Thirteen Indians with camptodactyly-arthropathy-coxa vara-pericarditis syndrome

Abstract

Camptodactyly-arthropathy-coxa vara-pericarditis (CACP) syndrome (MIM# 208250) is a rare monogenic disorder, characterized by early onset of camptodactyly, progressive coxa vara, bilateral arthropathy and constrictive pericarditis. The syndrome is caused by biallelic loss-of-function variants in PRG4. Deficiency of PRG4 results in progressive worsening of joint deformity with age. Thirteen individuals with CACP syndrome from eight consanguineous Indian families were evaluated. We used exome sequencing to elucidate disease-causing variants in all the probands. These variants were further validated and segregated by Sanger sequencing, confirming the diagnosis of CACP syndrome in them. Seven females and six males aged 2−23 years were studied. Camptodactyly (13/13), coxa vara (11/13), short femoral neck (11/13) and arthritis in large joints (12/13) [wrists (11/13), ankle (11/13), elbow (10/13) and knee (10/13)] were observed commonly. Five novel disease-causing variants (c.3636G>T, c.1935del, c.1134dup, c.1699del and c.962T>A) and two previously reported variants (c.1910_1911del and c.2816_2817del) were identified in homozygous state in PRG4. We describe the phenotype and mutations in one of the large cohorts of patients with CACP syndrome, from India. Clin Dysmorphol 33: 152−159 Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.

Introduction

Camptodactyly-arthropathy-coxa vara-pericarditis (CACP) syndrome, (MIM# 208250) is an autosomal recessive disorder, characterized by camptodactyly, noninflammatory arthropathy, progressive coxa vara deformity and sterile pericarditis (Johnson et al., 2021), caused by biallelic disease-causing variants in PRG4. The PRG4 gene encodes for a chondroprotective glycoprotein, proteoglycan 4 (lubricin), involved in the lubrication of the boundaries of cartilage surfaces. Loss-of-function variants in PRG4 result in the deficiency of lubricating properties in the human synovial fluid, causing severe joint deformities and cardiovascular dysfunction (Yilmaz et al., 2018). To date, 40 disease-causing variants in PRG4 have been reported (Stenson et al., 2020). In the present study, we report 13 patients from 8 unrelated Indian families with CACP syndrome and describe the genetic variants in PRG4.

Methods

Patients

Thirteen individuals (P1−P13) with CACP syndrome from eight Indian families were recruited as part of ongoing studies on rare diseases and autoinflammatory diseases. The medical family history, clinical and radiological findings of all individuals with CACP syndrome were documented, and written informed consents were obtained from the affected individuals and their family members. Peripheral blood samples from probands, siblings and their parents were collected for genetic testing. The study has the approval of the Institutional Ethics Committee of Kasturba Medical College and Hospital, Manipal.

Genetic testing

Exome sequencing was performed on probands from all the eight families. Genomic DNA was isolated from the blood using QIAmp DNA Blood Mini Kit (Cat# 51106, QIAGEN, Germantown, Maryland,, USA). The genomic DNA capture was performed using the TWIST Human Core exome capture kit (TWIST Biosciences, South San Francisco, California, USA). Exome sequencing had an average coverage of 100×, 95% of bases covered at a minimum of 20× with 97% sensitivity (Girisha et al., 2019). Retrieval of the raw data was done in FASTQ format and was aligned to GRCh38 assembly using Burrows−Wheeler Aligner (v2.2.1) and our in-house pipeline based on “Genome Analysis Toolkit Best Practices” (McKenna et al., 2010). These data were annotated by “Annotate Variation (ANNOVAR)” (Wang et al., 2010) and our in-house scripts (Kausthubham et al., 2021). The filtered variants were then analyzed using in silico pathogenicity prediction tools, CADD phred, REVEL and M-CAP and interpreted using the clinical information. Sanger sequencing was used for the validation and segregation of the rare disease-causing variants identified in the family. Clinical interpretation of identified variants was as per the American College of Medical Genetics and Genomics and the Association for Molecular Pathology guidelines (Richards et al., 2015).

Results

Clinical findings and radiographical findings

All eight unrelated families were consanguineous. Seven females and six males between the ages of 2 and 23 years were symptomatic. We noted camptodactyly (13/13) and arthropathy of large joints (12/13) [wrists (11/13), ankle (11/13), elbow (10/13) and knee (10/13)] as common manifestations. Pericardial effusion was seen only in one affected individual. Erythrocyte sedimentation rate, C-reactive protein and antinuclear antibodies were found to be within reference range in tested individuals. None of the patients had congenital cataracts, pleural effusions or temporomandibular joint arthropathy.

Radiographical evaluation revealed coxa vara deformity (11/13), short femoral neck (11/13), short iliac wings (4/13), reduced hip joint space (4/13), delayed ossification of carpal bones (4/13), squaring of metacarpals (4/13) and mild lumbar lordosis (1/13). Acetabular cysts were noted in (7/13) affected individuals. Exostosis was noted in one individual and narrow sacrosciatic notch in another individual. Detailed clinical and radiological findings are out-lined in Figs. 1–3 and Table 1.

Fig. 1.

Hand radiographs of the patients (arranged chronologically) show camptodactyly in all. Wrist joint spaces are reduced in older individuals (P6, P9, P8 and P12). We observed delayed carpal ossification in P4, P10, P11 and P13. Squaring of metacarpals is more evident in P3, P6, P10 and P13. Flexion deformities at interphalangeal joints were noted in several individuals progressively.

Fig. 3. Knee radiographs show the progression of arthritis with age. An exostosis can be observed at the proximal end of left tibia in P3.

Table 1. Clinical, radiographical and genetic findings of 13 Indians with camptodactyly-arthropathy-coxa vara-pericarditis syndrome.

Family 1D	F1		F2		F3			F4		F5	F6	F7	F8
Patient ID	P1	P2	P3	P4	P5	P6	P7	P8	P9	P10	P11	P12	P13
Demographics
Age at time of examination	1 year	6 years	2 years	4 years	2 years	19 years	22 years	23 years	21 years	6 years	9 years	18 years	3 years
Age of onset	6 months	6 months	6 months	8 months	NA	2 years	NA	By birth	By birth	3 years	Early infancy	3 years	1 year
Gender	Male	Female	Male	Male	Female	Female	Female	Female	Female	Male	Male	Female	Male
Consanguinity	+	+	+	+	+	+	+	+	+	+	+	+	+
Ethnicity	Indian		Indian		Indian			Indian		Indian	Indian	Indian	Indian
Anthropometry at age of examination
Height in cm (SD)	NA	110 (−0.95)	98.5 (+3.2)	108 (+1.36)	NA	NA	NA	145 (−2.81)	144.6 (−2.87)	105 (−2.05)	136 (+0.40)	151 (−1.84)	NA
Weight in kg (SD)	NA	17 (−1.14)	10.66 (−2.08)	16.7 (+0.19)	NA	NA	NA	70 (+0.77)	62.8 (+0.33)	14.75 (−2.62)	34 (+0.34)	56 (+0.05)	NA
Head circumference in cm (SD)	NA	48 (−2.29)	45.4 (−2.43)	48 (−1.62)	NA	NA	NA	53.4 (−1.23)	53 (−1.23)	48.5 (−2.26)	53.3 (+0.72)	NA	46 (−2.2)
Clinical features
Generalized morning stiffness	+	+	+	+	+	+	NA	+	+	NA	+	−	−
Camptodactyly of hands/feet	+	+	+	+	+	+	NA	+	+	+	+	+	+
Polyarticular large joint arthritis
Wrist contractures	+	+	+	+	+	+	NA	+	+	+	+	+	−
Elbow contractures	+	+	−	−	+	+	NA	+	+	+	+	+	+
Knee contractures	+	+	+	+	+	+	NA	−	−	+	+	+	+
Hip arthropathy	−	−	−	−	−	+	NA	−	−	+	+	−	−
Ankle arthropathy	+	+	+	+	+	+	NA	+	+	+	+	+	−
Radiological features
Squaring of meta- carpal heads	−	−	+	−	−	+	NA	−	−	+	−	−	+
Lumbar lordosis	−	−	−	−	−	−	NA	−	−	NA	+	NA	NA
Short iliac wings	+	+	−	−	−	−	NA	+	+	−	−	−	−
Short femoral neck	−	+	+	+	+	+	NA	+	+	+	+	+	+
Coxa vara deformity	−	+	+	+	+	+	NA	+	+	+	+	+	+
Synovial hypertrophy	+	+	−	+	−	−	NA	+	+	+	+	+	NA
Acetabular cyst	−	+	+	−	+	−	NA	+	−	+	NA	+	+
Other findings
Joint pain	+	+	+	+	+	+	NA	+	+	+	+	+	−
Pericardial effusion	−	−	−	−	NA	+	NA	NA	NA	NA	NA	−	−
Molecular findings in PRG4
Nucleotide change (NM 005807.6)	c.962T>A		c.1910_1911del		c.1935del			c.1134dup		c.1910_1911del	c.3636G>T	c.2816_2817del	c.1699del
Amino acid change (NP-005798.3)	p.(Leu321 Ter)		p.(Pro637ArgfsTer9)		p.(Glu646ArgfsTer266)			p.(Lys379GilnfsTer260)		p.(Pro637ArgfsTer9)	p.(Lys1212Asn)	p.(Lys939ArgfsTer38)	p.(Glu567Serf-sTer345)
Exon					Exon 7						Exon 9	Exon 7
Domain					Mucin-like domain						HX repeats	Mucin-like domain
Zygosity	Hom		Hom		Hom			Hom		Hom	Hom	Hom	Hom
Parental origin	Maternala		Maternal and paternal		Maternal and paternal			Maternal and paternal		Unknownb	Maternal and paternal	Unknownb	Maternal and paternal
Known/novel	Novel		Known		Novel			Novel		Known	Novel	Known	Novel
ACMG classification	Pathogenic (PVS1 + PM2 + PP1 + PP3)		Pathogenic (PVS1 + PM2 + PP1)		Pathogenic (PVS1 + PM2 + PP1)			Pathogenic (PVS1 + PM2 + PP1)		Pathogenic (PVS1 + PM2 + PP5)	VUS (PM2 + PP3)	Pathogenic (PVS1 + PM2 + PP4 + PP5)	Pathogenic (PVS1 + PM2 + PP4)
In silico prediction tools
Mutation Taster	Disease causing		Disease causing		Disease causing			Disease causing		Disease causing	Disease causing	Disease causing	Disease causing
REVEL	NA		NA		NA			NA		NA	0.379	NA	NA
CADD Phred	25.5		NA		NA			NA		NA	23	NA	NA
SIFT Indel	NA		0.858		0.858			0.858		0.858	NA	0.858	0.858
GERP	0.139		NA		NA			NA		NA	4.86	NA	NA

⁺present; −, absent; Hom, homozygous; NA, not available; VUS, variant of uncertain significance.

^aPaternal samples were not available.

^bParental samples were not available.

Molecular findings

Seven variants were identified in PRG4 (NM_005807.6; NP_005798.3) in the eight families described here (13 individuals affected with CACP syndrome) in homozygous state. Families 2 and 5 harbored the same frameshift deletion variant c.1910_1911del. Five of them are being reported for the first time: c.962T>A, p.(Leu321Ter); c.1134dup, p.(Lys379GlnfsTer260); c.3636G>T, p.(Lys1212Asn); c.1699del, p.(Glu567SerfsTer345); c.1935del, p.(Glu646ArgfsTer266). The two previously reported variants were c.1910_1911del, p.(Pro637ArgfsTer9) and c.2816_2817del, p.(Lys939ArgfsTer38) (Basit et al., 2011; Yilmaz et al., 2018). Sanger sequencing of these variants confirmed the segregation in the family members in autosomal recessive manner (Supplementary Figure 1, Supplemental digital content 1, http://links.lww.com/CD/A41). Six disease-causing variants consisting of one stop gain and five frameshifts were located in exon 7, encoding the mucin-like domain and one missense variant in exon 9, encoding the hemopexin-like domain (Fig. 4). Multiple in silico tools predicted that all the variants interfered in the functioning of PRG4 protein. All the variants were absent in gnomAD population database and our in-house dataset of 2970 exomes Kausthubham et al., (2021). According to the American College of Medical Genetics and Genomics (ACMG) 2015 criteria, six of these variants were classified as pathogenic and one as variant of uncertain significance (Richards et al., 2015). The variant details are listed in Table 1. The novel variants were submitted to ClinVar database (SCV002547319.1, SUB13803580, SCV002507164.1, SUB13803617, SCV002507145.1, SCV002553183.1, SUB13803603).

Fig. 4.

(a) Cartoon illustration of the structure of the gene (PRG4) and (b) domains of PRG4 protein with biallelic disease-causing variants identified in individuals with camptodactyly-arthropathy-coxa vara-pericarditis (CACP) syndrome in this study; novel variants are shown in red. HX, hemopexin-like repeats; SO, somatomedin-like domain.

Discussion

CACP syndrome comprises a constellation of camptodactyly, arthropathy, coxa vara and pericarditis. The condition was first introduced as a triad of camptodactyly, pericarditis and arthropathy (Martínez-Lavín et al., 1983). Bulutlar et al. (1986) characterized it further and included coxa vara as a feature of CACP syndrome, which was later confirmed by Bahabri et al. (1998).

Approximately 149 individuals affected with CACP syndrome have been reported in literature to date (Maniscalco et al., 2022; Bağrul et al., 2023). The study by Yilmaz et al. (2018) described the largest cohort of 35 individuals affected with CACP syndrome from Turkey. Herein, we report 13 Indian individuals affected with CACP syndrome.

The camptodactyly was the most consistently observed manifestation of CACP syndrome in the majority of the studies, occurring in 97% (158/162) of individuals affected with CACP syndrome, followed by coxa vara in 94% (153/162), including 13 patients and 11 patients, respectively, from our cohort (Maniscalco et al., 2022). Camptodactyly had an infantile onset, most notably camptodactyly of thumb, and was usually present bilaterally. These features were evident in our cohort as well.

Arthropathy typically affects large joints including hips, knees, elbows, wrists and ankles characterized by joint swelling, warmth, limited range of motion, synovial hypertrophy or pain (Yilmaz et al., 2018; Maniscalco et al., 2022; Bağrul et al., 2023). Arthropathy develops postnatally and in most of the cases bilaterally and becomes prominent around 1 year of age (Albtoush et al., 2018). In our cohort, the most commonly affected joints were wrists (12/13) and ankles (12/13) followed by elbows (10/13) and knees (10/13). Hip arthropathy was seen in only three individuals in our cohort. In previously described patients, however, hip arthropathy was found to be the most common clinical manifestation and ankle arthropathy was the least frequent manifestation (Maniscalco et al., 2022).

Although the presence of pericarditis can be a key diagnostic feature, it is seen only in a few affected individuals (Maniscalco et al., 2022; Bağrul et al., 2023). Pericarditis with mild thickening of the pericardium was documented in only one patient (P6) in this study.

Spinal abnormalities such as lumbar lordosis and thora-columbar scoliosis were described as rare clinical findings with severity varying between mild to moderate among affected individuals with CACP syndrome (Faivre et al., 2000; Emad et al., 2013; Maniscalco et al., 2022). In this study, lumbar lordosis was observed only in one patient (Supplementary Figure 2, Supplemental digital content 1, http://links.lww.com/CD/A41).

Other rare manifestations such as congenital cataracts, pleural effusions and temporomandibular joint arthropathy were not found in our cohort (Albtoush et al., 2018; Kisla Ekinci et al., 2021; Maniscalco et al., 2022). Exostosis, however, was noted in P3 as an incidental finding.

Bone age in all affected individuals with CACP syndrome appeared to be normal (Albtoush et al., 2018; Kisla Ekinci et al., 2021; Bağrul et al., 2023) whereas, in this study, delayed ossification of carpal bones was noted in four affected individuals: 3-year-old (P13), 4-year-old (P4), 6-year-old (P10) and 8-year-old (P11) patients.

As reported in most of the studies, the CACP syndrome had different clinical presentations depending on the age of affected individuals. Shortening of the femoral neck is observed as age progresses and this is apparent in our cohort (Faivre et al., 2000; Albtoush et al., 2018; Maniscalco et al., 2022). Severity in the degree of curvature of camptodactyly and pain in large joints might worsen with age (Albtoush et al., 2018; Kisla Ekinci et al., 2021).

Moreover, intraosseous cysts are the characteristic feature of CACP syndrome and may develop over time at any age affecting radio-ulnar joint or acetabular surface (Albtoush et al., 2018; Yilmaz et al., 2018; Kisla Ekinci et al., 2021; Bağrul et al., 2023). Acetabular cysts were evident at 6 years (P2), 2 years (P3), 2 years (P5), 23 years (P8), 3 years (P13), 6 years (P10) and 18 years (P12) in the affected individuals in our cohort.

Childhood onset of arthropathy can also be an overlapping feature of juvenile idiopathic arthritis (JIA), which can lead to misdiagnosis and delay in necessary treatment (Maniscalco et al., 2022). In this study, two families (F6 and F8) were initially diagnosed to have JIA. They were treated with disease-modifying antirheumatic drugs such as methotrexate. The distinct clinical features including early onset of camptodactyly, bilateral arthropathy and normal inflammatory markers and unique radiological features such as flattening of the metacarpal and phalangeal heads, short femoral neck, short iliac wings, coxa vara, irregular acetabular roof, intraosseous cysts, osteoporosis and osteopenia can help in differentiating JIA from CACP syndrome (Kakkar et al., 2013).

The study by Bagrul et al. emphasized that the presence of consanguinity in family can also act as a clue for the diagnosis of CACP syndrome in affected individuals (Albtoush et al., 2018; Kisla Ekinci et al., 2021; Bağrul et al., 2023).

Proteoglycan 4 protein is also known as lubricin. The lubricin is a glycoprotein involved in lubrication and protection of synovial joints. It is composed of three domains: a central mu cin-like domain, an N-terminal somatomedin-like domain and a C-terminal hemopexin-like domain. The extensively glycosylated mucin-like-domain interacts with various proteins involved in cell signaling or homeostasis of articular cartilage, while the N- and C-terminal domains bind to the cell receptors, providing PRG4 protein a brush-like appearance. This protein can also attach with each other at the N-terminal to reduce the friction in synovial joints (Flannery et al., 1999; Jay and Waller, 2014; Elsaid et al., 2023). The pathogenic alteration in the protein structure could reduce its lubricating properties.

The deletion of Prg4 in mice does not affect skeletal development in the neonatal period, but it does produce abnormalities at the surface of the articular cartilage with aging (Coles et al., 2010). Although PRG4 is expressed during joint cavitation, it is anticipated to be less crucial during joint development and helps in homeostasis and protection of articular cartilage (Rhee et al., 2005; Takahata et al., 2022; Yao et al., 2023). This might explain the development of arthropathy postnatally and its worsening with age.

To date, 40 different variants have been identified (Stenson et al., 2020), mostly in exons 4 and 7−13 in PRG4 (NM_ 005807.6). Most of the pathogenic variants are truncating and are located in exon 7, encoding for mucin-like domain of lubricin protein. Five of the six identified biallelic variants in this study are likely to affect the mucin-like domain disrupting the normal function of protein.

Previously, 10 individuals affected with CACP syndrome from six Indian families with five mutations have been reported (Kakkar et al., 2013; Nandagopalan et al., 2014; Madhusudan et al., 2016; Patil et al., 2016; Vutukuru and Reddy, 2016; Johnson et al., 2021) and with the current study, we add a large cohort of individuals affected with CACP from India, enabling in cataloging the causative variants in PRG4.

Fig. 2.

Pelvic radiographs of the patients at different ages (arranged chronologically) demonstrate short femoral neck and coxa vara in all. Small iliac wings were seen in P1, P2, P8 and P9. Hip joint space is reduced in older patients. Narrow sacrosciatic notch in P13. Intraosseous cysts are evident at acetabular surface in P2, P3, P5, P8, P10, P12 and P13 (white arrows).