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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.
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Abstract |
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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.
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Introduction |
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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 2
of Ref. 16 , the clinical, radiographical, histological, and biochemical differences between type I and type II BOCD are described.
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Subjects and Methods |
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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. 1A). 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. 1B). 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. 1, 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 .
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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 
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 1.
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Results |
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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. 2A). The presence of the mutation was verified by restriction fragment length analysis (Fig. 2B). 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.
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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. 3A). 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. 3B). 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. 3C). 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. 3E). 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. 3D, 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. 3E).
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Dermal fibroblasts of case 4 showed no cAMP accumulation after a challenge with a high dose of PTH or PTHrP (Fig. 4A), 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. 4B). The presence of the mutation was verified by restriction fragment length analysis (Fig. 4C). 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. 4D, 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. 4D). 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. 4E). 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.
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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. 4C
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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 2. 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.
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Discussion |
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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.
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Acknowledgments |
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Footnotes |
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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.
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References |
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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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