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Department of Pediatrics, Chiba University Graduate School of Medicine (M.M., T.Y., T.W., K.M., Y.T., Y.K.), Inohana, Chuo-ku, Chiba 260-8670, Japan; Department of Pediatrics, National Chiba Hospital (T.Y.), Tsubakimori, Chuo-ku, Chiba 260-8606, Japan; Departments of Physiology and Medicine (D.G., J.H.W., G.N.H.) and Human Genetics (G.N.H.), McGill University, Montréal, Québec, Canada H3G 1Y6; and Calcium Research Laboratory, McGill University, Royal Victoria Hospital (D.G., G.N.H.), Montréal, Québec, Canada H3A 1A1
Address all correspondence and requests for reprints to: Toshiyuki Yasuda, M.D., Department of Pediatrics, National Chiba Hospital, Tsubakimori, Chuo-ku, Chiba 260-8606, Japan. E-mail: . toshi{at}chiba.hosp.go.jp
Abstract
The PTH/PTHrP receptor (PTHR1) plays an essential role in skeletal development and mediates many other functions of PTH and PTHrP. Human PTHR1 gene transcription is controlled by three promoters, P1–P3. The most proximal promoter, P3, is active in bone and osteoblast-like cell lines and accounts for the majority of renal transcripts in adults. We have identified a tetranucleotide repeat (AAAG)n polymorphism in the P3 promoter. In 214 unrelated Japanese, the repeat number (n) ranged from 3–8, with the AAAG5 allele being the most frequent (59%). In 55 unrelated Caucasians, n ranged from 5–7, and the frequency of the AAAG5 allele was 78%. The most frequent genotypes in a cohort of 85 young (18–20 yr) female Japanese were 5/5, 5/6, and 6/6. The 6/6 genotype was associated with greater height (5/5 vs. 6/6; P < 0.02) and lower urinary deoxypyridinoline and pyridinoline (P < 0.02), which are markers of bone resorption. The height of an additional 71 healthy female Japanese subjects, aged 14–17 yr, having genotype 5/5, 5/6, or 6/6 was also in the order of genotype 5/5 < 5/6 < 6/6 (5/5 vs. 6/6, P < 0.05). There was no significant difference in lumbar and femoral bone mineral density between genotypes. Likewise, there was no difference in circulating intact PTH levels between groups. The activity of P3 promoter-luciferase reporter constructs in transcription assays in 2 human osteoblast-like cell-lines varied according to repeat number, with AAAG6 being the least active. In conclusion, the P3 promoter (AAAG)n polymorphism is frequent in both Japanese and Caucasians and has potential as a linkage marker for the PTHR1 locus. In addition, it may influence the expression of the receptor in target tissues and have functional consequences on the developing skeleton.
PTH AND THE hormonally active metabolite of vitamin D, 1,25-dihydroxyvitamin D, are the principal regulators of calcium homeostasis. PTH exerts its calciotropic effects by acting on target tissues, bone and kidney (1). The PTHrP, originally discovered as the cause of hypercalcemia of malignancy, is a second member of the PTH family (2). PTHrP acts in many tissues as an autocrine/paracrine factor to regulate both cell proliferation and differentiation. In the bone growth plate, PTHrP regulates the differentiation of prehypertrophic chondrocytes into hypertrophic chondrocytes and inhibits their apoptosis, which precedes bone synthesis in the process of endochondral bone formation (3).
The actions of both PTH and PTHrP are mediated through the PTH/PTHrP receptor (PTHR1) with similar efficacy (4). This receptor belongs to the vast family of G protein-coupled receptors containing seven transmembrane domains. Binding of ligand(s) can stimulate the production of intracellular cAMP and IP3 (5).
We have cloned and characterized well conserved promoters (P1 and P2) of the mouse and human PTHR1 gene (6, 7, 8) and, more recently, have identified and characterized a third promoter, P3, that is highly expressed in the human, but not in the mouse (9). The human P3 promoter is (G+C) rich and contains Sp1 consensus binding sequences as well as an A-rich sequence. The P3 promoter in the human PTHR1 gene is the main promoter in kidney and bone (9).
Recently, we have identified an (AAAG)n polymorphism in the P3 promoter region of the PTHR1 gene (10). As the PTHR1 plays major roles in mediating both endocrine PTH actions in bone and kidney and also the paracrine/autocrine PTHrP action in endochondral bone growth (2), we examined the frequency of this polymorphism and its relation to adult height, bone mineral density (BMD), bone resorption markers, and PTH levels in a Japanese population. We found that this polymorphism is prevalent in both Japanese and Caucasians and that there are significant relationships between adult height and bone resorption markers and the repeat number.
Subjects and Methods
Subjects
Eighty-five healthy Japanese women, aged 18–20 yr (height SD score, -2.2 to +2.4; body mass index, 18.4–27.3), were recruited (group A). All of them had regular menses and undertook normal physical activity. After overnight fasting, blood was drawn between 0700–0800 h for measurement of biochemical parameters and DNA isolation, and the second voided morning urine was collected. Lumbar and femoral neck BMDs were measured by dual energy x-ray absorptiometry using QDR-1000 (Hologic, Inc., Waltham, MA) as previously described (11). Genomic DNA was also obtained from 129 healthy Japanese (group B) and 55 normal Caucasians (group C) for analysis of the PTHR1 gene polymorphism. Among group B, we analyzed height in 71 healthy female subjects, aged 14–17 yr, with genotypes 5/5, 5/6, and 6/6. Informed consent was obtained from each participant.
Biochemical parameters
In group A, we measured serum PTH, urinary deoxypyridinoline, and pyridinoline as markers for parathyroid function and bone resorption, respectively, and we also measured serum calcium, phosphorus, alkaline phosphatase, and 1,25-dihydroxyvitamin D. Serum PTH was measured in two assays by a midregion-specific RIA (High Sensitive PTH, Yamasa Shoyu Co. Ltd., Chiba, Japan) and an immunoradiometric assay (Intact PTH, Nichols Institute Diagnostics, San Juan, CA). Urinary deoxypyridinoline and pyridinoline were measured by HPLC and were expressed as a ratio of urinary creatinine (deoxypyridinoline/creatinine, pyridinoline/creatinine; nanomoles per mmol). Serum calcium and phosphorus were measured colorimetrically in a Hitachi 764 autoanalyzer (Hialeah, FL).
DNA isolation and PCR amplification
DNA was isolated from peripheral blood using standard procedures, and nested PCR was employed for amplification of the minimal region of the P3 promoter including the A-rich region (Fig. 1A
). PCR was performed using 20 ng genomic DNA or 0.4 µl of the first PCR product, 15 pmol of each primer, 200 µmol/liter dNTPs, 1.5 mmol/liter MgCl2, 4% dimethylsulfoxide, 1 x Expand HF buffer, and 1 U enzyme mix of the Expand High Fidelity PCR System (Roche Molecular Biochemicals, Tokyo, Japan) in a total volume of 20 µl. The sequences of the forward primer and reverse primer for the first PCR are: P3(-254), 5'-AATAACAGGTTCCTGCGCGC-3'; and P3R(+205), 5'-GGGTGCAGAGCTGCGTCAGG-3', respectively. Samples were cycled at 95 C for 50 sec, 65 C for 1 min, and 72 C for 1 min for 15 cycles, and then at 95 C for 50 sec, 63 C for 1 min, and 72 C for 1 min and 15 sec for 25 cycles, followed by 10 min at 72 C. The sequences of the forward primer and reverse primer for the second PCR are: P3(-175), 5'-GAAGCCACAGCTCCCATTTC-3'; and P3R(-34), 5'-TGCCTCGGAGCGAAGAAATC-3', respectively. Samples were cycled at 95 C for 50 sec, 64 C for 30 sec, and 72 C for 20 sec for 40 cycles, followed by 6 min at 72 C. The final PCR products were separated on an 8% polyacrylamide gel (Fig. 1B) and were purified with a Qia-Quick gel extraction kit (QIAGEN, Hilden, Germany). Nucleotide sequencing was performed on several different samples from each individual using a model 373A automated sequencer (PE Applied Biosystems, Foster City, CA) with a Taq DyeDeoxy terminator cycle sequencing kit (PE Applied Biosystems) to confirm the AAAG repeat number.
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The sequences of the forward primer and reverse primer of the PCR are P3(-181), 5'-CGCGGATCCTGGGGCGAAGCCACAGCTCC-3' and P3R(+205), 5'-GCTCTAGAGGGTGCAGAGCTGCGTCAGG-3', respectively (underlined sequences represent restriction enzyme sites added to facilitate subcloning). The PCR conditions were as described above. Samples were cycled at 95 C for 50 sec, 69 C for 1 min, and 72 C for 1 min for 15 cycles, and then at 95 C for 50 sec, 67 C for 1 min, and 72 C for 1 min 15 sec for 25 cycles, followed by 10 min at 72 C. PCR products were subcloned into the pBluescript SK- plasmid and sequenced [T7 sequencing kit (Pharmacia Biotech, Uppsala, Sweden)]. The PCR fragments that included different (AAAG)n repeats (where n = 3, 5, 6, 7, or 8) were inserted into the polylinker of the luciferase reporter plasmid pXP2 (9). Human osteosarcoma SaOS2 and HOS cell lines were propagated in DMEM (Life Technologies, Inc., Tokyo, Japan) in 10% FBS (Life Technologies, Inc.). Cells were seeded in six-well plates at approximately 35% confluence, and the next day transfections were performed with Effectene Transfection Reagent (QIAGEN, Tokyo, Japan) using 0.6 µg luciferase reporter plasmid DNA and 0.2 µg ß-galactosidase expression vector P610AZ. Cells were harvested 48 h later, and extracts were prepared by lysing cells in 250 µl reporter lysis buffer (Promega Corp., Tokyo, Japan), of which 50 µl were used for both ß-galactosidase assay (for normalization of transfection efficiency) and luciferase assay (Promega Corp.). Results are the mean ± SEM of three independent experiments.
Statistical analysis
Statistical significance was determined by one-way ANOVA and Fisher’s protected least significance difference using StatView J 4.02 software (Abacus Concepts, Inc., Berkeley, CA), and the data are presented as the mean ± SD unless otherwise noted.
Results
The (AAAG)n polymorphism in Japanese and Caucasian populations
In the Japanese population (groups A and B) the following allele frequencies were found: n = 3, 1.9%; n = 4, 0%; n = 5, 59.3%; n = 6, 36.2%; n = 7, 2.3%; and n = 8, 0.2%. The genotype frequencies were 3/3, 0.5%; 3/5, 1.9%; 3/6, 0.9%; 5/5, 35.0%; 5/6, 44.0%; 5/7, 2.8%; 6/6, 13.1%; 6/7, 1.4%; and 7/8, 0.5%, with a heterozygote frequency of 32.7%. In the Caucasian population (group C) the allele frequencies were: n = 5, 78.2%; n = 6, 20.0%; and n = 7, 1.8%. The genotype frequencies were: 5/5, 63.6%; 5/6, 29.1%; 6/6, 3.6%; and 6/7, 3.6%, with a heterozygote frequency of 59.5%. Thus, the AAAG5 is the most common in both populations and is more frequent in Caucasians.
Genotype, anthropometric characteristics, and serum biochemistries
The genotypes 5/5, 5/6, and 6/6 comprise 92% of Japanese individuals (see above). The genotypes, height, weight, and serum biochemistries of group A subjects are shown in Table 1. Individuals of the 6/6 genotype were of significantly greater height (5/5 vs. 6/6, P < 0.02; Fig. 2A), whereas serum calcium, phosphate, intact PTH, 1,25-dihydroxyvitamin D, and alkaline phosphatase levels were not different between groups (Table 1). However, genotype 6/6 individuals had significantly higher circulating midregion PTH levels (5/5 vs. 6/6, P < 0.05). The height in 71 female subjects, aged 14–17 yr, in group B was 5/5 < 5/6 < 6/6 (5/5 vs. 6/6, P < 0.05; Fig. 2B). The height in group A did not correlate with biochemical parameters (PTH, urinary pyridinoline and deoxypyridinoline).
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Individuals with the 6/6 genotype excreted significantly lower concentrations of the bone resorption markers deoxypyridinoline and pyridinoline than those with 5/5 and 5/6 genotypes (Table 1
and Fig. 3). However, there was no significant difference in either lumbar spine or femoral neck BMD shortly after achieving peak bone mass between genotypes (Fig. 4).
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The activities of PTHR1 P3 promoter-luciferase constructs representing all identified polymorphic variants were analyzed by transiently transfecting recombinants into human osteoblast-like SaOS-2 and HOS cell lines. Significant differences were observed in promoter activity between various constructs (Fig. 5) demonstrating that the AAAG repeat number influences the P3 promoter activity. Promoter activity was inversely related to repeat numbers from 3–6, and an increase in transcriptional activity was noted for AAAG8.
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The P3 promoter in the human PTHR1 gene is active in target tissues, bone and kidney. In the present study we identified a tetranucleotide repeat (AAAG)n polymorphism in this promoter region in both Japanese and Caucasians, with the AAAG5 allele being the most frequent in both. The 5/6 genotype comprises 44% of Japanese subjects and is predominant, whereas the genotype 5/5 accounts for 64% of Caucasians. Thus, this polymorphism is frequent, and it could be a useful tool in linkage analysis of quantitative traits to the PTHR1 locus.
Our previous studies have shown that the AAAG repeat region represses P3 promoter activation in gene transfer experiments performed in human osteoblast-like SaOS-2 and HOS cells (12). In the present study the functionality of the expansion and contraction of the AAAG repeat number was demonstrated in vitro by transfecting P3 promoter/reporter constructs into SaOS-2 and HOS cells. Promoter function varied according to repeat number, with the AAAG6 variant exhibiting the least activity. If this finding is reflected by the relative expression level of the PTHR1 alleles in chondrocytes and bone cells in vivo, then the predicted alterations in PTHR1 levels could be associated with parameters such as adult height and bone formation and resorption in young adults.
Longitudinal growth is under strong genetic control, as evidenced by the effect of parental height on child height and final height attained, which is manifested by developmental canalization in normal height in children (13, 14). The PTHR1, which mediates the effects of PTHrP in the transitional zone of the growth plate, is critical in ensuring the appropriate temporal and spatial control of endochondral bone formation (2, 15, 16). Hence, the PTHR1 gene is a strong candidate to be one of the important factors influencing bone growth and final height. The importance of the PTHR1 in bone growth is well illustrated in two rare genetic disorders, Jansen’s metaphyseal chondrodysplasia (17, 18) and Blomstrand lethal chondrodysplasia (BLC) (19, 20). Jansen’s metaphyseal chondrodysplasia is characterized by short-limbed dwarfism secondary to severe growth plate abnormalities and increased bone resorption and is due to heterozygous gain of function mutations in the PTHR1 gene giving rise to constitutively active receptors. BLC is characterized by advanced endochondral bone maturation, short-limbed dwarfism, and fetal death and is due to inactivating mutations in the PTHR1 gene. The majority of BLC cases were born to phenotypically normal, consanguineous parents, suggesting an autosomal recessive mode of inheritance. Thus, both loss and gain of function mutations in the PTHR1 give rise to extreme short-limbed dwarfism, which may indicate that fine regulation of PTHR1 expression in the growth plate is critical for optimum height growth. Indeed, in the present study it was found that the PTHR1 genotype was associated with adult stature, with the 6/6 genotype group having a significantly greater mean adult height compared with other groups. In functional studies we demonstrated that this genotype is associated with lower promoter activity, suggesting that a small, but significant, difference in PTHR1 promoter activity could contribute to the difference in adult height.
There was no difference in serum alkaline phosphatase levels among groups, indicating that osteoblast activities with respect to bone formation were not different. However, differences were found with respect to bone resorption markers, and the 6/6 genotype group had lower urinary levels of the collagen breakdown products, deoxypyridinoline and pyridinoline (21). This is likely to represent the role that osteoblastic PTHR1 signaling plays in modulating osteoclastogenesis and/or osteoclast activity via osteoclast differentiation factor and its receptor (22). Cyclical PTH administration in postmenopausal women leads to increased trabecular BMD (23). In addition, suggestive linkage of the PTHR1 locus to BMD was found in subjects (mean age, 50 yr) from families with osteoporosis (24). However, in the present study no differences were observed in BMD at lumbar spine and femoral neck sites among genotype groups. The balance between bone resorption and formation is important in maintaining peak BMD once achieved, but the contribution that PTHR1 makes to the overall process may only become obvious later in life. Therefore, this would not be apparent in the population studied here shortly after achieving peak bone mass.
Serum intact PTH levels were not different among genotypes, suggesting that there was no alteration in parathyroid gland function. However, lower circulating midregion PTH levels were found in the 5/5 genotype group relative to others. This could point to a link between PTHR1 activity in tissues that metabolize PTH (kidney and liver) and the rate at which PTH is cleared from the circulation (25).
In summary, we have identified a polymorphism in a tetranucleotide AAAG repeat region of the human PTHR1 gene promoter that functions as a repressor sequence. Gene transfer experiments showed that promoter recombinant containing the AAAG6 variant displayed the lowest promoter activity. The 6/6 genotype group of a cohort of young adult Japanese women was of significantly greater mean adult height compared with the 5/5 group and had significantly lower levels of urinary markers of bone resorption. These results point to the PTHR1 gene as one of the important genetic factors influencing bone growth and final height.
Acknowledgments
Footnotes
This work was supported by grants from the Japanese Ministry of Education, Culture, Sports, Science, and Technology (12670728); the Japanese Ministry of Health, Labor, and Welfare; the 5th and 6th Novo Nordisk Awards; and the Novo Nordisk fund for Pediatric Study Group of Molecular Endocrinology.
Abbreviations: BLC, Blomstrand lethal chondrodysplasia; BMD, bone mineral density; PTHR1, PTH/PTHrP receptor.
Received February 13, 2001.
Accepted January 10, 2002.
References
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