Broadening the Phenotypic Spectrum of POP1-Skeletal Dysplasias: Identification of POP1 Mutations in a Mild and Severe Skeletal Dysplasia

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Article: Barraza-García, J., Rivera-Pedroza, C.I., Hisado-Oliva, A. et al. (10 more authors) (2017) Broadening the phenotypic spectrum of POP1-skeletal dysplasias: identification of POP1 mutations in a mild and severe skeletal dysplasia. Clinical Genetics. ISSN 0009-9163 https://doi.org/10.1111/cge.12964

This is the peer reviewed version of the following article: Barraza-García, J. et al (2017), Broadening the phenotypic spectrum of POP1-skeletal dysplasias: identification of POP1 mutations in a mild and severe skeletal dysplasia. Clin Genet., which has been published in final form at https://doi.org/10.1111/cge.12964. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.

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Jimena Barraza-García Jimena Key Words: 7. 9. 8. 6. 3. 2. 1. Kristina Ibáñez-Garikano Kristina Heath Martínez 5. 4. article as doi: 10.1 differences version between lead to this may been through the copyediting, typesetting, pagination andproofreading process, which This article hasbeen accepted for publication andundergone full peer review but has not

Necker-EnfantsMalades, Paris, France. AP-H Descartes-Sorbonne CitéUniversity, Paris Paris ofOsteochondrodysplasia, Bases Physiopathological Molecular and of Laboratory Spain. Salamanca, Salamanca, de Biomédica UK. Sheffield, Bank, Western ofSheffield, University Health, Child of Ho Children's Sheffield Department, Radiology Australia. Brisbane, Hospital, AlexandraInstitute, Princess Department of Medical Genetics, Reference Center for Skeletal Dysplasia, INSERM UMR 1163, UMR 1163, INSERM Dysplasia, for Skeletal Center Reference Medical Genetics, of Department Unit.Pediatric Endocrinology HospitalUniversita Queenslan of University Group, Genetics Human Multidisciplinary Multidisciplinary Unit (UMDE), Skeletal dysplasia III, Madrid, Spain. de En Red en Biomédica Investigación Centro de Universidad Autónoma de Madrid, IdiPAZ, Madrid, Spain. Spain. Madrid, IdiPAZ, Madrid, de Autónoma Universidad Genetics &Molecular ofMedical Institute Dept. of Endocrinology, Royal Brisbane Brisbane Royal Endocrinology, of Dept. Dept. of Pediatric Endocrinology, Hospital Universitario Infanta Leonor, Madrid, Spain. Spain. Madrid, Infanta Leonor, Hospital Universitario of PediatricEndocrinology, Dept. Broadening the phenotypic spectrum of of POP1-ske spectrum thephenotypic Broadening 1,2,3* 1,2,3 , Lucia Sentchordi-Montané POP1, RMRP, skeletalPOP1, dysplasia anauxetic RMRP, dysplasia, bone, This article is protected by copyright. All rights reserved. rights All bycopyright. protected is article This 111 1,2,3 1 / , Carlos I.Rivera-Pedroza , Amaka Offiah cge.12964 mutations inamutations mild and severedysplasia skeletal

1,3,4 7 , Pablo Prieto-Matos , Emma L. Duncan and Women's Hospital,Herston, Australia. (INGEMM), Hospital Universitario La Paz,

spital NHSFoundation Trust and Academic Unit 1,3 and the Versionof Record. Please cite this P, Institut Imagine, an fermedades Raras (CIBERER), Instituto Carlos Carlos Instituto (CIBERER), Raras fermedades d Diamantina Institute, Translational Research Translational d Institute, Diamantina , AlfonsoHisado-Oliva rio de Salamanca, Instituto deInvestigación Hospital Universitario La letal dysplasias: identification of POP1 identificationofPOP1 letaldysplasias: 5 , Graeme R. Clark R. , Graeme 8 , Valerie Cormier-Daire d HôpitalUniversitaire 1,2,3 , Alberta Belinchón- 6 Paz, Madrid, Paz, Spain. , Angeladel Pozo 9 , Karen E. , KarenE. 1,2 , This article is protected by copyright. All rights reserved. rights All bycopyright. protected is article This Accepted Article +34 91 207 1010 Ext 269; Fax: +34 91 207 1040 207 91 Fax: +34 +34 1040 91 207 1010 Ext269; † support. CoresWe alsothank fortheir technicalhelp. Andr We woul Country. Basque the from studentship Ph.D. aFPI of wasa recipient A.H-O and fellowship CIBERER apredoctoral of G wasa recipient 66831-R, SAF2012-30871) andJ.B- the S2010/BMD-2396). (ENDOSCREEN: de Madrid Comunidad Acknowledgements: of Conflict interest Universitario La Paz, Pº Castellana 261, 28046 PºCastellana 261, Paz, La Universitario Corresponding author: Corresponding

– The –authors The have nothing disclose. to This work was supported in part by the following grants: MINECO (SAF2015- grants: following the by part in workwassupported This

Karen Heath, InstituteofMedical Karen Heath, Madrid, Spain. [email protected]. Tel: [email protected]. Spain. Madrid, d like to thank the INGEMM NGS and Sequencing Sequencing and NGS INGEMM thankthe d liketo eas Zankl and Matthew Brown for their scientific Brownfor their Zankland Matthew eas & Genetics, HospitalMolecular This article is protected by copyright. All rights reserved. rights All bycopyright. protected is article This Accepted Article suspected. Biallelic with andsevere short mildstature a relatively dyspla to anauxetic families withfeatures similar cartilage hair hypoplasiacartilage hair (CHH) POP1 novel 2, ahomozygous proband In wasobserved. pre5.8s rRNA of levels elevated and of RMRP Ma p.[Pro582Ser]:[Glu870fs*5]. 1, proband in detected were deletion base stability of bothstability complexes. Numerous or the assembly in participate it appearsto isunknown, function precise its Although complexes. RNase- the to common protein large POP1 isa Abstract skeletal dysplasias caused by mutations in mutations by caused dysplasias skeletal presenta the phenotypicthe clinical extremes in knowledge about the function of the RMRP complex in skeletal development. in skeletal complex RMRP of the function about the knowledge We discuss the and past present assumption. biogenesisstudies question this disorders, recent mutation was identified POP1 POP1 mutations were identified in both. A missense mutation and a novel single andnovel mutation a missense both. A identifiedin mutations were , p.[(Asp511Tyr)];[(Asp511Tyr)]. p.[(Asp511Tyr)];[(Asp511Tyr)]. but, to date, only three RMRP RMRP mutations have been mutations reported in individuals with skeletal dysplasia and another in whom AD was ADwas in whom and another dysplasia skeletal MRP and RNase-P (RMRP) endoribonucleoprotein MRP and RNase-Pendoribonucleoprotein (RMRP) tion of POP1-dysplasias. Although CHH tion ofPOP1-dysplasias. Although and other sia (AD). We presenttwo sia (AD). or POP1 POP1 are commonly cited as ribosomal citedasribosomal commonly are mutations mutations have been described in two These two individuals demonstrate demonstrate twoindividuals These rkedly reduced abundance abundance reduced rkedly further individuals,onefurther This article is protected by copyright. All rights reserved. rights All bycopyright. protected is article This Accepted Article Currently, weknow that alterations in RNA wasthefirstnon-coding nu subunit untranslated Domain-only Protein, also called POP1. called POP1. also Protein, Domain-only ta must be Care (14). complexes RMRP and RNase-P P/MRP ribonuclease homolog 1 (POP1) of Precursor Processing isthe bothby complexes shared proteins of the One (5,7-12). functions specialized perform (2-6). It is essential for cell viability and appear proteins different and 10 a RNA subunit of composed detectionand expanding the phenotypic spectrum of knowndisorders. (NGS) based sequencing has generation mutationa central diagnostics become tool improving in increased withimplementation the of whole exom 42groups, associated with alterations in >364 ge Introduction cartilage-hair hypoplasia (CHH, w mild dysplasia metaphyseal severity: varying lumbar region and show delayed headsbone maturation.lumbar Femoral and delayed region and show necks as are hypoplastic, well as hypoplasia and mild intellectual disability.Vertebrmild intellectual and hypoplasia very onset, prenatal of short stature with severe conserved(18,20,21). aregenerally andskull spine moremetaphyseal severeaffectatio by dysplasias ar alterations Skeletal plates. growth ofthe borders bones, with longof the thickeningand shortening 607095)(16-19). The RNAse for mitochondrial RNA processing RNA mitochondrial for RNAse The The RNAse formitochondrial RNA processing (RMRP) endoribonuclease complex, In the 2015 nosology ofIn 2015 the nosology skeletal (SD), wereclassifieddysplasias 436 different dysplasias into CHH is a metaphyseal dysplasia in which the limbs and ribs are mostly affected. There is There affected. mostly are ribs and limbs the inwhich dysplasia metaphyseal a is CHH MIM 250250), andan MIM250250), severe RMRP are related to several autosomal SDswith recessive several to are related s to have evolved from the complexRNase-P to ithout hypotrichosis (MIM 250460);moderate short adult height (less than 1m), hypodontia, midface midface hypodontia, 1m), than (less adult height short nes (1). Discovery of new genes has considerably nes (1).Discovery of new genes has considerably n of the knee compared to the proximal femur. The femur. proximal the kneen of to the compared e/genome sequencing and at the same time, Next time, same atthe and sequencing e/genome wide, irregular, and partially sclerotic metaphyseal metaphyseal sclerotic partially irregular,and wide, ae are ovoid with concave concave are ovoidwith ae e present at birth and are distinguished from other from e present and at are distinguished birth AD is a severe spondyloepimetaphyseal dysplasia AD is a spondyloepimetaphyseal severe ken in order to avoid confusion with the PYRIN withthe avoid confusionorder to ken in subunit, a large proteinthatseems alarge tostabilize subunit, , has been identified in virtually all eukaryotes clear RNA associated with human disease (15). disease associated clear RNA human with

gene ( auxetic dysplasia (AD, dysplasia MIM auxetic RMRP, RMRP, MIM 157660), encoding the dorsal surfaces in the dorsal surfacesin This article is protected by copyright. All rights reserved. rights All bycopyright. protected is article This Accepted Article extremes extremes in clinical presentationof 51.8cm (1.7 SDS)(Fig. 2). On physical examination,On physical 51.8cm (1.7SDS)(Fig. 2). were normal. studies hormonal and biochemical Karyotype, cm (-3.1SDS) andheadcircumference (HC) 34cm sect caesarean by weeks 37.5 at wasdelivered child 180 cm (0.4 SDS) mother’sand was without 164 (0.1 complications and thecm SDS).Pregnancy suspected, in whombiallelic 2(25). type Proband 1 Clinical cases disease. with relationship its and of POP1 role aboutthe literature theavailable review also We previously. and the authors suggested that they should be classified as classified be should they that suggested authors the and diminished has of zone alsobeencolumnisation the hypertrophic observed (19,22,23). in number the ofA large phalanges. reduction chondroc are short metacarpals and widewith small, la bodies.the iliac Longbones haveirregularminera More recently, ahomozygous two compound heterozygous heterozygous two compound At 4.6 years of age, she weighed 11.8 kg (-2.47 SDS), height 84.4 cm (-5.57 SDS) and HC and cm(-5.57 SDS) height 84.4 SDS), (-2.47 kg 11.8 she weighed of age, 4.6 years At We present two We present cases, index one an with unknown SD and inanother AD whom was Female of Moroccan origin, born to healthy, no healthy, bornto origin, of Moroccan Female In 2011, two sisterswith aSD AD to similar POP1 POP1 POP1 mutations were identified. These twoprobands demonstrate the mutations, a gene previously not disease. withany associated previously mutations, a gene POP1 mutation was identified in another similarly affectedindividual inmutation another identified was mutations, and mutations, we compare then to them those reported te-ossifying epiphyseste-ossifying andbullet-shapedmiddle lization of the metaphyses. Thefirstandfifth lization of the (-1.4 SDS). Short limbs were noted limbs were at birth. (-1.4 SDS).Short ion, weight 2535g (-2.3 SDS), body length(BL) 45 body 2535g (-2.3SDS), weight ion, she had relative macrocephaly, short neck, broad broad neck, short macrocephaly, relative had she were described (24). Exome analysis revealed revealed analysis Exome (24). described were n-consanguineous parents. Father’s height was parents. Father’s n-consanguineous ytes in the resting and pr intherestingand ytes POP1 -associated skeletal phenotype AD phenotype skeletal -associated oliferating zones, witholiferating zones, This article is protected by copyright. All rights reserved. rights All bycopyright. protected is article This Accepted Article growth plate. tibial metaphyses, and chevron deformity of distal thoracolumbarmetaphyses scoliosis, flared withir at7analysis agenew revealed (Fig. 1O-Q), radiological A bullet-shaped. were phalanges Middle the proximal metaphyses, bowing of the ulna, short femora short ulna, of the bowing metaphyses, proximal narrow iliac wingsmild with flaring ofthe aceta pelvis showed tall vertebral The bodies. vertebral of lumbar bodies with posterior lower the scalloping (Fig. analysis Radiological limbs. and lower upper was able (-2.73 SDS).He HC 46cm was born at 38 weeks, weight 2210g ( 2210g weight weeks, 38 at born was SDS). Intrauterinegrowth retarda parents werefrom thesamevillage Proband 2 Table S1. and bullet-shapedmiddle phalanges (Fig. 1A-J). C phalanges, middle and ofallproximal cone-shapedepiphyses and metaphyses distal irregular tibiae, and and proximal femur distal of metaphyses irregular angle, widened iliac slightly position, valgus femoralnecks ossification, of bodies, the lumbarhypoplastic vertebral and inscalloping delayed lower (Fig. 2). developmentPsychomotor was normal. heels prominent and extension, at with difficulties cubitus valgus brachydactyly, chest, hyperlordosis, proximal femoral physes He was first seen at 2 years old; height 59.5 height old; 2 years at seen first was He Male of Senegalese origin born to healthy parents. Although consanguinity was both denied, parents. origin bornMale consanguinity of Senegalese to healthy Although Radiographic analysis showed mild irregularity of the vertebral end plates with posterior posterior with endplates vertebral the of mild irregularity showed analysis Radiographic and and irregularity sclerosis of tion wasnoted onultrasoundperform . Father’s 178cmheight (0.09 to walk with help. Clinical exam revealed major deformitiesmajor of Clinicalexam walkwithhelp. revealed to -2.32 SDS bula. He had short,broa ), BL 38 ( cm ), BL femoral epiphyses with premature fusion of the of premature fusion with epiphyses femoral regular marginsregular in the long bones, cupped distal linical and radiological data are summarized in dataaresummarized linical andradiological 1K-Q) showed severely delayed bone age. He had boneage. delayed severely 1K-Q) showed cm (-10.18 SDS), weight 6200g (-4.73 SDS) and and (-4.73SDS) 6200g SDS),weight (-10.18 cm l necks, coxa vara with vara coxa l necks, the femoral and tibial metaphyses the femoral and tibial -6.83 SDS SDS), and mother’s 165 cm (0.15 ed at 24-gestational weeks. He weeks. at 24-gestational ed ) and HC31( cm d humeri with irregular vertical orientation of vertical orientation -1.9 SDS . ). ). This article is protected by copyright. All rights reserved. rights All bycopyright. protected is article This Accepted Article compared to the limbs, with pointed fingers and smal and fingers pointed with tothelimbs, compared when weresmaller His hands theelbows. of withflexion contracturesvalgus cubitus hypodontia, examination3.51 reveal 2).Physical SDS)(Fig. sequence hg19. sequence hg19. referencegenomic andhuman dbSNPv137, v2.7, ClinVar dbNSFP 3.5e, SnpE v2.6-5, TK Analysis Genome 0.6.1-r104-tpx, BWA 2.4.60.8, Isis v2.16.1; Bedtools v0.1.19-44428cd; 1.27: Samtools analysis MiSeqIn house bioinformatic (Illumina). EZ SeqCap using SDgenes) 315 (SkeletalSeq.V3, parents. and 1 institutions.respective DNA Materials & Methods &Methods Materials sequencing. brachydactyly with delayed carpal ossification. The absenceof The ossification. carpal withdelayed brachydactyly with shortening long-bone severe kyphoscoliosis, ex onhis based CHH/AD wassuspected, diagnosis of and hearing normal including found were disease on abnormality withoutanassembly fibroblasts, of them of complex I Deficiency spectroscopy. bitemporal subcortical non-specific with associated ofth hyperintensity showed imaging resonance heels(Fig.2,kyphosis andprominent S1). He At six years, his height was 62.5 cm (-10.99 SDS), weight 7200g (-4 SDS) and HC 47.5 cm (- andHC 47.5 SDS) (-4 SDS),weight7200g (-10.99 cm was62.5 height hissix years, At

Proband 1, with anSD, with acustom-designed unknown was analyzed NGS targeted panel All participants providedAll in POP1 direct sequencing was undertaken for proband 2 and his parents. parents.his 2andproband for wasundertaken direct sequencing was extracted from all participants formedconsent and a ethical has cognitive developmental delay. has cognitiveBrain developmental delay. magnetic e subcortical and periventricular white matter white e subcortical andperiventricular ed sparse and apparently hypopigmented hair, hypopigmented apparently and edsparse itochondrial respiratory chain respiratory itochondrial alsowas found in the BN-PAGE. nothe But, BN-PAGE. features ofmitochondrial eye-survey and metabolic screening. A clinical clinical A screening. andmetabolic eye-survey irregular metaphyses irregular and deformed epiphyses, and was performed using: Bowtie2 v2.0;Picard-tools Bowtie2 was performed using: technology (Roche Nimblegen) and sequenced on a andsequenced (Roche Nimblegen) technology edema (Fig. S2). edema (Fig. noThere were lactate peaks at l, dysplastic nails.He had hip flexion deformity, treme short stature, lumbar hyperlordosis and hyperlordosis lumbar stature, short treme RMRP and RNAwasextractedfromproband pproval was obtained from the the from obtained was pproval mutations, led to POP1 POP1 direct This article is protected by copyright. All rights reserved. rights All bycopyright. protected is article This Accepted Article (http://cadd.gs.washington.edu/), conservation us conservation (http://cadd.gs.washington.edu/), (http://db.systemsbiology.net/kaviar). GO Exome Sequencing Project (ESP;https://e for frequency data: ExomeConsortium Aggregation Results three and intriplicate undertaken were Allreactions (24). described aspreviously controls, sex-matched and age- and parents, unaffected compared rRNA pre-5.8S unprocessed RMRP and homozygosity in an individual from Morrocco (25). It affects a highly conserved amino acidand conserved a highly (25). affects from Morrocco It inan individual homozygosity mi The databases. population from absent are p. exondetected, 16 were c.[1744C>T];[2607delC]; misse a 1, In proband S2). (Table 2, respectively frommother. the The paternallyinherited single base silico premature stop codon. RMRP RMRP sex-matched controls (Fig. 3). 3). (Fig. controls sex-matched population databasesand is classifiedall by aspathogenic affects mutation missense This 3). (Fig. parents in and the in unaffected heterozygosity p.[(Asp511Tyr)];[(Asp511Tyr)]) c.[1531G>T];[1531G>T]; pathogenicity predictions classify predictions thevariantpathogenicity and elevated levels of pre5.8S rRNA when comp when rRNA pre5.8S of levels elevated and Variants were evaluated with the help of Alamut V2.8 and CADD V1.3 V1.3 CADD and V2.8 Alamut of help the with evaluated were Variants Compound heterozygous and homozygous andhomozygous heterozygous Compound To determine the functional impact of the muta impactof the thefunctional To determine Gene expression that pa demonstrated assays A homozygous mutation mutation was inexon 11 found A homozygous of biological biological replicas wereperformed. ssense variant has been recently ssense variant reported hasbeen recently in sp.gs.washington.edu/drupal/) and Kaviar andKaviar sp.gs.washington.edu/drupal/) a highly conserveda highly amino absent acid, from the nse mutation in exon 13 and a one base deletion in basedeletion one anda 13 exon mutation in nse ing ing GerpN and databases population were consulted to endogenous GAPDH in to endogenous proband 1, her POP1 as highly as highly deleterious. Thismutation was inherited [Pro582Ser];[Glu870fs*5] (Fig. 3). Bothvariants[Pro582Ser];[Glu870fs*5] (ExAC, http://exac.broad tient 1 had a markedly reduced abundancehad a tient 1 of deletion in exon 16 is predicted to resultin a ted alleles, we measured relative abundance of abundance measured relative we ted alleles, ared to his ared parents and unaffected to his and age- mutations wereidentified in probands1 and POP1 in silico in patient 2:patient in predictors. predictors. institute.org), NHLBI- in This article is protected by copyright. All rights reserved. rights All bycopyright. protected is article This Accepted Article Discussion Discussion patient hasclinical feat as a good candidate gene. Indeed, direct sequencing identified a homozygous homozygous a identified sequencing direct Indeed, gene. candidate as agood found in found (24-25). cases described milder, tothepreviously three similar, arevery although findings notdidsusp thus, we 1were lesssevere proband Howe ancestor. share acommon probably individuals two the origin, of Moroccan 1 isalso proband As (25). features AD-like with individual Moroccan ina homozygosity reportedin recently been has mutation the p.Pro582Ser Interestingly, mutation. Prob of different degrees severity. of the protein which is unlikely to be exposed tobeexposed is unlikely which protein of the truncation in premature the results and exon16, lastexon, the islocatedmutation in C-terminal most Interestingly, the Ser584 isone proximal of four are mutations located exon 13,in Pro582 and encoding missense Gly583 in POPLD domain. the mutantmutationtwo protein Another ispredictedthedomain. in a lacking result POPLD to nonsense 511codons and 513, in a conserved highly between thePOP1region The and domains. POPLD families mutations, a missense (this3). Two anonsense, 24-25)(Fig. and study, located are in exon 11, abnormalities, observed by MRI. also additional features, such as cognitive de reported nonsense variant (24), his phenot variant(24), reported nonsense previously the closeto very located mutation missense a 2 has proband that, although noteworthy itsloss of domains, both func POP1 contains protein In this work,biallelic Incontrast, ADdirectly was suspected inproba Thus, to date, five different RMRP and given the previous report of theprevious given and ures common to CHH such as hypodontia such as CHH to common ures POP1 and 1 is a compound heterozygote for a missense and a frameshift and a frameshift missense for a heterozygote is a 1 compound and POP1 mutations mutations have been identified in ype appears to be much more severe. severe. more much tobe appears ype mutations have been reported in five individuals from four fromfive individuals four mutations beenreported in have lay, forearm and handabnormalities, and brain to nonsense mediated decay. Although this mutant mutant this Although mediated decay. to nonsense ect AD. However,retrospectively, her skeletal amino acids shown to be phosphorylated (26). The (26). be phosphorylated to shown acids amino POP1 tion may be due toa conformational bedue may defect.Itis tion ver, theclinicaland radiological data for mutations mutations in 2, AD type nd2 but, whenno path , sparse and hypopigmented hair, but hypopigmented , sparse and two children with aSDwith twochildrenwith POP1 ogenic mutations were POP1 mutation. This was considered was This article is protected by copyright. All rights reserved. rights All bycopyright. protected is article This Accepted Article The RNase-MRP complex originallywas identified in studies be requiredwill thetoclarify function of POP1. the effects of these mutations on the function and biogenesis of RNase-P and MRP and of RNase-P biogenesis and function onthe mutations these of the effects to (27).itsinsolubility due Mutagenizingfoure 30). roles in (4,7,27- RNase-P/MRP of the thefunctions have mutated positions similar the that suggesting affect many processing ofmore oneor of the pr extension of the elbows. elbows. of the extension in some observedwith patients AD, wasonly in one patient whilst, probands 1 and 2had limited describedabnormalities in were two casesdescribedstudy inthis (T RMRP or of RNA mutations inthe by CHHandotherSDscaused why explain P/MRP (27,30). studies These producing component, RNA ofthe destabilization two previously reported siblings with reported siblings two previously 5.8S increased of levels However, species (31-32). of POP1,and, the depletionby affected significantly alterednot (31) were significantly 5.8SS/5.8SL HeLa cells using interference RN ref 21). However, recent studies have laid doubton these assumptions.When POP1 was de Finally, in Finally, pe ofthe involvement four individuals, on going to have shortened BL. I nvolvement ofmetaphysis the of the and nvolvement and bones epiphysis long Pop1 In proband 2, the deficiency of complex I of the respiratory In chain proband I the 2, was of complex of thedeficiency an finding. respiratory unexpected Attempts to characterize thefunctionof yeast Pop1 We compared available clinical and radiological da and radiological clinical available We compared POP1 mutations have also been shown to beimpl all patients except patient 1, the initial suspected diagnosis was AD. was1,thesuspected diagnosis all patientsexceptpatient initial are commonly cited are commonly asdiseases alterawith all individuals (Table S1), allindividuals (Table Severe postnatal growth retardat Severe postnatal A (RNAi), the ofA (RNAi), pre-rRNA levels able S1). Intrauterine growth rest able S1).Intrauterine POP1 lvic bones was notedin threecases, lvic bones mutations (24), and in CHH . Furthermore, mature rRNA levels were not volutionarily conserved Pop1 regions and examiningconserved Pop1regions and volutionarily ecursors of tRNA and the 5.8S rRNA in yeast, yeast, in rRNA 5.8S the and tRNA of ecursors substrate processing defe processing substrate rRNA precursor have been found in patient 1,the patient in found havebeen rRNAprecursor most importantly, there in was no 5.8S importantly, change most icated in holoenzymeicated in leading assembly, to murine cells by its ability itsability to processanRNA murine by cells bullet-shaped phalanges were identifiedin were bullet-shapedphalanges ta of ta the three previous individuals with the tions in ribosomal biogenesis (reviewed in in vitro in ion wasidentifiedinion allcases. have been not mainly successful, intermediate or in intermediate rate the v riction wasnoted cases, 3/5 in all arious vertebral bodies bodies vertebral arious patients (19). Thus, further and cts in both the RNase- inboththe cts

Joint laxity, Jointdescribed laxity, two had coxa vara coxa two had in vivo showed that showed that pleted in . This article is protected by copyright. All rights reserved. rights All bycopyright. protected is article This Accepted Article essential for cell growth an cell growth essential for another protein, involved in the MAGMAS, ofpre-proteins importing into mitochondriathe and it will thus observation, be interesting to ifthere investigate is a functional connection. Recently this explain can of POP1 functions known of the none However, impairment. mitochondrial homozygous homozygous tothe berelated could 1deficiency thecomplex Alternatively, gene. mutationanother in a reported in individuals with reported inindividuals compartment, and the mitochondrial activities ar activities mitochondrial andthe compartment, relevant (33-34).This would suggest that RNase-P/ too P tolow RNA, amounts be ofRNase 175molecules functionally than more not and molecules It been thathas shown mito mitochondria. human link may not extend to RNase-MRP that withinteracts the complex TERT and of RNAsubunit itisthe However, CHH. in mechanism pathogenic betheprimary may of siRNA that suggesting the others; alteredproduction differentiation among and hematopoietic development, gene target may which siRNAs, produce to Dicer by pr complexes RMRP-TERT RNA (40-41). telomerase ofcyclin RMRP, and degradation that RMRP- (37). replication transcriptstrand to complementary to thelight and RMRP/TERT complexes have also been localized, localized, been also have complexes RMRP/TERT Mitochondrial disease features, such as complex I deficiency, have not been previously have notbeen previously such disease Mitochondrial I deficiency, as complex features, Other functions of the RMRP complex may provide of an Other may insightmolecular the into of functions the pathology RMRP complex in vitro POP1- POP1 . Currently, there is discussion about whet about there isdiscussion Currently, . associated dysplasias. It is known that mR that It is known dysplasias. associated mutation ofand beone thefirstrepor POP1 d development, has been linked with a POP1 associated dysplasias. dysplasias. associated or RMRP B canregulate (38-39).cycle thecell mutations. This may be may andmutations. a coincidental findingThis due to generate RNA primers for the heavy-strand DNA heavy-strand the for generate RNAprimers e associated with other complexes (35-36). with (35-36). other e associated complexes chondria RNA chondria of aHeLa cell RMRP 6-15 contain MRP complexes reside exclusively in the nuclear inthe exclusively reside MRP complexes s implicated in skeletal developmental, hair hair developmental,s implicated skeletal in oduce double-stranded RNA that are processed that are processed RNA double-stranded oduce not the protein subunits of the complex, soproteinnot ofthecomplex, this the subunits ts of anassociation betweenSD and mainly to the nucleus of cells expressingcells mainly tothenucleusof her RMRP exertsher RMRP in the somefunction NA levels of cyclin B2 are regulated by by B2 regulated are ofcyclin NA levels severe spondylodysplastic dysplasia severe This article is protected by copyright. All rights reserved. rights All bycopyright. protected is article This Accepted Article 3. 2. 1. References SDs. these of mechanism molecular the todefine needed, POP1 is and complex theidentificationlead to 6. 5. 4. be considered in AD patients without inAD be considered severity, expanding the phenotypic spectrum of spectrum expanding the phenotypic severity, chondrogenic regulator (42). (42). regulator chondrogenic duri murine chondrocytes observed in been has mRNAsubstrate isa for cleavage activity of the RNase-MRP complex. Recently, viperin expression RNAi(31,42). Viperinby down areknocked POP1, subunits, including complex its RMRP and

Clayton DA. A nuclear function for RNase MRP. for MRP. RNase nuclear function A DA. Clayton mitochondrial DNA replication. EMBO J 1987: 6(2): 409-417. 409-417. J1987: DNAreplication. EMBO 6(2): mitochondrial Chang of DD, at DA. mouse site Clayton a priming A novel cleaves endoribonuclease Bonafé L, Cormier-Daire V, Hall C et al. NosologyV, HallCet Bonafé L,Cormier-Daire 2015 revision. Am J Med Genet A 2015: 167(12): 2869-2892. 2869-2892. 167(12): A2015: Genet AmMed J revision. 2015 between thelargest RNasebetween Pop1eukaryotic protein andP/MRP RNase P/MRP components. RNA FagerlundRD, Perederina A, Berezin I,Krasilni 1998: 12 (11): 1678-1690. 12 (11): 1998: Dev Genes MRP. RNase with overlap subunit extensive reveals complex P holoenzyme RNase nuclear of the characterization and Purification DR. WS, JR, Lee Y, Lane Engelke Chamberlain by the human RNase P and RNase MRP ribonucleoproteins. EMBO J 1996: 15(21): 5936-5948. 5936-5948. 15(21): J 1996: EMBO ribonucleoproteins. MRP RNase RNase Pand the human by shared subunit protein hPop1: anautoantigenic B. WJ, Séraphin Venrooij van PlukH, Z, Lygerou Increased viperin expression has also been observed in patients CHHand with in been observed viperinIncreased also expression has In summary, POP1 of additional Finally, further cases. research thefunctions into of RMRP the mutations have been identified in tw RMRP mutations. Our description of a milder phenotype may may phenotype a milder Our of description mutations. ng several developmental stages, and may actasa may and stages, developmental ng several POP1 kov AS. Footprinting of analysis interactions Proc Natl Acad Sci USA 1994:91:Proc NatlAcadSci 4615-4617. -associated AD type 2. ADtype -associated and classification of genetic skeletal disorders: disorders: skeletal of genetic and classification o individuals with SDs of different of with SDs o individuals POP1 mutations should in vivo when This article is protected by copyright. All rights reserved. rights All bycopyright. protected is article This Accepted Article 16. 15. 14. 13. 12. 11. 10. 9. 8. 7.

Ridanpää M, van Eenennaam H,PelinK, vanet Ridanpää M, al. Eenennaam pre-rRNA-processing system. Trends Biochem Sci 1995: 20(2): 78-82. 78-82. 20(2): Sci 1995: Biochem Trends system. pre-rRNA-processing eukaryotic and the MRP RNase of evolution the snoRNPs: Birthof the D. JP, Tollervey Morrissey 7084-7091. 7084-7091. 2013: 41(14): Res Acids Nucleic substrate. its with interactions in ribonucleaseMRP areinvolved I, Krasilni A,Berezin Esakova O, Perederina 1725-1747. 1725-1747. and RN Ofproteins AS. Krasilnikov O, Esakova 2005: 280(12): 11352-11360. 11352-11360. 280(12): 2005: J Biol Chem component. protein unique new MRPreveals a RNase cerevisiae Saccharomyces of and purification Characterization ME. Schmitt L, WierzbickiZhou Salinas K, S, 11(21): 2926-2937. 11(21): 2926-2937. GenesDev1997: cerevisiae. rRNA inSaccharomyces precursor 35S and precursor tRNA of s protein anessential S.Rpp1, Stolc V,Altman ribonucleoproteins in vivo. EMBO J 1997: 16(2): 417-429. 417-429. 16(2): J1997: EMBO vivo. in ribonucleoproteins D. Pop3p Dichtlfo is essential B, Tollervey 3(4): 382-391. 3(4): 382-391. RNase P.RNA1997: and MRP RNase by shared L. Anovelprotein Lindahl S, ZengelJM, Chu rRNA in Saccharomyces cerevisiae. MolCell Biol 1993:13(12): 7935-41. Schmitt ME, Clayton DA. Nuclear RNase MRP is re MRP RNase SchmittNuclear ME,Clayton DA. 1423-1433. 1423-1433. RNase MRPand RNase the to common component protein a encodesgene POP1 The D. SéraphinB,Tollervey E, Petfalski Mitchell P, Z, Lygerou RNA 2015: 21(9): 1591-1605. r the activity of the RNase MRP and RNase P RNase and MRP theRNase of activity the r kov ConservedAS. regionsof ribonucleoprotein ubunit of nuclear RNase P required forprocessing required P nuclear RNase of ubunit Mutations in the RNA component ofRNase MRP A: the RNase P/MRP family. RNA A: the 2010: 16(9): family. P/MRP RNase P ribonucleoproteins. Ge P ribonucleoproteins. quired for correct processing of pre-5.8S nes Dev 1994: 8(12):1994: Dev nes This article is protected by copyright. All rights reserved. rights All bycopyright. protected is article This Accepted Article 27. 25. 24. 23. 22. 26. 21. 17. 20. 19. 18.

Xiao S, Hsieh J, Nugent RL, Coughlin DJ, Fier Coughlin RL, XiaoS, HsiehJ,Nugent extensive protein networks responsive to responsive D protein extensivenetworks Elalaoui SC. Laarabi FZ, Mansouri M, Mrani NA, Nishimura G, Sefiani A. Further evidence of of evidence G, Further A. Sefiani M,Mrani Nishimura NA, Mansouri FZ, Laarabi SC. Elalaoui POP1 mutations as the cause of a novel skeletal dysplasia. PLoS Genet 2011: 7(3): e1002027. e1002027. 7(3): 2011: PLoS Genet dysplasia. skeletal novel a causeof asthe mutations POP1 identifies quartet afamily in re-sequencing Whole-exome et al. M, Donskoi A, Zankl EA, Glazov dysplasia with dysplasia extreme dwarfism. 262-265. Genet J 2001:38: Med spondylometaepiphyseal a dysplasia, Anauxetic J. Spranger J, Kunze E, Rupprecht D, Horn dysplasia in sibs with extreme short stature. Am J Med Genet 1996: 63(1): 80-83. 1996: Genet JMed extreme Am with stature. short sibs in dysplasia AnMenger spondylo-meta-epiphyseal S, Becker K, H, Mundlos J, Zabel unknown B. Spranger Matsuoka S, Ballif BA, SmorgoMatsuoka S,BallifBA, 769-778. Acta disorders. 1842(6): Biophys 2014: skeletal Biochim Trainor Merrill PA, AE. of Ribosome biogenesis pathogenesis and development the in skeletal Boer M,Petersde Ridanpaa Kuijpers TW, cause a pleiotropic human disease, cartilage-hair hypoplasia. Cell 2001: 104(2): 195-203. 195-203. Cell 2001: hypoplasia. 104(2): disease, cartilage-hair human cause apleiotropic POP1 mutations Am as POP1 mutations cause the dysplasia. Jof anauxetic Med Genet 2016: 170 (9):2462-2465., 1992: 22(6): 434-439. Radiol Pediatr of82patients. study MarttinenMäkitie O, E,KaitilaI.Skeletal grow Am J Hum Genet 2015: 77(5): 795-806. 77(5): 795-806. 2015: Genet Am JHum endoribonuclease RNA-processing identify stature Thielincapaal. Severely CT,Horn D,Zabel et B, pathogenicity of RMRP mutations. PLoS Genet 2005: 1(4): e47. 1(4):e47. 2005: Genet PLoS mutations. of RMRP pathogenicity Bonafé comparison Unger L,DermitzakisEvolutionary etET, al. S, provides evidence for anaemia caused by novel anaemia caused by mutations the in (sic) 40:gene. JGenet 2003: RMPR 761-766. Med aplastic onset late and deficiency, immune bowing, combined with dwarfism Short-limbed RCM. rzewska A, et al.ATMandAT rzewska A, NA damage. Science 2007: 316 (5828): 1160-1166. 2007: 1160-1166. 316 (5828): Science NA damage. I, VossenJMJJ, Pals ke CA, Engelke DR. Functional characterization of characterization DR.Functional Engelke CA, ke th in cartilage-hair hypoplasia. A radiological citating mutationscitating in patients with extreme short RMRP as an essential cell growthregulator. R substrate analysis reveals analysis reveals R substrate ST, Kaitila I,Hennekam, This article is protected by copyright. All rights reserved. rights All bycopyright. protected is article This Accepted Article 30. 31. 29. 32. 28. 33. 34. 35. 36. 37.

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Cai T, Reilly Reilly T, TR, Mut CerioM,SchmittCai ME. plasmid segregation. Mol Cell Biol 1999: 19 (11): 7857-7869. 7857-7869. 19 (11): 1999: CellBiol Mol segregation. plasmid in endoribonuclease this ribonucleoprotein arolefor reveals RNase MRP, the of yeast component Gill T,Cai T, WierzbickiAulds J, S, Sc 945-953. 945-953. Mol Cell Biol 2004: 24(3): degradation. ofmRNA method progression: novel cell cycle promote TERT and the RMRP RNA. Nature 2009: 461 (7261): 230-235. 230-235. 461 (7261): Nature2009: RMRPRNA. and the TERT Maida Y, Yasukawa M, Furuuchi M, et al. al. et M, Furuuchi M, Yasukawa Y, Maida Jarrous N, Gopalan V. Archaeal/eukaryal RNase RNase V. Archaeal/eukaryal N, Gopalan Jarrous Nucleic Acids Res 2010: 38 (22): 7885-7894. 7885-7894. 38 (22): 2010: Res Acids Nucleic Osteoarthritis and Cartilage 2015: 23(2): A148–A149. A148–A149. 2015: 23(2): and Cartilage Osteoarthritis al. F, et Eckmann CaronMM, MM, Steinbusch hmitt ME. RNase MRP cleaves the CLB2 mRNA CLB2 the to MRP cleaves hmitt ME.RNase An RNA-dependent RNA polymerase formed by by formed polymerase RNA An RNA-dependent agenesis of SNM1, which encodes a protein Viperin; A novel chondrogenic regulator. Viperin; A regulator. novel chondrogenic P: subunits, functions and RNA diversification. diversification. andRNA subunits, functions P: This article is protected by copyright. All rights reserved. rights All bycopyright. protected is article This Accepted Article phalanges 5 phalanges to CHH,bullet-shaped in similar middle that phalanges, ivory phalanges, epiphysis proximal phalanges, middle and proximal all and metacarpals proband proband 2 atage 2. and 1 ofprobands 1: Radiological analysis Figure Figure legends Thoracolumbar scoliosis centered at T12. Relatively scoliosis broad at ribs. Relatively centered Thoracolumbar T12. of with posteriorscalloping vertebral bodies femoral epiphyses with premature fusion of the growth plates, P-Q. Normal segmenta metaphyses with margins.irregular distalmetaphyses. tibial Cupped Chevron deformity of distal varadefo O. Bilateralcoxa metaphyses, femoral necks, coxa vara with vertical orientation of orientation vertical vara with necks, coxa dev = N. iliac bone 4m, age Narrow wings = birth), boneof agewithnoossified delayed the epiphyses hand and (chronological wrist age= 2 male yrs, std proximalwith irregular metaphysis. Bowingof th of metaphys and Bowedsclerosis femur. Irregularity metaphyses,vertebral K.Tall bodies posterior and of and sclerosis Irregularity J. metaphyses, femoral of theproximalsclerosis humeralmetaphyses,linear I. years, female std dev = 8m, bone age = 2yrs). Cupped, sclerotic, irregular metaphyses of 2 metaphyses sclerotic,irregular Cupped, = 2yrs). bone age = 8m, std dev female years, F.Signi bodies, vertebral lowerlumbar scalloping vertebral end plates,D. Narrowpedicles lower th fingers andmild proximal pointing of of2 base

2 (K-N) (O-Q).7 and y A:Macrocephaly, B. lower thoracic and lumbar vertebral lower bodies. and lumbar thoracic femoral physes femoral thoracic and upper lumbar spine, E.Posterior spine, lumbar and upper thoracic rmity. Short, broad and bowed femora. Flared bowedfemora. and broad Short, rmity. e ulna. Bullet-shapedmiddle phalanges.Severely ficantly delayed bone age (chronological age = 4.6 =4.6 age age (chronological delayed bone ficantly scalloping of lower lumbar vertebral bodies, L. bodies, vertebral lumbar lower of scalloping distal femoral, and proximal tibial distal tibial cone-shaped epiphyses cone-shaped epiphyses with mild of theflaring acetabula. Short femoral es of femur and tibia, M. Short, broad humerus broadhumerus Short, and tibia,M. ofes femur Coxa valga. Irregularity and of Coxa sclerosis Irregularity distalvalga. Radiographs of 1Radiographs proband at (A-J) and 4.6y and medial fragmentation of the proximal proximal of the fragmentation medial and nd to 5 Narrow pedicles, C. of Mild irregularity th metacarpals, G. & H. Patchy Patchy G. &H. metacarpals, of proximal and middle of proximal andmiddle tion. Tall nd to4 th

This article is protected by copyright. All rights reserved. rights All bycopyright. protected is article This Accepted Article showing marked growth delay, more severely in proband 2 compared to proband 1. proband to 2compared inproband more severely delay, marked growth showing kyphosis andprominent curvesof The growth deformity, 1 heels. proband (I) and proband 2 (J) locate abnormally seemedthumbs his and fingers His hands were smaller when compared to his limbs and he had deep palmar creases. He hadpointed 2 Proband at age 5 (B, 7 D) and of years age (F,H) wi withstubbyvalgus fingers, cubitus brachydactyly macrocand withrelative individual E G):Female Figure 2: Clinical photographsClinical and growth 1 curve Proband at 4.6 of probands. of age years the (A, C, d. He also had prominent thorax, hipflexion thorax, prominent d. Healsohad ephaly, short neck, broad chest, hyperlordosis, hyperlordosis, broadchest, short neck, ephaly, : hehas and sparse appare th difficulties at extension, and prominent heels. ntly hypopigmentedntly hair. This article is protected by copyright. All rights reserved. rights All bycopyright. protected is article This Accepted Article

This article is protected by copyright. All rights reserved. rights All bycopyright. protected is article This Accepted Article Figure 3: Genetic characterization of the of the characterization 3: Genetic Figure Chromatograms of the the of Chromatograms D: Expression levelsof for homozygous 2was Proband c.2607delC. father, his from deletion base compound heterozygote for two two for heterozygote compound been deposited in PhenomeCentral. box: ref25). Orange (p.(Pro582Ser);(Pro582Ser), ref24),arrows red arrows(p.(Arg513*);(Gly583Glu), blue mutations, described previously those mutations of the showing thelocalization structure threeunrelated age andsex-matched controls (C1, POP1 RMRP mutations detected in probands 1 (A,B) and 2 (C). Proband 1 wasa Proband and 2(C). 1(A,B) probands detectedin mutations and pre-5.8S rRNA in proband 1 (P), probandpre-5.8S rRNAin and POP1 mutations: c.1744C>T, inherited from the mother, and a single a single and mother, the from inherited c.1744C>T, mutations: POP1 POP1 domain; POP1 blue box: Data has POPLD domain. mutations detected in probands 1 and 2. probands 1and in detected mutations C2, C3). E: Schematic diagram of POP1 protein protein POP1 of diagram Schematic E: C3). C2, identified 1identified in probands 2 and (green and arrows) his mother (M) and father (F) and and (F) father and (M) mother his the c.1531G>T mutation. mutation. c.1531G>T the A,B,C: