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Whole Exome Sequencing of Patients with Diffuse Idiopathic Skeletal Hyperostosis and Calcium Pyrophosphate Crystal Chondrocalcinosis

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Whole Exome Sequencing of Patients with Diffuse Idiopathic Skeletal Hyperostosis and Calcium Pyrophosphate Crystal Chondrocalcinosis ORIgInAL PAPERS Whole exome sequencing of patients with diffuse idiopathic skeletal hyperostosis and calcium pyrophosphate crystal chondrocalcinosis Parreira B 1,2 , Couto AR 1,2 , Rocha F 1,2 , Sousa M 1,2 , Faustino V 1, Power DM 3, Bruges-Armas J 1,2 ACTA REUMATOL PORT. 2020;45:116-126 ABSTRACT could be involved in this phenotype in an as yet un - known way. Objectives: DISH/CC is a poorly understood pheno - type characterised by peripheral and axial entheso - Keywords: Rheumatic and musculoskeletal diseases ; pathic calcifications, frequently fulfilling the radiolog - Genetic association; Rheumatology. ical criteria for Diffuse Idiopathic Skeletal Hyperostosis (DISH, MIM 106400), and in some cases associated with Calcium Pyrophosphate Dihydrate (CPPD) Chon - INTRODUCTION drocalcinosis (CC). The concurrence of DISH and CC suggests a shared pathogenic mechanism. In order to Previous studies undertaken by our group, identified identify genetic variants for susceptibility we performed and characterized twelve families affected with Diffuse whole exome sequencing in four patients showing this Idiopathic Skeletal Hyperostosis (DISH, MIM 106400) phenotype. and/or Calcium Pyrophosphate Dihydrate (CPPD) Materials and methods: Exome data were filtered in Chondrocalcinosis (CC), hereafter designated, order to find a variant or a group of variants that could DISH/CC. DISH/CC is a poorly understood phenotype be associated with the DISH/CC phenotype. V ariants characterised by peripheral and axial enthesopathic cal - of interest were subsequently confirmed by Sanger se - cifications, frequently fulfilling the radiological criteria quencing. Selected variants were screened in a cohort for DISH, and in some cases associated with CPPD of 65 DISH/CC patients vs 118 controls from Azores. Chondrocalcinosis. A common pathogenic mechanism, The statistical analysis was performed using PLINK shared by the two conditions, has been suggested 1. V1.07. DISH is a common skeletal disorder characterized by Results: We identified 21 genetic variants in 17 genes progressive calcification and ossification of ligaments that were directly or indirectly related to mineraliza - and entheses 2-3 . The exact prevalence and incidence of tion, several are predicted to have a strong effect at a DISH is unknown, however it is well known that it is protein level. Phylogenetic analysis of altered amino more frequent in males and its prevalence rapidly in - acids indicates that these are either highly conserved creases with age, mainly affecting subjects over the age in vertebrates or conserved in mammals. In case-con - of 40 4. The prevalence of DISH in patients over 50 years trol analyses, variant rs34473884 in PPP2R2D was sig - of age is 25% in males and 15% in females, 5 and this nificantly associated with the DISH/CC phenotype disease is now becoming a serious problem in aging so - (p=0.028; OR=1.789, 95% CI= 1.060 - 3.021) ). cieties. Several lines of evidence suggest that genetic Conclusion: The results of the present and preceding factors might play a part in the etiology of DISH, such studies with the DISH/CC families suggests that the as the existence of familial cases with early onset (in the phenotype has a polygenic basis. The PPP2R2D gene third decade of life) 6 and the higher frequency of DISH in the boxer dogs relative to other dog breeds 7-8 . So far, however, no single gene has been conclusively associa - 1. Serviço Especializado de Epidemiologia e Biologia Molecular ted with the disease. Chondrocalcinosis is characteri - (SEEBMO) / Hospital de Santo Espírito da Ilha Terceira (HSEIT) 2. Comprehensive Health Research Center, CHRC, Lisbon, Portugal zed by deposition of crystals of calcium pyrophosphate 3. Centre of Marine Science (CCMAR) / University of Algarve dihydrate (CPPD) in articular hyaline and fibro-carti - ÓRgÃO OfICIAL dA SOCIEdAdE PORTUgUESA dE REUMATOLOgIA 116 PARREIRA B ET AL lage 9. For the moment two genes, ANKH (CCAL2) and analysis with an Agilent 2100 Bioanalyzer and Qubit. TNFRSF11B , are known to be involved in the devel - Each library went through a process of emulsion PCR opment of Chondrocalcinosis 10-11 . Previously, we per - for clonal amplification of the fragments, followed by formed genetic studies of DISH/CC using a “Whole an enrichment process and chemical modification to genome wide linkage study” and an “Identity by allow loading into the reaction chamber. The quality State/Identity by Descent” association study and two and quantity of the beads obtained for each library genes were identified as good candidate genes for were estimated taking into account the parameters giv - DISH/CC; RSPO4 on chromosome 20, and LEMD3 on en by Work Flow Analysis. Then, ligation sequencing chromosome 12. Several RSPO4 gene variants were was done to obtain sequences of 50nt +35nt (Paired- identified and nucleotide modifications located in the end) reads using a SOLiD4 sequencer. The data qual - regulatory region were more frequent in control indi - ity was estimated using the parameters provided by viduals than in the DISH/CC group 12 . Even though the the software SETS (SOLiD Experimental Tracking Sys - genetic basis of DISH/CC is unknown, it is considered tem). Single Nucleotide Variants (SNVs) were classified to be a bone forming disease and genes related to cal - using Ensembl’s nomenclature and grouped using the cification and ossification are considered good candi - following scheme: Known and Novel (Coding, Splic - dates. In the present study, we took advantage of whole ing, Others). The coding variants were divided into exome sequencing (WES) so that even in the absence nonsynonymous and synonymous. The “Others” in - of sufficient pedigrees and samples for traditional link - cluded intronic, UTR, regulatory region, intergenic, age approaches we could identify rare protein-coding downstream and upstream variants. variants that are the cause of the majority of mono - genic diseases 13-14 . We performed targeted exome se - WES fIlTERINg quencing on four DISH/CC patients, with an appar - The segregation analysis of these families indicated that ently autosomal dominant DISH/CC phenotype, in the most likely model for this disease is an autosomal order to capture rare and pathogenic variants expect - dominant model 1. However, given that a previous ed to have potentially damaging effects on protein linkage study did not identify a major dominant lo - function that lead to modified calcification and/or os - cus, the data were analyzed assuming both a recessive sification. To our knowledge this is the first report of and a dominant model. WES analysis in patients affected with DISH. Genetic variants were identified in 815, 917, 872 and 593 genes under a dominant model, for indivi - duals AZ1 to AZ4. Assuming that the genetic cause is MATERIAlS AND METHODS the same for the individuals AZ1 to AZ4, identified genes were then filtered to select common, shared This study was approved by the “Comissão de Ética genes (supplementary Table I, available in supple - do Hospital de Santo Espírito da Ilha Terceira”. All mentary file). A group of 52 genes were common to the methods were performed in accordance with the ap - four patients; candidate genes for testing were select - proved guidelines, obtaining informed consent from ed taking into account their involvement in calcifica - each subject before conducting the experiments. tion and/or ossification or related conditions that could be associated with the DISH/CC disease. Gene vari - ExOME CApTURE ants shared between the genes common to the four pa - The selection of patients was made after ruling out mu - tients were identified and nonsynonymous, splice site, tations in ANKH and secondary causes for Chondro - stop lost/gain and frameshift variants were targeted, in calcinosis. DNA was extracted from peripheral blood anticipation that synonymous and intronic variants samples using a standard salting out procedure. Sam - would be far less likely to be relevant in the ples were resequenced, using an ABI-SOLiD platform pathogenicity. and Agilent’s SureSelect Target Enrichment System for 38 Mb, by “Sistemas Genómicos, S.L.” in Valencia, CANDIDATE gENES – SElECTION By fUNCTION Spain. After standard DNA quality control, SOLiD A group of 20 candidate genes was selected and in - Fragment libraries were prepared and enriched with cluded genes that are reported to be involved in bone SureSelect All Human Enrichment Target Exon. The metabolism and/or related conditions. These genes quality and quantity of the libraries were assessed by were Alkaline Phosphatase ( ALPL/TNAP ), the Calcium ÓRgÃO OfICIAL dA SOCIEdAdE PORTUgUESA dE REUMATOLOgIA 117 EcToPIc cALcIfIcATIon Sensing Receptor ( CASR ), Bone Morphogenetic s s u d t e Protein Receptor Type 1B ( BMPR1B ), Osteopon - i n s l , , a a l d a i s s l y y e c n i i e tin ( OPN/SPP1 ), Integrin Binding Sialoprotein t t a a s s a i i m s c i m i s s a a i s h i i t d i e e t d s r (IBSP ), Fibroblast Growth Factor 2 ( FGF2 ), Inor - r s h h b b y e e a t t r o t r i i r V c e O O r e e : L L ganic Pyrophosphate Transport Regulator a l h b s : t c i a y s s i O h , o (ANKH ), Collagen Type XI Alpha 2 ( COL11A2 ), t t D g a y n p i h Nucleotide Pyrophosphatase 1 ( ENPP1 ), Runt- o p w s o o e r t h related Transcription Factor ( ), Dickkopf t RUNX2 m r t a s a e 0 0 A n e 0 s / n e 4 4 d E 3 WNT Signaling Pathway ( DKK-1 ), Insulin Like n g e < < N n ) c o y A 4 a S ; Growth Factor 1 ( IGF1 ), Matrix Gla Protein p e ) s 2 u t s ; n (MGP ); Vitamin D ( VDR ), Bone Morphogenetic s i s i e t o i j e s s s s d Protein 4 ( BMP-4 ), Collagen Type 1 Alpha 1 f r r r r n o n n n n o a a a a l l l l o b o o o o e i i i i b u u u u l (COL1A1 ), Transforming Growth Factor Beta 1 c t t t t w a c c c c f n a a a a i i i i r o o t t t t e c c c c b b r r r r s (TGF 1), Solute Carrier Family 29 Member 1 i i i i s e l f f f f e a a a a t i i i i r i i i i w r E c c c c r r r r p e o l l l l (SLC29A1 ), Bone Morphogenetic Protein 2 ( BMP- r e e e e v : a a a a g s P P P P s c c c c t i s u u -2 ) and Collagen Type VI Alpha 1 ( COL6A1 ).
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