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Identification of novel mutations in WISP3 gene in two unrelated Chinese families with progressive pseudorheumatoid dysplasia
Bone 44 (2009) 547–554
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Bone j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b o n e
Identification of novel mutations in WISP3 gene in two unrelated Chinese families with progressive pseudorheumatoid dysplasia Hua Yue, Zhen-Lin Zhang ⁎, Jin-Wei He Department of Osteoporosis, Metabolic Bone Disease and Genetic Research Unit, Shanghai Jiao Tong University Affiliated the Sixth People's Hospital, Shanghai 200233, 600 Yi-Shan Rd., PR China
a r t i c l e
i n f o
Article history: Received 2 August 2008 Revised 30 October 2008 Accepted 5 November 2008 Available online 21 November 2008 Edited by: B. Olsen Keywords: Progressive pseudorheumatoid dysplasia WISP3 Mutation
a b s t r a c t Introduction: Progressive pseudorheumatoid dysplasia (PPD) is an autosomal recessive genetic disease and it has been reported that PPD is caused by mutations of the Wnt1-inducible signaling pathway protein 3 (WISP3) gene which is located on chromosome 6q22. Up to date, 16 different mutations in the WISP3 have been identified in patients with PPD in different countries previously, but only two mutations in exon 5 were previously identified from Asian origin. Our study aimed to characterize the clinical manifestations and features of PPD and screen the mutations of the disease causing WISP3, and try to elucidate the molecular pathogenesis of PPD. Materials and methods: Altogether, 153 persons, including 4 affected individuals, 49 unaffected individuals from two unrelated Chinese families, and 100 healthy donors were recruited and genomic DNA was extracted. PPD was diagnosed based on the clinical manifestations, physical examination, characteristics of their bones on X-ray and laboratory results. All 5 exons and their exon–intron boundaries of the WISP3 gene were amplified by polymerase chain reaction (PCR) and sequenced directly. Results: In family 1, we identified that the proband (IV4) carried a novel non-sense mutation (G46X) which consisted of a homozygous C to T transition at c.8004 in exon 3. This mutation changed codon CAG to TAG and resulted in a subsequent change of the glutamine codon to stop codon and truncation at p. 46. In family 2, a novel missense mutation (C114Y) was found in the three patients (IV6, IV7, IV8), namely, a homozygous G to A transition at c.8209 in exon 3, which resulted in a cysteine (TGT) to tyrosine (TAT) substitution at p.114. Neither G46X nor C114Y was found in 100 normal controls. Meanwhile, we found that these patients had some different phenotypes, compared with the affected individuals with PPD from cases reported previously. Conclusions: Our study suggests that the novel G46X and C114Y mutations in exon 3 in WISP3 gene are responsible for PPD in Chinese patients. Furthermore, many heterozygous carriers (c.8004CNT and c.8209GNA) are found in the two families, suggesting the existence of a founder effect in the locality where they live, respectively. © 2008 Elsevier Inc. All rights reserved.
Introduction Progressive pseudorheumatoid dysplasia (PPD) (OMIM 208230), also referred to as spondyloepiphyseal dysplasia tarda with progressive arthropathy (SEDT-PA) or progressive pseudorheumatoid arthropathy of childhood (PPAC), is an autosomal recessive genetic disease, and its population incidence has been estimated at 1 per million in the UK [1], but it is likely to be higher in the Middle East and Gulf states [2]. It is a rare disease in the world and there is no prevalence data documented yet in China. Patients with this disorder are asymptomatic in early childhood [3,4]. Signs and symptoms of disease typically develop between 3 and 8 years of age [5], whose typical clinical manifestations and radiographic findings consist of progressive
⁎ Corresponding author. Fax: +86 21 64081474. E-mail address: [email protected] (Z.-L. Zhang). 8756-3282/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bone.2008.11.005
deformities of multiple joints associated with stiffness, swelling without any inflammatory context, limitation of motion, bone pain, short stature, widened epiphyses, vertebral flattening, narrow joint spaces and osteoporosis. Although both genetic and environmental factors have been implicated in the pathogenesis of PPD, PPD has been attributed to loss-of-function mutations of Wnt1-inducible signaling pathway protein 3 (WISP3) gene located on chromosome 6q22 [5–7]. The WISP3 gene is a member of the connective tissue growth factor (CCN) gene family. The latter comprises cysteine-rich 61 (CYR61/CCN1), connective tissue growth factor (CTGF/CCN2), nephroblastoma overexpressed (NOV/CCN3), and Wnt-induced secreted proteins-1 (WISP-1/CCN4), -2 (WISP-2/CCN5) and -3 (WISP-3/CCN6) [8,9]. These proteins stimulate mitosis, adhesion, apoptosis, extra-cellular matrix production, growth arrest and migration of multiple cell types. WISP3 encodes Wnt1-inducible signaling protein 3 which is a cysteine-rich, multi-domain, secreted protein and consists of 354 amino acids.
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Because the population incidence of PPD is very low, it is not very easy for us to find such patients, in that case, which hindered us to recognize this disease to some extent. Up to now, there are about 16 different mutations that have been identified in the WISP3 gene, including: A15fsX, g.IVS1 + 2insT (splicing defect), C52X, C78R, P82fsX104, G83E, C145Y, C179fsX, A197fsX201, C247fsX277, F280fsX312, T288fsX313, Q289fsX301, W331X, Q338L and S334P [6,10,11]. However, only two WISP3 gene mutations in exon 5 (F280fsX312 and S334P) were previously identified in the Asian population [12,13]. Moreover, the expression profiles and characteristics of mutation are known to be influenced by ethnic background. Because population incidence would be vary greatly in different races of people, it is very important to make clear the forms of mutant in Chinese PPD patients. Furthermore, we also want to know whether PPD from Chinese origin is caused by the mutation of the WISP3 gene. Until now, the relationship between phenotype and genotype of PPD is still unclear, so it is urgent for us to find more patients with PPD to develop further studies. In this study, we performed mutation analysis of the WISP3 gene in two unrelated Chinese families affected with PPD and revealed one novel nonsense mutation (G46X) and the other one novel missense mutation (C114Y) which consisted of a homozygous C to T transition at c.8004 and a G to A transition at c.8209 in exon 3, respectively. Meanwhile, we found that these patients had some different phenotypes, compared with the affected individuals with PPD from cases reported previously.
Materials and methods Patients The study was approved by the Ethics Committee of the Shanghai Jiao Tong University Affiliated the Sixth People's Hospital. All the subjects involved in this study were recruited by the department of osteoporosis from outpatients presented to our department within 2 years and signed informed consent documents before entering the project. Altogether, four patients with PPD from two unrelated families and their family members were investigated in this study (Fig. 1). Family 1 came from Liaoning province, located on the east territory of China. Family 2 came from Henan province, located on the central territory of China. All the subjects are Han ethnicity. The proband (IV4) in family 1 is a 13-year-old female, the only daughter of non-consanguineous and healthy parents. She was born with full term pregnancy and normal delivery. Birth weight and length were within normal limits. Her current height and weight are 138.5 cm and 33 kg, respectively. She came to our department with an 11-year history of joint deformities. When she was 2 years old, swelling of joints emerged, fingers were affected first, and then hands, carpal. Whereafter, elbows, knees, hip and spine were all affected, associated with pain and deformities. She progressed to ankylosis at the age of 10. Last year, she presented obvious limitation of motion, and almost lost her ability of self-care. X-rays manifestations revealed a general decrease of bone density involving vertebrae and pelvis;
Fig. 1. Pedigrees of the two families in our study. Patients with PPD are shown by darkened symbols. Carriers are shown by half-darkened symbols.
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Fig. 2. Photographs and X-rays of the patient (Family 1, IV4). (A) the enlargement of the interphalangeal joints (B) enlargement of the epiphyseal and metaphyseal portions of the metacarpals and phalanges; (C) intervertebral and flattened vertebral bodies with rounded end plates; (D) short and wide femoral neck and large capital femoral epiphyses with narrow joint spaces of hip.
enlargement of the epiphyseal and metaphyseal portions of the metacarpals and phalanges; short and wide femoral neck and large capital femoral epiphyses with narrow joint spaces of hip and intervertebral and flattened vertebral bodies with rounded end plates (Fig. 2). Laboratory data showed normal serum sodium, potassium, phosphorus, calcium, creatinine, glucose, blood gas, and hemoglobin. Serum alkaline phosphatase (ALP), parathyroid hormone (PTH), antistreptolysin “O” (ASO), rheumatoid factor (RF), C-reactive protein (CRP), and erythrocyte sedimentation rate (ESR) levels were within the normal range. Moreover, urine sample of the proband had been tested to rule out the congenital metabolic diseases, such as abnormal
metabolism of amino acid, urea cycle, amino acid absorption, fatty acid, purine, pyrimidine and glycometabolism. Simultaneously, hyperlactacidemia, hyperpyruvemia, and peroxisomal disease were also eliminated. The rest thirty-eight members of this family including her parents didn't have any abnormal clinical, biochemical and radiographic manifestations. The proband (IV7) in family 2 is a 14-year-old male, the third son of consanguineous parents, term birth with normal length and weight. His current height and weight are 141 cm and 35 kg, respectively. His first clinical abnormality was found at the age of 4 with swelling of his articulationes interphalangeae, and looked like “clawhands”. Until age
Fig. 3. X-rays of the manifestations in patient (IV7) in family 2. (A) Short and wide femoral neck and large capital femoral epiphyses with narrow joint spaces of hip; (B) right fingers narrow joint spaces and narrow spaces between the carpal bones; (C) flattening and anterior beaking on the thoraco-lumber spine with irregular upper and lower end plates.
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Fig. 4. Photographs and X-rays of the patient (Family 2, IV6). (A) Deformity of bilateral elbow joints; (B) hump back; genu valgum resulted in “X” appearance; (C) arthrogryposis of elbow joint; (D) enlargement of the epiphyseal and metaphyseal portions of the metacarpals and phalanges; (E) left femoral neck fracture; femoral head necrosis; the pelvis severely out of shape; (F) flattened vertebral bodies.
14, he didn't feel any upset, and could walk, run and do common activities like other healthy persons. One month ago, he complained of hip pain and had difficulty in elevating his legs and associated with instability of gait. He has two sisters, one (IV5, 19 years old) with normal phenotype, however, the other sister (IV6, 16 years old), had similar manifestations with him. Her age of onset was 3 years, swelling of articulationes interphalangeae were also her first physical sign. Deformities of joints associated with pain and function loss progressed as she aged. At the age of 12, her lower limbs were found asymmetry with the left shorter than the right. About 2 years ago, she couldn't walk without a crutch. Another patient in this family is the proband's older female cousin (IV8), who is 15 years old. She is the first daughter of non-consanguineous parents and has a normal little brother. Her age of onset was 3 years with almost the same symptoms as the other two patients in the family. She lost her walking ability at 13 years of age. X-ray manifestations of the three patients revealed a general decrease of bone density involving vertebrae and pelvis; enlargement of the epiphyseal and metaphyseal portions of the metacarpals and phalanges; short and wide femoral neck and large capital femoral epiphyses, unsmooth and irregularity of joint surface; flattened vertebral bodies. In addition, some different features among them are listed as follows: The proband (IV7): his articulationes interphalangeae and articulatio metacarpophalangea could not be straightened and bent, and are relatively fixed like “clawhands” (Fig. 3). His sister (IV6): her pelvis is severely out of shape associated with arthrogryposis of elbow joint, hump back, genu valgum resulted in “X” appearance (Fig. 4). His older female cousin (IV8): her pelvis is out of shape with left hand side femoral head damaged just like IV6 (Fig. 5). Laboratory data showed normal serum sodium, potassium, phosphorus, calcium, creatinine, glucose, blood gas, hemoglobin,
PTH, RF, CRP and ESR, except for the mildly elevated levels of serum ALP and ASO. No clinical, biochemical and radiographic abnormalities were found in the rest of the 11 members (II1, II2, II3, II4, III2, III3, III5, III6, III7, III8 and IV5) of this family. In addition, III4, III9, III10, IV1–4 and IV9–11 didn't apply their blood samples, so we just observed their phenotypes and didn't analyze their DNA. General features, laboratory data, clinical and radiological findings of the 4 patients in the 2 families are listed in Table 1 and Table 2, respectively.
Fig. 5. X-rays of the manifestations in patient (IV8) in family 2. (A) Left femoral neck fracture, femoral head necrosis and the pelvis severely out of shape; (B) flattened vertebral bodies.
H. Yue et al. / Bone 44 (2009) 547–554 Table 1 General features and laboratory findings in patients of 2 families
Patients Gender Age (years) Height (cm) Weight (kg) ALP (U/l) BUN (mmol/l) Cr (μmol/l) Calcium (mmol/l) Phosphate (mmol/l) PTH (pg/ml) ESR (mm/h) CRP (mg/l) ASO (IU/ml) RF (IU/ml) HLA-B27
Family 1
Family 2
Proband (IV4) Female 13 138.5 33 278 3.30 35 2.51 1.65 14.07 12 0.10 243 8 −
Proband (IV7) Male 14 141 35 206 2.90 35 2.45 1.61 15.2 9 0.20 364 9.5 −
Table 3 The primers sequence of exons in WISP3 gene Normal value
Sister (IV6) Female 16 140 41 64 7.4 48 2.53 1.26 33.4 15 0.25 125 8.5 −
Older female cousin (IV8) Female 15 139 39 110 5.6 51 2.39 1.42 29.4 11 0.92 95 11 −
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15–112 2.5–6.4 53–115 2.08–2.6 0.8–1.6 15–65 0–20 b 3 mg/l 0–200 0–15 −
“−” negative.
Sequence analysis of the WISP3 gene Informed consents were obtained from each family and 100 healthy volunteers before blood sampling and DNA analysis. Altogether, 153 DNA samples, including 4 affected individuals, 49 unaffected individuals from two families, and 100 healthy donors. DNA was extracted from peripheral white blood cells with conventional methods. We sequenced the entire coding region (five exons with several alternatively spliced exons) of the WISP3 (GeneBank accession number: NC_000006) to screen mutations using 6 pairs of primers (Table 3). Primers were designed using Primer 3 software (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi). All 5 exons and their exon–intron boundaries of the WISP3 gene were amplified by polymerase chain reaction (PCR). Direct nucleotide sequencing was performed using the Big-Dye Terminator Cycle Sequencing Ready Reaction Kit version 3.1 (Applied by Bio-systems, CA, USA) and analyzed by an ABI 3700 automatic sequencer (Applied by Bio-systems, CA, USA) with standard methods. Results In family 1, the proband (IV4, 13-year-old female) and 38 unaffected subjects of the family were genotyped by direct nucleotide sequencing. We identified that the proband carried a novel non-sense mutation (G46X) which consisted of a homozygous C to T transition at c.8004 in exon 3 (Fig. 6). This mutation changed codon CAG to TAG and
Exon
Primers (5′–3′) sense
Primers (5′–3′) anti-sense
1 2 3 4 5
TCA CTG CGA AGG CAG GTT ATT CTG CAC GCT GTT GCT ATG AAG CCT GTT TGG GGG AAA TCT TCT TCC TGT GAA GGA GGT TCC AAA AGG CAA AGC AGA AAA ATG CAA AAG GGT AAA GAG AGT GCT GGA
TGC CAT TAC CTG AAA GGG AGA CCTTGC TCC TTT GTC CAC TTG TAC AAT GGA GCC AGT CCC ACT TCC CTG TCT GAG GCA AAG ATT ATC CCA CCC TCC AAA ACA CAC AAA CAA AGT AGA TTT GCC ACC A
resulted in a subsequent change of the glutamine codon to stop codon and truncation at p. 46. In all other members of this PPD kindred, the proband's father (III7), mother (III8), her father's brother (III9), her mother's younger brother (III19) and her older male cousin (IV1) are the heterozygous (T/C) carriers. DNA samples from 100 normal volunteers were subsequently tested for this mutation, but no G46X was found in all chromosomes, and normal individuals had C/C genotype. In family 2, the proband (IV7, 14-year-old male), two patients (IV6 and IV8) and the other 11 unaffected subjects (II1, II2, II3, II4, III2, III3, III5, III6, III7, III8 and IV5) of the family were genotyped by direct nucleotide sequencing. A novel missense mutation was found in the three patients, namely, a homozygous G to A transition at c.8209 in exon 3 (Fig. 7), which resulted in a cysteine (TGT) to tyrosine (TAT) substitution at p.114. This C114Y mutation was also found in the heterozygous state in his father's mother (II2), mother's mother (II4), father's older sister (III3), father (III5), mother (III6) and father's younger sister (III8), aunt's husband (III7) and his sister (IV5). No C114Y mutation was found in all chromosomes from 100 normal controls and normal individuals had G/G genotype. In this study, we found that G46X mutant had more severe symptoms and the age of onset was earlier than the C114Y mutants (the proband in family 1, whose age of onset was 2 years, and had limited flexion and extension at 10). Moreover, we observed some intra-familial clinical variability and symptoms that became more severe with the age in patients with C114Y mutation (the females both had arthrogryposis of elbow joint and genu valgum resulted in “X” appearance). Meanwhile, it seemed that the symptoms in a female were more severe than in a male in C114Y mutant (two females both have left hand side femoral neck fracture, femoral head necrosis and the pelvis severely out of shape, but the male had none of the symptoms). Furthermore, many heterozygous carriers (c.8004CNT and c.8209GNA) are found in the two families, suggesting the existence of a founder effect in the locality where they live, respectively. Discussion
Table 2 Clinical and radiological findings in patients of the 2 families
Age at examination (years) Age of onset (years) Age of joint swelling (years) Limited flexion and extension (years) Painful joints (years) Short stature Vertebral changes Osteoarthrosis of joints Widened epiphyses Osteoporosis Muscle wasting Intelligence, acouesthesia Eyesight, smell Femoral head necrosis
Family 1 (IV4)
Family 2 (IV7, IV6, IV8)
13 2 2 10
14, 16 and 15 4, 3 and 3 4, 3 and 3 14, 12 and 13
5 + Platyspondy and wedging of vertebra + + + + − − −
14, 11 and 11 + Platyspondy and wedging of vertebra + + + + − − −, + and +
“+”: positive manifestations; “−”: normal.
PPD is a rare inherited metabolic disease. In our study, we reported 4 patients in two unrelated Chinese families and found the 4 patients all had bone pain, and multiple joints deformities (stiffness and swelling) as they age. Radiographs showed progressive loss of joint space, widened epiphyses, vertebral flattening and generalized decrease of bone density. The patients had almost normal blood laboratory findings. And the molecular findings in the 4 patients from two unrelated families are in complete agreement with the clinical diagnosis of PPD. That is to say, PPD was diagnosed based on the clinical presentations, the characteristics of radiographic examinations and molecular findings. Unlike other congenital skeleton diseases, the patients with PPD are all asymptomatic in the first years of life and often mimic early juvenile rheumatoid arthritis and mucolipidosis type IV. The latter two are the common causes of misdiagnosis, which can be excluded by genetic counseling and laboratory findings. In this study, direct sequencing of the WISP3 gene was performed in two PPD families. We identified that the proband in family 1 carried
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Fig. 6. Automated sequencing traces of the mutation in WISP3 gene in family 1 with PPD. (A) Mutation screening was performed in patient (IV4) by direct sequencing of PCR products, revealing a CNT transition in exon 3 of WISP3, which resulted in G46X change. (B) Mutation screening was performed in heterozygous carriers (III7, III8, III9, III19 and IV1).
a novel non-sense mutation (G46X) which consisted of a homozygous C to T transition at c.8004 in exon 3. This mutation changed codon CAG to TAG and resulted in a subsequent change of the glutamine codon to stop codon and truncation at p. 46. Another novel missense mutation (C114Y) was found in the proband and the other two patients in family 2, namely, a homozygous G to A transition at c.8209 in exon 3, which resulted in a Cysteine (TGT) to Tyrosine (TAT) substitution at p. 114. To date, apart from our findings, over 16 mutations in WISP3 have been identified in 22 different PPD families from USA, Jordan, Italy, France, Lebanon, Syria, Iran, Saudi Arabia, Caucasian, and China [6,10,11], including exon-deletion, frameshift, non-sense, and missense mutations [5,6,10,11] (Fig. 8), which suggests that PPD results from a loss of WISP3 protein function [5]. Among the families with WISP3 gene mutations, 14 families are from consanguineous mating, and 8 are from non-consanguineous mating. Patients from nonconsanguineous families all appeared to be compound-heterozygotes, except one is heterozygous. And patients from consanguineous families were all homozygous [6]. In this study, we identified the molecular defect responsible for PPD in two unrelated families. We described two novel mutations and found many heterozygous carriers within families. However, the 4 patients are all homozygotes, and we found no compound-heterozygotes. It is interesting that the parents of the proband (IV4) in family 1 and the patient (IV8) in family 2 are both non-consanguineous mating and they are all in the heterozygous states harboring the disease-causing T or A allele, respectively. Since the two couples are all natives, we can conclude that the numbers of risk gene carriers in the place they live must be higher than from anywhere else and suggest the existence of a founder effect. In that
case, the two carriers could meet more frequently in the circumstances of random marriage, and then the incidence of PPD would be higher in the locality. Therefore, the next thing we should do is to go to the districts they live in, and investigate the local people in order to find the carriers harboring the disease-causing gene as soon as possible and take methods to interfere, so as to decrease the incidence of the disease to the utmost extent. WISP3 is a member of the CCN family of secreted growth factors. CCN proteins are multimodular mosaic proteins containing four conserved modules which are present in other unrelated extracellular proteins [14]. Module 1 is an insulin-like growth factor (IGF) binding domain which is encoded by exon 2. Module 2 is a von Willebrand type C domain which is encoded by exon 3. WISP3, unlike all other CCN proteins identified to date, lacks 4 of 10 conserved cysteine residues in this domain [6,7]. Module 3 is a thrombospondin domain which is encoded by exon 4. Module 4 is a C-terminal domain containing a putative cysteine knot which is encoded by exon 5 [5,6,15–19]. The CCN proteins demonstrate a wide variety of biological activities regulating cell adhesion, proliferation, survival, migration, invasion in vitro and tumorigenesis and angiogenesis in vivo. At present, only WISP3 among CCN family members has been linked to a Mendelian genetic human disease [5,20]. Among the mutations in the WISP3 gene, one mutation was found in exon 1, 4 in exon 2, 5 in exon 3, 1 in exon 4, 6 in exon 5 and 1 in intron 1, respectively (Fig. 8). From the above, we can learn that mutations are located in all coding exons that can result in PPD. Mutations occurred in exon 3 and exon 5 have been found in multiple studies, indicating that exon 3 and exon 5 may be hotspots for mutations.
Fig. 7. Automated sequencing traces of the mutation in WISP3 gene in family 2 with PPD. (A) Mutation screening was performed in patients (IV6, IV7 and IV8) by direct sequencing of PCR products, revealing a GNA transition in exon 3 of WISP3, which resulted in C114Y change. (B) Mutation screening was performed in heterozygous carriers (II2, II4, III3, III5, III6, III7, III8 and IV5). (C) Mutation screening was performed in normal persons.
H. Yue et al. / Bone 44 (2009) 547–554
Fig. 8. Domain structure of WISP3, with locations of the disease-causing PPD mutations noted (G46X and C114Y are novel mutations found in this study). Signal peptide (SP), insulin-like growth factor-binding domain (IGFBP), von Willebrand factor type C domain (VWC), thrombospondin domain (TSP), and C-terminal cysteine knot-like domain (CT) are shown. The homozygous g.IVS1 + 2insT (Splicing defect) mutation in intron 1 is not shown. The mutations are reported according to mutation nomenclature guidelines. Numbering begins at the initiation codon and is referring to the cDNA sequence with the following GeneBank: accession number: AF 100781.1.
WISP3 was cloned and characterized as the gene locates on the downstream in the Wnt signaling pathway in 1998 [7]. A recent study found that overexpression of WISP3 inhibited bone morphogenetic protein (BMP) and Wnt signaling during zebrafish development. ZebraWISP3 and human WISP3 have 49% identity and 54% similarity at the amino acid level. Zebrafish and human WISP3 inhibited BMP and Wnt signaling in mammalian cells by binding to BMPs and to the Wnt coreceptor low density lipoprotein receptor related protein 6 (LRP6) and frizzled respectively [5]. Therefore, they concluded that dysregulation of BMP and/or WNT signaling contributes to cartilage failure in humans with PPD. WISP3 gene is composed of 5 coding exons, with several alternatively spliced exons described in the database. It encodes a 354-amino acid protein, member of the WISP family of growth modulators, expressed in adult/fetal kidney, testis, placenta, ovary, prostate, small intestine, as well as in skeletally derived cells, such as synoviocytes, chondrocytes, and bone narrow-derived mesenchymal progenitor cells [6,7,21]. Sen et al. [21] found that WISP3 gene can regulate the synthesis of chondrocyte collagen type II and saccharan, and the latter two are the major components of cartilaginous tissue. So any depletion or dysfunction of WISP3 can result in cartilage lesion. As above mentioned, the population incidence is likely to be higher in the Middle East and Gulf states [2]. Hence, some differences must exist among different ethnics in the forms of WISP3 gene mutation. In addition, the prognosis of the patients with PPD is definitely ominous with the probability of losing their abilities of selfcare. Therefore, it is very important to identify the general forms of WISP3 gene mutation in Chinese decent, and provide a possible method to interfere in. On the whole, the phenotypes of the patients in our study were mainly in accordance with the literature. Although mutations located in different exons, the patients had almost the same phenotypes, confirming that mutations in different WISP3 domains are all associated with similar clinical features, and that the integrity of the WISP3 gene are very important for proper skeletal growth and cartilage protection [10]. But we found little different clinical manifestations from the cases reported previously, which are listed as follows: 1. in family 2, two female patients presented arthrogryposis of elbow joints and genu valgum resulted in “X” appearance, which were seldom mentioned in the previously reported cases. 2. An intra-familial variability was noted: symptoms tended to be more severe with advancing age in patients with C114Y mutation. At the meanwhile, it seemed that the symptoms in female were more severe than that in male with C114Y mutation. 3. Compared with C114Y mutant, G46X mutant seemed to have more severe symptoms and the age of onset was earlier than the C114 mutant. 4. Many heterozygous carriers (c.8004CNT and c.8209GNA) were found in the two families,
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suggesting the existence of a founder effect in the locality where they live, respectively. In this study, we observed some features other than the characteristics reported previously, and through which we seemed to find some relations between genotype and phenotype, but we reviewed all the previously published mutations, we could hardly draw a conclusion that non-sense or splice mutation is generally more severe than missense mutation right now. In any event, analysis of other more patients with PPD will be necessary to further identify the relationship between genotype and phenotype. In conclusion, our present findings suggest that the novel nonsynonymous mutations (G46X and C114Y) in exon 3 in the WISP3 gene are critical for PPD in Chinese patients. Furthermore, many heterozygous carriers (c.8004CNT and c.8209GNA) are found in the two families, suggesting the existence of a founder effect in the locality where they live, respectively. Therefore, a further study that we will do is to investigate the local people in order to screen the carriers harboring the disease-causing gene and take methods to interfere to decrease the incidence of the disease. Acknowledgments The authors are grateful to the patients for their collaboration. We also give thanks to the anonymous reviewers for their good comments which helped us improve the manuscript. The study was supported by the National Natural Science Foundation of China (No 30570891, 30771019) and Program of Shanghai subject chief scientist (No 08XD1403000). References [1] Wynne-Davies R, Hall C, Ansell BM. Spondylo-epiphysical dysplasia tarda with progressive arthropathy. A “new” disorder of autosomal recessive inheritance. J Bone Joint Surg Br 1982;64:442–5. [2] Teebi AS, Al Awadi SA. Spondyloepiphyseal dysplasia tarda with progressive arthropathy: a rare disorder frequently diagnosed among Arabs. J Med Genet 1986;23:189–91. [3] el-Shanti HE, Omari HZ, Qubain HI. Progressive pseudorheumatoid dysplasia: report of a family and review. J Med Genet 1997;34:559–63. [4] Rezai-Delui H, Mamoori G, Sadri-Mahvelati E, Noori NM. Progressive pseudorheumatoid chondrodysplasia: a report of nine cases in three families. Skeletal Radiol 1994;23:411–9. [5] Nakamura Y, Weidinger G, Liang JO, Aquilina-Beck A, Tamai K, Moon RT, et al. The CCN family member Wisp3, mutant in progressive pseudorheumatoid dysplasia, modulates BMP and Wnt signaling. J Clin Invest 2007;117:3075–86. [6] Hurvitz JR, Suwairi WM, Van Hul W, El-Shanti H, Superti-Furga A, Roudier J, et al. Mutations in the CCN gene family member WISP3 cause progressive pseudorheumatoid dysplasia. Nat Genet 1999;23:94–8. [7] Pennica D, Swanson TA, Welsh JW, Roy MA, Lawrence DA, Lee J, et al. WISP genes are members of the connective tissue growth factor family that are up-regulated in Wnt-1-transformed cells and aberrantly expressed in human colon tumors. Proc Natl Acad Sci USA 1998;95:14717–22. [8] Rachfal AW, Brigstock DR. Structural and functional properties of CCN proteins. Vitam Horm 2005;70:69–103. [9] Leask A, Abraham DJ. All in the CCN family: essential matricellular signaling modulators emerge from the bunker. J Cell Sci 2006;119:4803–10. [10] Delague V, Chouery E, Corbani S, Ghanem I, Aamar S, Fischer J, et al. Molecular study of WISP3 in nine families originating from the Middle-East and presenting with progressive pseudorheumatoid dysplasia: identification of two novel mutations, and description of a founder effect. Am J Med Genet A 2005;138A:118–26. [11] Ehl S, Uhl M, Berner R, Bonafé L, Superti-Furga A, Kirchhoff A. Clinical, radiographic, and genetic diagnosis of progressive pseudorheumatoid dysplasia in a patient with severe polyarthropathy. Rheumatol Int 2004;24:53–6. [12] Liao EY, Peng YQ, Zhou HD, Mackie EJ, Li J, Hu PA, et al. Gene symbol: WISP3. Disease: spondyloepiphyseal dysplasia tarda with progressive arthropathy. Hum Genet 2004;115:174. [13] Peng YQ, Liao EY, Gu HM, Wei QY, Zhou HD, Li J, et al. Pathology and molecular pathogenesis of spondyloepiphyseal dysplasia tarda with progressive arthropathy caused by compound CCN6 heterogeneous gene mutations. Zhonghua Yi Xue Za Zhi 2004;84:1796–803. [14] Bork P. The modular architecture of a new family of growth regulators related to connective tissue growth factor. FEBS Lett 1993;327:125–30. [15] Brigstock DR. The CCN family: a new stimulus package. J Endocrinol 2003;178:169–75. [16] Perbal B. CCN proteins: multifunctional signalling regulators. Lancet 2004;363:62–4. [17] Itasaki N, Jones CM, Mercurio S, Rowe A, Domingos PM, Smith JC, et al. Wise, a context-dependent activator and inhibitor of Wnt signalling. Development 2003;130:4295–305.
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[18] Avsian-Kretchmer O, Hsueh AJ. Comparative genomic analysis of the eightmembered ring cystine knot-containing bone morphogenetic protein antagonists. Mol Endocrinol 2004;18:1–12. [19] Piccolo S, Agius E, Leyns L, Bhattacharyya S, Grunz H, Bouwmeester T, et al. The head inducer Cerberus is a multifunctional antagonist of Nodal, BMP and Wnt signals. Nature 1999;397:707–10.
[20] Ivkovic S, Yoon BS, Popoff SN, Safadi FF, Libuda DE, Stephenson RC, et al. Connective tissue growth factor coordinates chondrogenesis and angiogenesis during skeletal development. Development 2003;130:2779–91. [21] Sen M, Cheng YH, Goldring MB, Lotz MK, Carson DA. WISP3 dependent regulation of type II collagen and aggrecan production I chondrocytes. Arthritis Rheum 2004;50:488–97.
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Bone j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b o n e
Identification of novel mutations in WISP3 gene in two unrelated Chinese families with progressive pseudorheumatoid dysplasia Hua Yue, Zhen-Lin Zhang ⁎, Jin-Wei He Department of Osteoporosis, Metabolic Bone Disease and Genetic Research Unit, Shanghai Jiao Tong University Affiliated the Sixth People's Hospital, Shanghai 200233, 600 Yi-Shan Rd., PR China
a r t i c l e
i n f o
Article history: Received 2 August 2008 Revised 30 October 2008 Accepted 5 November 2008 Available online 21 November 2008 Edited by: B. Olsen Keywords: Progressive pseudorheumatoid dysplasia WISP3 Mutation
a b s t r a c t Introduction: Progressive pseudorheumatoid dysplasia (PPD) is an autosomal recessive genetic disease and it has been reported that PPD is caused by mutations of the Wnt1-inducible signaling pathway protein 3 (WISP3) gene which is located on chromosome 6q22. Up to date, 16 different mutations in the WISP3 have been identified in patients with PPD in different countries previously, but only two mutations in exon 5 were previously identified from Asian origin. Our study aimed to characterize the clinical manifestations and features of PPD and screen the mutations of the disease causing WISP3, and try to elucidate the molecular pathogenesis of PPD. Materials and methods: Altogether, 153 persons, including 4 affected individuals, 49 unaffected individuals from two unrelated Chinese families, and 100 healthy donors were recruited and genomic DNA was extracted. PPD was diagnosed based on the clinical manifestations, physical examination, characteristics of their bones on X-ray and laboratory results. All 5 exons and their exon–intron boundaries of the WISP3 gene were amplified by polymerase chain reaction (PCR) and sequenced directly. Results: In family 1, we identified that the proband (IV4) carried a novel non-sense mutation (G46X) which consisted of a homozygous C to T transition at c.8004 in exon 3. This mutation changed codon CAG to TAG and resulted in a subsequent change of the glutamine codon to stop codon and truncation at p. 46. In family 2, a novel missense mutation (C114Y) was found in the three patients (IV6, IV7, IV8), namely, a homozygous G to A transition at c.8209 in exon 3, which resulted in a cysteine (TGT) to tyrosine (TAT) substitution at p.114. Neither G46X nor C114Y was found in 100 normal controls. Meanwhile, we found that these patients had some different phenotypes, compared with the affected individuals with PPD from cases reported previously. Conclusions: Our study suggests that the novel G46X and C114Y mutations in exon 3 in WISP3 gene are responsible for PPD in Chinese patients. Furthermore, many heterozygous carriers (c.8004CNT and c.8209GNA) are found in the two families, suggesting the existence of a founder effect in the locality where they live, respectively. © 2008 Elsevier Inc. All rights reserved.
Introduction Progressive pseudorheumatoid dysplasia (PPD) (OMIM 208230), also referred to as spondyloepiphyseal dysplasia tarda with progressive arthropathy (SEDT-PA) or progressive pseudorheumatoid arthropathy of childhood (PPAC), is an autosomal recessive genetic disease, and its population incidence has been estimated at 1 per million in the UK [1], but it is likely to be higher in the Middle East and Gulf states [2]. It is a rare disease in the world and there is no prevalence data documented yet in China. Patients with this disorder are asymptomatic in early childhood [3,4]. Signs and symptoms of disease typically develop between 3 and 8 years of age [5], whose typical clinical manifestations and radiographic findings consist of progressive
⁎ Corresponding author. Fax: +86 21 64081474. E-mail address: [email protected] (Z.-L. Zhang). 8756-3282/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bone.2008.11.005
deformities of multiple joints associated with stiffness, swelling without any inflammatory context, limitation of motion, bone pain, short stature, widened epiphyses, vertebral flattening, narrow joint spaces and osteoporosis. Although both genetic and environmental factors have been implicated in the pathogenesis of PPD, PPD has been attributed to loss-of-function mutations of Wnt1-inducible signaling pathway protein 3 (WISP3) gene located on chromosome 6q22 [5–7]. The WISP3 gene is a member of the connective tissue growth factor (CCN) gene family. The latter comprises cysteine-rich 61 (CYR61/CCN1), connective tissue growth factor (CTGF/CCN2), nephroblastoma overexpressed (NOV/CCN3), and Wnt-induced secreted proteins-1 (WISP-1/CCN4), -2 (WISP-2/CCN5) and -3 (WISP-3/CCN6) [8,9]. These proteins stimulate mitosis, adhesion, apoptosis, extra-cellular matrix production, growth arrest and migration of multiple cell types. WISP3 encodes Wnt1-inducible signaling protein 3 which is a cysteine-rich, multi-domain, secreted protein and consists of 354 amino acids.
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Because the population incidence of PPD is very low, it is not very easy for us to find such patients, in that case, which hindered us to recognize this disease to some extent. Up to now, there are about 16 different mutations that have been identified in the WISP3 gene, including: A15fsX, g.IVS1 + 2insT (splicing defect), C52X, C78R, P82fsX104, G83E, C145Y, C179fsX, A197fsX201, C247fsX277, F280fsX312, T288fsX313, Q289fsX301, W331X, Q338L and S334P [6,10,11]. However, only two WISP3 gene mutations in exon 5 (F280fsX312 and S334P) were previously identified in the Asian population [12,13]. Moreover, the expression profiles and characteristics of mutation are known to be influenced by ethnic background. Because population incidence would be vary greatly in different races of people, it is very important to make clear the forms of mutant in Chinese PPD patients. Furthermore, we also want to know whether PPD from Chinese origin is caused by the mutation of the WISP3 gene. Until now, the relationship between phenotype and genotype of PPD is still unclear, so it is urgent for us to find more patients with PPD to develop further studies. In this study, we performed mutation analysis of the WISP3 gene in two unrelated Chinese families affected with PPD and revealed one novel nonsense mutation (G46X) and the other one novel missense mutation (C114Y) which consisted of a homozygous C to T transition at c.8004 and a G to A transition at c.8209 in exon 3, respectively. Meanwhile, we found that these patients had some different phenotypes, compared with the affected individuals with PPD from cases reported previously.
Materials and methods Patients The study was approved by the Ethics Committee of the Shanghai Jiao Tong University Affiliated the Sixth People's Hospital. All the subjects involved in this study were recruited by the department of osteoporosis from outpatients presented to our department within 2 years and signed informed consent documents before entering the project. Altogether, four patients with PPD from two unrelated families and their family members were investigated in this study (Fig. 1). Family 1 came from Liaoning province, located on the east territory of China. Family 2 came from Henan province, located on the central territory of China. All the subjects are Han ethnicity. The proband (IV4) in family 1 is a 13-year-old female, the only daughter of non-consanguineous and healthy parents. She was born with full term pregnancy and normal delivery. Birth weight and length were within normal limits. Her current height and weight are 138.5 cm and 33 kg, respectively. She came to our department with an 11-year history of joint deformities. When she was 2 years old, swelling of joints emerged, fingers were affected first, and then hands, carpal. Whereafter, elbows, knees, hip and spine were all affected, associated with pain and deformities. She progressed to ankylosis at the age of 10. Last year, she presented obvious limitation of motion, and almost lost her ability of self-care. X-rays manifestations revealed a general decrease of bone density involving vertebrae and pelvis;
Fig. 1. Pedigrees of the two families in our study. Patients with PPD are shown by darkened symbols. Carriers are shown by half-darkened symbols.
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Fig. 2. Photographs and X-rays of the patient (Family 1, IV4). (A) the enlargement of the interphalangeal joints (B) enlargement of the epiphyseal and metaphyseal portions of the metacarpals and phalanges; (C) intervertebral and flattened vertebral bodies with rounded end plates; (D) short and wide femoral neck and large capital femoral epiphyses with narrow joint spaces of hip.
enlargement of the epiphyseal and metaphyseal portions of the metacarpals and phalanges; short and wide femoral neck and large capital femoral epiphyses with narrow joint spaces of hip and intervertebral and flattened vertebral bodies with rounded end plates (Fig. 2). Laboratory data showed normal serum sodium, potassium, phosphorus, calcium, creatinine, glucose, blood gas, and hemoglobin. Serum alkaline phosphatase (ALP), parathyroid hormone (PTH), antistreptolysin “O” (ASO), rheumatoid factor (RF), C-reactive protein (CRP), and erythrocyte sedimentation rate (ESR) levels were within the normal range. Moreover, urine sample of the proband had been tested to rule out the congenital metabolic diseases, such as abnormal
metabolism of amino acid, urea cycle, amino acid absorption, fatty acid, purine, pyrimidine and glycometabolism. Simultaneously, hyperlactacidemia, hyperpyruvemia, and peroxisomal disease were also eliminated. The rest thirty-eight members of this family including her parents didn't have any abnormal clinical, biochemical and radiographic manifestations. The proband (IV7) in family 2 is a 14-year-old male, the third son of consanguineous parents, term birth with normal length and weight. His current height and weight are 141 cm and 35 kg, respectively. His first clinical abnormality was found at the age of 4 with swelling of his articulationes interphalangeae, and looked like “clawhands”. Until age
Fig. 3. X-rays of the manifestations in patient (IV7) in family 2. (A) Short and wide femoral neck and large capital femoral epiphyses with narrow joint spaces of hip; (B) right fingers narrow joint spaces and narrow spaces between the carpal bones; (C) flattening and anterior beaking on the thoraco-lumber spine with irregular upper and lower end plates.
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Fig. 4. Photographs and X-rays of the patient (Family 2, IV6). (A) Deformity of bilateral elbow joints; (B) hump back; genu valgum resulted in “X” appearance; (C) arthrogryposis of elbow joint; (D) enlargement of the epiphyseal and metaphyseal portions of the metacarpals and phalanges; (E) left femoral neck fracture; femoral head necrosis; the pelvis severely out of shape; (F) flattened vertebral bodies.
14, he didn't feel any upset, and could walk, run and do common activities like other healthy persons. One month ago, he complained of hip pain and had difficulty in elevating his legs and associated with instability of gait. He has two sisters, one (IV5, 19 years old) with normal phenotype, however, the other sister (IV6, 16 years old), had similar manifestations with him. Her age of onset was 3 years, swelling of articulationes interphalangeae were also her first physical sign. Deformities of joints associated with pain and function loss progressed as she aged. At the age of 12, her lower limbs were found asymmetry with the left shorter than the right. About 2 years ago, she couldn't walk without a crutch. Another patient in this family is the proband's older female cousin (IV8), who is 15 years old. She is the first daughter of non-consanguineous parents and has a normal little brother. Her age of onset was 3 years with almost the same symptoms as the other two patients in the family. She lost her walking ability at 13 years of age. X-ray manifestations of the three patients revealed a general decrease of bone density involving vertebrae and pelvis; enlargement of the epiphyseal and metaphyseal portions of the metacarpals and phalanges; short and wide femoral neck and large capital femoral epiphyses, unsmooth and irregularity of joint surface; flattened vertebral bodies. In addition, some different features among them are listed as follows: The proband (IV7): his articulationes interphalangeae and articulatio metacarpophalangea could not be straightened and bent, and are relatively fixed like “clawhands” (Fig. 3). His sister (IV6): her pelvis is severely out of shape associated with arthrogryposis of elbow joint, hump back, genu valgum resulted in “X” appearance (Fig. 4). His older female cousin (IV8): her pelvis is out of shape with left hand side femoral head damaged just like IV6 (Fig. 5). Laboratory data showed normal serum sodium, potassium, phosphorus, calcium, creatinine, glucose, blood gas, hemoglobin,
PTH, RF, CRP and ESR, except for the mildly elevated levels of serum ALP and ASO. No clinical, biochemical and radiographic abnormalities were found in the rest of the 11 members (II1, II2, II3, II4, III2, III3, III5, III6, III7, III8 and IV5) of this family. In addition, III4, III9, III10, IV1–4 and IV9–11 didn't apply their blood samples, so we just observed their phenotypes and didn't analyze their DNA. General features, laboratory data, clinical and radiological findings of the 4 patients in the 2 families are listed in Table 1 and Table 2, respectively.
Fig. 5. X-rays of the manifestations in patient (IV8) in family 2. (A) Left femoral neck fracture, femoral head necrosis and the pelvis severely out of shape; (B) flattened vertebral bodies.
H. Yue et al. / Bone 44 (2009) 547–554 Table 1 General features and laboratory findings in patients of 2 families
Patients Gender Age (years) Height (cm) Weight (kg) ALP (U/l) BUN (mmol/l) Cr (μmol/l) Calcium (mmol/l) Phosphate (mmol/l) PTH (pg/ml) ESR (mm/h) CRP (mg/l) ASO (IU/ml) RF (IU/ml) HLA-B27
Family 1
Family 2
Proband (IV4) Female 13 138.5 33 278 3.30 35 2.51 1.65 14.07 12 0.10 243 8 −
Proband (IV7) Male 14 141 35 206 2.90 35 2.45 1.61 15.2 9 0.20 364 9.5 −
Table 3 The primers sequence of exons in WISP3 gene Normal value
Sister (IV6) Female 16 140 41 64 7.4 48 2.53 1.26 33.4 15 0.25 125 8.5 −
Older female cousin (IV8) Female 15 139 39 110 5.6 51 2.39 1.42 29.4 11 0.92 95 11 −
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15–112 2.5–6.4 53–115 2.08–2.6 0.8–1.6 15–65 0–20 b 3 mg/l 0–200 0–15 −
“−” negative.
Sequence analysis of the WISP3 gene Informed consents were obtained from each family and 100 healthy volunteers before blood sampling and DNA analysis. Altogether, 153 DNA samples, including 4 affected individuals, 49 unaffected individuals from two families, and 100 healthy donors. DNA was extracted from peripheral white blood cells with conventional methods. We sequenced the entire coding region (five exons with several alternatively spliced exons) of the WISP3 (GeneBank accession number: NC_000006) to screen mutations using 6 pairs of primers (Table 3). Primers were designed using Primer 3 software (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi). All 5 exons and their exon–intron boundaries of the WISP3 gene were amplified by polymerase chain reaction (PCR). Direct nucleotide sequencing was performed using the Big-Dye Terminator Cycle Sequencing Ready Reaction Kit version 3.1 (Applied by Bio-systems, CA, USA) and analyzed by an ABI 3700 automatic sequencer (Applied by Bio-systems, CA, USA) with standard methods. Results In family 1, the proband (IV4, 13-year-old female) and 38 unaffected subjects of the family were genotyped by direct nucleotide sequencing. We identified that the proband carried a novel non-sense mutation (G46X) which consisted of a homozygous C to T transition at c.8004 in exon 3 (Fig. 6). This mutation changed codon CAG to TAG and
Exon
Primers (5′–3′) sense
Primers (5′–3′) anti-sense
1 2 3 4 5
TCA CTG CGA AGG CAG GTT ATT CTG CAC GCT GTT GCT ATG AAG CCT GTT TGG GGG AAA TCT TCT TCC TGT GAA GGA GGT TCC AAA AGG CAA AGC AGA AAA ATG CAA AAG GGT AAA GAG AGT GCT GGA
TGC CAT TAC CTG AAA GGG AGA CCTTGC TCC TTT GTC CAC TTG TAC AAT GGA GCC AGT CCC ACT TCC CTG TCT GAG GCA AAG ATT ATC CCA CCC TCC AAA ACA CAC AAA CAA AGT AGA TTT GCC ACC A
resulted in a subsequent change of the glutamine codon to stop codon and truncation at p. 46. In all other members of this PPD kindred, the proband's father (III7), mother (III8), her father's brother (III9), her mother's younger brother (III19) and her older male cousin (IV1) are the heterozygous (T/C) carriers. DNA samples from 100 normal volunteers were subsequently tested for this mutation, but no G46X was found in all chromosomes, and normal individuals had C/C genotype. In family 2, the proband (IV7, 14-year-old male), two patients (IV6 and IV8) and the other 11 unaffected subjects (II1, II2, II3, II4, III2, III3, III5, III6, III7, III8 and IV5) of the family were genotyped by direct nucleotide sequencing. A novel missense mutation was found in the three patients, namely, a homozygous G to A transition at c.8209 in exon 3 (Fig. 7), which resulted in a cysteine (TGT) to tyrosine (TAT) substitution at p.114. This C114Y mutation was also found in the heterozygous state in his father's mother (II2), mother's mother (II4), father's older sister (III3), father (III5), mother (III6) and father's younger sister (III8), aunt's husband (III7) and his sister (IV5). No C114Y mutation was found in all chromosomes from 100 normal controls and normal individuals had G/G genotype. In this study, we found that G46X mutant had more severe symptoms and the age of onset was earlier than the C114Y mutants (the proband in family 1, whose age of onset was 2 years, and had limited flexion and extension at 10). Moreover, we observed some intra-familial clinical variability and symptoms that became more severe with the age in patients with C114Y mutation (the females both had arthrogryposis of elbow joint and genu valgum resulted in “X” appearance). Meanwhile, it seemed that the symptoms in a female were more severe than in a male in C114Y mutant (two females both have left hand side femoral neck fracture, femoral head necrosis and the pelvis severely out of shape, but the male had none of the symptoms). Furthermore, many heterozygous carriers (c.8004CNT and c.8209GNA) are found in the two families, suggesting the existence of a founder effect in the locality where they live, respectively. Discussion
Table 2 Clinical and radiological findings in patients of the 2 families
Age at examination (years) Age of onset (years) Age of joint swelling (years) Limited flexion and extension (years) Painful joints (years) Short stature Vertebral changes Osteoarthrosis of joints Widened epiphyses Osteoporosis Muscle wasting Intelligence, acouesthesia Eyesight, smell Femoral head necrosis
Family 1 (IV4)
Family 2 (IV7, IV6, IV8)
13 2 2 10
14, 16 and 15 4, 3 and 3 4, 3 and 3 14, 12 and 13
5 + Platyspondy and wedging of vertebra + + + + − − −
14, 11 and 11 + Platyspondy and wedging of vertebra + + + + − − −, + and +
“+”: positive manifestations; “−”: normal.
PPD is a rare inherited metabolic disease. In our study, we reported 4 patients in two unrelated Chinese families and found the 4 patients all had bone pain, and multiple joints deformities (stiffness and swelling) as they age. Radiographs showed progressive loss of joint space, widened epiphyses, vertebral flattening and generalized decrease of bone density. The patients had almost normal blood laboratory findings. And the molecular findings in the 4 patients from two unrelated families are in complete agreement with the clinical diagnosis of PPD. That is to say, PPD was diagnosed based on the clinical presentations, the characteristics of radiographic examinations and molecular findings. Unlike other congenital skeleton diseases, the patients with PPD are all asymptomatic in the first years of life and often mimic early juvenile rheumatoid arthritis and mucolipidosis type IV. The latter two are the common causes of misdiagnosis, which can be excluded by genetic counseling and laboratory findings. In this study, direct sequencing of the WISP3 gene was performed in two PPD families. We identified that the proband in family 1 carried
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Fig. 6. Automated sequencing traces of the mutation in WISP3 gene in family 1 with PPD. (A) Mutation screening was performed in patient (IV4) by direct sequencing of PCR products, revealing a CNT transition in exon 3 of WISP3, which resulted in G46X change. (B) Mutation screening was performed in heterozygous carriers (III7, III8, III9, III19 and IV1).
a novel non-sense mutation (G46X) which consisted of a homozygous C to T transition at c.8004 in exon 3. This mutation changed codon CAG to TAG and resulted in a subsequent change of the glutamine codon to stop codon and truncation at p. 46. Another novel missense mutation (C114Y) was found in the proband and the other two patients in family 2, namely, a homozygous G to A transition at c.8209 in exon 3, which resulted in a Cysteine (TGT) to Tyrosine (TAT) substitution at p. 114. To date, apart from our findings, over 16 mutations in WISP3 have been identified in 22 different PPD families from USA, Jordan, Italy, France, Lebanon, Syria, Iran, Saudi Arabia, Caucasian, and China [6,10,11], including exon-deletion, frameshift, non-sense, and missense mutations [5,6,10,11] (Fig. 8), which suggests that PPD results from a loss of WISP3 protein function [5]. Among the families with WISP3 gene mutations, 14 families are from consanguineous mating, and 8 are from non-consanguineous mating. Patients from nonconsanguineous families all appeared to be compound-heterozygotes, except one is heterozygous. And patients from consanguineous families were all homozygous [6]. In this study, we identified the molecular defect responsible for PPD in two unrelated families. We described two novel mutations and found many heterozygous carriers within families. However, the 4 patients are all homozygotes, and we found no compound-heterozygotes. It is interesting that the parents of the proband (IV4) in family 1 and the patient (IV8) in family 2 are both non-consanguineous mating and they are all in the heterozygous states harboring the disease-causing T or A allele, respectively. Since the two couples are all natives, we can conclude that the numbers of risk gene carriers in the place they live must be higher than from anywhere else and suggest the existence of a founder effect. In that
case, the two carriers could meet more frequently in the circumstances of random marriage, and then the incidence of PPD would be higher in the locality. Therefore, the next thing we should do is to go to the districts they live in, and investigate the local people in order to find the carriers harboring the disease-causing gene as soon as possible and take methods to interfere, so as to decrease the incidence of the disease to the utmost extent. WISP3 is a member of the CCN family of secreted growth factors. CCN proteins are multimodular mosaic proteins containing four conserved modules which are present in other unrelated extracellular proteins [14]. Module 1 is an insulin-like growth factor (IGF) binding domain which is encoded by exon 2. Module 2 is a von Willebrand type C domain which is encoded by exon 3. WISP3, unlike all other CCN proteins identified to date, lacks 4 of 10 conserved cysteine residues in this domain [6,7]. Module 3 is a thrombospondin domain which is encoded by exon 4. Module 4 is a C-terminal domain containing a putative cysteine knot which is encoded by exon 5 [5,6,15–19]. The CCN proteins demonstrate a wide variety of biological activities regulating cell adhesion, proliferation, survival, migration, invasion in vitro and tumorigenesis and angiogenesis in vivo. At present, only WISP3 among CCN family members has been linked to a Mendelian genetic human disease [5,20]. Among the mutations in the WISP3 gene, one mutation was found in exon 1, 4 in exon 2, 5 in exon 3, 1 in exon 4, 6 in exon 5 and 1 in intron 1, respectively (Fig. 8). From the above, we can learn that mutations are located in all coding exons that can result in PPD. Mutations occurred in exon 3 and exon 5 have been found in multiple studies, indicating that exon 3 and exon 5 may be hotspots for mutations.
Fig. 7. Automated sequencing traces of the mutation in WISP3 gene in family 2 with PPD. (A) Mutation screening was performed in patients (IV6, IV7 and IV8) by direct sequencing of PCR products, revealing a GNA transition in exon 3 of WISP3, which resulted in C114Y change. (B) Mutation screening was performed in heterozygous carriers (II2, II4, III3, III5, III6, III7, III8 and IV5). (C) Mutation screening was performed in normal persons.
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Fig. 8. Domain structure of WISP3, with locations of the disease-causing PPD mutations noted (G46X and C114Y are novel mutations found in this study). Signal peptide (SP), insulin-like growth factor-binding domain (IGFBP), von Willebrand factor type C domain (VWC), thrombospondin domain (TSP), and C-terminal cysteine knot-like domain (CT) are shown. The homozygous g.IVS1 + 2insT (Splicing defect) mutation in intron 1 is not shown. The mutations are reported according to mutation nomenclature guidelines. Numbering begins at the initiation codon and is referring to the cDNA sequence with the following GeneBank: accession number: AF 100781.1.
WISP3 was cloned and characterized as the gene locates on the downstream in the Wnt signaling pathway in 1998 [7]. A recent study found that overexpression of WISP3 inhibited bone morphogenetic protein (BMP) and Wnt signaling during zebrafish development. ZebraWISP3 and human WISP3 have 49% identity and 54% similarity at the amino acid level. Zebrafish and human WISP3 inhibited BMP and Wnt signaling in mammalian cells by binding to BMPs and to the Wnt coreceptor low density lipoprotein receptor related protein 6 (LRP6) and frizzled respectively [5]. Therefore, they concluded that dysregulation of BMP and/or WNT signaling contributes to cartilage failure in humans with PPD. WISP3 gene is composed of 5 coding exons, with several alternatively spliced exons described in the database. It encodes a 354-amino acid protein, member of the WISP family of growth modulators, expressed in adult/fetal kidney, testis, placenta, ovary, prostate, small intestine, as well as in skeletally derived cells, such as synoviocytes, chondrocytes, and bone narrow-derived mesenchymal progenitor cells [6,7,21]. Sen et al. [21] found that WISP3 gene can regulate the synthesis of chondrocyte collagen type II and saccharan, and the latter two are the major components of cartilaginous tissue. So any depletion or dysfunction of WISP3 can result in cartilage lesion. As above mentioned, the population incidence is likely to be higher in the Middle East and Gulf states [2]. Hence, some differences must exist among different ethnics in the forms of WISP3 gene mutation. In addition, the prognosis of the patients with PPD is definitely ominous with the probability of losing their abilities of selfcare. Therefore, it is very important to identify the general forms of WISP3 gene mutation in Chinese decent, and provide a possible method to interfere in. On the whole, the phenotypes of the patients in our study were mainly in accordance with the literature. Although mutations located in different exons, the patients had almost the same phenotypes, confirming that mutations in different WISP3 domains are all associated with similar clinical features, and that the integrity of the WISP3 gene are very important for proper skeletal growth and cartilage protection [10]. But we found little different clinical manifestations from the cases reported previously, which are listed as follows: 1. in family 2, two female patients presented arthrogryposis of elbow joints and genu valgum resulted in “X” appearance, which were seldom mentioned in the previously reported cases. 2. An intra-familial variability was noted: symptoms tended to be more severe with advancing age in patients with C114Y mutation. At the meanwhile, it seemed that the symptoms in female were more severe than that in male with C114Y mutation. 3. Compared with C114Y mutant, G46X mutant seemed to have more severe symptoms and the age of onset was earlier than the C114 mutant. 4. Many heterozygous carriers (c.8004CNT and c.8209GNA) were found in the two families,
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suggesting the existence of a founder effect in the locality where they live, respectively. In this study, we observed some features other than the characteristics reported previously, and through which we seemed to find some relations between genotype and phenotype, but we reviewed all the previously published mutations, we could hardly draw a conclusion that non-sense or splice mutation is generally more severe than missense mutation right now. In any event, analysis of other more patients with PPD will be necessary to further identify the relationship between genotype and phenotype. In conclusion, our present findings suggest that the novel nonsynonymous mutations (G46X and C114Y) in exon 3 in the WISP3 gene are critical for PPD in Chinese patients. Furthermore, many heterozygous carriers (c.8004CNT and c.8209GNA) are found in the two families, suggesting the existence of a founder effect in the locality where they live, respectively. Therefore, a further study that we will do is to investigate the local people in order to screen the carriers harboring the disease-causing gene and take methods to interfere to decrease the incidence of the disease. Acknowledgments The authors are grateful to the patients for their collaboration. We also give thanks to the anonymous reviewers for their good comments which helped us improve the manuscript. The study was supported by the National Natural Science Foundation of China (No 30570891, 30771019) and Program of Shanghai subject chief scientist (No 08XD1403000). References [1] Wynne-Davies R, Hall C, Ansell BM. Spondylo-epiphysical dysplasia tarda with progressive arthropathy. A “new” disorder of autosomal recessive inheritance. J Bone Joint Surg Br 1982;64:442–5. [2] Teebi AS, Al Awadi SA. Spondyloepiphyseal dysplasia tarda with progressive arthropathy: a rare disorder frequently diagnosed among Arabs. J Med Genet 1986;23:189–91. [3] el-Shanti HE, Omari HZ, Qubain HI. Progressive pseudorheumatoid dysplasia: report of a family and review. J Med Genet 1997;34:559–63. [4] Rezai-Delui H, Mamoori G, Sadri-Mahvelati E, Noori NM. Progressive pseudorheumatoid chondrodysplasia: a report of nine cases in three families. Skeletal Radiol 1994;23:411–9. [5] Nakamura Y, Weidinger G, Liang JO, Aquilina-Beck A, Tamai K, Moon RT, et al. The CCN family member Wisp3, mutant in progressive pseudorheumatoid dysplasia, modulates BMP and Wnt signaling. J Clin Invest 2007;117:3075–86. [6] Hurvitz JR, Suwairi WM, Van Hul W, El-Shanti H, Superti-Furga A, Roudier J, et al. Mutations in the CCN gene family member WISP3 cause progressive pseudorheumatoid dysplasia. Nat Genet 1999;23:94–8. [7] Pennica D, Swanson TA, Welsh JW, Roy MA, Lawrence DA, Lee J, et al. WISP genes are members of the connective tissue growth factor family that are up-regulated in Wnt-1-transformed cells and aberrantly expressed in human colon tumors. Proc Natl Acad Sci USA 1998;95:14717–22. [8] Rachfal AW, Brigstock DR. Structural and functional properties of CCN proteins. Vitam Horm 2005;70:69–103. [9] Leask A, Abraham DJ. All in the CCN family: essential matricellular signaling modulators emerge from the bunker. J Cell Sci 2006;119:4803–10. [10] Delague V, Chouery E, Corbani S, Ghanem I, Aamar S, Fischer J, et al. Molecular study of WISP3 in nine families originating from the Middle-East and presenting with progressive pseudorheumatoid dysplasia: identification of two novel mutations, and description of a founder effect. Am J Med Genet A 2005;138A:118–26. [11] Ehl S, Uhl M, Berner R, Bonafé L, Superti-Furga A, Kirchhoff A. Clinical, radiographic, and genetic diagnosis of progressive pseudorheumatoid dysplasia in a patient with severe polyarthropathy. Rheumatol Int 2004;24:53–6. [12] Liao EY, Peng YQ, Zhou HD, Mackie EJ, Li J, Hu PA, et al. Gene symbol: WISP3. Disease: spondyloepiphyseal dysplasia tarda with progressive arthropathy. Hum Genet 2004;115:174. [13] Peng YQ, Liao EY, Gu HM, Wei QY, Zhou HD, Li J, et al. Pathology and molecular pathogenesis of spondyloepiphyseal dysplasia tarda with progressive arthropathy caused by compound CCN6 heterogeneous gene mutations. Zhonghua Yi Xue Za Zhi 2004;84:1796–803. [14] Bork P. The modular architecture of a new family of growth regulators related to connective tissue growth factor. FEBS Lett 1993;327:125–30. [15] Brigstock DR. The CCN family: a new stimulus package. J Endocrinol 2003;178:169–75. [16] Perbal B. CCN proteins: multifunctional signalling regulators. Lancet 2004;363:62–4. [17] Itasaki N, Jones CM, Mercurio S, Rowe A, Domingos PM, Smith JC, et al. Wise, a context-dependent activator and inhibitor of Wnt signalling. Development 2003;130:4295–305.
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