A retrospective study of nine patients with progressive pseudorheumatoid dysplasia: to explore early diagnosis and further treatment

Most patients with progressive pseudorheumatoid dysplasia (PPRD) are initially misdiagnosed because of disease rarity and lack of awareness by most clinicians. The purpose of this study was to provide further early diagnostic options and potential treatment to patients with PPRD. A retrospective study was performed by collecting and organizing clinical manifestations, radiographic features, laboratory test results, genetic test outcome, treatment, and follow-up records of the patients with PPRD. Age of diagnosis and genotype-phenotype correlation were further analyzed. Nine PPRD children with causative CCN6 mutation were included. There were 3 pairs of siblings and 1 patient from inbred family. Five patients were primarily misdiagnosed as juvenile idiopathic arthritis (JIA). The interval between onset of symptoms and definite diagnosis of 8 patients varied from 3.6 to 20 years. Symptoms at the onset included enlarged and stiff interphalangeal joints of the fingers, gait disturbance, or joint pain. Laboratory tests revealed normal range of inflammatory parameters. Radiographic findings disclosed different degrees of abnormal vertebral bodies and epiphyseal enlargement of the interphalangeal joints. After the treatment of calcitriol in 5 patients with low level 25-hydroxyvitamin D3 for around 1.25 years to 1.75 years, 2 patients kept stable, while 3 of them improved gradually. Combining the patient’s family history, clinical features, normal inflammatory markers, and the characteristic radiographic findings, the clinical diagnosis of PPRD for the patients could be obtained at very early stage of the disease. The patients with PPRD carrying c.624dupA variant in CCN6 may have delayed onset. Underlying vitamin D deficiency should be sought and corrected in patients with PPRD. Key Points • Children with progressive pseudorheumatoid dysplasia (PPRD) are easily misdiagnosed with juvenile idiopathic arthritis (JIA). • PPRD is different from JIA with normal range of inflammatory parameters and abnormal vertebral bodies. • Underlying vitamin D deficiency should be sought and corrected in patients with PPRD.


Introduction
Progressive pseudorheumatoid dysplasia (PPRD) (MIM 208230) is a rare disease first described by Wynne-Davies et al. in 1982 [1]. This genetic skeletal disorder with autosomal recessive inheritance is characterized by progressive noninflammatory arthropathy, with articular cartilages primarily and predominantly affected. [2]. The prevalence of PPRD has been estimated at one per million in the UK [1]. PPRD can be probably underdiagnosed or misdiagnosed because of its overlapping with other pediatric musculoskeletal disorders in clinical and imaging features, such as juvenile idiopathic arthritis (JIA), Scheuermann's disease, spondyloepiphyseal dysplasia, Stickler syndrome, and mucopolysaccharidosis [3][4][5][6]. The onset of symptoms is usually in childhood between 3 and 6 years of age, and symptoms progressively worsen with time [2]. As the disease is so rare and clinicians are thus lacking awareness, most patients are initially misdiagnosed [7]. The average delay in diagnosis is 6 to 13 years [2]. The patients are most often referred to a pediatrician, rheumatologist, orthopedic surgeon, and very few to a medical geneticist [8]. We hope to find further clues to aid earlier and correct diagnosis based on the information between the clinical manifestations and genotypes of the patients with PPRD. PPRD is caused by mutant Wnt1inducible signaling protein 3 (CCN6) gene which maps to chromosome 6q22 [9,10]. The CCN6 gene expresses in synoviocytes and chondrocytes, encoding a member of the connective tissue growth factor [2,9]. This growth factor involves in the cartilage homeostasis and skeletal growth [9,10]. Homozygous or compound heterozygous CCN6 variants will cause the loss of articular cartilage, then narrow the articular spaces, and reduce mobility of all joints progressively [10]. Although the molecular basis of PPRD has been illustrated for more than 10 years, there has been no specific treatment for patients with PPRD by now [2]. 1,25(OH) 2 D 3 (calcitriol) has been used to treat hypocalcemia and osteoporosis and prevent corticosteroidinduced osteoporosis [11]. Recently, in the rheumatoid arthritis rat models, it was demonstrated that 1,25(OH) 2 D 3 could accelerate chondrocyte apoptosis and restore the joint function through suppressing the NF-κB signaling pathway [12]. Moreover, 1,25(OH) 2 D 3 could diminish the cytotoxic effects of TNF-α on chondrocytes and showed its chondro-protective effects in human chondrocytes [13]. Zhou et al. found that articular chondrocytes in PPRD performed increased cell proliferation and decreased cell apoptosis [14]. Articular cartilage (AC) tissues in PPRD are significantly stiffer than normal tissues. The larger elastic modulus of PPRD chondrocytes might reduce their migration capability and decelerate the growth and repair of cartilage [15]. Here we suppose that calcitriol may maintain the normal proliferation of articular chondrocytes by accelerating chondrocyte apoptosis. In the present study, we systematically evaluated the clinical history and characteristics, laboratory test results, radiological data, and CCN6 gene variants in 9 patients with PPRD recruited by our special consultation clinic and evaluated the effects of calcitriol in PPRD patients with vitamin D deficiency.

Patients
Research subjects' blood samples and pedigree data were obtained from August 2011 to December 2019 from individuals with a genetic confirmed diagnosis of progressive pseudorheumatoid dysplasia in our center. According to the principles of the Declaration of Helsinki, informed written consent was obtained from the patients and their parents. This study was approved by the Ethics Committee of the Shanghai Children's Medical Center, Shanghai Jiao Tong University School of Medicine, Shanghai, China. The medical history, results of physical examination, other relevant laboratory investigations, and radiological data were obtained from the patients during clinical visits/routine clinical care and systematically analyzed.

Laboratory investigations
Laboratory tests of inflammatory parameters were performed including complete blood count, C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), and the tests to exclude other diseases including liver function test, serum blood urea and creatinine, rheumatoid factor, antinuclear antibody, HLA-B27, electrolytes, thyroid function tests, and parathyroid hormone. Serum level of 25-hydroxyvitamin D3 was tested in some of the patients.

Sanger sequencing
Genomic DNA was extracted from 2 mL peripheral blood samples using the QIAamp Blood DNA Mini Kit® (Qiagen, Hilden, Germany) according to the manufacturer's instructions. All 5 coding exons and the exon-intron boundaries of the CCN6 gene (NM_003880.3) were amplified by polymerase chain reaction (PCR) using the primers described preciously [16]. Sanger sequencing was performed on an Applied Biosystems ABI3730XL sequencer (Thermo Fisher Scientific, Waltham, MA, USA), and the data were then analyzed using Mutation Surveyor v.4.0.4 software (SoftGenetics LLC, State College, PA, USA).

Results (Table 1)
Nine patients (2 males and 7 females, 7 children and 2 adults) introduced to our center for diagnostic assistance by local hospitals from 4 provinces of China during the period of August 2011 to December 2019 were diagnosed with PPRD. Five of them (case 3,4,6,7,9) were initially diagnosed with juvenile idiopathic arthritis (JIA). Case 9 had been treated with methotrexate and cyclophosphamide for 3 months without improvement in local hospital. Case 5 was diagnosed with ankylosing spondylitis because of positive HLA-B27. Case 8 had the suspected diagnosis of congenital spondyloepiphyseal dysplasia. Six patients (cases 1 and 2, cases 3 and 4, cases 6 and 7) were 3 pairs of siblings. Case 5 had parental consanguinity. After reviewing the family histories, clinical symptoms, and laboratory investigations of the patients, we found that there were several clues which guide us to the diagnosis of PPRD. Key indicators were enlargement of the interphalangeal joints of the fingers and varying degrees of limitation of joint movement, the lack of increased inflammatory parameters (ESR and CRP) in the blood (Table 1), and the radiological findings of the abnormal vertebral bodies. The patients and their parents were subjected to DNA sequencing of CCN6 to investigate the genetic cause of this anomaly and for appropriate genetic counseling.

History of diagnosis and clinical findings (Table 1)
Eight of the 9 patients had an age of onset between 3 and 6 years. Only case 8 first got sick from 14 years old. The interval between onset and diagnosis with PPRD for all patients except case 2 varied from 3.6 to 20 years. Case 2 got the definite diagnosis in 1 month without obvious joint enlargement or movement limitation (Fig. 1a, b) since the patient's older brother (case 1) had been already diagnosed with PPRD. The onset symptoms for the patients included enlarged and stiff interphalangeal joints of the fingers (2/9), gait disturbance (5/9), or joint pain (3/9). Causes for clinic visits were the worsening of symptoms, most (7/9) had progressive abnormal gait, one patient (case 1) had short stature, and one patient (case 2) visited the clinic for further investigation upon her brother's positive PPRD diagnosis. At the time of diagnosis, all the patients except case 2 had manifested enlargement of the interphalangeal joints of the fingers (Figs. 2a, 3a) and various degrees of limitation of joint movement, especially for the fingers and hips (Figs. 2e, f and 3b). No symptoms or signs of inflammatory arthritis such as redness, heat, effusion, or soft tissue swelling was observed. Two patients had progressed to walking difficulties that required the aid of crutches (Fig. 3b) or wheels.

Laboratory investigations (Table 1)
The normal range of ESR and CRP were lower than 20 mm/h and 8 mg/L, respectively. The normal range of serum level of 25-hydroxyvitamin D3 was higher than 30 ng/mL. All the patients' ESR and CRP levels were in normal range. Their liver function test, serum blood urea and creatinine, rheumatoid factor, antinuclear antibody, HLA-B27, electrolytes, thyroid function, and parathyroid hormone level were all normal (data not shown) except for case 5 who had positive HLA-B27. After finding that case 2 showed low level of serum 25-hydroxyvitamin D3, we analyzed this parameter in other 5 patients (case 1, case 3, case 6, case 8, and case 9). Their serum 25-hydroxyvitamin D3 levels ranged from 6.16 to 22.1 ng/mL, all below the normal range.

Radiographic features
All the patients' radiographs were collected and analyzed except case 7, where radiographs could not be obtained. All (8/8) patients had different degrees of abnormal vertebral bodies, severity of which ranging from platyspondyly and anterior blunt of the vertebral bodies (Fig. 1c) to anterior beak-like of the vertebral bodies (Fig. 2c). Hand X-rays of 4 patients were obtained which consistently showed epiphyseal enlargement of the interphalangeal joints with juxta-articular osteopenia (Fig. 2b). Other radiological findings included osteopenia, reduction in hip articular space (Fig. 2d), and cyst-like structures in the left femoral head (Fig. 3c).

Treatment and outcomes
After obtaining the definite diagnosis of PPRD, the patients were treated with physical therapy and occasional NSAIDs for relieving pain. Upon finding low level of serum 25-hydroxyvitamin D3 in 6 patients, combined with their normal serum calcium, phosphate, alkaline phosphatase, parathyroid hormone, and osteopenia, we treated these 6 patients using calcitriol with the dosage of calcitriol ranging from 0.25 to 0.5 μg each day except case 2 who accepted 0.25 μg calcitriol every other day. Five of them had been treated for 1.25 years to 1.75 years. The patients were asked to describe the severity of joint pain on a scale of 0-10 (0 is no pain and 10 is severe pain) and how they are doing on daily life because of the stiffness and restricted movement of the joints on a scale of 0-10 (0 is very well and 10 is very poor) before and after the treatment (Table 3). After the treatment of calcitriol, case 2 kept stable without manifesting any symptoms and signs, while case 6 neither improved nor worsened. The other 3 patients' joint pain, stiffness, and restricted movement of their joints were improved gradually during the treatment instead of getting worse as before.
During the treatment of calcitriol, they continued to accept physical therapy and home-based exercise to improve mobility and daily function. In addition, case 8 was treated with left hip arthroplasty at 32 years old and right hip arthroplasty at 35 years old. Case 7 was planning to receive hip arthroplasty treatment.

Discussion
PPRD is a rare and inheritable skeletal dysplasia [23]. The clinical and radiological phenotypes of patients with PPRD are rather homogeneous with progressive worsening of symptoms and radiological changes during childhood and adolescence [2]. The unspecific symptoms often cause a significant delay in diagnosis [2], or even lead to misdiagnosis resulting in incorrect treatment [24]. In 9 patients of PPRD presented here, 5 were misdiagnosed with juvenile idiopathic arthritis (JIA) before they came to our center, and 1 patient even had received immunosuppressants despite their autoimmunity markers and inflammatory parameters were all within normal range. Moreover, in the 2 pairs of sisters, when the younger sisters were diagnosed with PPRD, their elder sisters had already suffered from symptoms for 3 and 11 years, respectively. One patient was misdiagnosed with ankylosing spondylitis only because of positive HLA-B27. Her parental consanguinity, normal inflammatory parameters, and cyst-like structures in the left femoral head were all neglected. Most of our patients (8/9) had an age of onset between 3 and 6 years, which was consistent with a previous review including large samples concerning the onset age [2]. Diagnosis of PPRD is often delayed, sometimes for decades, because only few clinicians are aware of the disease [2]. The interval between onset and definite diagnosis for most of our patients (7/9) was longer than 6 years. Most of our patients presented with the characteristic manifestations of PPRD including gait abnormalities, symmetric stiffness in multiple joints (particularly the hip), and enlarged interphalangeal joints of the hands [10]. Radiological features of PPRD include spondyloepiphyseal dysplasia with platyspondyly, anterior blunt or beak-like of the vertebral bodies, short and wide femoral necks, large epiphyses, narrow joint spaces, and osteopenia [2,25]. The early images show metaphyseal enlargement of interphalangeal joints [2]. In our patients, we found that anterior blunt of the vertebral bodies could be an early radiological finding when the patient had not developed obvious clinical symptoms and signs yet. If the family history, clinical features presenting with abnormal gait, or enlarged and stiff interphalangeal joints of the fingers but with normal inflammatory markers could be combined with the characteristic radiographic findings, all the patients could get the clinical diagnosis of PPRD at very early stage of the disease. PPRD is due to a variant in CCN6 gene encoding a signaling factor that regulates chondrocyte proliferation and differentiation involving cartilage homeostasis and bone growth [10]. CCN6 expression in PPRD articular chondrocytes (ACs) is reduced. Increased proliferation activity and decreased apoptosis are the characteristics of PPRD ACs, accounting for the metaphyseal enlargement in PPRD patients [14]. The highly mineralized and significantly stiffer PPRD articular cartilage may decelerate growth and repair of cartilage [15]. All of our 9 patients had CCN6 variants. Four patients including a pair of brother and sister had homozygous variants of the CCN6 gene. Five patients including 2 pairs of sisters had compound heterozygous variants of the CCN6 gene. There is a low prevalence for Fig. 1 Images of case 2 (girl, 4 years old). a Normal fingers. b Normal squatting. c Radiograph of the thoracolumbar spine showing anterior blunt of the vertebral bodies. d Normal radiograph of the pelvis PPRD in the UK, while previous reports indicate it is more common in the Middle East, Gulf States, Arabic countries, and countries of the Mediterranean basin, possibly because of the high consanguinity rate in these populations [2,3,9]. Case 5 had parental consanguinity and carried homozygous missense variants c.667 T > G (p.Cys223Gly) in the CCN6 gene. The c.667 T > G variant, located in exon 4, had been reported before in Chinese and other ethnic origins' children affected by PPRD [9,24]. The variant c.624dupA had been reported in patients from China, India, and countries of North and West Africa [16,20,21]. The onset ages of patients with PPRD carrying homozygous or heterozygous c.624dupA variants in CCN6 gene were much older (from 7 to 18 years old) than the common onset age of PPRD [16,21]. In line with this, case 8, who presented with compound heterozygous variants (c.624dupA and c.756C > A), had late and atypical symptoms with an age of onset at 14 years old. Based on the information of onset age and genotype of the patients, we suspect that the patients with PPRD having c.624dupA variant in CCN6 may have delayed onset. One of our pair of sisters had compound heterozygous variants (c.646 T > C and c.1000 T > C). The variant c.646 T > C (p.Trp216Arg) had not been reported previously, and its allele frequency was 0.00081% in gno-mAD database. However, the missense variant c.1000 T > C (p.Ser334Pro) had been reported in Chinese patients [18,19]. Patient with this variant presented marked abnormalities of cartilage chondrocytes and matrix in morphology and function [19].
Although the molecular basis of PPRD has been elucidated for more than 10 years, no progress has been made toward a specific treatment [2]. The current treatment options can only alleviate symptoms. Pain due to secondary osteoarthritis may respond to NSAIDs. Physical therapy and rehabilitation may help preserve joint mobility. Surgical intervention, including realignment of the lower limbs, joint arthroplasty, and/or treatment of spinal stenosis when necessary, is the mainstay of treatment today [4,25]. Total hip arthroplasty (THA) is a treatment option for PPRD patients to restore the joint function and improve life quality. It has been proven that THA is an effective treatment for PPRD patients with hip arthropathy, with satisfactory clinical and radiological outcome after follow-up for 18 months to 93 months (mean 47.9 months) [26]. Case 8 has received bilateral hip arthroplasty, and he is able to continue his job as a driver. The other adult patient is preparing to receive this treatment.
Six of our 9 patients were tested for the level of serum 25-hydroxyvitamin D3 which were all below the normal range. Concerning their normal liver function, serum creatinine, serum calcium, phosphate, alkaline phosphatase, and parathyroid hormone, we treated these patients with oral 1,25(OH) 2 D 3 (calcitriol). In the followup appointment between 1.25 and 1.75 years, we found that 2 of 5 patients' symptoms stopped progressing and 3 of 5 patients' symptoms improved gradually. These 3 patients also accepted the treatment with physical therapy. 1,25(OH) 2 D 3 works on the key role in calcium homeostasis and bone mineralization, which is actually the most active form of vitamin D [27]. The PPRD articular chondrocytes are in an immature, hyper-proliferative, and decreased apoptosis state [14]. 1,25(OH) 2 D 3 could accelerate chondrocyte apoptosis by impairing activation of the NF-κB signaling pathway in rats with rheumatoid arthritis [12], and it showed chondro-protective  [13]. The murine model also showed that 1,25(OH) 2 D 3 controlled chondrocyte proliferation and differentiation directly [28]. So we suppose that calcitriol may maintain the normal proliferation of articular chondrocytes by accelerating chondrocyte apoptosis in patients with PPRD. Our preliminary data with 5 patients for more than 1 year of treatment suggest that calcitriol may provide a potential treatment option for PPRD patients with vitamin D deficiency. In addition, physical therapy may have played a role in those 3 patients whose symptoms improved besides calcitriol. Of course, more cases should be included into the study, and long-term follow-up is needed for a more comprehensive analysis of the potential effectiveness of calcitriol in PPRD.

Conclusion
PPRD is a rare disease characterized by progressive noninflammatory arthropathy affecting primarily the articular cartilage. Anterior blunt of the vertebral bodies could be an early radiological sign in the patient even without obvious clinical symptoms and signs yet. The patient with c.624dupA variant in CCN6 may have delayed onset of PPRD symptoms. These findings may aid in earlier diagnosis in such patients. Calcitriol can improve the clinical symptoms in the PPRD patients with vitamin D deficiency which implies that clinicians should seek and correct underlying vitamin D deficiency in patients with PPRD. Further studies are needed to investigate the related mechanism.  Without pain of the joints, continues to have claudication