTH/PTHrP receptor gene and
the PTH gene. Using polymorphic microsatellite markers that flank the
PTH2 receptor gene, we excluded linkage to PHP 1b. We report here the
exclusion of PTH and its receptors as candidate genes for PHP 1b.
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Subjects and Methods |
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Informed consent was obtained from each subject for
participation in these studies. These studies were approved by the
joint committee on clinical investigation of The Johns Hopkins
University School of Medicine. Criteria used to establish the diagnosis
of PHP 1b include 1) biochemical hypoparathyroidism (hypocalcemia and
elevated serum levels of intact PTH), 2) absence of features of AHO, 3)
absence of TSH or LH resistance, 4) no evidence of vitamin D deficiency
or hypomagnesemia, 5) defective nephrogenous cAMP and phosphaturic
responses to exogenous PTH infusion, and 6) normal expression (12) or
activity of erythrocyte Gs (4) and normal
GNAS1 genes. Two multigenerational families with PHP 1b were analyzed
in linkage studies of the PTH/PTHrP receptor and the PTH gene. Family M
contains 5 affected and 7 unaffected members (Fig. 1
). The biochemical characteristics are
summarized in Table 1. Detailed clinical
characteristics, biochemical data, and normal
Gs levels of this family have been previously
described (9, 13). In some cases the diagnosis of PHP 1b was confirmed
by the infusion of PTH, which produced negligible increases in urinary
excretion of nephrogenous cAMP. In 1 case (MIII-2), serum levels of
calcium and phosphorous were normal, but serum levels of PTH were
elevated, and this patient showed a deficient nephrogenous cAMP
response to iv infusion of human (h) PTH-(1–34). In another case
(MIII-7), the subject had normal serum calcium levels and an elevated
PTH level, but a normal response to PTH infusion. We have classified
this individual as unaffected for the purposes of linkage analysis.
Family K consisted of 4 affected members, 2 obligate gene carriers, and
12 unaffected members (Fig. 1). Members of family K were also
characterized biochemically and phenotypically, and levels of
erythrocyte Gs protein were normal by
immunoblot analysis (Table 1). Two subjects (KII-3 and KII-5) were
obligate carriers and had normal serum levels of calcium, phosphorous,
and PTH as well as normal nephrogenous cAMP responses to infusion of
PTH-(1–34). Family R is comprised of 5 affected members, 1 obligate
gene carrier, and 10 unaffected members (Fig. 1). Affected members of
family R lacked stigmata of AHO, exhibited PTH resistance without TSH
or LH resistance, and had a structurally normal GNAS1 gene (coding
region and intron/exon boundaries). Combined these 3 families had a
total of 14 affected individuals, 29 unaffected individuals, and 3
obligate gene carriers. These 3 families are unrelated; families M and
R are of Western European origin, and family R is of Eastern European
origin. No common ancestors among these families have been
identified.
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Infusion of PTH and collection of blood and urine samples were performed according to the protocol of Chase et al.(1) with the exception that subjects were infused iv with 35 µg synthetic PTH-(1–34) (Parathar, Rorer Pharmaceuticals, King of Prussia, PA). Urinary cAMP was measured and expressed as nanomoles per dL glomerular filtrate.
PCR conditions and oligonucleotide primers
High molecular weight DNA was isolated from peripheral blood leukocytes of PHP 1b patients and unaffected family members as previously described (3) PCR was performed using the method described by Saiki et al. (14) with an automatic DNA thermocycler (MJ Research, Inc., Cambridge, MA). Genomic DNA (200 ng) was amplified in a 50-µL volume containing 10 mmol/L Tris (pH 8.4); 50 mmol/L KCl; 1.5 mmol/L MgCl2; 0.01% gelatin; 50 pmol of each primer; 100 µmol/L each of deoxy (d)-ATP, dCTP, dGTP, and dTTP; and 2.5 U Taq polymerase (Perkin-Elmer Corp.-PE Applied Biosystems, Norwalk, CT). Exon 13 of the PTH/PTHrP receptor gene exon 13 was amplified using sense oligonucleotide 5'-[GC]40GAGTCCAGATGCACTATGAGATGCT-3' and antisense oligonucleotide 5'-TGGAAGAATGGAGAAATGAGCCTT-3'. After an initial denaturation step at 95 C for 4 min, samples underwent 40 cycles of amplification consisting of denaturation at 95 C for 30 s, annealing at 57 C for 30 s, and extension at 72 C for 40 s. The final extension step was at 72 C for 5 min. The PTH gene exon 3 was amplified using sense oligonucleotide 5'-[GC]40AGCTAATGGGAAGTGGCCCTCTCTG-3' and antisense oligonucleotide 5'-TTGCCCTACACTGTCTAGAGC-3'. After an initial denaturation step of 94 C for 4 min, samples underwent 40 amplification cycles consisting of denaturation at 94 C for 40 s, annealing at 55 C for 30 s, and extension at 72 C for 1 min. The final extension step was at 72 C for 5 min. After PCR, the amplified products were analyzed by electrophoresis through 5% PAGE in Tris-borate/EDTA electrophoresis buffer and visualized by ethidium bromide staining and an UV light illuminator.
Denaturing gradient gel electrophoresis
Amplified DNA samples were analyzed by denaturing gradient gel electrophoresis as previously described (15). Samples were electrophoresed at 60 C for 16 h at 85 V in 7.5% polyacrylamide gels (37.5% acrylamide and 1% bisacrylamide) containing a denaturing gradient parallel to the direction of electrophoresis (100% denaturant is 7 M urea and 40% polyacrylamide). After electrophoresis, the gels were stained with ethidium bromide, and the bands were visualized with a UV light source. The optimal gradient for each amplified fragment was determined empirically. The identity of abnormally migrating DNA fragments was confirmed by direct sequence analysis (15) using glycerol-tolerant gels.
PTH2 receptor genotyping
Chromosome 2 microsatellite markers flanking the PTH type 2 receptor (PTH2R) locus (i.e. D2s117, D2s325, and D2s164) were amplified in 10-µL reaction volumes containing 0.1 µmol/L primers, 0.1 mmol/L dNTPs, 10 mmol/L Tris (pH 8.3), 50 mmol/L KCl, 1.5 mmol/L MgCl2, 0.6 U Taq DNA polymerase (Perkin-Elmer Corp., Foster City, CA), and 60 ng DNA. One of each primer pair was labeled with fluorescent dyes (6-FAM, TET, and HEX). PCR was performed using a GeneAmp 9600 (Perkin-Elmer Corp.) or a PTC-225 DNA Engine Tetrad (MJ Research, Inc.) with an initial denaturation of 4 min at 94 C followed by a touchdown PCR program (16) consisting of a series of two cycle steps with the first two-cycle step was denaturation at 94 C for 20 s, annealing at 62 C for 20 s, and extension at 72 C for 20 s. The annealing temperature in each subsequent two-cycle step was reduced by 1 C until 52 C, at which point 10 cycles were performed, with a final 10-min extension at 72 C. Labeled products were pooled by multiplexing markers of different size ranges and dyes when possible and were electrophoresed on a model 373 DNA Sequencer (PE Applied Biosystems, Foster City, CA) on 6% denaturing polyacrylamide gels. Data were collected and analyzed with Genescan software (PE Applied Biosystems), which calculates fragment length in reference to an internal lane standard (Genescan-500 labeled with TAMRA) and quantifies the amount of fluorescence in each fragment. The data were then imported into Genotyper (PE Applied Biosystems) to identify alleles.
Linkage analysis statistical methods
The PHP 1b disease locus was modeled under both autosomal dominant and recessive models, assuming 80% penetrance of the disease allele and allowing for 10% phenocopies. The population prevalence of the disorder was estimated to be 5/100,000. Two-point linkage analyses, including maximum log of odds (LOD) scores, were conducted using the MLINK and ILINK programs with FASTLINK (version 4.0p) (17, 18, 19), a faster version of the general pedigree programs of LINKAGE (20). Standard exclusion criteria (i.e. LOD score -2.0 or less) (21) were used to exclude linkage to a genomic region under the parameters of the model tested. Nonparametric linkage analyses were conducted using the simulation-based, identity by descent method (SIMIBD) described by Davis et al. (22) and require no a priori assumptions with respect to the underlying genetic model parameters.
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Results |
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Inheritance of PHP 1b was consistent with autosomal dominant transmission, as affected members are present in each generation. Families K and R contained members who were obligate gene carriers with no evidence of PTH resistance, similar to the pseudopseudohypoparathyroidism seen in PHP 1a kindreds. This pattern of inheritance is consistent with genetic imprinting of the paternal allele.
Gs analysis
Affected and unaffected members from family M (9;13) had normal
Gs activity in erythrocyte membranes (Table 1
). Affected and unaffected members of family K had normal
Gs protein levels (Table 1). Moreover, GNAS1
exons 1–13 and the intron/exon borders from one affected member of
each family were screened for mutations using DGGE, and no mutations
were identified.
Linkage analysis of the PTH/PTHrP receptor in PHP 1b
Subjects from families 1 and 2 were genotyped using a PTH/PTHrP
receptor intragenic polymorphism, a C to T transition at base 1417 in
exon 13 (Fig. 2, A and C) (23). The
segregation pattern of informative alleles of the exon 13 polymorphism
was compared with the segregation of the PHP 1b phenotype (Fig. 3). The PTH/PTHrP receptor polymorphism
was informative for linkage in these two families. In family M, the two
affected siblings in the second generation (MII-1 and MII-3) share the
C allele. If the PTH/PTHrP receptor were segregating with the PHP 1b
locus, then the C allele would be present in all affected individuals.
In the third generation of this family, there are two affected
individuals with the C/C genotype (MIII-2 and MIII-4). However, MIII-6
is affected, yet has not inherited a C allele from her affected mother.
This pattern of inheritance is discordant with linkage of PHP 1b to the
PTH/PTHrP receptor. In family K, the two obligate gene carriers in the
second generation share the C allele (KII-3 and KII-5). In the
offspring of KII-5, an obligate gene carrier, both affected and
unaffected individuals have the C/C genotype (KIII-5, KIII-8, and
KIII-9). KIII-9 is unaffected, yet inherited the C allele from her
mother. This pattern of segregation is discordant with linkage of PHP
1b to the PTH/PTHrP receptor.
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An abnormal PTH molecule was evaluated as a candidate for PHP 1b.
Direct sequencing demonstrated normal prepro-PTH genes in affected
individuals from families M, K, and R. We next evaluated whether
intragenic polymorphisms in exons 2 and 3 of the prepro-PTH gene would
be useful for linkage analysis (Fig. 2B) (24). Only family M was
informative for a PTH gene polymorphism (Fig. 4), the exon 3 polymorphism that
conserves the arginine residue at codon 52 (CGA to AGA) (24). The
segregation pattern of the polymorphic alleles was compared to the
segregation pattern of the PHP 1b phenotype. Two affected siblings in
the second generation share an A allele (MII-1 and MII-3), which should
segregate with the disease phenotype if the PTH gene and PHP 1b are
linked. However, there are no individuals in the third generation with
an A allele despite the fact that there are three affected siblings
(MIII-2, MIII-4, and MIII-6). This pattern of segregation is discordant
with linkage to the PHP 1b locus.
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Subjects from families M, K, and R were genotyped with three
polymorphic microsatellite markers flanking the PTH2R locus and
spanning 20.5 cM. A total of 14 affected, 3 obligate carriers, and 29
unaffected were genotyped. As there was a minor mapping discrepancy
(GeneMap ’98), the PTH2R could be localized only to a 4.1-cM area
encompassing D2S325 (Fig. 5). The marker
closest to the PTH2 receptor locus, D2S325, had a LOD score of -6.51
(
= 0) and excluded 3 cM around the marker. According to 1 map
position for the PTH2R, this would definitively exclude this locus as a
candidate for PHP 1b. However, it does not definitively exclude the
entire 4-cM region that includes the second possible locus for the
PTH2R. The marker D2S117 is 10.2 cM centromeric to the PTH2R. The LOD
score observed for this marker was -6.09 ( = 0), with an
exclusion of 0.4 cM on each side of the marker. The maximum LOD score
for D2S117 was 0.46 ( = 0.16). The marker D2s164 was 6 cM
telomeric of the PTH2 receptor. The LOD score was -10.49 ( =
0), with 4.7 cM of exclusion on each side of the marker. The amount of
exclusion around these markers paired with the negative LOD scores for
each of these markers in a 20-cM region provide evidence excluding the
PTH2 receptor as the PHP 1b locus. Furthermore, analysis of 11 markers
(D2S151 to D2S125) covering 110 cM of chromosome 2q revealed no areas
of linkage, with a maximum LOD score of only 0.34 achieved for marker
D2S364, located 20 cM centromeric to the PTH2R.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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A second form of the PTH receptor, type 2 (PTH2R), has a 51% amino acid sequence identity with the PTH/PTHrP receptor and is potently activated by PTH, but not by PTHrP (39). The PTH2R is most abundantly expressed in the brain, with lower levels present in the pancreas, testis, and placenta (39). The physiological role of the receptor is currently unknown; however, the PTH2R is not abundant in bone or kidney, where PTH exerts its most potent effects and where PTH resistance is manifested clinically. Usdin et al. mapped the chromosomal location of the PTH2 receptor to chromosome 2q33 using fluorescence in situ hybridization (40). Although a less likely candidate disease gene, the compelling hypothesis that a PTH receptor mutation would explain the pathogenesis of PHP 1b led us to examine markers flanking the PTH2R locus for evidence of linkage to PHP 1b. This analysis revealed no evidence of linkage of PHP 1b to the PTH2R locus. Although the significantly negative LOD scores are strong evidence against the PTH2R as the PHP 1b locus, our studies exclude only one PTH2R map locus. The alternative map locus for the PTHR2 gene, 4 cM telomeric to D2S325, cannot be definitively excluded without analyzing markers closer to that locus.
The PTH gene itself is a plausible candidate disease gene for PHP 1b, as PTH resistance could result from a biologically inactive PTH that acts as a competitive inhibitor of the PTH/PTHrP receptor. A circulating inhibitor of PTH action has been proposed as a cause of PTH resistance on the basis of studies showing an apparent dissociation between plasma levels of endogenous immunoreactive and bioactive PTH in subjects with PHP type 1. Despite high circulating levels of immunoreactive PTH, the levels of bioactive PTH in many patients with PHP type 1 have been found to be within the normal range when measured with highly sensitive renal (41) and metatarsal (42) cytochemical bioassay systems. Furthermore, plasma from many of these patients has been shown to diminish the biological activity of exogenous PTH in these in vitro bioassays (43). Currently, the nature of this putative inhibitor or antagonist remains unknown. The observation that prolonged hypercalcemia can remove or significantly reduce the level of inhibitory activity in the plasma of patients with PHP has suggested that the parathyroid gland may be the source of the inhibitor. In addition, analysis of circulating PTH immunoactivity after fractionation of patient plasma by reverse phase high performance liquid chromatography has disclosed the presence of aberrant forms of immunoreactive PTH in many of these patients (44). The recent identification of circulating amino-terminal-truncated PTH fragments in serum from normal subjects and patients with primary and uremic hyperparathyroidism now provide at least a theoretical basis for this hypothesis (45, 46, 47). These fragments consist of at least the 7–84 sequence of hPTH and probably lack biological activity. The existence of these fragments, which are also present in parathyroid tissue (46, 47), raises the possibility that mutant forms of PTH that lack bioactivity may bind to the PTH/PTHrP receptor and thereby inhibit binding by physiologically active hPTH-(1–84). Our data exclude the PTH gene as the disease locus in PHP 1b based on a discordant pattern of inheritance using a PTH gene intragenic polymorphism and direct sequencing of the prepro-PTH gene in our PHP 1b kindreds.
Linkage discordance of PHP 1b to the PTH/PTHrP and PTH2 receptors remind us that receptor mutations do not account for all forms of hormone resistance. For example, although mutations in the TSH receptor gene have been reported in many families with TSH resistance (48), affected members of other kindreds have structurally normal TSH receptor genes (49) or show linkage discordance with clinical TSH resistance (50).
Our data do not exclude candidate genes that might reduce the
responsiveness of the PTH/PTHrP receptor to PTH in the kidney. Although
proteins such as G protein receptor kinases are attractive candidates,
the complexity of PTH signaling makes comprehensive evaluation using a
candidate gene approach daunting. An alternative approach is to perform
a genome scan to identify genes that are tightly linked to PHP 1b.
Recently, Jueppner et al. published the linkage data of four unrelated
PHP 1b kindreds. The criteria for establishing the diagnosis of PHP 1b
was based only on the presence of PTH resistance and the absence of
AHO; Gs levels were not measured (51). They
established linkage to 20q13.3, a region that includes the GNAS1 gene
encoding Gs, the defect in PHP 1a. The
chromosomal region most tightly linked to the phenotype in these
kindreds was an area centromeric to GNAS1. Although these data are
consistent with the existence of a second gene involved in mineral ion
homeostasis in close proximity to GNAS1 on chromosome 20q, their
linkage analysis did not exclude GNAS1 (51) or defects in the promoter
region of GNAS1 that might limit Gs deficiency
to the kidney. Alternatively, some (or all) of these families might
represent unusual forms of PHP 1a in which GNAS1 gene mutations do not
lead to AHO. In the families presented here, we have excluded a
generalized defect in Gs as evidenced by
normal Gs protein levels and structurally
normal GNAS1 genes. In a preliminary study we undertook a systematic
search for linkage in affected members of these three families with PHP
1b by genotyping subjects with 384 polymorphic microsatellite markers
at an average intermarker distance of 10 cM. Linkage analysis suggested
linkage to the region containing GNAS1 on chromosome 20q13. Thus, our
data suggest that PHP 1b may be due to unusual defects in GNAS1, or to
defects in as yet undisclosed genes that specifically alter PTH
signaling. Additional genetic studies of PHP 1b will be necessary to
identify the gene locus or loci and the pathophysiology of PTH
resistance in this disorder.
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Acknowledgments |
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Footnotes |
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2 These authors contributed equally to this work.
Received June 2, 1999.
Revised November 8, 1999.
Revised February 14, 2000.
Accepted February 21, 2000.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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subunit of the guanine nucleotide-binding protein
Gs as the molecular basis for Albright hereditary
osteodystrophy. Proc Natl Acad Sci USA. 85:617–621.-subunit of the stimulatory G-protein of adenylyl cyclase in
patients with Albright’s hereditary osteodystrophy. J Clin
Endocrinol Metab. 71:1208–1214.[Abstract]
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