Download Recessive mutations in PTHR1 cause contrasting skeletal

Human Molecular Genetics, 2005, Vol. 14, No. 1
doi:10.1093/hmg/ddi001
Advance Access published on November 3, 2004
1–5
Recessive mutations in PTHR1 cause contrasting
skeletal dysplasias in Eiken and Blomstrand
syndromes
Sabine Duchatelet1, Elsebet Ostergaard2, Dina Cortes3, Arnaud Lemainque4 and Cécile Julier1,*
1
Genetics of Infectious and Autoimmune Diseases, Pasteur Institute, INSERM E102, 28 rue du docteur Roux, 75724
Paris Cedex 15, France, 2Department of Medical Genetics, The John F. Kennedy Institute, Gl. Landevej 7, 2600
Glostrup, Denmark, 3Department of Pediatrics, Glostrup University Hospital, Ndr. Ringvej 57, 2600 Glostrup, Denmark
and 4Centre National de Génotypage, 2 rue Gaston Crémieux, CP 5721, 91057 Evry Cedex, France
Received September 25, 2004; Revised and Accepted October 20, 2004
Eiken syndrome is a rare autosomal recessive skeletal dysplasia. We identified a truncation mutation in the
C-terminal cytoplasmic tail of the parathyroid hormone (PTH)/PTH-related peptide (PTHrP) type 1 receptor
(PTHR1 ) gene as the cause of this syndrome. Eiken syndrome differs from Jansen and Blomstrand chondrodysplasia and from enchondromatosis, which are all syndromes caused by PTHR1 mutations. Notably, the
skeletal features are opposite to those in Blomstrand chondrodysplasia, which is caused by inactivating
recessive mutations in PTHR1. To our knowledge, this is the first description of opposite manifestations
resulting from distinct recessive mutations in the same gene.
INTRODUCTION
Eiken syndrome is a rare familial skeletal dysplasia which has
been described in a unique consanguineous family, where it
segregates as a recessive trait (1). It is characterized by
multiple epiphyseal dysplasia, with extremely retarded ossification, principally of the epiphyses, pelvis, hands and feet, as
well as by abnormal modeling of the bones in hands and feet,
abnormal persistence of cartilage in the pelvis and mild
growth retardation (1). On the basis of the genetic study of
this original family, we report here that a truncation mutation
in the C-terminal tail of the parathyroid hormone (PTH)/PTHrelated peptide (PTHrP) type 1 receptor (PTHR1 ) gene is
responsible for this syndrome.
RESULTS
We have studied the family originally described by Eiken,
from which six individuals were available (Fig. 1). In addition
to Eiken syndrome, one of the patients developed type 1 diabetes at 9 years of age. The association of multiple epiphyseal
dysplasia and insulin-dependent diabetes in this case would
strictly classify her as having Wolcott – Rallison syndrome
(WRS) (2), although the later age at onset, the absence of a
systematic association of skeletal dysplasia and diabetes in
this family and the unique clinical features, particularly in
the pelvis, hands and feet, make it clearly distinct. By
linkage analysis and mutation screening, we excluded the
implication of the pancreatic eIF2a kinase gene (EIF2AK3 ),
which is responsible for WRS (3, 4), in this patient (data not
shown).
Despite the limited size of this family (five informative
individuals), the maximum expected LOD score, assuming
full genetic information in the region of linkage, would
reach a value of 3.3. We therefore performed a genomewide scan using 400 microsatellite markers, and identified a
single region of linkage, located on chromosome 3p, with a
maximum multilocus LOD score of 3.2 (Fig. 1). As expected,
the region of linkage is broad, 50 cM, between markers
D3S2338 and D3S1285. This region may contain in the
order of 500 genes, and mutation screening of all these genes
would not be practical. Using Mapviewer interface (http://
www.ncbi.nlm.nih.gov/mapview), we found one gene in this
region that has been previously implicated in some forms of
chondrodysplasias: the PTHR1 gene; this is a G proteincoupled receptor, which is involved in the regulation of chondrocyte proliferation and differentiation and plays a major role in
bone development (5, 6). We therefore considered this gene
as a good candidate for Eiken syndrome. We screened the
totality of the coding region of the gene in two heterozygous
*To whom correspondence should be addressed. Tel: þ33 140613701; Fax: þ33 145688929; Email: [email protected]
Human Molecular Genetics, Vol. 14, No. 1 # Oxford University Press 2005; all rights reserved
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Human Molecular Genetics, 2005, Vol. 14, No. 1
Figure 2. PTHR1 gene mutation screening and identification. (A) Sequence of
two heterozygous parents (individuals 5 and 6) and one homozygous child
(individual 1), showing a C . T substitution at position 1656 on the cDNA
(GenBank: accession no. NM_000316), which results in an ARG485STOP
nonsense mutation in the C-terminal tail of the protein. (B) Mutation genotyping by PCR–RFLP assay, showing co-segregation of the mutation with the
disease in the family.
Figure 1. Eiken syndrome family and linkage analysis. Individuals shown in
black are affected by Eiken syndrome. Individual 3 is affected by type 1 diabetes, in addition to Eiken syndrome. Segregating haplotypes in the region of
linkage are shown. Undetermined genotypes are shown as ‘0’. The region
boxed is found in the homozygous status in all affected individuals.
individuals (individuals 5 and 6) and their homozygous
affected child (individual 1) and identified a C . T substitution in the last exon, resulting in a nonsense mutation
ARG485STOP (Fig. 2A). We confirmed the cosegregation
of this mutation in the homozygous status with Eiken syndrome in this family, both by sequencing (data not shown)
and by a PCR –RFLP assay (Fig. 2B). Using the same assay,
we confirmed the absence of this mutation in 160 Caucasian
controls. The mutation is located in the C-terminal cytoplasmic tail of the protein, resulting in a variant truncated of
its last 108 amino acids. This domain contains a cluster of
serine residues which are phosphorylated upon ligand
binding; it is able to bind to several proteins, including G
protein receptor kinases and b-arrestin, and has been shown
to be involved in the desensitization/internalization process
of the receptor and in its regulation (5).
DISCUSSION
Mutations in PTHR1 have been reported in two types of
skeletal dysplasias: metaphyseal dysplasia in Jansen chondrodysplasia, a dominant disorder resulting from constitutively
activating mutations (7), and osteosclerosis and advanced
skeletal maturation in Blomstrand chondrodysplasia, a recessive lethal disorder resulting from inactivating mutations (8).
A mutation in PTHR1 has also been reported in enchondromatosis, a dominant disorder characterized by multiple cartilage
tumors, frequently associated with skeletal deformity (9). In
addition, PTHR1 knock-out mice exhibit a phenotype similar
to Blomstrand syndrome (10). In contrast to Blomstrand chondrodysplasia patients, patients with Eiken syndrome have a
severely delayed skeletal maturation, although both disorders
are caused by recessive mutations in PTHR1. The phenotype
in Eiken syndrome is more similar to Jansen chondrodysplasia, although the syndromes clearly differ by the mode of
inheritance, specific skeletal features and calcium and phosphate concentrations; calcium and phosphate levels were
found to be within normal range in Eiken patients (1), while
Jansen patients have severe hypercalcemia and hypophosphatemia (6). In addition, the serum PTH level was found to be
slightly elevated in Eiken patient 3, while it was normal in
Human Molecular Genetics, 2005, Vol. 14, No. 1
patients with Jansen syndrome (6). Circulating levels of
1,25-(OH)2VitD were normal in the Eiken patient, while the
levels were found to be elevated in patients with Jansen syndrome (6). To our knowledge, this is the first description of
opposite manifestations resulting from distinct recessive
mutations in the same gene.
PTHR1 activates several signal transduction pathways,
including adenyl cyclase (AC)/protein kinase A (PKA) and
phospholipase C (PLC)/protein kinase C (PKC). Recent
studies have shown opposite effects of these two pathways
on chondrocyte differentiation, the former increasing the proliferation of chondrocytes and delaying their differentiation,
opposite to the effect of the latter (6). This was evidenced in
a knock-in mouse model expressing solely a mutant form of
PTHR1 (DSEL) modified in the second intracellular loop,
that activates AC/PKA normally, but not PLC/PKC; this
mouse shows a recessive phenotype with delayed ossification,
particularly marked in the tail, metatarsal and digital bones,
expansion of columnar proliferating chondrocytes and
normal calcium and phosphate levels (11). Despite the different nature and location of the mutations, these abnormalities
are remarkably similar to the distinctive features observed in
Eiken patients, who also have delayed ossification, principally
of the hands and feet, abnormal development of some cartilage
areas and normal calcium and phosphate levels.
Extended in vitro studies have been performed on PTHR1
variants carrying truncations in the C-terminal cytoplasmic
tail. PTHR1 variants truncated at positions 480 and 513
showed a marked increase of the AC/PKA signaling activity,
particularly for the 480STOP variant, while the PLC/PKC
activity was unaltered (12). In addition, these truncated variants showed decreased expression, so that the net effect
may be an unchanged AC/PKA activity and a decreased
PLC/PKC activity in vitro (12), leading to similar overall consequences as the DSEL variant (13). We therefore propose that
the expected unbalanced AC/PKA versus PLC/PKC activity
caused by the Eiken mutation is responsible for a phenotype
similar to the DSEL mouse. Alternatively, or in addition,
part of the biological functions that are mediated by PTHR1
and altered in Eiken syndrome may occur in the cytoplasm
or the nucleus, where PTHR1 has also been localized, and
for which there is increasing evidence for a role in mediating
biological effects (5,14). The C-terminal tail of PTHR1 is
likely to be involved in these functions, because of its role
in the receptor conformation, in the stabilization of some
protein complexes and because of the presence of a predicted
nuclear localization signal at positions 471 – 487 (15), which is
disrupted in the ARG485STOP mutant.
One of the four Eiken syndrome patients (individual 3)
developed type 1 diabetes at 9 years of age. This patient presented GAD autoantibodies at onset of diabetes, and had the
high risk HLA (DRB1 03/DRB1 04) and insulin (INS23HphI A/A) genotypes. The diabetes in Eiken syndrome
therefore differs from that in typical WRS, which manifests
early, usually before 6 months of age, is not autoimmune
and is systematically associated with the specific epiphyseal
dysplasia in patients with EIF2AK3 mutations (4). Interestingly, PTHrP has been shown to mediate pancreatic b-cell
growth (5), and we hypothesize that the PTHR1 mutation in
Eiken syndrome may be responsible for a reduced b-cell
3
mass, which may increase the risk of diabetes in genetically
predisposed individuals.
Despite the extreme rarity of Eiken syndrome (a small
unique family), we were able to characterize the molecular
defect underlying it. This is the fourth disease associated
with mutations in the PTHR1 gene, and our observation provides further insight into the multiple functions mediated by
this receptor, which has important therapeutic potentials for
many diseases, including osteoporosis and diabetes.
MATERIALS AND METHODS
Patients and family
We studied the family originally reported by Eiken et al. (1).
Six individuals were available for study, four of whom were
affected with this syndrome (Fig. 1). Individuals 1 and 4
belong to the original sibship of three affected children
described in this previous report (cases 3 and 1, respectively),
where they were still in childhood. Individual 2 was an
affected cousin. A previous child from this couple (individuals
1 and 2), affected with the same syndrome, died earlier and
samples were not available for study. Adult patients’ height
was slightly decreased (153.5 and 154 cm for individuals 1
and 2, respectively), as was their child (individual 3) at 10
years of age (130.2 cm, 21 SD). In the original article, all
standard biochemical analyses were normal in all the patients
examined, including calcium and phosphate levels. In addition
to these, serum PTH level was measured in child 3 and found
to be slightly elevated (63 ng/l; normal range: 10– 50 ng/l);
1,25-(OH)2VitD was normal in this patient. In addition to
Eiken syndrome, individual 3 developed type 1 diabetes,
with onset at 9 years of age. GAD autoantibodies were positive at onset of the diabetes. She was treated by subcutaneous
insulin injections. All individuals participating in this study
gave their informed consent.
Microsatellite genotyping
Genome scan was performed by semi-automated fluorescent
genotyping, using 400 microsatellite markers (Linkage
Mapping Set 2, Applied Biosystems), as described (http://
www.cng.fr/fr/teams/microsatellite/index.html).
Mutation screening by sequencing of genomic DNA
Mutation screening was performed on genomic DNA from an
affected individual (individual 1) and his unaffected parents
(individuals 5 and 6) using primers shown in Table 1.
Sequencing reactions were performed using big-dye terminator chemistry using standard protocols and run on an
Applied Biosystems Sequencer ABI3700.
PCR – RFLP genotyping of the mutation
The region containing the mutation was first amplified from
genomic DNA with primers used for sequencing (fragment
24, Table 1); a nested PCR was then performed with
primers 50 -cactggcactggacttcacg-30 and 50 -gtggcagtgggcag
tagg-30 . The forward primer includes a mismatched base in
4
Human Molecular Genetics, 2005, Vol. 14, No. 1
Table 1. Sequencing primers, size and position of the fragments
PCR
fragment
Forward
Amplification/sequencing primers
Reverse
Size of PCR
product (bp)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
ctgaggagacacccttctgg
cccaaagggtttatgggtct
cagggatccacaggtcaaag
gcctgcctccctgactaact
gctcgttctacagacccacag
gggctgtgttcatacctcgt
cgtcccaaggaggctacata
aatacatggggaatgcacct
gtaccgggaggtggtaggtt
cctagggccgagaggaac
ccgtgctttccagaagagaat
ggtatcccgagagctccat
gacaggcagaccgacagag
catagagcagattccccaca
cctcacccatcgtctcagat
gagcagagagaaaggcgaga
ggaatgggaccacatcctg
caaccttaccctggcctctt
agcagacctgggagtctgaa
gagagttcccctgtgctctg
agcacattccagctgtagcc
atctgactccttgcccactg
cacgtgtgtgtgcctcaata
atagcacggcccaggtattt
ccaaccctcagtgcaaatct
gaccctcgacttaggggaag
gcgacgctgtcagtccac
gagtgtggagaggccgagag
ctgacaccgagacagagcag
cagccacactgagccctta
gaggggactctcacccaaag
tctgggcctcttcagttgat
gtagccctgggtccactctt
cactgaacccctagctccaa
gatgagcacagctacggtga
tgaggtaggagccttgatgg
566
556
563
564
521
586
773
617
584
504
595
593
997
550
543
856
588
799
19
20
21
22
23
24
gcatccccctgagagagc
gagtgtggctctgtcaccaa
acttccaaagaggcctgtga
gaccagctgatccacactcc
aacgggccctattagcactt
cctgtagccaaacaccctgt
ctgaatcttggcctgatggt
ttgaggcattagctcccatc
cccggacgatattgatgaag
cactcaccgcctacctgttc
cagaatgtcctcaggggtgt
gatttccacatgggtccact
585
530
550
566
582
920
order to create an artificial discriminative BsaAI restriction
site. PCR fragments were then digested with BsaAI, yielding
fragments of 148 bp (non-mutated allele) or 129 þ 19 bp
(mutated allele), which were resolved by agarose gel
electrophoresis.
Linkage analyses
Parametric multilocus linkage analysis was performed using
SIMWALK program (16).
5.
6.
7.
ACKNOWLEDGEMENTS
We thank Dr Lise Lykke Thomsen for initial follow-up of the
family. S.D. was a recipient of a Ministery of Research PhD
training Grant. This work was supported in part by grants
from the Pasteur Institute, INSERM and a JDRF/INSERM/
FRM grant to C.J.
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123611–124196
126380–127159
127034–127650
127508–128091
127911–128414
128226–128820
128598–129190
129014–130010
140003–140552
141830–142372
143426–144281
144123–144710
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