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Department of Pediatrics, Chiba University School of Medicine (M.M., T.W., Y.K., T.Y.), Inohana, Chuo-ku, Chiba 260-8670, Japan; Division of Endocrinology and Metabolism, Saitama Children’s Medical Center (H.M.), Iwatsuki, Saitama 339-8551, Japan; Department of Physiology, McGill University (G.N.H., D.G., J.H.W.), Montréal, Québec, Canada H3G 1Y6; and Department of Medicine and Calcium Research Laboratory, McGill University (G.N.H., D.G.), and Royal Victoria Hospital, Montreal, Québec, Canada H3A 1A1
Address all correspondence and requests for reprints to: Toshiyuki Yasuda, M.D., Department of Pediatrics, Chiba University School of Medicine, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan. E-mail: toshi{at}med.m.chiba-u.ac.jp
Abstract
Hypocalcemia and hyperphosphatemia caused by PTH resistance are the only discernible abnormalities in pseudohypoparathyroidism type 1b (PHP-1b). Because of the selective resistance toward PTH, inactivating mutations in its receptor, the PTH/PTH-related peptide receptor (PTHR1), were thought to be responsible for PHP-1b. However, gene abnormalities responsible for PHP-1b have not been identified in the coding region and well conserved promoters (P1 and P2) of the PTHR1 gene. The purpose of the present study was to analyze the structure of the P3 promoter, the main promoter of the human PTHR1 gene in kidney, in patients with PHP-1b. Southern analysis of genomic DNA from lymphoblastoid cell lines of eight nonfamilial patients with PHP-1b revealed neither gross rearrangements nor methylation abnormalities in the P3 promoter region of the PTHR1 gene. Sequencing revealed no abnormalities in the P3 promoter region, although one patient was homozygous for an (AAAG)n polymorphic variant.
In conclusion, despite the selective resistance toward PTH in the kidney, which mainly uses the PTHR1 P3 promoter, PHP-1b in eight cases is not associated with structural abnormalities in this promoter. This study also indicates that inactivation of the P3 promoter is not achieved by methylation as tested in patients’ genomic DNA from lymphoblastoid cell lines. The influence of alterations in the polymorphic A-rich repeat sequence on promoter activity warrants further study.
PSEUDOHYPOPARATHYROIDISM (PHP) type 1 is a
group of disorders characterized by hypocalcemia and hyperphosphatemia
despite high PTH levels, and patients with this condition fail to show
an appropriate increase in urinary cAMP in response to exogenous PTH
infusion. It is usually classified into two groups, PHP-1a and PHP-1b.
Patients with PHP-1a have, in addition to PTH resistance, multiple
hormone resistance and Albright’s hereditary osteodystrophy,
manifested as short stature, brachydactyly and sc ossifications
(1). PHP-1a is caused by maternally transmitted
heterozygous inactivating mutations in the GNAS1 gene encoding the
-subunit of the stimulatory G protein
(Gs) (2), whereas the
paternally transmitted condition termed pseudo-PHP shows Albright’s
hereditary osteodystrophy without hypoparathyroidism.
Gs mutations lead to PTH resistance through
loss of Gs expression and thus explain at
least partially the resistance toward PTH and other hormones that
mediate their actions through G protein-coupled receptors. In contrast,
hypocalcemia and hyperphosphatemia caused by PTH resistance are the
only discernible abnormalities in PHP-1b. Because of the selective
resistance toward only this hormone, inactivating mutations in the
PTH/PTH-related peptide (PTHrP) receptor (PTHR1) could be responsible
for PHP-1b. However, such mutations were excluded for all coding and
noncoding exons of the PTHR1 gene (3, 4).
The PTHR1 belongs to the vast family of G protein- coupled
receptors containing seven transmembrane domains (5).
Binding of ligand can stimulate the production of intracellular cAMP
and inositol 1,4,5-trisphosphate (6). Silve et
al. (7) reported reduced levels of PTHR1 messenger
ribonucleic acid (mRNA) in some patients with PHP-1b in skin fibroblast
cells, suggesting an impairment in transcriptional regulation of the
PTHR1 gene. We previously cloned the 5'-regulatory regions of the mouse
(8, 9) and human PTHR1 genes, characterized well conserved
promoters (P1 and P2; see Fig. 1
), and
examined the structure of both P1 and P2 promoters in genomic DNA from
seven nonfamilial cases with PHP-1b. We found no structural abnormality
in those regions (10).
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Subjects and Methods
Patients and assay methods
The study subjects were eight Japanese PHP-1b patients, as shown
in Table 1. Serum PTH was measured by a
two-site immunoradiometric assay (Allegro intact PTH kit, Nichols Institute Diagnostics, San Juan Capistrano, CA) and/or by a
midregion specific RIA (high sensitive PTH, Yamasa Shoyu Co. Ltd.,
Chiba, Japan). The diagnostic criteria of five patients were reported
previously (10). All were apparently sporadic. In each
case a careful family history was made. Normal serum calcium,
phosphorus, and PTH levels of their siblings and parents were
documented in all families. In the family of patient 4, normal serum
calcium, phosphorus, and PTH levels were found in grandfathers
and grandmothers of both sides and the patient’s two daughters.
Lymphoblastoid cell lines derived from PHP-1b patients were established
by Epstein-Barr virus transformation. All patients provided informed
consent.
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DNA was isolated from whole blood and lymphoblastoid cell lines
using QIAGEN DNA kits (QIAGEN, Hilden,
Germany). Ten micrograms of genomic DNA were digested to
completion with ApaI, ApaI-HpaII, or
ApaI-MspI restriction endonuclease under the
conditions recommended by the supplier (New England Biolabs, Inc., Beverly, MA), electrophoresed on 2% agarose gels, and
transferred to Hybond-N+ membrane (Amersham Pharmacia Biotech, Arlington Heights, IL). Genomic DNA in the P3
promoter region was detected by probing with a
32P-labeled 515-bp SmaI fragment
extending from a naturally occurring SmaI site located 111
bp downstream of the 5'-P3 major transcription start site upstream to a
second site representing the 5'-end of an exonuclease III digestion
(see Fig. 1
). Conditions for hybridization and autoradiography were
described previously (12).
PCR amplification of PHP-1b patient genomic DNA and PCR-single strand conformation polymorphism (SSCP)
PCR was performed using 0.5 µg genomic DNA, 15 pmol of each
primer, 200 mmol/L deoxy-NTPs, 1.5 mmol/L MgCl2,
1 x Expand HF buffer, and 2.6 U enzyme mix of Expand High
Fidelity PCR System (Roche Molecular Biochemicals,
Mannheim, Germany) in a total volume of 50 µL. PCR primers are
designed to encompass the A-rich region and the minimal promoter region
(see Fig. 1). The forward primer and reverse primer are: P3(-181),
5'-CGCGGATCCTGGGGCGAAGCCACAGCTCC-3'; and P3R(+205),
5'-GCTCTAGAGGGTGCAGAGCTGCGTCAGG-3', respectively (see Fig. 1; underlined sequences represent restriction enzyme sites
added to facilitate subcloning). Samples were cycled at 95 C for
50 s, 69 C for 1 min, and 72 C for 1 min for 15 cycles, and then
at 95 C for 50 s, 67 C for 1 min, and 72 C for 1 min and 15 s
for 25 cycles, followed by 72 C for 10 min. For PCR-SSCP
[32P]deoxy-CTP was added to the PCR reaction
and electrophoresed on 5% glycerol containing 7% acrylamide gels, and
the gels were subjected to autoradiography.
Sequencing
Amplified DNA fragments were purified with a QIA Quick-Gel extraction kit (QIAGEN). Nucleotide sequencing was performed using a mode 373A automated sequencer with a Taq DyeDeoxy terminator cycle sequencing kit (PE Applied Biosystems, Foster City, CA). PCR products that showed possible heterozygosity were subcloned into pBluescript SK- plasmid and sequenced with a T7 sequencing kit (Pharmacia Biotech, Uppsala, Sweden). At least six independent clones from each patient DNA were sequenced.
Results
Southern blot analysis of PHP-1b patient genomic DNA
The genomic DNAs of eight PHP-1b patients were compared with those
of normal individuals using Southern blotting and hybridization with a
PTHR1 P3 promoter-specific probe. ApaI digestion generated
restriction fragments of 784 and 178 bp, as predicted from the map of
the normal PTHR1 gene, and did not reveal any differences between
samples (Fig. 2A). As the P3 promoter
region lies within the GC-rich region, and methylation in this region
reduces the promoter activity in vitro (12),
digestion with either methylation- sensitive (HpaII;
Fig. 2B) or methylation-insensitive (MspI; Fig. 2C)
restriction enzymes was performed to explore differences in methylation
state. Both Southern blots generated restriction fragments of
approximately 209, 78, 89, and 99 bp in size, and the latter three
fragments are identified as a single relatively broad band (Fig. 2, B
and C; also see Fig. 1). None of the samples showed any difference
between the two endonucleases (Fig. 2, B and C).
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PCR amplification of PHP-1b patient genomic DNA and PCR-SSCP
showed some different patterns in both patients and controls (data not
shown), indicating that there may be some (sequence) differences
between -181 and +205. PCR-direct sequencing revealed no difference in
the sequence of minimal promoter region (between -115 and +63) of P3
(13). We originally reported that an A-rich region reduced
P3 promoter activity (see Fig. 1) and had five repeats of AAAG
(underlined in Fig. 3A)
(11, 14, 15). A homozygous (AAAG)n repeat, where n =
6, was identified in patient 3 (Table 1 and Fig. 3B); however, the
unaffected mother had the same genotype, and the unaffected brother was
heterozygous.
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In previous studies mutations for all coding and noncoding exons of the PTHR1 gene were excluded as a cause of PHP-1b, and analysis of the receptor’s mRNA provided no evidence for splice variants (3, 4, 5). The homozygous loss of function in the PTHR1 in both mouse (13, 16, 17) and human (18, 19) induced lethal-type bone dysplasia, and the severity of symptoms manifested by loss of function of PTHR1 might also indicate that mutation of the PTHR1 is unlikely as the cause of PHP-1b. Taken together, all available data imply that PHP-1b is caused by a tissue- or cell-specific defect in PTHR1 expression or by a defect in a protein that mediates the PTH-dependent signaling events downstream. With respect to the tissue- or cell-specific defect in PTHR1 expression, an abnormality in the third promoter (P3) in the human PTHR1 gene that has recently been identified is a candidate for the cause of PHP-1b (11).
The P3 promoter consists of both GC-rich and A-rich regions, and the former may be subjected to methylation. We have previously shown that methylation of the P3 promoter abolished its promoter activity completely (12). We showed in this study that the P3 promoter is not methylated in normal subjects and PHP-1b patients. Thus, this is not likely to be the cause of renal resistance to PTH in these patients with PHP-1b, although we have not ruled out that there is kidney-specific methylation of the P3 promoter in them.
We examined the P3 promoter by PCR-SSCP and nucleotide sequencing. We have identified a homozygous AAAG repeat polymorphism in the A-rich area of P3 promoter in one patient with PHP-1b. Other sequences in the P3 promoter region, including the GC-rich region, did not vary between individuals. As we have previously shown that the deletion of this A-rich sequence increases promoter activity (14), this change may affect promoter activity. However, the same polymorphism was identified in normal subjects. A subsequent study revealed that there is an AAAG repeat polymorphism in this region, with the repeat ranging in number from three to eight in the Japanese population. Further studies will be required to elucidate the significance of this polymorphism. Jüppner et al. (20) conducted a genome-wide search with four familial PHP-1b kindreds and established linkage to a small telomeric region on chromosome 20q13.3 that overlaps the stimulatory G protein gene. In addition, in two PHP-1b families the PTHR1 gene was excluded as a candidate gene by linkage analysis (21). The PHP-1b patients examined in the present study were all sporadic cases, and as familial PHP-1b is extremely rare, it is possible that the cause of most sporadic PHP-1b is different from that of familial PHP-1b.
In summary, PHP-1b in eight cases is not associated with structural abnormalities in the PTHR1 P3 promoter, and its methylation status is not different in PHP-1b patients relative to that in normal subjects. The significance of an additional A-rich repeat on promoter activity warrants further study.
Footnotes
1 This work was supported by grants from the Japanese Ministry of
Education, Science, and Culture (10670706 and 12670728); the
Investigation Committee on Abnormalities in Hormone Reception Mechanism
by the Japanese Ministry of Health and Welfare, and the Third and Fifth
Novo Nordisk Awards. 
Received August 9, 2000.
Revised October 24, 2000.
Revised December 1, 2000.
Accepted December 4, 2000.
References
This article has been cited by other articles:
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  M. Minagawa, T. Yasuda, T. Watanabe, K. Minamitani, Y. Takahashi, D. Goltzman, J. H. White, G. N. Hendy, and Y. Kohno Association between AAAG Repeat Polymorphism in the P3 Promoter of the Human Parathyroid Hormone (PTH)/PTH-Related Peptide Receptor Gene and Adult Height, Urinary Pyridinoline Excretion, and Promoter Activity J. Clin. Endocrinol. Metab., April 1, 2002; 87(4): 1791 - 1796. [Abstract] [Full Text] [PDF]
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