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The Journal of Clinical Endocrinology & Metabolism Vol. 85, No. 6 2239-2246
Copyright © 2000 by The Endocrine Society

Original Studies

Pseudohypoparathyroidism 1b: Exclusion of Parathyroid Hormone and Its Receptors as Candidate Disease Genes1

Suzanne M. Jan de Beur2, Chang-Lin Ding2, Michele C. LaBuda, Ted B. Usdin and Michael A. Levine

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.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Pseudohypoparathyroidism 1b (PHP 1b) is characterized by specific resistance of target tissues to PTH, but no mutations in the PTH/PTH-related peptide (PTHrP) receptor gene have been identified. To investigate the basis for defective PTH signaling, we used polymorphic markers in or near the genes encoding PTH and its receptors to perform linkage analysis between these loci and PHP 1b. Two multiplex PHP 1b families (families M and K) were informative for an intragenic polymorphism in exon 13 of the PTH/PTHrP receptor gene detected by PCR amplification and resolved by denaturing gradient gel electrophoresis. Linkage analysis revealed discordance of the PTH/PTHrP receptor with PHP1b. One PHP 1b kindred (family M) was informative for a intragenic polymorphism in exon 3 of the PTH gene detected by PCR amplification and resolved by denaturing gradient gel electrophoresis. The PTH gene polymorphism segregation was discordant with PHP 1b. Probands from each family had normal PTH genes by direct sequence analysis. In three PHP 1b kindreds, we analyzed simple sequence polymorphisms in three microsatellite markers flanking the PTH type 2 receptor locus located at 2q33. Linkage analysis demonstrated no linkage. In conclusion, neither the PTH gene nor the PTH receptor genes (type 1 and 2) are linked to PHP 1b.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
PSEUDOHYPOPARATHYROIDISM (PHP) is a heterogeneous disorder characterized by resistance to the biological actions of PTH. Patients with PHP type 1 fail to show an increase in urinary excretion of nephrogenous cAMP and phosphate in response to PTH infusion (1). These observations suggested that the molecular defect in PHP type 1 resides within the PTH receptor, adenylyl cyclase, or the heterotrimeric GTP-binding protein (Gs) that couples the PTH receptor to activation of adenylyl cyclase (2).

Tissues that have been analyzed from patients with PHP 1a have a 50% reduction in the expression and activity of Gs{alpha} (3, 4) due to heterozygous mutations in the gene encoding the {alpha}-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{alpha} 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{alpha} 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{alpha} 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.


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

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{alpha} (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. 1Go). The biochemical characteristics are summarized in Table 1Go. Detailed clinical characteristics, biochemical data, and normal Gs{alpha} 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. 1Go). Members of family K were also characterized biochemically and phenotypically, and levels of erythrocyte Gs{alpha} protein were normal by immunoblot analysis (Table 1Go). 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. 1Go). 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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  		Figure 1. Family pedigrees. Circles indicate females; squares indicate males. Symbols for unaffected individuals are shaded gray, and solid symbols indicate affected individuals. Individuals that were not analyzed in this study are represented by open symbols, and obligate gene carriers without clinical PTH resistance are represented by cross-hatched symbols. The pattern of inheritance is consistent with an autosomal dominant inheritance, with affected individuals in each generation.

 

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  		Table 1. Biochemical characteristics of PHP 1b families

 
PTH infusion

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.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Pedigree analysis

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{alpha} analysis

Affected and unaffected members from family M (9;13) had normal Gs{alpha} activity in erythrocyte membranes (Table 1Go). Affected and unaffected members of family K had normal Gs{alpha} protein levels (Table 1Go). 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. 2Go, 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. 3Go). 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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  		Figure 2. A, Denaturing gradient gel electrophoresis of the PTH/PTHrP receptor (PTH1R) gene exon 13 polymorphism. The C allele migrates more quickly than the T allele. The C/C genotype is shown in the left lane, the C/T genotype is shown in the middle lane, and the T/T genotype is shown in the far right lane. Heteroduplexes, which migrate more slowly than homoduplexes, are formed in individuals with the C/T genotype. B, Denaturing gradient gel electrophoresis of the PTH gene exon 3 polymorphism. The C allele migrates more quickly than the A allele. Heteroduplexes of the C and A alleles migrate more slowly than either the C homoduplexes or the A homoduplexes. The A/A genotype is shown in the left lane, the C/C genotype is shown in the middle lane, and the A/C genotype is shown in the right lane. C, Sequence analysis of the PTH/PTHrP receptor gene polymorphism demonstrating both the C and T alleles at base 1417.

 


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  		Figure 3. A, Pedigree of family M labeled with the PTH/PTHrP receptor genotype. Affected individuals in generation MII share the C allele. Therefore, affected individuals in generation MIII must have a C allele, and unaffected individuals must not have two C alleles (C/C). The affected individual with the T/T genotype (MIII-6) is inconsistent with linkage to the PHP 1b phenotype. Individuals that are discordant with linkage are marked with an asterisk. B, Pedigree of family K labeled with the PTH/PTHrP receptor genotype. Obligate gene carriers in generation KII share the C allele. Therefore, if there is linkage, then the PHP 1b phenotype must segregate with the C allele. As both affected and unaffected individuals in generation KIII have the C/C genotype (KIII-5, -8, and -9), there is no linkage of the C allele to the PHP 1b trait. Individuals that are discordant with linkage are marked with an asterisk. Specifically, individual KIII-9 (C/C) excludes linkage of PHP 1b to the C allele.

 
Linkage analysis of the PTH gene in PHP 1b

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. 2BGo) (24). Only family M was informative for a PTH gene polymorphism (Fig. 4Go), 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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  		Figure 4. Pedigree of family M labeled with the PTH gene genotype. Affected individuals in generations HII-1 and HII-3 share an A allele. Because none of the affected individuals in generation HIII has an A allele (MIII-2, MIII-4, and MIII-6), there is linkage discordance with the PHP 1b phenotype (individuals marked with an asterisk).

 
Linkage analysis of the PTH2 receptor

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. 5Go). The marker closest to the PTH2 receptor locus, D2S325, had a LOD score of -6.51 ({theta} = 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 ({theta} = 0), with an exclusion of 0.4 cM on each side of the marker. The maximum LOD score for D2S117 was 0.46 ({theta} = 0.16). The marker D2s164 was 6 cM telomeric of the PTH2 receptor. The LOD score was -10.49 ({theta} = 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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  		Figure 5. A, Fine map of chromosome 2q33. An enlargement of the area around the PTH2R locus is shown in the right panel. The relationship of the enlarged area to the entire chromosome 2 is shown on the left. Microsatellite markers are labeled with intervening genetic map distances (centiMorgans). The two published PTH2R locations (minor discrepancies exist in the physical map) are depicted in shaded gray boxes near D2S325 and approximately 4 cM telomeric to D2S325. Areas with cross-hatched bars represent the area definitively excluded based on a LOD score of -2 or less. B, TableGo of the LOD scores at each of the microsatellite markers at 0-, 5-, and 10-cM intervals. The exclusion around each marker is determined by the greatest distance at which a LOD score of -2 is observed. The calculated exclusions are in the right column.

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
PTH exerts its cellular effects on bone and kidney by binding to transmembrane receptors that are coupled via G proteins to signal generators adenylyl cyclase and phospholipase C. These enzymes produced second messengers that ultimately activate pathways that result in target tissue hormone action. Failure of patients with PHP 1b to generate cAMP in response to PTH infusion suggests that the signaling defect lies between the PTH/PTHrP receptor and generation of cAMP. Mutations in genes that encode G protein-coupled receptors are the molecular basis for several other syndromes of hormonal resistance, including some forms of nephrogenic diabetes insipidus (type 2 vasopressin receptor) (25), inherited nongoitrous hypothyroidism (TSH receptor) (26), and familial dwarfism (GHRH receptor) (27, 28, 29, 30). Evidence supporting the hypothesis that PTH/PTHrP receptors are defective in PHP 1b comes from studies of skin fibroblasts from PHP 1b patients. Fibroblasts from some patients with PHP 1b accumulated less cAMP in response to PTH than fibroblasts from normal subjects (31); furthermore, cultured fibroblasts from some PHP 1b patients have reduced levels of messenger ribonucleic acid encoding the PTH/PTHrP receptor (32). These observations led to the hypothesis that reduced PTH responsiveness in PHP 1b patients might be due to defects in the gene encoding the PTH/PTHrP receptor. However, despite extensive analysis of the complementary DNA sequence (33, 34), coding exons and exon/intron boundaries (23), and the promoter regions of the PTH/PTHrP gene in patients with PHP 1b (35, 36), no mutations have been identified in PHP 1b. By contrast, inactivating mutations in the PTH/PTHrP receptor have recently been demonstrated in patients with Blomstrand chondrodysplasia, a lethal genetic syndrome characterized by advanced endochondral bone maturation (37, 38). Heterozygous subjects were phenotypically normal, suggesting that the loss of one receptor allele was insufficient to cause PTH resistance or abnormal mineral metabolism (38). Because PHP 1b appears to be inherited in an autosomal dominant manner, these observations make it unlikely that heterozygous haploinsufficiency of the PTH/PTHrP receptor could account for the PHP 1b phenotype. Our present molecular genetic studies provide additional experimental evidence that PHP 1b is not due to uncharacterized defects in the PTH/PTHrP receptor gene that might lead to the production of abnormal PTH receptors. Specifically, our data exclude the possibility of rearrangments in the upstream regulatory elements, alternative promoter regions, or intronic sequences of the PTH/PTHrp receptor that might cause PHP 1b. We cannot exclude the possibility that the PHP 1b gene regulates expression of the PTH/PTHrP receptor, however.

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{alpha} levels were not measured (51). They established linkage to 20q13.3, a region that includes the GNAS1 gene encoding Gs{alpha}, 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{alpha} 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{alpha} as evidenced by normal Gs{alpha} 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.


   Acknowledgments
 
We gratefully acknowledge the excellent technical assistance of Dr. Zhichao Deng, Laura Kasch, and the Methods Development Laboratory and DNA Analysis Facility of Johns Hopkins Genetic Resources Core Facility. We thank Drs. William Schwindinger and Dan Rogers for thoughtful review of the manuscript.


   Footnotes
 
1 This work was supported by NIH Grants R01-DK-46720 and T32-DK-07751, The Pearl M. Stettler Award for Women Physicians (to S.M.J.), and The Johns Hopkins University School of Medicine General Clinical Research Center (NIH/NCRR M01-RR00052). Back

2 These authors contributed equally to this work. Back

Received June 2, 1999.

Revised November 8, 1999.

Revised February 14, 2000.

Accepted February 21, 2000.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

  1. Chase LR, Melson GL, Aurbach GD. 1969 Pseudohypoparathyroidism: defective excretion of 3',5'-AMP in response to parathyroid hormone. J Clin Invest. 48:1832–1844.
  2. Levine MA. 1998 Pseudohypoparathyroidism. In: Avioli LV, Krane SM, eds. Metabolic bone disease, 3rd Ed., San Diego: Academic Press.
  3. Levine MA, Ahn TG, Klupt SF, et al. 1988 Genetic deficiency of the {alpha} subunit of the guanine nucleotide-binding protein Gs as the molecular basis for Albright hereditary osteodystrophy. Proc Natl Acad Sci USA. 85:617–621.[Abstract/Free Full Text]
  4. Levine MA, Eil C, Downs Jr RW, Spiegel AM. 1983 Deficient guanine nucleotide regulatory unit activity in cultured fibroblast membranes from patients with pseudohypoparathyroidism type I: a cause of impaired synthesis of 3':5'-cyclic AMP by intact and broken cells. J Clin Invest. 72:316–324.
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