Systematical analysis of nine Chinese patients with progressive pseudorheumatoid dysplasia and assessment of calcitriol treatment

Background: Progressive pseudorheumatoid dysplasia (PPRD) (MIM 208230) is a rare genetic disease characterized by progressive noninflammatory arthropathy affecting primarily the articular cartilage. Most patients are initially misdiagnosed because of the rarity of the disease and lack of awareness by most clinicians. Therefore, the purpose of the present study was to provide further diagnostic options to help clinicians make early precise diagnosis, and to investigate a new potential treatment for patients with PPRD. Methods: Clinical manifestations, radiographic features, Sanger Sequencing to determine WISP3 gene mutation and follow-up records were collected in 9 Chinese patients with PPRD. The time until diagnosis, relationship between clinical phenotypes and genotypes, and treatment effects were analyzed. Results: The clinical history and characteristics, mutations in the WISP3 gene, radiological data, treatment and outcome of 9 PPRD children were collected and reviewed. There were 3 pairs of siblings in this group and one patient had parental consanguinity. Five patients were misdiagnosed with juvenile idiopathic arthritis (JIA). The onset of disease in most patients (8/9) was between 3 and 6 years of age. The interval between onset of symptoms and obtaining the diagnosis for 8 of the patients varied between 3.6 years to 20 years. The onset of symptoms included enlarged and stiff interphalangeal joints of the fingers, gait disturbance or joint pain. Blood analysis revealed normal range of inflammatory parameters (erythrocyte sedimentation rate, C-reactive protein). Serum levels of 25-hydroxyvitamin D3 were analyzed in six patients and ranged from 6.16 to 22.1ng/ml, all lower than the normal range. Radiographic findings included different degree of abnormal vertebral bodies, epiphyseal enlargement of the interphalangeal joints with juxta-articular osteopenia, or reduction in hip articular space and cyst-like structures femoral head. A total of 7 variants in the WISP3 gene were identified in the 9 cases, presenting with three homozygous variants of 25-hydroxyvitamin D3 for 1.25 years to 1.75 years, two cases showed stable clinical disease activity and the other four patients joints’ pain and abnormal gait had improved. Conclusions: Combining the patient’s family history, clinical features presenting with abnormal gait or enlarged and stiff interphalangeal joints of the fingers, normal inflammatory markers, and the characteristic radiographic findings, we can obtain the clinical diagnosis of PPRD for the patients at a very early stage of the disease. The patients with PPRD having c.624dupA mutation in WISP3 may have delayed onset. Calcitriol showed treatment benefit by improving symptoms and decelerating progression of PPRD disease.


Introduction
Progressive pseudorheumatoid dysplasia (PPRD) (MIM 208230) is a rare genetic disease initially described by Wynne-Davies et al in 1982 1 . This autosomal recessive inherited skeletal dysplasia is characterized by progressive noninflammatory arthropathy affecting primarily the articular cartilage 2 . The prevalence of PPRD has been estimated at one per million in the United Kingdom 3 , but it may be underdiagnosed due to the overlap of clinical and imaging features with other pediatric musculoskeletal disorders, such as juvenile idiopathic arthritis (JIA), Scheuermann's disease, spondyloepiphyseal dysplasia, Blount disease, Stickler syndrome, and mucopolysaccharidosis 4,5 . The onset of symptoms is usually in childhood between 3 to 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 6 . 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 7 .
PPRD is caused by mutations in the WISP3 gene which maps to chromosome 6q22, and encodes a protein in the connective tissue growth factor that is expressed in synoviocytes and chondrocytes involved in bone and cartilage growth and development 8 . Homozygous or compound heterozygous WISP3 mutations cause the loss of articular cartilage, which leads to the progressive narrowing of all articular spaces and reduced joint mobility 9 . While this molecular basis was elucidated over 10 years ago, no progress has been made towards a specific treatment for PPRD 2 .
1,25(OH)2D3 (calcitriol), which is used to treat hypocalcemia, osteoporosis, and prevention of corticosteroid-induced osteoporosis 10 , has showed chondro-protective effects in human chondrocytes 11 . Combined with the results of a murine model to study the role of 1,25(OH)2D3, the patients and rat model with rheumatoid arthritis, and the properties of patient's cartilage and chondrocytes under PPRD, we suspected that calcitriol may maintain the normal proliferation of articular chondrocytes by accelerating chondrocyte apoptosis [12][13][14] and could thus be used to treat PPRD. In the present study, we systematically evaluated the clinical history and characteristics, radiological data and WISP3 gene mutations in 9 patients with PPRD recruited by our special consultation clinic, and evaluated potential treatment with calcitriol for PPRD.
Furthermore, based on the information between the age of onset and genotype of the patients, we suggest that patients with PPRD with underlying c.624dupA mutation in the WISP3 gene may have delayed onset.

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.

Whole blood analysis
Blood tests of inflammatory parameters were performed including complete blood count, and 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, parathyroid hormone.

Sanger Sequencing
Genomic DNA was extracted from 2-mL peripheral blood samples using the QIAamp

Whole blood analysis
All the patients' ESR and CRP levels were in normal range. The normal range of ESR and CRP were lower than 20mm/hr and 8mg/L, respectively. The normal range of serum level of 25-hydroxyvitamin D3 was higher than 30ng/ml(table1). Their liver function test, serum blood urea and creatinine, rheumatoid factor, antinuclear antibody, HLA-B27, electrolytes, thyroid function tests, parathyroid hormone were all normal (data not shown) except for case5 who had positive HLA-B27. After we found that case2 had low serum level of 25-hydroxyvitamin D3, we also analyzed this parameter in case1, case3, case6, case8 and case9. The results of serum level of 25-hydroxyvitamin D3 ranged from 6.16 to 22.1ng/ml, all below normal.

Radiographic features
All the patients' radiographs were collected and analyzed except case7, where radiographs could not be obtained. All

Discussion
PPRD is a rare and inheritable skeletal dysplasia 21 . The clinical and radiological phenotype of patients with PPRD is 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 22 . In our nine patients with PPRD presented here, five were misdiagnosed with juvenile idiopathic arthritis (JIA) before they came to our center, and one patient (case9) even had received treatment with immunosuppressants despite their autoimmunity markers and inflammatory parameters all within normal range. Moreover, in the two pair of sisters (case3 and 4, case6 and 7), when the younger sisters (case3, case6) were diagnosed with PPRD, their older sisters had already suffered from symptoms for 3 or 11 years, respectively. One patient was Although the molecular basis of PPRD has been elucidated for more than 10 years, no progress has been made towards a specific treatment 2 . The current treatment options 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, are the mainstay of treatment today 3,5 .
Total hip arthroplasty is a treatment option for PPRD patients to restore the joint function and improve life quality. It has been shown 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) 20 . Case8 has received bilateral hip arthroplasty and since has been able to continue his job as a driver.
The other adult patient is preparing to receive this treatment. The murine model also showed that 1,25(OH)2D3 controlled chondrocyte proliferation and differentiation directly 13 . Concerning their normal liver function, serum creatinine, serum calcium, phosphate, alkaline phosphatase, parathyroid hormone, we treated our patients that presented with low serum level of 25-hydroxyvitamin D3 with oral calcitriol and in the follow-up appointment found that 2/6 patient's symptoms stopped progressing, while 4/6 patient's symptoms improved gradually. We suspected that calcitriol may maintain the normal proliferation of articular chondrocytes by accelerating chondrocyte apoptosis [11][12][13] in patients with PPRD. Therefore, our preliminary data with six patients suggest that calcitriol may provide a new beneficial treatment option for PPRD. However, more cases will have to be included into the study and long-term follow up is needed for a more comprehensive analysis of the effectiveness of calcitriol in PPRD.

Conclusion
PPRD is a rare disease characterized by progressive noninflammatory arthropathy affecting primarily the articular cartilage. Despite symptoms severely affecting quality of life, no current treatment consensus exists. In this study we found that calcitriol, which is used in osteoporosis, significantly improved clinical symptoms in 4/6 patients with PPRD. These findings suggest a great potential for implementing calcitriol in the treatment of this disease, and further study with larger case numbers are needed to validate these findings. Furthermore, we observed a connection between the c.624dupA mutation in WISP3 and delayed onset of PPRD symptoms. These findings may aid in earlier diagnosis in such patients.