Progressive Pseudorheumatoid Dysplasia of Childhood (PPRD)—A Case Series with Recurrent c.740_741del Variant

Abstract

Progressive pseudorheumatoid dysplasia (PPRD) is an autosomal recessive arthropathy, affecting school-aged children. It is characterized by progressive degeneration of the articular cartilage. The majority of the pathogenic variations are found in exon 2, exon 4, and exon 5 of the putative gene, CCN6 (WISP3). Three unrelated individuals with clinical diagnosis of PPD were included in this study. Detailed clinicoradiological evaluation was attempted with brief literature review. Exome sequencing was performed in all three cases. All the pathogenic variations detected in our cohort were located in exons 2 and 4 of WISP3 gene. Though the clinicoradiological features are already well described, this study in north India highlights the occurrence of a recurring pathogenic variant. The c.740_741del variant was a recurrent pathogenic variant seen in all three patients in this cohort. This may be a common pathogenic variant in the North Indian population; however, a larger cohort needs to be studied before drawing final conclusions. A proper molecular diagnosis is a must to end the diagnostic odyssey, safeguarding patients with PPRD from unnecessary use of drugs like corticosteroids.

Introduction

Progressive pseudorheumatoid dysplasia (PPRD) (OMIM #208230) is a type of skeletal dysplasia involving articular cartilage. This debilitating ailment was first described by Spranger et al ¹ as a progressive connective tissue disease that involves painless progressive joint stiffness and expansion with typical absence of inflammation. It is caused by homozygous or compound heterozygous mutation in the CCN6 gene (OMIM *603400), encoding the cellular communication network factor 6 (CCN6; also termed WNT1-Inducible Signaling Pathway Protein 3) on chromosome 6q21. ² It is inherited in an autosomal recessive manner. Age of onset is 3 to 6 years and begins with the involvement of the interphalangeal joints. ³ As time progresses, there is involvement of large joints and the spine, causing typical joint contractures, gait abnormality, with scoliosis and/or kyphosis, thus resulting in abnormal posture and morbidities associated with muscle weakness. Commonly involved joints include hips, knees, wrists, and fingers. ³ Physiotherapy helps in preserving joint mobility. Surgical interventions like realignment of the lower limbs, joint arthroplasty, and/or treatment of spinal stenosis can be done. Immobilization and casting should be avoided. Pain due to secondary osteoarthritis is best treated with nonsteroidal anti-inflammatory drugs (NSAIDs). ⁴ Steroids and other anti-inflammatory drugs like methotrexate should be avoided. We analyzed the phenotype and mutation detected by exome sequencing in three unrelated individuals with PPRD. Pretest counselling describing the goals of the genetic test, turnaround time, costs, limitations, the yield of diagnostic success, and the potential to identify variants of uncertain clinical significance was also discussed. Exact molecular diagnosis is necessary, as such skeletal disorders are genetically heterogenous and misdiagnosis might lead to inappropriate treatment, assuming it to be an inflammatory arthropathy. Also, the common mutations in WISP3 (CCN6) may vary in different populations and the Indian subcontinent having a diverse population, this study on a North Indian cohort highlights the occurrence of a recurring mutation in nonconsanguineous families.

Patient Information, Clinical Findings, and Diagnostic Assessment

This study was conducted at a tertiary care hospital in India. Three unrelated probands from North Indian families were studied in this case series. Details of clinical features, three generation family pedigree, and radiographs were acquired from the case sheets and electronic health records. Detailed phenotype and mutation spectrum are depicted in Table 1 .

Table 1. Clinical features and mutation spectrum of patients in our cohort.

Subject	Patient 1	Patient 2	Patient 3
Gender	Male	Male	Male
Age at diagnosis	11 y	9 y	16 y
Height at diagnosis (WHO Z-score)	125 cm (−2.75)	122cm (−1.82)	158 cm (−1.95)
Age of onset	2 y	4 y	5 y
Joint involvement	IPJ, hip joint, elbow joint, and spine	IPJ, right knee joint, elbow joint, and spine	IPJ, hip joint and spine
Radiography	Hand—decreased small joint spaces with widened joints Spine—kyphoscoliosis with platyspondyly with anterior beaking Hip joint—bilateral femoral head avascular necrosis	Hand—decreased small joint spaces with widened joints Spine—platyspondyly present	Spine—platyspondyly present Hip joint—reduced hip joint spaces, large capital femoral epiphyses, short and broad femoral necks, and irregular acetabular roofs
Laboratory investigations	25 hydroxy vitamin D: 24.67nmol/L (50–250); calcium: 8.8 mg/dL (8.5–10.8); phosphorous: 5.7 mg/dL (2.5–4.5); albumin: 4.0 gm/dL (3.5–5.5); potassium: 4.3mmol/L (3.8–5.4); creatinine: 0.7 mg/dL (0.5–1.6)	25 hydroxy vitamin D: 38 nmol/L (50–250); calcium: 9.9mg/dL (8.5–10.8); phosphorous: 4.6 mg/dL (2.5–4.5); alkaline phosphatase: 242 IU/L Younger sibling-25 hydroxy vitamin D: 67 nmol/L (50–250); calcium: 9.1 mg/dL (8.5–10.8); alkaline phosphatase: 205 IU/L	Hb: 8.1 gm/dL (12.5–16.5) with microcytic hypochromic anemia; TLC-12320/cu.mm (4,000–10,500); platelet: 2.11 lakh/cu.mm (1.5–4.5); potassium: 4.3 mmol/L (3.8–5.4). 25 hydroxy vitamin D: 55 nmol/L (50–250); calcium: 9.5 mg/dL (8.5–10.8)
Mutation in CCN6 (WISP3) (Variants detected)	1. Exon 4, homozygous variant (c.740_741del) 6 7 (p.Cys247LeufsTer31) (Sanger validated) ( Fig. S1 )	1. Exon 4, A heterozygous two base pair deletion (c.740_741del) 6 7 (p.Cys247LeufsTer31) 2. Exon 2, A heterozygous nonsense variation c.156C > A (p.Cys52Ter) 6 (Sanger validated) ( Fig. S2 )	1. Exon 4, A heterozygous single base pair duplication c.624dup (p.Cys209MetfsTer21) 8 2. Exon 4, A heterozygous 2 base pair deletion (c.740_741del) 6 7 (p.Cys247LeufsTer31) ( Fig. S3 )
MAF in population databases	c.740_741del in gnomAD: 0.00000798 and 1000Genomes: absent	c.156C > A in gnomAD: 0.00003 and 1000Genomes: absent	c.624dup in gnomAD: absent and 1000Genomes: absent
Protein domain affected	c.740_741del: TSP	c.156C > A: IGFBP	c.624dup: TSP

Abbreviations: Hb, hemoglobin; IGFBP, insulin-like growth factor-binding domain; IPJ, interphalangeal joint; TLC, total leucocyte count; TSP, thrombospondin domain.

Patient 1

An 11-year-old boy presented to our outpatient department (OPD) with swelling and pain at interphalangeal joints along with bilateral hip pain. He also had significant muscle weakness. He had a history of waddling gait, noticed since the age of 2 years. Since then, he developed gradually progressive pain and restricted mobility of small joints of hands, elbow, and hip, resulting in difficulty in getting from squatting position. He was being managed as juvenile idiopathic arthritis (JIA) by community physician. At the age of 5 years, he also underwent a bilateral trochanteric valgus osteotomy. However, he continued to have progressive deformity of joints. He was second in birth order, born out of nonconsanguineous marriage. There was no history of similarly affected family members. His developmental milestones were achieved as per age. He had normal intellect, and had no facial dysmorphism or other chronic health issue. At the time of diagnosis, his height was 125 cm (−2.75 Z-score), while his present height was 133 cm (−3.92 Z score) at the age of 14 years. His X-rays showed changes suggestive of PPRD ( Fig. 1 ).

Fig. 1.

Phenotypic features and radiographs in patient 1 showing ( A ) interphalangeal joint (IPJ) involvement, ( B ) elbow joint involvement, ( C ) reduced IPJ space with large epiphyses and widened metaphyses of metacarpals and phalanges, ( D ) genu varum at right knee joint, ( E ) platyspondyly at the lumbosacral spine, ( F ) typical hip joint involvement with reduced joint space, large capital femoral epiphyses, short femoral neck, and irregular acetabular roofs. Skeletal changes as in Figure legends are indicated by red arrows.

Patient 2

This is a 12-year-old boy who was brought to our OPD at the age of 9 years with the inability to maintain firm grip and hold objects with hands. He also had deformity at bilateral knee joints and difficulty in walking since the age of 4 years. On examination, he was found to have joint contractures at interphalangeal joints and deformed knee joints. There was abnormal gait and difficulty in getting up. His height at the time of diagnosis was 122 cm (−1.82 Z score), while his present height was 133cm (−2.27 Z score).

He was second in birth order (nonconsanguineous family) with a similarly affected younger male sibling (5-year-old) who also had gradually progressive joint deformities since the age of 2 years and his present height is 112 cm (0.38 Z score). Both of them were otherwise intellectually normal. The skeletal phenotype and radiographs are shown in Fig. 2 .

Fig. 2.

Multiple joint deformities in patient 2 showing ( A ) genu valgum, ( B ) deformity at left elbow joint, ( C ) typical interphalangeal joint involvement, ( D ) platyspondyly seen in spinal radiograph, ( E ) reduced IPJ space with epiphyseal changes at phalanges suggestive of progressive pseudorheumatoid dysplasia, ( F ) characteristic varus deformity in younger male sibling of patient 2.

Patient 3

This is an 18-year-old male who presented to our OPD at the age of 16 years with similar complaints as the previous two patients, with somewhat slower onset of the disease, that is, progressive stiffness of multiple joints since the age of 5 years and limping on walking by the age of 9 years. He had progressive swelling in multiple joints (bilateral elbow, distal interphalangeal, and proximal interphalangeal joints). He also complained of pain in right hip joint on walking/running and proximal muscle weakness. He was also being managed as JIA by the primary care physician and had been on prolonged course of NSAIDs and occasional oral steroids. After the proper diagnosis and counselling at our center, he also underwent orthopaedic correction for his joint deformities. He had normal intellect, and has been symptom-free after the surgical correction. He was also found to have microcytic hypochromic anemia at the time of diagnosis and responded well to oral iron supplementation. His parents were from a nonconsanguineous family. He was second in birth order with no similar morbidity in the family. The skeletal changes are depicted in Fig. 3 .

Fig. 3.

Radiographs in patient 3 showing ( A ) interphalangeal joint involvement typical of progressive pseudorheumatoid dysplasia; ( B ) reduced hip joint spaces, large capital femoral epiphyses, short and broad femoral necks, and irregular acetabular roofs. Irregular iliac crests are also seen; ( C ) platyspondyly with erosive changes at the superior and inferior articular cartilages.

In our cohort, the mean age at diagnosis was 12 years, (range: 9–16 years), while the mean age of symptom onset was 3.66 years (range: 2–5 years). Bilateral interphalangeal joint contracture was the most common presenting symptom followed by difficulty in walking. All the three patients had significant muscle wasting involving the axial and appendicular skeletal system. Typical interphalangeal joint swelling was seen in patient 3, while joint pain was reported by patient 1 and patient 3, possibly due to secondary osteoarthritis.

Genetic Testing

For each patient, 3 mL peripheral blood was collected in ethylene-diamine-tetraacetic acid vacutainer. Genomic DNA was extracted from blood leucocytes using standard procedures of QIAamp DNA blood Mini Kit (Qiagen, Hilden, Germany) and quantified using Qubit DNA Assay, while its integrity was checked on agarose gel. Targeted exome sequencing was performed using Illumina sequencing platform following enrichment and capture using customized capture kit targeting ∼8.000 genes known to be associated with Mendelian disorders. The nucleotide sequence numbering for CCN6 (WISP3) is based on NCBI Reference Sequence (NM_003880.4). Variants with a minor allele frequency (MAF) of >0.01 in the population databases like 1000 Genome project phase 3 ( https://www.internationalgenome.org/ ) and gnomAD ( https://gnomad.broadinstitute.org/ ) were filtered out. Subsequently, The American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG) criteria were utilized for the classification of detected variants into likely pathogenic/pathogenic/variant of uncertain significance. Disease-causing variants in CCN6 (WISP3) gene were detected in all the three individuals in exon 4 and exon 2 with c.740_741del being a recurring mutation (homozygous in patient 1 and compound heterozygous in patient 2 and patient 3. Standard polymerase chain reaction and Sanger sequencing were performed to confirm the presence of the candidate variants in patient 1 and patient 2, while patient 3 did not give consent for Sanger sequencing. The pathogenic variants detected in patient 2 were also found in compound heterozygous state in the similarly affected younger sibling (Sanger sequencing).

The c.740_741del variant was classified as pathogenic (PVS1, PM2, PP3, PP5) according to the ACMG guidelines. ⁵ It is predicted to cause a frameshift, altering the amino acid sequence from the position 247 and premature termination codon 31 amino acids downstream in the last exon of CCN6 . The c.624dup variant results in a frameshift and premature truncation of the protein 21 amino acids downstream to codon 209, while the c.156C > A variant results in a stop codon and premature truncation of the protein at codon 52. The c.156C > A variant is the most commonly detected pathogenic variant in PPRD worldwide. ⁶ All of the disease-causing variants in our cohort ( Table 1 ) have previously been reported in patients affected with progressive pseudorheumatoid dysplasia. ^{6 7 8} The MAF of these variants in the population databases is depicted in Table 1 . The in-silico prediction of the variants was disease causing by MutationTaster2 ( http://www.mutationtaster.org/ ) and other computational evidences for pathogenicity and classified as “pathogenic” (PVS1, PM2, PP3, PP5) according to the ACMG guidelines. ⁵

Discussion

PPRD is often misdiagnosed as JIA owing to the distinctive joint deformity of the hands (enlargement of the interphalangeal joints). However, in PPRD, there is absence of inflammation and normal erythrocyte sedimentation rate and C-reactive protein levels are typically seen. On radiography, joint destruction is seen in JIA, while dysplasia along with epiphyseal enlargement and platyspondyly are characteristic for PPRD. Early diagnosis with specific molecular etiology in a child with PPRD protects them from the prolonged use and side effects of medications like methotrexate and corticosteroids employed in the management of JIA. As seen in our cohort in two of the patients, there might be history of steroid intake and eventual myopathy. Around 50% of children with PPRD are found to have short stature owing to the skeletal deformities at knees, hip, and spine. ⁸ One of the children in our cohort (patient 1) was found to have short stature at diagnosis. Serial follow-up is necessary as the typical adult height is less than 3rd percentile. ³

Wynne-Davies et al ⁹ estimated a frequency of 1 per million in the United Kingdom, but the disorder is likely to be higher in the Middle East and Gulf states. ¹⁰ The gene CCN6 ( WISP3 ) belongs to CCN family of growth factors that harbor highly conserved cysteine residues across different species, both in position and number. ¹¹ Thirty-four cysteine residues are present in the CCN6 gene. Mutations frequently affect these residues and result in altered protein structure and function. ^{6 12 13} CCN6, being a multi-domain protein, interacts with several proteins involved in cellular functions and musculoskeletal growth. The function of this protein was largely unclear until recently. Some reports suggested that CCN6 maintains the expression of cartilage-specific matrix proteins (collagen II and aggrecan) in the chondrocytes and controls the generation of reactive oxygen species and cartilage hypertrophy. ¹⁴ However, recent functional studies demonstrated a role of CCN6 in the mitochondrial respiratory complex assembly/activity and ATP production. ¹⁵ Hence, mutations in CCN6 result in mitochondrial defects and deprived cell survival. Sengupta et al ¹⁶ used a zebrafish model to study functional effects of CCN6 protein. They demonstrated morpholino-mediated reduction in CCN6 in zebrafish musculoskeletal system resulting in significant depletion of respiratory complex assembly and activity. This loss of mitochondrial milieu in turn affects muscle architecture with a reduced muscle function in zebrafish. Thus, defects in the mitochondrial respiratory complex assembly/activity and loss of mitochondrial integrity were proposed as an underlying cause of muscle weakness in PPRD.

Hurvitz et al ² identified nine different mutations (OMIM 603400.0001–603400.0009) in the CCN6 gene in unrelated affected individuals, indicating that the gene is essential for normal postnatal skeletal growth and cartilage homeostasis. Of the nine mutations found, those in patients from Italy, France, and the United States were present in compound heterozygous state; those from Saudi Arabia, Jordan, and Iran were present in homozygous state, reflecting the differences in the frequency of consanguinity in the different populations. Nearly 100% of the mutations detected in CCN6 ( WISP3 ) are sequence variations and an exome sequencing was offered in our cohort, considering common differentials like atypical forms of COL2A1 -related spondyloepiphyseal dysplasias (OMIM #184250), X-linked spondyloepiphyseal dysplasia tarda (OMIM #313400), LACC1 -related juvenile arthritis (OMIM #618795), and spondylometaphyseal dysplasia corner fracture type (OMIM #184255). ⁴ However, rare case reports with a microdeletion, involving CCN6 (WISP3), have been reported in PPRD patients. ¹⁷ Dalal et al ⁶ studied the clinical features of 35 patients with this disorder and reported 11 different homozygous mutations and only one case of compound heterozygosity. One missense mutation (c.1010G > A; p.Cys337Tyr) appeared to be the most common in the Indian population being seen in 10 unrelated families, while Bhavani et al ¹⁸ identified 16 causative mutations in 54 families with progressive pseudorheumatoid dysplasia. Most families (49/54, 90.7%) demonstrated homozygous and only five families showed compound heterozygous mutations. However, two out of three individuals in our cohort showed compound heterozygous mutations; hence, such compound heterozygotes may not be so uncommon and a large cohort analysis is required before drawing final conclusions. Also, the variant c.740_741del (p.Cys247Leu fs *31) was found to recur in all three unrelated individuals (homozygous in one case and heterozygous in the other two cases) in our cohort. Similar truncating variants have been reported downstream of this position in children with PPRD. ^{2 3 6} The c.740_741del variant has been reported previously in India ^{6 18} and also in Caucasians ⁷ and German ³ populations. The occurrence of a homozygous mutation in a nonconsanguineous family, as seen in patient 1, can be due to the hidden consanguinity (custom of endogamy and same caste marriages in this part of the country). The recurrent c.740_741del variant may be a founder mutation in North India. Most of the published studies from India involve patients from the southern part of India. A larger cohort along with STR marker analysis in those of North Indian ethnicity may further validate or negate this observation. The mean age at symptom onset in our cohort with the recurring c.740_741del variant is 3.66 years (younger sibling of patient 2 not taken into account), while a previous study in India on PPRD had reported a mean age of 5.08 years. ⁶ This variant has also been reported in the homozygous state in three unrelated families in another Indian study ¹⁸ however, the mean age of symptom onset is not known in that cohort. Also, the patient 1 in our cohort has an early age of symptom onset and more aggressive course, thus indicating that the homozygous presence of this variant is more penetrant when compared with the compound heterozygous form with another variant.

The variants detected in our cohort are located in important functional domains of the WISP3 protein ( Table 1 ). The WISP3 protein has four important cysteine-rich domains, with most of the mutations (34%) detected in insulin-like growth factor-binding domain followed by the thrombospondin domain (31%) and the carboxy-terminal cystine knot-like domain (25%). ¹⁹

Whole exome sequencing identifies DNA variants within the 1% of the genome, that is, those coding proteins (exons) and also regions flanking the exons (splice junctions). By analyzing virtually all protein-coding regions in the genome with this test, the cost of multiple single-gene sequencing tests is drastically reduced and the odds of identifying a high-risk variant are also enhanced, which can then be used to guide clinical management and genetic counselling. Since the sample size was limited, we could not draw any definite phenotypic correlation with the exon in which the mutation was identified.

Conclusion

In this era of genetic testing for monogenic disorders by techniques like whole exome sequencing, a genotype–phenotype correlation can be better established. Definitive risk of recurrence and proper genetic counselling can be provided to the proband and predictive genetic testing can be done in at risk relatives. Getting a diagnosis is paramount in children with skeletal deformities, as it avoids unnecessary medications, which create additional problems due to prolonged inadvertent use. Prenatal testing in the family is possible if the disease-causing mutation has been identified and also aids in therapeutic trials and precision medicine for such rare skeletal dysplasia.