help button home button Endocrine Society JCEM
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2006-0300
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hoogendam, J.
Right arrow Articles by Karperien, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hoogendam, J.
Right arrow Articles by Karperien, M.
Related Collections
Right arrow Calcium and Bone Metabolism
The Journal of Clinical Endocrinology & Metabolism Vol. 92, No. 3 1088-1095
Copyright © 2007 by The Endocrine Society

Novel Mutations in the Parathyroid Hormone (PTH)/PTH-Related Peptide Receptor Type 1 Causing Blomstrand Osteochondrodysplasia Types I and II

J. Hoogendam, H. Farih-Sips, L. C. Wÿnaendts, C. W. G. M. Löwik, J. M. Wit and M. Karperien

Departments of Pediatrics (J.H., J.M.W., M.K.) and Endocrinology and Metabolic Diseases (H.F.-S., C.W.G.M.L., M.K.), Leiden University Medical Center, 2300 RC Leiden, The Netherlands; and Department of Pathology (L.C.W.), Free University Amsterdam, 1007 MB Amsterdam, The Netherlands

Address all correspondence and requests for reprints to: M. Karperien, Leiden University Medical Center, Department of Endocrinology, C4R89, Albinusdreef 2, 2333 ZA Leiden, The Netherlands. E-mail: karperien{at}lumc.nl.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Context: The PTH/PTHrP receptor type 1 (PTHR1) has a key role in endochondral ossification, which is emphasized by diseases resulting from mutations in the PTHR1 gene. Among these diseases is Blomstrand osteochondrodysplasia (BOCD).

Objective: BOCD can be divided into two types, depending on the severity of the skeletal abnormalities. The molecular basis for this heterogenic presentation is unknown.

Design and Patients: We performed mutation analysis in two families with type I and in three families with the less severe form of BOCD type II.

Results: In one of the type I BOCD cases, a homozygous nonsense mutation (R104X) was found, resulting in a truncated PTHR1. In the second type I BOCD case, no mutation was found. A homozygous nucleotide change (intron M4+27C>T) was demonstrated in one of the type II BOCD cases creating a novel splice site. In dermal fibroblasts of the patient, this novel splice site was preferentially used, resulting in an aberrant transcript. The wild-type transcript remained, however, present, albeit at low levels. In the other two families with type II BOCD, a previously identified homozygous missense mutation (P132L) was found. Functional analysis demonstrated that the P132L mutant had low residual activity.

Conclusions: In combination with data presented in literature, we conclude that type I BOCD is caused by a complete inactivation of the PTHR1, whereas low levels of residual activity due to a near complete inactivation of the PTHR1 result in the relatively milder presentation of type II BOCD.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
THE PTH/PTHrP receptor type 1 (PTHR1) and its ligand PTHrP have a key role in endochondral bone formation during embryonic development (1, 2, 3). This is exemplified by the identification of mutations in various human skeletal dysplasias. Mutations in the PTHR1 can be divided into dominant and recessive mutations. Dominant mutations are found in Jansen-type metaphyseal chondrodysplasia (JMC) and Ollier disease (4, 5). Four different heterozygous missense mutations have been identified in JMC patients. The mutations result in ligand-independent constitutive activation of the receptor. The syndrome is associated with severe dwarfism and a marked metaphyseal thickening (4, 6, 7, 8). In a few patients with enchondromatosis (Ollier disease), a disease characterized by the presence of multiple benign tumors of cartilage that develop into bone in close proximity of the growth plate, a heterozygous mutation in the PTHR1 gene was identified (5). It is postulated that this mutation results in up-regulation of Indian hedgehog signaling in growth plate chondrocytes (5). In a different panel of patients, however, this finding could not be confirmed (9).

Recessive mutations in the PTHR1 have been identified in Eiken syndrome and in Blomstrand osteochondrodysplasia (BOCD) (10, 11, 12, 13). Eiken syndrome is an extremely rare syndrome presently reported in one family only. It is caused by a homozygous nonsense mutation in the C terminus of the receptor. This mutation results in a truncated receptor missing a small part of the C terminus. It is postulated that the mutation results in a disturbed balance between the activation of the adenylate/protein kinase A and the phospholipase C ß/protein kinase C signaling pathways that underlies the clinical presentation (13, 14).

BOCD is a rare autosomal recessive disorder characterized by advanced maturation and premature ossification of all skeletal elements. In addition, BOCD is characterized by extraskeletal manifestations, like hypoplastic lungs, tooth abnormalities, aortic coarctation, and absence of breast development (15, 16, 17, 18, 19, 20, 21, 22, 23). The skeletal and extraskeletal abnormalities resemble the defects observed in PTHR1 knockout mice (3, 24). It has been proposed that BOCD presents in two forms, type I and type II (16). Type I BOCD is the classical and most severe form, characterized by extremely short and malformed bones. Although also lethal, the skeletal manifestations in type II BOCD are less severe compared with type I. In Table 2Go of Ref. 16 , the clinical, radiographical, histological, and biochemical differences between type I and type II BOCD are described.


View this table:
[in this window]
[in a new window]

  		TABLE 2. Haplotype analysis of families with the P132l mutation

 
In this study, we addressed the molecular basis for the heterogenic presentation of BOCD by performing mutation analysis in two type I and three type II BOCD cases.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Cases included in this study

Case 1 was the first reported case of BOCD described in detail by Blomstrand et al. (25). The female fetus was born after 29 wk gestation to consanguineous parents. The case is classified as type I BOCD (16). The second case, a female fetus born to consanguineous Caucasian parents at 32 wk gestation, is described in detail elsewhere and is also classified as type I BOCD (case I, Ref. 16). Case 3 is described for the first time in the present study (for a detailed description, see next paragraph). Based on the criteria in Ref. 16 , it is classified as type II BOCD. Case 4 is one of the three affected siblings born to Asian parents who where first-degree cousins and who had no family history of skeletal dysplasia (case II in Ref. 16). The first two fetuses were described in detail before (16). A third pregnancy resulted in an affected fetus, showing the same characteristics as its siblings. All three fetuses were classified as type II BOCD (16). The fifth case was obtained from a terminated pregnancy at 26 wk gestation (case III in Ref. 16). The Asian parents were nonconsanguineous. Postmortem radiography and osteochondral histopathology classified the skeletal dysplasia as type II BOCD (16). Parental informed consent was obtained for all cases described in this study.

Case 3 clinical report and family history

Case 3 was a male infant whose mother presented with polyhydramnion at 32 wk. Fetal ultrasound at 32 wk revealed furthermore a relatively large head, a small thorax, lung hypoplasia, and very short dense tubular bones. Because of the gross polyhydramnion and the probable lethal prognosis labor was initiated. At birth the infant did not breathe and died within minutes. Birth weight was 1800 g. The boy showed a short crown–rump length (28.5 cm), a large head (head circumference, 32 cm), and a hypoplastic viscerocranium (Fig. 1AGo). The face showed typical abnormalities: severe micrognathia with a protruding tongue, a hypoplastic nose, and low-set ears. The infant also had a narrow thorax and no nipples. The limbs were symmetrically shortened. Autopsy revealed hypoplastic lungs and a preductal aortic coarctation. Abdominal and pelvic organs showed no abnormalities. No details were available of the size of the parathyroid glands or the presence or absence of cataract. Radiography showed generalized osteosclerosis and advanced skeletal maturation, a small viscerocranium, short ribs, ossification of laryngeal cartilage, and patella (Fig. 1BGo). The tubular bones were short with metaphyseal broadening. Most carpal and tarsal bones were ossified. Histology of tubular bones showed a reduction of the resting and proliferative zone of the growth plate (Fig. 1Go, C and D). The epiphysis showed irregular columnization of the hypertrophic chondrocytes. The overall picture resembles the clinical manifestations of BOCD. Given the relatively well-developed long bones, a recognizable epiphysis, and a normal diaphyseal bone marrow space, this case was classified as type II BOCD according to the criteria in Ref. 16 .


Figure 1
View larger version (44K):
[in this window]
[in a new window]

  		FIG. 1. Case 3, Phenotypic presentation. A, External view of a fetus delivered at 32 wk gestation, showing short stature and micromelia. B, Whole-body radiograph, showing advanced skeletal maturation, a small viscerocranium, short ribs, ossification of laryngeal cartilage and patella. The tubular bones are short with metaphyseal broadening. Most carpal and tarsal bones are ossified. C, Histology of a femoral head, showing a reduction in size of the epiphysis. D, High magnification of the epiphysis shows a decrease in resting chondrocytes and a near-complete absence of column-wise orientated proliferating chondrocytes. Note the irregular border between the growth plate and the primary spongiosum.

 
The parents both originated from a 250-person village in Turkey, but they deny any consanguinity. This fetus was the seventh pregnancy of these parents. Two sons are healthy, one child died at 1 wk of age of unknown cause, one child had been healthy until the age of death at 11 months of unknown cause, one earlier pregnancy had ended in the birth of a stillborn boy at 28 wk of pregnancy, and one pregnancy ended in a spontaneous miscarriage at 12 wk. No medical information on these deceased children is available.

Sequence analysis and site-directed mutagenesis

From cases 1, 2, and 5, only tissue blocks embedded in paraffin were present. Sections from these blocks were dewaxed and hydrated through graded ethanols. Subsequently, DNA was extracted by proteinase K (Invitrogen, Breda, The Netherlands) digestion and ethanol precipitation. From cases 3 and 4, genomic DNA was isolated from cultured fibroblasts by sequential proteinase K treatment and high salt precipitation.

Primer sets used for the amplification of the coding exons of human (h)PTHR1 were previously described (26, 27). PCR products were sequenced using an ABI thermal sequencer (PE Applied Biosystems, Foster City, CA).

A mutant P132L PTHR1 receptor cDNA was created by PCR-based site-directed mutagenesis, using the wild-type receptor as a template. The mutant construct was verified by sequencing and cloned in the pcDNA3.1 expression vector.

Haplotype analysis was performed using three microsatellite markers proximally (D3S3624, D3S3646, D3S3582) and three microsatellite markers distally (D3S3729, D3S3629, D3S3688) of the PTHR1 gene. The length of the dinucleotide CA-repeats was determined on an ABI system. DNA of the three siblings of case 4, case 5, and a previously described case with the P132L mutation (DNA was kindly provided by Dr. C. Silve, Faculte de Medecine Xavier Bichat, Paris, France) was used in haplotype analysis.

Cell culture, transient transfection assays, and cAMP production

At autopsy of cases 3 and 4, a skin biopsy was performed, and a culture of dermal fibroblast was established in {alpha}MEM, containing 100 U/ml penicillin (Invitrogen), 100 U/ml streptomycin (Invitrogen), and 10% fetal calf serum (Integro BV, Zaandam, The Netherlands). Cells were seeded at a density of 15,000 cells/cm2 in a 24-well tissue culture plate. As controls, previously established cultures of dermal fibroblasts of healthy children were used (28). After 4 d, cells were used for intracellular cAMP measurement after a challenge with hPTHrP(1–34) or bNle8,18PTH(1–34) (Bachum Holding AG, Bubendorf, Switzerland) as described before (26). cAMP was measured using an enzyme immunoassay (Amersham, Freiburg, Germany) according to the manufacturer’s protocol. All experiments were performed in triplicate and repeated at least twice.

COS-7 cells were cultured in bicarbonate-buffered DMEM, containing 7.5% fetal calf serum, 100 U/ml penicillin, and 100 U/ml streptomycin. For transient transfection assays, cells were seeded at a density of 15,000 cells/cm2 in a 24-well tissue culture plate. The next day, cells were transiently transfected with a dose range of the pcDNA3.1 expression vector (Invitrogen), containing either the wild-type or mutant P132L PTHR1 cDNA using Fugene-6 (Roche, Indianapolis, IN). After 2 d, cells were challenged with 10–7 M PTHrP(1-34), and cAMP accumulation was measured as described. cAMP accumulation was expressed as fold induction compared with nonstimulated cells. Each experiment was performed in triplicate at least twice.

For luciferase measurements, COS-7cells were cotransfected with a dose range of the pcDNA3.1 expression vector (Invitrogen), containing either the wild-type or P132L mutant PTHR1 cDNA and 1 µg of a cAMP responsive promoter reporter construct CRE-Luc (Stratagene, La Jolla, CA). To correct for transfection efficiency, 100 ng of renilla luciferase (pRL-SV40; Promega, Madison, WI) was cotransfected. The next day medium was changed, and cells were stimulated with 10–7 M PTHrP(1-34) for 24 h. Luciferase assays were performed using the Dual-Luciferase Reporter assay system (Promega) according to the protocol. 10 µl of cell lysate was first assayed for firefly luciferase and then for Renilla luciferase activity using the Wallac 1450 Microbeta Trilux luminescence counter (PerkinElmer, Boston, MA). Firefly luciferase activity was corrected for Renilla luciferase activity. Each experiment was performed in triplicate and repeated at least twice.

RT-PCR analysis

Dermal fibroblasts were cultured as described. Cells were seeded at a density of 15,000 cells/cm2 in a 56-cm2 tissue culture disk. After confluence, total RNA was isolated according to the method of Chomczynksi and Sacchi (29). Total RNA was reverse-transcribed into cDNA using random hexamer primers (Amersham, Freiburg, Germany). To correct for variations in RNA content and cDNA synthesis between the different samples, cDNAs were equalized on the basis of their content of the housekeeping gene ß-2-microglobulin as described in detail elsewhere (30). Semiquantitative PCR was performed for the PTHR1 or for various parts of the PTHR1 under the following conditions: cDNA was denatured at 94 C for 5 min, followed by 35 cycles of 30 sec at 94 C, 30 sec at 56 C, and 30 sec at 72 C, and final extension for 10 min at 72 C. Primer combinations are indicated in Table 1Go.


View this table:
[in this window]
[in a new window]

  		TABLE 1. Primer sets used for PCR analysis in case 3

 

   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Case 1

Sequence analysis of the coding exons and the flanking exon-intron boundaries of the PTHR1 gene revealed a homozygous point mutation (338C>T), causing a premature stop codon at position 104 (R104X) (Fig. 2AGo). The presence of the mutation was verified by restriction fragment length analysis (Fig. 2BGo). DNA corresponding to exon 1 from the fetus was amplified by PCR and was, as expected, resistant to KpnI enzyme digestion. Due to the substitution a truncated protein was formed, only consisting of the signal peptide and the first 79 amino acids. This mutant protein lacks all functional domains of the PTHR1, including a large part of the extracellular N terminus, the transmembrane domains, and the intracellular C terminus, and is therefore completely inactivating.


Figure 2
View larger version (24K):
[in this window]
[in a new window]

  		FIG. 2. Case 1, Premature stop codon in the PTHR1. A, Comparison of the DNA and amino acid sequences of the wild-type (wt) and mutant (mt) receptors. Sequence analysis of PTHR1 gene revealed a 338C>T substitution (uppercase and bold), resulting in a premature stop codon in the extracellular part of the receptor. The position of the mutation is shown in a schematic representation of the structure of the PTHR1. B, Exon E2 was amplified and subjected to restriction enzyme analysis. The PCR product of the patient but not of a control was resistant to KpnI enzyme digestion. Marker, 100-bp marker.

 
Case 2

DNA was extracted from tissue blocks embedded in paraffin. Subsequently, all coding exons and flanking exon-intron boundaries of the PTHR1 gene were amplified by PCR and sequenced. For each amplicon, three reliable sequences were analyzed. Sequence analysis, however, did not reveal a mutation (data not shown).

Case 3

The dermal fibroblasts of the affected fetus showed no cAMP accumulation after a challenge with a high dose of PTH or PTHrP in contrast to control (Fig. 3AGo). To establish whether this abnormality was caused by a mutation in the PTHR1, all coding exons and flanking exon-intron boundaries of the PTHR1 gene were sequenced. In none of the coding exons was a mutation found. However, a homozygous point mutation was identified in the intron between exon M4 and exon EL2 (intron M4 + 27C>T) (Fig. 3BGo). This mutation creates a perfect match with the consensus sequence of an exon-intron boundary with higher homology than the native splice site. RT-PCR analysis of the PTHR1 mRNA expression revealed a slight decrease in gene expression in the patient (Fig. 3CGo). Furthermore, the PCR product consisted of two amplicons. Sequence analysis showed that the predominant larger PCR product contained an insertion of 27 nucleotides between exon M4 and EL2 (Fig. 3EGo). The smaller transcript resembled the wild-type transcript. This suggested the preferential use of the aberrant splice site. To substantiate this further, RT-PCR was performed using a reverse primer, spanning the wild-type exon M4 and EL2 splice site. This primer can only result in amplification of wild-type, but not of mutant transcripts. As shown in Fig. 3DGo, a small amount of wild-type PTHR1 mRNA was expressed in fibroblasts of the patient compared with control samples. This provided further evidence for the preferential but not exclusive use of the aberrant splice site. The preferential use of the aberrant splice site and the extension of exon M4 resulted in a premature stop codon, which creates a truncated protein, lacking the fifth, sixth, and seventh transmembrane domains, the intervening intracellular and extracellular domains, as well as the cytoplasmatic tail (Fig. 3EGo).


Figure 3
View larger version (21K):
[in this window]
[in a new window]

  		FIG. 3. Case 3, Identification of a novel RNA splice site. A, Dermal fibroblasts of the affected fetus and control dermal fibroblasts were treated with a high dose (10–7 M) hPTH(1-34) or hPTHrP(1-34), and cAMP accumulation was measured. In contrast to control, dermal fibroblasts of the affected fetus showed no cAMP accumulation. B, A homozygous point mutation was identified 27 bp downstream of exon M4 (intron M4 + 27C>T), creating a new exon-intron boundary with higher homology to the consensus sequence than the wild-type splice site. Native and mutated RNA splicing sequences are boxed. C, RT-PCR of the PTHR1 using a sense primer (S8) located in exon M4 and an antisense primer (AS9) located in the downstream exon EL2 revealed a bigger transcript in case 3 compared with control samples, which is indicative for aberrant splicing. Also, a low amount of a transcript of normal size was present (arrows). The size of the PCR products (194 and 167 bp, respectively) is indicated in the margin. M, Molecular weight marker. D, RT-PCR was performed with a sense primer (S8) specific for exon M4 and an antisense primer (AS8) overlapping the wild-type exon-exon boundary of exon M4 and EL2. Only in the case of wild-type mRNA, this primer combination results in an amplicon of 100 bp. A small amount of wild-type PTHR1 mRNA was expressed in the affected sample compared with control samples. M, Molecular weight marker. E, Comparison of the DNA and amino acid sequences of the wild-type (wt) and the mutant (mt) receptors. In case the alternative splice site is used, the last amino acid of exon M4 will be followed by a stop codon. This results in a truncated protein, lacking the transmembrane domains 5, 6, and 7 and the cytoplasmatic C terminus. STP, Stop codon.

 
Case 4

Dermal fibroblasts of case 4 showed no cAMP accumulation after a challenge with a high dose of PTH or PTHrP (Fig. 4AGo), indicating abrogated PTHR1 signaling. We subsequently sequenced the coding exons and flanking exon-intron boundaries of the PTHR1 gene. A homozygous point mutation (395C>T) in exon E3 was identified, resulting in a missense mutation at position 132 (P132L) (Fig. 4BGo). The presence of the mutation was verified by restriction fragment length analysis (Fig. 4CGo). DNA corresponding to exon 3 from the fetus was amplified by PCR and was, as expected, resistant to MspI enzymatic activity. The same mutation was identified in the two other siblings with BOCD. To examine further the consequences of the P132L mutation, transient transfection experiments were performed. As shown in Fig. 4DGo, stimulation with hPTHrP(1-34) resulted in a small but significant increase in cAMP accumulation in cells transfected with the mutant construct, although the response was markedly lower than the response observed in wild-type transfected cells (Fig. 4DGo). In wild-type transfected cells, there was an increase in activation of a cAMP-responsive promoter reporter construct with increasing concentration of transfected receptor (Fig. 4EGo). This was also found in P132L transfected cells but only at high concentrations of transfected receptor and at a much lower level. This demonstrates that the P132L mutation created a receptor, which still possesses low levels of residual activity. Particularly at high levels of receptor expression, this may result in a blunted functional response.


Figure 4
View larger version (23K):
[in this window]
[in a new window]

  		FIG. 4. Cases 4 and 5, P132L missense mutation. A, Dermal fibroblast of case 4 and control dermal fibroblasts were treated with a high dose (10–7 M) hPTH(1-34) or hPTHrP(1-34), and cAMP accumulation was measured. Dermal fibroblasts of the affected fetus showed no cAMP accumulation, whereas in control fibroblasts cAMP accumulation was induced. B, Comparison of the DNA and amino acid sequences of the wild-type (wt) and the mutant (mt) receptors. Sequence analysis of PTHR1 gene revealed a homozygous 395C>T substitution (uppercase and bold), resulting in an amino acid change in the extracellular part of the receptor (P132L). The position of the mutation is shown in a schematic representation of the PTHR1. C, Exon E3 was PCR amplified and subjected to restriction enzyme analysis. The mutation resulted in a loss of an MspI restriction site in case 4 (three siblings) and case 5 (arrow) in contrast to control (con). M, Molecular weight marker. The size of the PCR products is indicated in the margins. D, COS-7 cells were transiently transfected with a dose range of a wild-type or P132L mutant PTHR1 expression vector. Subsequently, cells were treated with 10–7 M hPTHrP(1-34). cAMP accumulation was measured and expressed as fold change compared with vehicle treated cultures. *, Significant vs. control (0 ng/ml construct) (P < 0.05). E, COS-7 cells were transiently cotransfected with a dose range of an expression vector encoding wild-type PTHR1 or mutant P132L PTHR1 and with a CRE-promoter reporter construct. Subsequently, cells were treated with 10–7 M hPTHrP(1-34). Luciferase activity was measured, corrected for transfection efficiency, and expressed as fold change compared with vehicle treated cultures. *, Significant vs. control (0 ng/ml construct) (P < 0.05).

 
Case 5

Sequence analysis of DNA extracted from paraffin blocks also revealed the same homozygous mutation 395C>T in the PTHR1 gene as found in case 4. The presence of the mutation was confirmed by restriction fragment length analysis (Fig. 4CGo).

Haplotype analysis

The P132L mutation has previously been identified in one other case of BOCD (11, 12). Limited pedigree analysis of the three different families with the P132L mutation did not reveal the presence of a common ancestor. This seems likely, however, because the three families belong to the same ethnic minority group. To test whether the three families have a common ancestor, haplotype analysis was performed using DNA of case 5, the three siblings of case 4, and the previously reported case (11, 12). The results are summarized in Table 2Go. The previously reported case and case 5 shared four microsatellite markers surrounding the PTHR1 gene. Both cases shared the three microsatellite markers located distally of the PTHR1 gene with the siblings of case 4. Interestingly, the siblings were heterozygous for the markers localized proximally of the PTHR1 gene. These markers were not or only partly shared with case 5 and the previously reported case. This suggested the occurrence of at least two independent recombination events between the upstream and downstream markers, which are located at a distance of approximately 2 cM. Due to absence of paternal DNA, no definitive haplotypes could be established. The results of the haplotype analysis are in line with the presence of a common ancient founder.


   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
In the present study, we have performed mutation and functional analysis of five cases with BOCD. The presence of homozygous inactivating mutations in four of five cases further confirms the causative role of the PTHR1 in BOCD. In one patient (case 2 in this study), we did not identify a homozygous mutation in the coding exons and flanking exon-intron boundaries of the PTHR1 gene. Due to the poor quality of the starting material, we cannot completely rule out the absence of a mutation. This seems, however, unlikely because a similar approach is followed for mutation analysis of cases 1 and 5, in which mutations were found. Because this case has all the typical characteristics of BOCD type I (16), we therefore favor the hypothesis that the disease is caused by a mutation in an intron or in the promoter region of the PTHR1 gene resulting in loss of PTHR1 expression. Such a mutation probably also underlies the absence of expression of the paternal allele in the case described by Jobert et al. (10). Although unlikely on the basis of the phenotypic characteristics of this case, which resemble the descriptions of cases in which mutations in the PTHR1 have been found, we cannot completely exclude the presence of a mutation in another gene, like, for example, the PTHrP gene as the underlying cause. Unfortunately, we were unable to test this hypothesis because sufficient DNA could not be isolated from the tissue blocks.

Including the data presented in this study, five different mutations have been described in the PTHR1 causing BOCD (10, 11, 12, 26). Each family has its own mutation, except for the P132L missense mutation, which is identified in three different families. This may be indicative for the presence of a hotspot for mutations at this position as described for the H223R mutation in JMC, although a founder effect appears more likely. This is supported by haplotype analysis performed on DNA from affected individuals of the three families. This analysis showed that the affected individuals share three microsatellite markers distally and two of three families also share three microsatellite markers proximally of the PTHR1 gene. Furthermore, the three families with P132L live in the same region of England and belong to the same ethnic population. These data are suggestive for an ancient founder effect.

The topic we wanted to address was the molecular basis of the differences in clinical presentation between type I and type II BOCD. Oostra et al. (16) postulated that type I BOCD is caused by a completely inactivating mutation in the PTHR1 gene, whereas the less severe abnormalities in type II BOCD are caused by incomplete inactivation of the PTHR1. We present evidence in concordance with this hypothesis. The mutations in the type I BOCD cases described in this study and in previous studies are all completely inactivating mutations (10, 26). In the three families classified by Oostra et al. as type II BOCD, the P132L mutation was found (Refs. 11, 12 , and 16 and this study). Functional analysis of the P132L mutation in transient transfection assays revealed a low residual activity using accumulation of intracellular cAMP as a read-out. These findings are in agreement with previous studies in which partial activity of the P132L mutation was noted with respect of the activation of the cAMP/protein kinase A pathway, but not of the activation of the phospholipase C ß/protein kinase C pathway (11, 12).

The residual activity of the P132L mutation in COS-7 cells is in contrast to our observation in dermal fibroblasts of case 4, in which no cAMP accumulation was found after stimulation with PTH or PTHrP (data not shown). This discrepancy is most likely explained by a substantial difference in the number of receptors present in dermal fibroblasts and transiently transfected COS-7 cells. Only in the presence of high receptor numbers might residual activity become evident. We found evidence for this in transient transfection assays. Using activation of a CRE-luc reporter as read-out, we demonstrate that P132L transfected cells can only activate the reporter when high concentrations of mutant receptor are transfected. Because chondrocytes and osteoblasts express higher levels of PTHR1 mRNA and, therefore, are more responsive than dermal fibroblasts, the residual activity of the P132L mutation in vivo may only become evident in the developing skeleton, resulting in a milder clinical presentation.

Alternatively, the milder phenotype in the three type II BOCD cases with the P132L phenotype may depend on the ethnic background of the individuals, which is identical. It has been described that the phenotype of PTHR1 knockout mice strongly depends on the genetic background of the mouse strains (3). This latter explanation seems unlikely, because we have identified a new case of type II BOCD in this study. This case has a different ethnic background and a novel mutation (intron M4 + 27C>T). As in case 4, dermal fibroblasts of this patient did not reveal any responsiveness to PTH or PTHrP, using intracellular cAMP accumulation as a read-out. However, by sensitive RT-PCR we showed that the native splice site was still used, although at low levels. This will most likely result in the presence of low levels of wild-type mRNAs and wild-type receptors. In dermal fibroblasts, a cell type with low levels of PTHR1 transcripts, this expression may be too low to result in a measurable functional response. In skeletal cells with high levels of mRNA and high responsiveness, low residual activity due to native splicing may become evident, resulting in the milder skeletal manifestations. Taken together, the relatively milder presentation of BOCD type II, particularly in the skeleton, is most likely caused by a partially inactivating mutation in the PTHR1 gene, resulting in low amounts of residual activity during endochondral bone formation.


   Acknowledgments
 
We thank Dr. H. J. van der Harten (Department of Pathology, Free University Amsterdam, Amsterdam, The Netherlands) for helpful discussion and Dr. L. G. Kindblom (Department of Pathology, Sahlgrenska University Hospital, Göteborg, Sweden) for kindly providing tissue blocks of case 1. We are grateful to Dr. C. Silve (Faculte de medecine Xavier Bichat, Paris, France) for providing DNA from her case with the P132L mutation, and to Dr. M. Losekoot (Department of Clinical Genetics, Leiden University Medical Center, Leiden, The Netherlands) for help with the haplotype analysis.


   Footnotes
 
Disclosure Statement: The authors have nothing to declare.

First Published Online December 12, 2006

Abbreviations: BOCD, Blomstrand osteochondrodysplasia; h, human; JMC, Jansen-type metaphyseal chondrodysplasia; PTHR1, PTH receptor type 1.

Received February 9, 2006.

Accepted December 4, 2006.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

  1. Mallette LE 1991 The parathyroid polyhormones: new concepts in the spectrum of peptide hormone action. Endocr Rev 12:110–117[Abstract/Free Full Text]
  2. Karaplis AC, Luz A, Glowacki J, Bronson RT, Tybulewicz VL, Kronenberg HM, Mulligan RC 1994 Lethal skeletal dysplasia from targeted disruption of the parathyroid hormone-related peptide gene. Genes Dev 8:277–289[Abstract/Free Full Text]
  3. Lanske B, Karaplis AC, Lee K, Luz A, Vortkamp A, Pirro A, Karperien M, Defize LH, Ho C, Mulligan RC, Abou-Samra AB, Juppner H, Segre GV, Kronenberg HM 1996 PTH/PTHrP receptor in early development and Indian hedgehog-regulated bone growth. Science 273:663–666[Abstract]
  4. Schipani E, Langman C, Hunzelman J, Le Merrer M, Loke KY, Dillon MJ, Silve C, Juppner H 1999 A novel parathyroid hormone (PTH)/PTH-related peptide receptor mutation in Jansen’s metaphyseal chondrodysplasia. J Clin Endocrinol Metab 84:3052–3057[Abstract/Free Full Text]
  5. Hopyan S, Gokgoz N, Poon R, Gensure RC, Yu C, Cole WG, Bell RS, Juppner H, Andrulis IL, Wunder JS, Alman BA 2002 A mutant PTH/PTHrP type I receptor in enchondromatosis. Nat Genet 30:306–310[CrossRef][Medline]
  6. Schipani E, Kruse K, Juppner H 1995 A constitutively active mutant PTH-PTHrP receptor in Jansen-type metaphyseal chondrodysplasia. Science 268:98–100[Abstract/Free Full Text]
  7. Schipani E, Langman CB, Parfitt AM, Jensen GS, Kikuchi S, Kooh SW, Cole WG, Juppner H 1996 Constitutively activated receptors for parathyroid hormone and parathyroid hormone-related peptide in Jansen’s metaphyseal chondrodysplasia. N Engl J Med 335:708–714[Abstract/Free Full Text]
  8. Bastepe M, Raas-Rothschild A, Silver J, Weismann I, Wientroub S, Juppner H, Gillis D 2004 A form of Jansen’s metaphyseal chondrodysplasia with limited metabolic and skeletal abnormalities is caused by a novel activating parathyroid hormone (PTH)/PTH-related peptide receptor mutation. J Clin Endocrinol Metab 89:3595–3600[Abstract/Free Full Text]
  9. Rozeman LB, Sangiorgi L, Briaire-de Bruijn IH, Mainil-Varlet P, Bertoni F, Cleton-Jansen AM, Hogendoorn PC, Bovee JM 2004 Enchondromatosis (Ollier disease, Maffucci syndrome) is not caused by the PTHR1 mutation p.R150C. Hum Mutat 24:466–473[CrossRef][Medline]
  10. Jobert AS, Zhang P, Couvineau A, Bonaventure J, Roume J, Le Merrer M, Silve C 1998 Absence of functional receptors for parathyroid hormone and parathyroid hormone-related peptide in Blomstrand chondrodysplasia. J Clin Invest 102:34–40[Medline]
  11. Zhang P, Jobert AS, Couvineau A, Silve C 1998 A homozygous inactivating mutation in the parathyroid hormone/parathyroid hormone-related peptide receptor causing Blomstrand chondrodysplasia. J Clin Endocrinol Metab 83:3365–3368[Abstract/Free Full Text]
  12. Karaplis AC, He B, Nguyen MT, Young ID, Semeraro D, Ozawa H, Amizuka N 1998 Inactivating mutation in the human parathyroid hormone receptor type 1 gene in Blomstrand chondrodysplasia. Endocrinology 139:5255–5258[Abstract/Free Full Text]
  13. Duchatelet S, Ostergaard E, Cortes D, Lemainque A, Julier C 2005 Recessive mutations in PTHR1 cause contrasting skeletal dysplasias in Eiken and Blomstrand syndromes. Hum Mol Genet 14:1–5[Abstract/Free Full Text]
  14. Eiken M, Prag J, Petersen KE, Kaufmann HJ 1984 A new familial skeletal dysplasia with severely retarded ossification and abnormal modeling of bones especially of the epiphyses, the hands, and feet. Eur J Pediatr 141:231–235[CrossRef][Medline]
  15. Wysolmerski JJ, Cormier S, Philbrick WM, Dann P, Zhang JP, Roume J, Delezoide AL, Silve C 2001 Absence of functional type 1 parathyroid hormone (PTH)/PTH-related protein receptors in humans is associated with abnormal breast development and tooth impaction. J Clin Endocrinol Metab 86:1788–1794[Abstract/Free Full Text]
  16. Oostra RJ, van der Harten JJ, Rijnders WP, Scott RJ, Young MP, Trump D 2000 Blomstrand osteochondrodysplasia: three novel cases and histological evidence for heterogeneity. Virchows Arch 436:28–35[CrossRef][Medline]
  17. Spranger J, Maroteaux P 1990 The lethal osteochondrodysplasias. Adv Hum Genet 19:1–103, 331–2.:1–2[Medline]
  18. Young ID, Zuccollo JM, Broderick NJ 1993 A lethal skeletal dysplasia with generalised sclerosis and advanced skeletal maturation: Blomstrand chondrodysplasia? J Med Genet 30:155–157[Abstract/Free Full Text]
  19. Leroy JG, Keersmaeckers G, Coppens M, Dumon JE, Roels H 1996 Blomstrand lethal osteochondrodysplasia. Am J Med Genet 63:84–89[CrossRef][Medline]
  20. Loshkajian A, Roume J, Stanescu V, Delezoide AL, Stampf F, Maroteaux P 1997 Familial Blomstrand chondrodysplasia with advanced skeletal maturation: further delineation. Am J Med Genet 71:283–288[CrossRef][Medline]
  21. den Hollander NS, van der Harten HJ, Vermeij-Keers C, Niermeijer MF, Wladimiroff JW 1997 First-trimester diagnosis of Blomstrand lethal osteochondrodysplasia. Am J Med Genet 73:345–350[CrossRef][Medline]
  22. Oostra RJ, Baljet B, Dijkstra PF, Hennekam RC 1998 Congenital anomalies in the teratological collection of Museum Vrolik in Amsterdam, The Netherlands. II: Skeletal dysplasias. Am J Med Genet 77:116–134[CrossRef][Medline]
  23. Galera MF, Silva Patricio FR, Lederman HM, Porciuncula CG, Lopes Monlleo I, Brunoni D 1999 Blomstrand chondrodysplasia: a lethal sclerosing skeletal dysplasia. Case report and review. Pediatr Radiol 29:842–845[CrossRef][Medline]
  24. Wysolmerski JJ, McCaughern-Carucci JF, Daifotis AG, Broadus AE, Philbrick WM 1995 Overexpression of parathyroid hormone-related protein or parathyroid hormone in transgenic mice impairs branching morphogenesis during mammary gland development. Development 121:3539–3547[Abstract]
  25. Blomstrand S, Claesson I, Save-Soderbergh J 1985 A case of lethal congenital dwarfism with accelerated skeletal maturation. Pediatr Radiol 15:141–143[CrossRef][Medline]
  26. Karperien M, van der Harten HJ, van Schooten R, Farih-Sips H, Den Hollander NS, Kneppers SL, Nijweide P, Papapoulos SE, Lowik CW 1999 A frame-shift mutation in the type I parathyroid hormone (PTH)/PTH-related peptide receptor causing Blomstrand lethal osteochondrodysplasia. J Clin Endocrinol Metab 84:3713–3720[Abstract/Free Full Text]
  27. Schipani E, Weinstein LS, Bergwitz C, Lida-Klein A, Kong XF, Stuhrmann M, Kruse K, Whyte MP, Murray P, Schmidkte J 1995 Pseudohypoparathyroidism type Ib is not caused by mutations in the coding exons of the human parathyroid hormone (PTH)/PTH-related peptide receptor gene. J Clin Endocrinol Metab 80:1611–1621[Abstract/Free Full Text]
  28. Walenkamp MJ, van der Kamp HJ, Pereira AM, Kant SG, van Duyvenvoorde HA, Kruithof MF, Breuning MH, Romijn JA, Karperien M, Wit JM 2006 A variable degree of intrauterine and postnatal growth retardation in a family with a missense mutation in the insulin-like growth factor I receptor. J Clin Endocrinol Metab 91:3062–3070[Abstract/Free Full Text]
  29. Chomczynski P, Sacchi N 1987 Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159[Medline]
  30. Van Bezooijen RL, Farih-Sips HC, Papapoulos SE, Lowik CW 1998 IL-1{alpha}, IL-1ß, IL-6, and TNF-{alpha} 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]



This article has been cited by other articles:


Home page
 Hum Mol GenetHome page
A. Couvineau, V. Wouters, G. Bertrand, C. Rouyer, B. Gerard, L. M. Boon, B. Grandchamp, M. Vikkula, and C. Silve
PTHR1 mutations associated with Ollier disease result in receptor loss of function
Hum. Mol. Genet., September 15, 2008; 17(18): 2766 - 2775.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hoogendam, J.
Right arrow Articles by Karperien, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hoogendam, J.
Right arrow Articles by Karperien, M.
Related Collections
Right arrow Calcium and Bone Metabolism

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals