Novel and recurrent mutations in WISP3 and an atypical phenotype

For Peer Review

Novel and Recurrent Mutations in WISP3 and an Atypical

Phenotype

Journal: American Journal of Medical Genetics: Part A

Manuscript ID: 15-0152

Wiley - Manuscript type: Research Letter

Date Submitted by the Author: 19-Feb-2015

Complete List of Authors: Bhavani, Gandham; kasturba Medical College, Department of Medical Genetics Shah, Hitesh; Kasturba Medical College, Orthopedics Dalal, Ashwin; Centre for DNA Fingerprinting and Diagnostics, Diagnostics Division Shukla, Anju; Kasturba Medical College, Manipal University, Medical

Genetics Danda, Sumita; Christian Medical College, Clinical Genetics Unit, Gastrointestinal Sciences; Aggarwal, Shagun; Institute of Liver and Biliary Sciences, Genetics Phadke, Shubha; Sanjay Gandhi Post Graduate Institute of Medical Sciences , Medical Genetics Gupta, Neeraja; All India Institute of Medical Sciences, Genetics Unit, Department of Pediatrics Kabra, Madhulika; AIIMS, Gowrishankar, Kalpana; Kanchi Kamakoti Childs Trust Hospital, Department of Medical Genetics Gupta, Anju; Post Graduate Institute of Medical Education and Research,

Pediatrics Bhat, Meenakshi; Centre for Human Genetics, Puri, Ratna; Sir Gangaram hospital, Center of Medical Genetics Bijarnia-Mahay, Sunita; Sir Gangaram hospital, Center of Medical Genetics Nampoothiri, Sheela; Amrita Institute Of Medical Sciences & Research Centre, Mohanasundaram, Kavitha; Madras Medical College, Department of Rheumatology Rajeswari, S; Madras Medical College, Department of Rheumatology Kulkarni, Akhil; SS Institute of Medical Sciences and Research Centre, Department of Radiodiagnosis

Kulkarni, Muralidhar; JJMMC, Pediatrics Ranganath, Prajnya; Center for DNA Fingerprinting and Diagnostics, Diagnostics Division Akella, Radha; Rainbow Hospital, Department of Genetics Sankar, VH; , SAT Hospital, Government Medical College, Department of Pediatrics Girisha, Katta; Kasturba Medical College, Department of Medical Genetics

Keywords: Progressive pseudorheumatoid arthropathy of childhood, WISP3, Mutation,

John Wiley & Sons, Inc.

American Journal of Medical Genetics: Part A

For Peer Review

Genetics, India

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Novel and Recurrent Mutations in WISP3 and an Atypical Phenotype

Gandham SriLakshmi Bhavania, Hitesh Shah

b, Ashwin B Dalal

c, Anju Shukla

a, Sumita Danda

d,

Shagun Aggarwale, Shubha R Phadke

f, Neerja Gupta

g, Madhulika Kabra

g, Kalpana

Gowrishankarh, Anju Gupta

i, Meenakshi Bhat

j, Ratna D Puri

k, Sunita Bijarnia-Mahay

k, Sheela

Nampoothiril, Kavitha M Mohanasundaram

m, S Rajeswari

m, Akhil M Kulkarni

n, Muralidhar L

Kulkarnio, Prajnya Ranganath

e,c, A Radha Ramadevi

p, Sankar V Hariharan

q, Katta Mohan

Girishaa

aDepartment of Medical Genetics, Kasturba Medical College, Manipal University, Manipal, India

bPediatric Orthopedics Services, Department of Orthopedics, Kasturba Medical College, Manipal

University, Manipal, India

cDiagnostics Division, Centre for DNA Fingerprinting and Diagnostics, Hyderabad, India

dDepartment of Clinical Genetics, Christian Medical College and Hospital, Vellore, India

eDepartment of Medical Genetics, Nizam's Institute of Medical Sciences, Hyderabad, India

fDepartment of Medical Genetics, Sanjay Gandhi Postgraduate Institute of Medical Sciences,

Lucknow, India

gDivision of Genetics, Department of Pediatrics, All India Institute of Medical Science, New Delhi,

India

hDepartment of Medical Genetics, Kanchi Kamakoti Childs Trust Hospital, Chennai, Tamil Nadu, India

iDepartment of Pediatrics, Post Graduate Institute of Medical Education and Research, Chandigarh,

India

jCentre for Human Genetics, Bangalore, India

kCentre of Medical Genetics, Sir Ganga Ram Hospital, New Delhi, India

lDepartment of Pediatric Genetics, Amrita Institute of Medical Sciences and Research Centre,

Ponekkara, Cochin, Kerala, India

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mDepartment of Rheumatology, Madras Medical College, Chennai, India

nDepartment of Radiodiagnosis, SS Institute of Medical Sciences and Research Centre, Davangere,

India

oDepartment of Pediatrics, Jagadguru Jayadeva Murugarajendra Medical College, Davangere, India

pDepartment of Clinical Genetics, Genetics Unit, Rainbow Children Hospital, Hyderabad, India

qDepartment of Pediatrics, Sree Avittom Thirunal Hospital, Government Medical College,

Trivandrum, India

Corresponding author:

Dr Girisha KM

Department of Medical Genetics,

Kasturba Medical College, Manipal

Manipal University, Manipal-576104,

India

Email: [email protected]

Phone number: +91-820-2923149

Key words:

Progressive pseudorheumatoid arthropathy of childhood, WISP3, mutation, genetics, India

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Progressive pseudorheumatoid dysplasia (PPD; OMIM 208230) is an autosomal recessive

skeletal disorder characterized by progressive degeneration of articular cartilage. PPD

manifests with progressive joint deformities, stiffness and swelling mainly in hips, knees,

wrists and fingers [Garcia Segarra et al., 2012; Yue et al., 2009]. PPD occurs due to loss of

function mutations in Wnt1-inducible signalling pathway protein 3 (WISP3) gene, located on

chromosome 6q22 [Hurvitz et al., 1999]. We had reported mutation spectrum in 25 Indian

families earlier [Dalal et al., 2012]. We now add mutations in further 54 families and report

9 novel mutations. We also describe an unusual phenotype of this disorder with severe

involvement of humerus resulting in rhizomelic upper limb shortening in two sibs as a result

of a novel mutation in the WISP3.

Additional 60 patients from 54 families were recruited in this study from 15 referral

centres across India. Clinical evaluation and molecular analysis were performed as described

earlier [Dalal et al., 2012]. We identified sixteen causative mutations in 54 families with

progressive pseudorheumatoid dysplasia (Table I and Table II). Most families (50/54, 92.5%)

demonstrated homozygous and only five families showed compound heterozygous

mutations.

Eight mutations are novel. Four of them were splice site mutations (c.49-1G>A,

c.347-2A>G, c.347-1_347-3delCAG and c.779_783+1delTAAAGG), two were deletions

(c.740_741delGT and c.804delC) and one each of nonsense mutation (c.530C>A) and

insertion (c.683_684insT). All these novel mutations either alter the reading frame or cause

premature termination of translation and were not present in 100 healthy control

individuals.

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We also observed two homozygous variants (c.296A>T; p.Y99F and c.298T>A;

p.C100S) in exon 2 in the proband in family 27 (Table II) and her parents were heterozygous

carriers for these variants. Her phenotype was typical for PPD. No other pathogenic

sequence variation was observed in other four exons and the flanking intronic regions of

WISP3. Both the mutations are present in the same insulin-like growth factor binding

proteins (IGFBP) domain. Pathogenicity prediction softwares Mutation Taster, PolyPhen2

and PROVEAN predicted both the mutations as pathogenic. SIFT predicted the variant

p.C100S to be damaging while the other variant as tolerated. The gene WISP3 belongs to

CCN family of growth factors which harbour highly conserved cysteine residues across

different species, both in position and number [Holbourn et al., 2008]. Thirty four cysteine

residues are present in the WISP3 gene. Mutations frequently affect these residues and

result in altered protein structure and function [Dalal et al., 2012; Nakamura et al., 2007;

Sen et al., 2004]. As the variant p.C100S alters one of these cysteine residues, this is more

likely to be pathogenic than the other variant. However we await further functional studies

or reports to determine the nature of these two sequence variants. Also, a rare occurrence

of two mutations in consanguineous families cannot be entirely ruled out in this case.

Eight previously reported mutations (c.156C>A, c.233G>A, c.348C>A, c.433T>C,

c.624_625insA, c.677G>T, c.739_740delTG and c.1010G>A) were observed in this study. The

most common mutations were p.C52* and p.C78Y in exon 2 and p.C337Y in exon 5. These

three mutations account for 70.7% of total mutations and 18.9%, 21.5% and 30.3% of cases

respectively when we combine the data from our complete work [Dalal et al., 2012].

Mutation p.C52* is a recurrent variation reported worldwide whereas the other two

variations appear specific to our population. They too alter the cysteine residues.

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In eleven families (and 13 patients) in the present cohort we noted consistent

segregation of the variant with p.C52* with p.G83E. We had described the variation p.G83E

as pathogenic in two families in our earlier report [Dalal et al., 2012]. Reanalysis of these

two families revealed p.C52* in homozygous state in one of them. The other family was

found to have compound heterozygosity for p.C337Y and p.R230Lfs*4 (a novel pathogenic

variation) along with p.G83E in heterozygous state. Taking all patients with p.G83E into

consideration, we observed its co-segregation with p.C52* in 14 Indian families. Two

families however showed independent occurrence of these two variations (p.C52* and

p.G83E). Hence p.G83E is likely to be a polymorphism and is due to a founder effect, as

suggested by Delague et al earlier [Delague et al., 2005].

An interesting phenotype was noted in a 16-years-old male, born to second degree

consanguineous parents (Family 23). He presented with multiple limb deformities, swelling

and pain of joints. He had disproportionate short stature, (height 124 cm, 6SD below the

mean), prominent acromian process and abnormal curvature of both the shoulders. Bowing

of both arms was evident leading to rhizomelia in upper limbs. There was wasting of

forearm and leg muscles. Swelling and contractures of small joints of both the hands was

present (Fig. 1). Flexion deformity was observed at hips and shoulders. Chest deformities

and kyphoscoliosis were present. Radiological examination of the upper limbs revealed

bilateral bent, short and severely deformed humeri with dislocation at the shoulder joint. All

the epiphyses were enlarged, metaphysis were widened and the humeri had severe

involvement of the diaphysis as well. Platyspondyly with anterior beaking characteristic of

PPD was evident in the spine. Severe metaphyseal dysplasia, diaphyseal involvement and

bowing of long bones of upper limbs were the novel findings identified in this family. This

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phenotype has not been reported earlier with progressive pseudorheumatoid dysplasia.

Interestingly, he carried a homozygous novel deletion of single nucleotide C at the

nucleotide position 804 in exon 5 resulting in frame shift mutation, p.Q269Nfs*44. His

younger brother aged 13 years also had clinical features of PPD and right sided rhizomelia

(Fig. 1). However the family did not consent for his radiographs and genetic testing.

To summarize, we present the mutations in the largest series of patients with PPD

with a report on total nine new mutations and seventeen mutations observed only in Indian

population. We also add a new clinical feature of the condition to the literature with this

work.

Acknowledgements:

We thank all the patients and their family members for participation in this study. We

acknowledge the support of Indian Council of Medical Research (BMS 54/2/2013) and Dr

TMA Pai endowment chair, Manipal University, Manipal for funding this work. The authors

declared no conflict of interest.

References:

Dalal A, Bhavani GS, Togarrati PP, Bierhals T, Nandineni MR, Danda S, Danda D, Shah H, Vijayan S,

Gowrishankar K, Phadke SR, Bidchol AM, Rao AP, Nampoothiri S, Kutsche K, Girisha KM.

2012. Analysis of the WISP3 gene in Indian families with progressive pseudorheumatoid

dysplasia. Am J Med Genet A 158A:2820-8.

Delague V, Chouery E, Corbani S, Ghanem I, Aamar S, Fischer J, Levy-Lahad E, Urtizberea JA,

Megarbane A. 2005. 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 138A:118-26.

Garcia Segarra N, Mittaz L, Campos-Xavier AB, Bartels CF, Tuysuz B, Alanay Y, Cimaz R, Cormier-Daire

V, Di Rocco M, Duba HC, Elcioglu NH, Forzano F, Hospach T, Kilic E, Kuemmerle-Deschner JB,

Mortier G, Mrusek S, Nampoothiri S, Obersztyn E, Pauli RM, Selicorni A, Tenconi R, Unger S,

Utine GE, Wright M, Zabel B, Warman ML, Superti-Furga A, Bonafe L. 2012. The diagnostic

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challenge of progressive pseudorheumatoid dysplasia (PPRD): a review of clinical features,

radiographic features, and WISP3 mutations in 63 affected individuals. Am J Med Genet C

Semin Med Genet 160C:217-29.

Holbourn KP, Acharya KR, Perbal B. 2008. The CCN family of proteins: structure-function

relationships. Trends Biochem Sci 33:461-73.

Hurvitz JR, Suwairi WM, Van Hul W, El-Shanti H, Superti-Furga A, Roudier J, Holderbaum D, Pauli RM,

Herd JK, Van Hul EV, Rezai-Delui H, Legius E, Le Merrer M, Al-Alami J, Bahabri SA, Warman

ML. 1999. Mutations in the CCN gene family member WISP3 cause progressive

pseudorheumatoid dysplasia. Nat Genet 23:94-8.

Nakamura Y, Weidinger G, Liang JO, Aquilina-Beck A, Tamai K, Moon RT, Warman ML. 2007. The CCN

family member Wisp3, mutant in progressive pseudorheumatoid dysplasia, modulates BMP

and Wnt signaling. J Clin Invest 117:3075-86.

Sen M, Cheng YH, Goldring MB, Lotz MK, Carson DA. 2004. WISP3-dependent regulation of type II

collagen and aggrecan production in chondrocytes. Arthritis Rheum 50:488-97.

Yue H, Zhang ZL, He JW. 2009. Identification of novel mutations in WISP3 gene in two unrelated

Chinese families with progressive pseudorheumatoid dysplasia. Bone 44:547-54.

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Table I: Mutations identified in patients with progressive pseudorheumatoid dysplasia

S.

No Location

Nucleotide

change

Amino acid

change

Mutation Taster Number of

patients

Allele

count Status Probabilit

y Value Prediction

1 Intron 1 c.49-1G>A - 1 Disease

causing 1 2 Novel

2 Exon 2 c.156C>A p.C52* - - 14 24 Known

3 Exon 2 c.233G>A p.C78Y - - 15 29 Known

4 Intron 2 c.347-2A>G - 1 Disease

causing 2 4 Novel

5 Intron 2 c.347-1_347-

3delCAG - 1

Disease

causing 1 2 Novel

6 Exon 2 c.296A>T p.Y99F 0.99 Disease

causing 1* 2 Novel

7 Exon 2 c.298T>A p.C100S 0.99 Disease

causing 1* 2 Novel

8 Exon 3 c.348C>A p.Y116* - - 4 7 Known

9 Exon 3 c.433T>C p.C145R - - 1 2 Known

10 Exon 3 c.530C>A p.S177* 0.99 Disease

causing 1 2 Novel

11 Exon 3 c.624_625insA

p.C209Mfs*2

1 - - 2 3 Known

12 Exon 4 c.677G>T p.G226V - - 1 2 Known

13 Exon 4 c.683_684insT p.N229* 0.99 Disease

causing 1 2 Novel

14 Exon 4 c.685_686insA

TCTA

p.R230Lfs*4 1 Disease

causing

(Reanalyze

d patient) 1 Novel

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15 Exon 4 c.739_740delT

G

p.C247Lfs*31 - - 3 6 Known

16 Exon 4 c.740_741del

GT

p.C247Lfs*31 1 Disease

causing 1 1 Novel

17 Exon 4 +

Intron 4

c.779_783+1d

elTAAAGG

- 1 Disease

causing 1 1 Novel

18 Exon 5 c.804delC

p.Q269Nfs*4

4 1

Disease

causing 1 2 Novel

19 Exon 5 c.1010G>A p.C337Y - - 15 29 Known

* These two variations were present in the same family in homozygous state and the

causative mutation could not be determined.

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Table II. Mutations in Indian families with Progressive Pseudorheumatoid dysplasia.

Family

number

Allele 1 Amino acid

change

Allele 2 Amino acid

change

State

Family 1 c.233G>A p.C78Y c.779_783+1delTAAA

GG

p.I260Nfs*17 Compound

heterozygous

Family 2 c.233G>A p.C78Y c.233G>A p.C78Y Homozygous

Family 3 c.156C>A p.C52* c.1010G>A p.C337Y Compound

heterozygous


Family 5 c.156C>A p.C52* c.156C>A p.C52* Homozygous





Family 10 c.739_740delTG p.C247Lfs*31 c.739_740delTG p.C247Lfs*31 Homozygous




Family 14 c.624_625insA p.C209Mfs*21 c.624_625insA p.C209Mfs*21 Homozygous


Family 16 c.348C>A p.Y116* c.348C>A p.Y116* Homozygous





Family 21 c.348C>A p.Y116* c.624_625insA p.C209Mfs*21 Compound

heterozygous


Family 23 c.804delC p. Q269Nfs*44 c.804delC p. Q269Nfs*44 Homozygous




Family 27 c.298T>A p.C100S c.298T>A p.C100S Homozygous

c.296A>T p.Y99F c.296A>T p.Y99F Homozygous

Family 28 c.49-1G>A - c.49-1G>A - Homozygous




Family 32 c.156C>A p.C52* c.740_741delGT p. C247Lfs*31 Compound

heterozygous





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Family 37 c.530C>A p.S177* c.530C>A p.S177* Homozygous

Family 38 c.156C>A p.C52* c.677G>T p.G226V Compound

heterozygous




Family 42 c.683_684insT p.N229* c.683_684insT p.N229* Homozygous


Family 44 c.347-2A>G - c.347-2A>G - Homozygous



Family 47 c.433T>C p.C145R c.433T>C p.C145R Homozygous

Family 48 c.347-1_347-

3delCAG

- c.347-1_347-3delCAG - Homozygous







Reanalyzed

patient 1

c.685_686insATCT

A

p.R230Lfs*4 c.1010G>A p.C337Y Compound

heterozygous

Reanalyzed

patient 2

c.156C>A p.C52* c.156C>A p.C52* Homozygous

Note: Family 27 had two mutations in the proband in homozygous state. Families 9, 22, 38,

44, 50 and 54 had two affected sibs. cDNA numbering +1 corresponds to the ‘A’ of the first

ATG translation initiation codon with RefSeq NM_003880.

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Figure Legends:

Fig 1. Rhizomelia of upper limbs was noted in one of our patients with PPD (A, B). The

corresponding radiographs show underlying bent humeri (C, D). The hands and bones of the

hands however show typical changes (E, F). His younger brother too has short right arm (G)

and swelling of interphalangeal joints (H).

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Fig 1. Rhizomelia of upper limbs was noted in one of our patients with PPD (A,B). The corresponding radiographs show underlying bent humeri (C, D). The hands and bones of the hands however show typical changes (E, F). His younger brother too has short right arm (G) and swelling of interphalangeal joints (H).

248x141mm (150 x 150 DPI)

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Novel and recurrent mutations in WISP3 and an atypical phenotype

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