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Original Studies |
Departments of Endocrinology and Metabolic Diseases (M.K., H.F.-S., S.E.P., C.W.G.M.L.), Pediatrics (M.K.), Clinical Genetics (S.L.J.K.), and Molecular Cell Biology (P.N.), Leiden University Medical Center, 2300 RC Leiden; the Departments of Pathology (J.J.v.d.H.) and Clinical Genetics (R.v.S.), Academic Hospital Free University, 1007 MB Amsterdam; and the Department of Obstetrics and Gynecology, Academic Hospital Rotterdam (N.S.d.H.), 3000 DR Rotterdam, The Netherlands
Address all correspondence and requests for reprints to: Dr. Marcel Karperien, Department of Endocrinology and Metabolic Diseases, Leiden University Medical Center, Building 1 C4-R, P.O. Box 9600, 2300 RC Leiden, The Netherlands. E-mail: karperien{at}rullf2.leidenuniv.nl
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Abstract |
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We describe here a novel inactivating mutation in the PTH/PTHrP receptor. Sequence analysis of all coding exons of the type I PTH/PTHrP receptor gene and complementary DNA of a case with BOCD identified a homozygous point mutation in exon EL2 in which one nucleotide (G at position 1122) was absent. The mutation was inherited from both parents, supporting the autosomal recessive nature of the disease. The missense mutation resulted in a shift in the open reading frame, leading to a truncated protein that completely diverged from the wild-type sequence after amino acid 364. The mutant receptor, therefore, lacked transmembrane domains 5, 6, and 7; the connecting intra- and extracellular loops; and the cytoplasmic tail. Functional analysis of the mutant receptor in COS-7 cells and of dermal fibroblasts obtained from the case proved that the mutation was indeed inactivating. Neither the transiently transfected COS-7 cells nor the dermal fibroblasts responded to a challenge with PTH or PTHrP with a rise in intracellular cAMP levels, in sharp contrast to control cells. Our results provide further evidence that BOCD is caused by inactivating mutations of the type I PTH/PTHrP receptor and underscore the importance of this receptor in mammalian skeletal development
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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These and other observations have led to the identification of a locally acting negative feedback loop that regulates the rate of chondrocyte differentiation in the embryonal growth plate (7). Besides the PTH/PTHrP receptor, this loop involves the morphogene Indian hedgehog (Ihh), its receptor complex Patched and Smoothened, and PTHrP. Chondrocytes making the transition from the proliferative to the hypertropic zone express Ihh. Via an as yet unknown mechanism, Ihh increases the expression of PTHrP in the periarticular perichondrium. PTHrP, in turn, binds to PTH/PTHrP receptor-expressing proliferating chondrocytes and inhibits their further differentiation. This results in fewer Ihh-producing cells, which closes the feedback loop. In this model, the level of PTHrP expression critically determines the rate of chondrocyte differentiation. This is underscored by observations in transgenic mice either lacking or overexpressing PTHrP, in which chondrocyte differentiation is respectively accelerated or delayed (6, 8).
The pivotal role of the PTH/PTHrP receptor in endochondral bone formation makes this receptor a potential candidate gene involved in the pathogenesis of human skeletal disorders. Indeed, constitutively activating mutations have been detected in the PTH/PTHrP receptor as the most likely cause of Jansen’s metaphyseal chondrodysplasia (9, 10). More recently, inactivating mutations were detected in the human PTH/PTHrP receptor gene as the most likely cause of Blomstrand lethal osteochondrodysplasia (BOCD) (11, 12). This rare dysplasia is characterized by advanced skeletal maturation and premature ossification of the skeleton (13, 14, 15). The phenotype of BOCD closely resembles the malformations in the skeleton observed in PTH/PTHrP receptor knockout mice. Here we describe and characterize a novel homozygous inactivating mutation in the type I PTH/PTHrP receptor in a third case of BOCD.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A 19-week-old fetus was obtained from a terminated pregnancy of a healthy Caucasian 31-yr-old primigravida who was referred for a second opinion ultrasound at 18.5 weeks gestation because of suspected fetal skeletal abnormalities. The parents were consanguineous (second cousins in a multilaterally related pedigree). Postmortem radiography and osteochondral histopathology classified the skeletal dysplasia as BOCD. A detailed description of the case was provided previously (16). At termination, a skin biopsy was taken and used for establishing a cell culture of dermal fibroblast according to standard protocols. For comparison of osteochondral histology, a humeral head of a normal 19-week gestation fetus was used. Parental consent was obtained for this study.
Cell culture, ribonucleic acid (RNA) extraction, transient transfection assays, and cAMP production
Dermal fibroblasts were cultured in 
MEM supplemented with
10% FCS and antibiotics (all from Life Technologies, Inc., Rockville, MD). For the isolation of total RNA, cells were
seeded at a density of 15,000 cells/cm2 in a
56-cm2 tissue culture disk. After confluence,
total RNA was extracted according to the method of Chomzynski and
Sacchi (17). COS-7 cells were cultured in bicarbonate-buffered DMEM
supplemented with 7.5% FCS and antibiotics. For transient transfection
assays, cells were seeded in a 75-cm2 disk. At
80% confluence, cells were transfected with 6 µg of the pcDNA3
expression vector (Invitrogen, San Diego, CA) containing
either the wild-type or mutant human PTH/PTHrP receptor complementary
DNA (cDNA) or no insert (mock) using Fugene (Roche Molecular Biochemicals, Indianapolis, IN) overnight. The next day, cells
were trypsinized and seeded at a density of 15,000
cells/cm2 in a 24-well tissue culture plate.
After 2 days, cells were used for determination of intracellular cAMP.
For this, cells were washed twice with prechilled phosphate-buffered
saline and covered with 500 µL stimulation medium [DMEM containing
20 mM HEPES (pH 7.5), 0.1% fat-free BSA (Sigma Chemical Co., St. Louis, MO), 0.5 µg/µL aprotinin, and 2
mM of the phosphodiesterase inhibitor
isobutylmethylxanthine (Sigma Chemical Co.)] in the
absence or presence of human (h) PTHrP-(1–34), bovine (b) PTH-(1–34)
(both from Bachem, Basel, Switzerland) or forskolin
(Sigma Chemical Co.; dissolved as a
10-2 mol/L in ethanol). After
incubation for 15 min at 37 C, the stimulation medium was removed, and
the reaction was stopped by quickly freezing the cells on dry ice.
Intracellular cAMP was released from the cells by the addition of 500
µL 50 mmol/L HCl, and 20 µL was used for determination of cAMP
content using a commercially available RIA (Innogenetics, Nijmegen, The
Netherlands) according to the protocol of the manufacturer. Samples
were measured in triplicate.
Semiquantitative RT-competitive PCR
Denatured deoxyribonuclease-treated total RNA (1 µg, 5 min at 70 C, and quickly chilled on ice) was reverse transcribed into cDNA in a 20-µL reaction volume containing first strand buffer (75 mmol/L KCl, 3 mmol/L MgCl2, and 50 mmol/L Tris-HCl, pH 8.3), 10 mmol/L dithiothreitol, 0.5 mmol/L deoxy-NTPs, 200 ng random hexanucleotide primers, 1 U RNAsin/µL, and 2.5 U Moloney murine leukemia virus reverse transcriptase/µL (Life Technologies, Inc.). RT was performed at 37 C for 60 min, after which fresh Moloney murine leukemia virus reverse transcriptase and RNAsin was added. Enzymes were inactivated by incubation at 70 C for 5 min, and samples were diluted to a theoretical concentration of 10 ng/µL (assuming 100% efficiency of RT), aliquoted, and stored at -20 C for later use.
To correct for variation in RNA content and cDNA synthesis between the
different samples, cDNAs were equalized on the basis of their content
of the ß2-microglobulin housekeeping gene by
competition PCR. This method has been described in detail previously
(18). In short, 5 ng cDNA were coamplified in the presence of 4-fold
serial dilutions of internal standard plasmid pQA1 (19). Competition
PCR was performed in a 25-µL reaction volume containing reaction
buffer [75 mmol/L Tris-HCl (pH 9.0), 20 mmol/L
(NH4)2SO4,
0.01% (wt/vol) Tween-20], 1.5 mmol/L MgCl2, 200
µmol/L dNTPs, 0.25 µmol/L sense and antisense primer, and 0.125 U
Goldstar TAQ DNA polymerase (Eurogentec, Seraing, Belgium). PCR was
performed on a Gene Amp 9700 Thermocycler (Perkin Elmer Corp., Norwalk, CT). Samples were analyzed by ethidium bromide
staining of agarose gels. The intensity of the PCR products was
determined by densitometry, and the ratio of cDNA/internal standard was
plotted against the number of copies of the internal standard added to
the PCR reaction. As the amplicons for
ß2-microglobulin cDNA and internal standard
amplified with similar efficiency, the point at which the cDNA/internal
standard ratio equals 1 indicated the exact number of copies of
ß2-microglobulin in the cDNA preparations. The
corrected cDNA preparations were used for a semiquantitative PCR
reaction (conditions described above) to detect various parts of
hPTH/PTHrP receptor cDNA. The primer combinations are listed in Table 1
and were ordered from Eurogentec
(Seraing, Belgium).
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Genomic DNA of fibroblasts was isolated by sequential proteinase
K treatment and high salt precipitation. Genomic DNA was isolated from
whole blood using a DNA isolation kit from Roche Molecular Biochemicals. Primer sets for the amplification of exons of the
hPTH/PTHrP receptor gene were previously described (20), except that T7
promoter sequences were incorporated in the sense primers, and Sp6
promoter sequences were incorporated in the antisense primers to
facilitate sequencing. Oligonucleotides were ordered from Eurogentec.
Different primer sets were used for the amplification and sequencing of
exons S and M2 (Table 1
). Automated sequencing was performed on an ABI
thermal sequencer (PE Applied Biosystems, Foster City,
CA). Some sequencing was performed by Eurogentec.
PCR-based site-directed mutagenesis was used to introduce the missense mutation in the wild-type human PTH/PTHrP receptor cDNA. The mutated receptor was controlled by partial sequencing and restriction enzyme digestion. The wild-type and mutant receptor cDNAs were cloned in the eukaryotic pcDNA3 (Invitrogen, San Diego, CA) expression vector.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BOCD is a lethal, short limbed, skeletal dysplasia. Our case had
the typical features of BOCD, namely generalized sclerosis and advanced
skeletal maturation, a hypoplastic viscero-cranium, a protruding
tongue, calcified laryngeal cartilage and hyoid bones, and extreme
short ribs and extremities. As shown in Fig. 1, A and B, the cartilage part of the
humeral head is extremely reduced compared to that of an age-matched
control. Histological analysis of the humeral growth plate demonstrated
a dramatically reduced number of chondrocytes in the resting zone, the
near absence of columnization of proliferating chondrocytes, and a
diminished zone of hypertrophic chondrocytes (Fig. 1, C and D).
Furthermore, there is an irregular boundary between the growth plate
and the metaphysis and a thickening of subcortical bone. These features
are similar to those found in PTH/PTHrP receptor gene knockout
mice.
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). This mutation caused a shift in the
open reading frame, resulting in a truncated protein that in sequence
completely diverged from the wild-type receptor C-terminal of amino
acid 364 (Fig. 2B).
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). The absence of this
restriction site in genomic DNA was confirmed by enzymatic digestion of
a PCR amplicon of 151 bp encoding exon EL2. As expected, DNA of an
unrelated control was completely digested by incubation with
BanI, resulting in restriction fragments of 64 and 87 bp
(Fig. 3A). The amplicon of the affected
proband was resistant to enzymatic digestion, whereas both the father
and mother were heterozygous, having one wild-type digested allele and
one BanI-resistant mutant allele (Fig. 3A). In addition, an
unaffected proband was homozygous for the wild-type allele. These
results were in agreement with the consanguinity of the parents and the
autosomal recessive mode of inheritance. We then verified whether the
BanI restriction site was also absent in PTH/PTHrP receptor
cDNA. For this, RT-PCR reaction was performed on RNA isolated from
dermal fibroblasts from the case resulting in an amplicon of 399 bp. As
shown in Fig. 3B, the amplicon of BOCD was resistant to
BanI, whereas digestion of an amplicon obtained from a
normal control resulted in the appearance of the expected restriction
fragments of 218 and 181 bp.
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In the wild-type receptor, amino acids C-terminal of residue 364
encode transmembrane domains 5, 6, and 7, the intervening extra- and
intracellular loops, and the C-terminal cytoplasmic tail. In contrast
to the wild-type receptor, the mutant receptor did not contain
hydrophobic domains capable of spanning the cell membrane beyond amino
acid 364 (Fig. 4), indicating that the
mutation created a truncated receptor containing only four instead of
seven transmembrane domains. The lacking structural domains play a
crucial role in the proper incorporation of a G protein-coupled
receptor in the membrane, in ligand binding, and in signal
transduction, and the mutation was, therefore, expected to inactivate
the receptor. To test this, we performed functional analysis of dermal
fibroblasts derived from the case with BOCD, as these cells are well
known to express functional type I PTH/PTHrP receptors (21). We first
tested whether the mutation affected PTH/PTHrP receptor messenger RNA
(mRNA) expression. For this, total RNA was isolated from BOCD and
control fibroblasts and reversed transcribed into cDNA. To correct for
differences in amounts of cDNA, a semiquantitative competition PCR
reaction was performed. A fixed amount of cDNA was mixed with a series
of 4-fold dilutions of an internal standard that coamplified with
ß2-microglobulin cDNA in a PCR reaction. As
shown in Fig. 5A, the intensity of the
PCR products of cDNA and internal standard were identical at the same
dilution of the internal standard (lane 3 for BOCD and lane 8 for
control fibroblasts). This indicated that both samples contained equal
amounts of cDNA. Subsequently, semiquantitative PCR reactions were
performed with primer sets amplifying various parts of the hPTH/PTHrP
receptor cDNA. Both BOCD and control fibroblasts expressed comparable
levels of PTH/PTHrP receptor mRNA, suggesting that the mutation did not
have major effects on gene expression (Fig. 5B).
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, dermal fibroblasts from the case did not
accumulate intracellular cAMP levels in response to hPTHrP-(1–34), in
sharp contrast to control fibroblasts, in which an approximately 8-fold
increase was observed. The absence of a rise in intracellular cAMP
levels was not caused by dysfunction of the cAMP signaling cascade, as
forskolin, an activator of adenylate cyclase, efficiently increased
cAMP levels. This increase was higher in BOCD fibroblasts and may
reflect an increased sensitivity of adenylate cyclase in cells lacking
a functional PTH/PTHrP receptor.
We finally introduced the frame-shift mutation in a wild-type PTH/PTHrP
receptor expression vector and tested its effect on receptor
functioning in transiently transfected COS-7 cells. Challenging COS-7
cells, transiently transfected with the wild-type PTH/PTHrP receptor
expression vector with either hPTHrP-(1–34) or bPTH-(1–34), induced a
dose-dependent increase in intracellular cAMP levels (Fig. 5, D and E,
respectively). In sharp contrast, COS-7 cells transfected with the
mutant receptor or an empty expression vector did not respond to
challenges with hPTHrP-(1–34) or bPTH-(1–34).
These results indicated that the mutation indeed inactivated the PTH/PTHrP receptor.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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More recently, inactivating mutations were detected in the PTH/PTHrP receptor gene in two cases with BOCD (11, 12). This severe lethal skeletal dysplasia has many features in common with the skeletal aberrations found in transgenic mice lacking both copies of the type I PTH/PTHrP receptor gene. This is most prominent in the physis and the meta-physis of the long bones. The phenotypic similarity suggested that BOCD was caused by an inactivating mutation of the PTH/PTHrP receptor. Analysis of the PTH/PTHrP receptor genes in these two cases identified a heterozygous nucleotide substitution in exon M5 coding for the fifth transmembrane domain in one case, whereas in the second case a homozygous point mutation was detected in exon E3 coding for part of the extracellular N-terminus of the receptor. In contrast to the second case, the first case was from nonconsanguineous parents. The point mutation in exon M5 created a novel splice acceptor site leading to a defect in mRNA splicing, resulting in a protein that lacks amino acids 373–383 compared to the wild-type receptor. Functional studies in transiently transfected COS-7 cells demonstrated that this mutation inactivated the receptor. The mutation was inherited from the mother, whereas the paternal allele did not contain mutations in the coding exons of the PTH/PTHrP receptor gene. Analysis of chondrocytes from this case demonstrated, however, that the paternal allele was not expressed. Which genetic defect underlies the absence of expression of the paternal allele is presently unknown, but it might be caused by a mutation in a region that is involved in regulation of PTH/PTHrP receptor gene expression (11).
The homozygous point mutation in the second case resulted in the replacement of a proline at position 132 for a leucine. Functional analysis in transiently transfected COS-7 cells demonstrated that this receptor was equally well expressed as wild-type receptors, but binding of either PTH-(1–34) or PTHrP-(1–34) was less than 10% compared to the wild-type receptor. Furthermore, PTH-induced cAMP accumulation was severely reduced, and inositol phosphate accumulation was not detectable. It seems likely that the proline at position 132 plays a crucial role in ligand binding (12).
In this study we describe a novel inactivating mutation in the PTH/PTHrP receptor gene in a third unrelated case of BOCD. The identified mutation was located in exon EL2 coding for the second extracellular loop and consisted of a loss of 1 nucleotide at position 1122 of the cDNA sequence. Consequently, a frame shift was induced in the open reading frame of the PTH/PTHrP receptor mRNA. The resulting mutant protein completely diverged from the wild-type sequence C-terminal of amino acid 364. As shown by analysis of hydrophobicity plots, the mutation created a truncated receptor containing only four instead of seven transmembrane domains. Structure-function analysis of the PTH/PTHrP receptor has indicated a pivotal role in receptor functioning for the domains that are lacking in the mutant receptor. For example, it has been shown that residues in the third extracellular loop are essential for ligand binding (23, 24) and that the lacking intracellular domains are involved in signal transduction (25, 26). Furthermore, it seems highly unlikely that a G protein-coupled receptor lacking three of the seven transmembrane domains can be normally incorporated in the cell membrane due to the complete disruption of its secondary and tertiary structures. The mutation is, therefore, expected to be inactivating. Functional analysis of the mutant receptor proved indeed that the mutation inactivated the receptor. This was shown by functional analysis of dermal fibroblasts from the case itself. Dermal fibroblasts are well known to express functional PTH/PTHrP receptors (21). Analysis of PTH/PTHrP receptor mRNA expression in dermal fibroblasts from BOCD and from a normal control demonstrated that both cells expressed comparable levels of PTH/PTHrP receptor mRNA, suggesting that the mutation did not have major effects on PTH/PTHrP receptor mRNA expression. In marked contrast to the control fibroblasts, BOCD dermal fibroblasts did not respond to a challenge with high doses of hPTHrP-(1–34). These observations were furthermore corroborated by analysis of the mutant receptor in COS-7 cells. Unlike the wild-type receptor, COS-7 cells transiently transfected with the mutant receptor did not respond to either PTH or PTHrP, providing further evidence for the inactivating nature of the mutation. Analysis of parental DNA demonstrated that the mutation was inherited from both parents, in agreement with the consanguinity and the autosomal recessive mode of inheritance of the disease.
The existence of both activating and inactivating mutations has been described for a number of G protein-coupled receptors and has been implicated as the underlying cause of a variety of human diseases (for recent reviews, see Refs. 27, 28). The mutations involve single amino acid substitutions, frame-shift mutations, or the introduction of premature stop codons. For example, loss of function mutations in the calcium-sensing receptor cause familial hypocalciuric hypercalcemia and neonatal severe primary hyperparathyroidism (29), whereas constitutively activating mutations cause familial hypoparathyroidism (30). In addition, loss of function mutations in the LH receptor cause male pseudohermaphroditism (31), whereas gain of function mutations cause familial precocious puberty (32). The identification of loss and gain of function mutations places the type I PTH/PTHrP receptor on the expanding list of G protein-coupled receptors involved in the pathogenesis of various human diseases.
In conclusion, our results in combination with the previously identified inactivating mutations in the PTH/PTHrP receptor clearly demonstrate that BOCD is the human mirror image of PTH/PTHrP receptor-ablated mice and underscore the importance of this receptor in human skeletal devel-opment.
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Acknowledgments |
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Received November 23, 1998.
Revised June 14, 1999.
Accepted June 23, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-1ß, IL-6, and TNF- steady-state mRNA
levels analyzed by reverse transcription-competitive PCR in bone marrow
of gonadectomized mice. J Bone Miner Res. 13:185–194.[CrossRef][Medline]
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