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European Journal of -18-0129 different origins.TheCaSR(OMIM +601199)isknownto calciummetabolismdisorderswith commonly observed conditions thatneedtobe distinguished fromthemore disorders arearareheterogeneous groupofgenetic Inherited humancalcium-sensing receptor(CaSR) Introduction from otherentities.OurgeneticanalysisconfirmedCaSR disordersin82patientsthestudycohort. or hypocalcemiawithhypercalciuria.Molecularstudies are importantforconfirmingthediagnosisanddistinguishingit Conclusions: p.Pro935_Gln945dup). p.Glu133Val, p.Gly222Glu,p.Phe351Ile,p.Cys542Tyr,p.Cys546Gly,p.Cys677Tyr,p.Ile816Val,p.Ala887Asp,p.Glu934*, mutations, ofwhich,14havenotbeenpreviouslyreported(p.Ala26Ser,p.Cys60Arg,p.Lys119Ile,p.Leu123Met, (median :10.6 mg/dL;medianPTH:65 pg/mL)withnootheralterationsin common polymorphismspreviouslyassociatedwithhighbloodionizedcalciumlevelswerefoundin27patients patients hadamedianserumcalciumlevelof7.4 mg/dLandhypoparathyroidism(median:PTH13 pg/mL).Further, mL). Ontheotherhand,presumedCaSR-activatingmutationsweredetectedin17patientsfromeightfamilies. These hypercalcemia (median:11.3 mg/dL)butnormalorabnormallyhighparathyroidhormone(PTH)levels52 pg/ Results: sequencing. Methods: from 82familieswithsuspectedalterationsinthe clinical, biochemicalandgeneticcharacterizationofsequenceanomaliesinthisreceptoracohort130individuals receptor (CaSR)isknowntoplayacentralroleintheregulationofextracellularcalciumhomeostasis.Weperformed Objective: Abstract *(A García-CastañoandLMadariagacontributedequallytothiswork) Barcelona, Spain UPV/EHU, Barakaldo,Spain,and and Sonia Gaztambide Alejandro García-Castaño A clinicalgeneticstudy human calcium-sensingreceptordisorders: Novel mutationsassociatedwithinherited 1 Biocruces BizkaiaHealthResearchInstitute,CIBERDEM,CIBERER,Barakaldo,Spain, https://doi.org/ https://eje.bioscientifica.com Clinical Study Renal TubeGroup PresumedCaSR-inactivatingmutationswerefoundin65patientsfrom26families.Thesehad The Moleculardiagnosisisausefuldiagnostictoolincalciummetabolismdisorders.Thecalcium-sensing 10.1530/EJE Patientswith CASR genewasscreenedformutationsbypolymerasechainreactionfolloweddirectSanger -18-0129 1 , 2 and 3 Hospital UniversitarioMaterno-InfantilValld’Hebron, AutonomousUniversityof CASR Luis Castaño 1 1 , *, Leire Madariaga © 2019EuropeanSociety ofEndocrinology mutationsmaynotfittheclassicclinicalpicturesofhypercalcemiawithhypocalciuria L Madariagaandothers A García-Castaño, 1 , 2 onbehalfofSpanishEndocrinologyGroup Printed inGreatBritain 1 , 2 CASR , *, Gustavo Pérez de Nanclares gene,oneofthelargestseriesdescribed. (PTH) ( of CaSRinhibitsthesecretion ofparathyroidhormone extracellular Ca ( play acentralroleintheregulation ofextracellularcalcium receptor gene Novel mutationsinthecalcium Published byBioscientifica Ltd. 2 Hospital UniversitarioCruces, 3 ), whereastheeffectisreversed underlow[Ca 1 , 2 +2 ); specifically, inthe presence ofahigh concentration[Ca CASR 1 , 2 , Gema Ariceta Downloaded fromBioscientifica.com at09/27/202104:57:07PM . Overall,wefound30different

(2019) Endocrinology European Journalof osakidetza.eus CASTANOGONZALEZ@ LUISANTONIO. Email to LCastaño should be addressed Correspondence 180 180 +2 3 ], theactivation :1 , , 59–70

59

–70 via freeaccess +2 ] European Journal of Endocrinology https://eje.bioscientifica.com families. Atotalof82index caseswerereferredtoour of 130 patients (79 females) from 82 different unrelated The Population Minors over12 yearsalsoprovidedtheirassent. written informedconsentbeforeinclusioninthestudy. the caseofminors,theirrelativesorguardiansprovided Clinical Research ofEuskadi(CEIC-E).Patientsor, in The studywasapprovedbytheEthicsCommitteefor Ethics statement Patients andmethods 397 mutationsinthe Human GeneMutationDatabase( knowledge of these rare entities. So far, according to the calciumand/orPTHlevels,inordertowidenour among a group of individuals presenting with abnormal characterization ofsequenceanomaliesinthe study wastoperformclinical,biochemicalandgenetic awareness ofthisgroupdisorders.Theaim of geneticdiagnosisunderlinestheneedforgreater published todate;however, theincreasingavailability various signalingpathways( cellular responsesthroughtheactivationorinhibitionof functions, someofthemnotwellknown,thatinduce in manyothertissuesandparticipatesnumerous all nephronsegments( the parathyroidglandandkidney, whereitisfoundin receptor CaSR. Thisgene is mostabundantly expressed in 3q13.33-q21.1 ( elevated riskofnephrolithiasis( calciumlevelsandan polymorphisms tohighurinary generalized epilepsy(OMIM#612899),andsomegene anomalies havebeenlinkedtosusceptibilityidiopathic (OMIM#601198)( described asautosomaldominanthypocalcemiawith #601198) andcanalsomimicasalt-wastingnephropathy in hypercalcemia. Conversely, gain-of-functionmutations I (OMIM#145980)andsomecasesofhypercalciuric (OMIM #239200),hypocalciurichypercalcemia type the conditions ( Clinical Study CASR Few seriesofpatientswith The CASR CASR causeautosomal dominant (OMIM CASR genewasscreenedformutations inacohort gene cause neonatal 3 , genewasmappedtochromosome 4 9 , ) andencodestheGprotein-coupled 5 , CASR 6 10 ). Loss-of-functionmutationsin ). Inaddition, genehavebeendescribed. 11 CASR ). 8 L Madariagaandothers A García-Castaño, ). mutationshavebeen www.hgmd.cf.ac.uk 7 ). Moreover, CaSR CASR isexpressed CASR gene ),

network. In all cases, the genetic diagnoses ware made network. Inallcases,thegeneticdiagnoseswaremade renaltube.com a research tubulopathies( networkfor primary when thereferringhospitalwasamemberofRenalTube, or nephrologydepartments,butinsomecases( were sentforgenetictestingdirectlybytheendocrinology them inSpain.Mostpatientbiologicalsamples( nephrologists workingin38differenthospitals,mostof were madebyadultandpediatricendocrinologistsor referred based on family screening. Clinical diagnoses hypocalcemia inthefamily. Theother48patientswere ofhypercalcemiahave beenexcluded)andahistory or maternal diabetes,prematurityorneonatalasphyxia neonatal conditionsassociatedwithhypercalcemia, thyroidism withhypocalcemia(surgicalcausesortransient recurrence ofhypercalcemia orhypopara­ aftersurgery hypocalcemia; hyperparathyroidism with calciuric the presence of hypocalciuric hypercalcemia or hyper­ hospital for genetic testing based on the following criteria: acetate)andsamples weredenaturedandloaded sequencing. exons, thesameprimersasin thePCRwereusedfordirect exon 7 (5primers) for the sequencing ofexon3(2primers),4(4primers)and Technologies). We usedinternal primers fordirect (BigDye Terminator v3.1CycleSequencing Kit,Life in bothdirectionswithfluorescentdideoxynucleotides according tothemanufacturer’s specifications. agarose gelsusingQIAquickGelExtractionKits(QIAGEN), (USB Cleveland,OH,USA)orbyextractingthemfrom request). PCRproductswerethenpurifiedwithExoSAP-IT (primer sequencesandPCRconditionsareavailableon performed inatotalvolumeof50 their splice sitesfor the Sanger sequencingofbothstrands. were screenedformutationsbyPCRfolloweddirect gene ENSG00000036828;transcript,ENST00000490131) intronic sequencesofthe Mini Kit,QIAGEN).Theexonregionsandflanking to themanufacturer’s instructions(QIAampDNABlood peripheral bloodleukocyteswereperformedaccording Extraction andpurificationofgenomicDNAfrom DNA analysis Health Research Institute, Barakaldo,Spain. atBiocrucesBizkaia in theMolecularGeneticLaboratory receptor gene Novel mutationsinthecalcium Excess dyeterminatorswere removed(ethanol-EDTA- Purified amplifiedproductsweredirectlysequenced We usedprimerstoamplifyexons2–7togetherwith ), samples were received through this ), sampleswerereceivedthroughthis CASR Downloaded fromBioscientifica.com at09/27/202104:57:07PM CASR CASR gene. All PCR analyses were gene(Ensemblidentifiers: gene. In the remaining µL, withDMSO10% 180 :1 n n 114) =114) www. 16), =16), 60 via freeaccess

European Journal of Endocrinology related highserumalkaline phosphataselevelsinmost normal range (median: 2.3 while serummagnesium levels remainedwithinthe lower limitofthenormal ranges (median:3.2 mL). Moreover, serumphosphatelevelswere atthe or abnormally high intact PTH levels (median: 52 serum calcium levels (median: 11.3 featuresincluded elevated years.Laboratory was 24 mutations, themedianageatdiagnosisofdisease hyperparathyroidism( diagnosed withprimary whereas theindexcaseinother6familieswasinitially diagnosis washypocalciurichypercalcemia typeI, finding of asymptomatic hypercalcemia and the clinical the indexcasein20familiespresentedwithincidental 26 families(65patients)( hypocalcemic hypocalciuria(1%). normocalciuria (7%);andfinally, oneindexcasehad with hypercalciuria; six indexcaseshadhypocalcemic hypocalcemia andlowornormalserumPTHlevels hand, theindexcasesof11families(13%)exhibited had hypercalcemic hypercalciuria (4%).Ontheother hypercalcemic normocalciuria (4%)andthreeindexcases with hyperparathyroidism (18%); three index caseshad levels andmultiglandhyperplasia,werediagnosed of PTHbyparathyroidglands,highserumcalcium 15 indexcases,characterizedbyexcessivesecretion normal orelevatedserumPTHlevelsandhypocalciuria; (53%) presentedwithhypercalcemia associatedwith In ourcohortof82families,theindexcases43families Results IBM SPSS,StatisticsforWindows, version23. using linearregression.Theanalysiswasperformedwith relationships between calcium, PTH and age were assessed In patientswithalterationsinthe Statistical analysis Variation Societyguidelines( mutations werenamedaccording to theHumanGenome SNPs&GO ( genetics.bwh.harvard.edu sting ( using pathogenicity prediction software: Mutation t@ The potentialeffectsofthemutationswereinvestigated onto anABI3130xlGeneticAnalyzer(LifeTechnologies). Clinical Study In thegroupwithpresumedCASR-inactivating We found presumed CASR-inactivating mutations in www.mutationtaster.org www.snps-and-go.biocomp.unibo.it ), SIFT ( Table 1 mg/dL). We age- also observed www.hgvs.org ), PolyPhen-2( L Madariagaandothers A García-Castaño, ). Amongthesefamilies, www.sift.jcvi.org mg/dL) but normal CASR ). gene,the Fig. 1 ). DNA mg/dL), ) and www. ). pg/ diagnosed withhyperparathyroidism(CA02,CA07, calcium excretion (CA73) orwere initially urinary calciumexcretion (CA57) orelevated normal urinary CASR-inactivating mutationshadhypercalcemia but data supplementary 109 also demonstratedlowrenalcalciumexcretion(median: finding wasconfirmedby24-hurinecollection,which lowdespitetheirhighserumcalciumlevels,andthis very calcium-to-creatinine ratio(UCa/Cr0.01 to CASRdisorders.Specifically, theirmedianurinary study patientsexhibitedhypocalciuria,whichpointed hyperparathyroidism,butcharacteristicallythe primary deficiency. Taken together, theseclinicalfindingssuggested excluding renalfailureornutritional25-OHvitaminD 25-OH vitaminDlevelswerewithinthereferencerange, phosphatase: 37–114 patients (median:259 and (2.6 hyperparathyroidism with high levels of PTH (518 lethargy. Moreover, analysisrevealedsevere laboratory to thrive, hypotonia, constipation, dehydration and characteristic ofneonatal hyperparathyroidism: failure while probandCA42presentedwithsymptomsthatare hyperparathyroidism thatnormalizedspontaneously, Proband CA34hadatransientepisodeofprimary cases, maternalhypoparathyroidismwasexcluded. of age, respectively. Inthose 23and20 days 6 months, with symptomsofneonatalhyperparathyroidism, at families (CA34,CA42andCA02),theindexcasepresented hyperplasia inbothcases.Moreover, inthreeadditional , andparathyroidglands showed Both oftheaforementionedpatientsweretreatedwith a but onlytwobeingfoundtocarry having hyperparathyroidismandvitaminDinsufficiency, pedigree ofthisfamilywasunusual(allmembersassessed high PTHlevel(154 D insufficiency, serumcalciumof10.6 the probandoffamilyME133presentedwithvitamin and anabnormallyhighPTHlevel(75 osteoporosis, togetherwithhypercalcemia (11.5 ofage,hadchondrocalcinosisand diagnosed at64 years cases, PTHlevelswerealsohigh.Firstly, probandCA19, in his51-year-oldsisterweresimilar. Further, insome calciumexcretion(160 urinary an abnormally high PTH level(54 hyperparathyroidismdueto yearswithprimary age 56 the indexcaseoffamilyCA07wasinitiallydiagnosedat CA19, CA34, CA42 and ME133) ( receptor gene Novel mutationsinthecalcium mg/24 Notably, someindividualsofthecohortwithconfirmed )(upeetr al 1,seesectionon Table h) (Supplementary givenattheendofthisarticle). pg/mL) andasshownin mg/mL), togetherwithlow levels of U/L). Further, serumcreatinineand U/L; referencerangeforalkaline Downloaded fromBioscientifica.com at09/27/202104:57:07PM https://eje.bioscientifica.com mg/24 al 1 Table pg/mL) but anormal 180 pg/mL). Secondly, h), andfindings :1 CASR ). For instance, mg/dL anda mg/mg) was mutation). Fig. 2 pg/mL) mg/dL) , the 61 via freeaccess European Journal of Endocrinology https://eje.bioscientifica.com

Table 1 Clinical characteristics, laboratory findings and molecular results for presumed CASR-inactivating mutations in the studied cohort. Clinical Study

Family Number CASR mutation† Polymor­ Clinical phenotype of the Gender * Age at Total serum Serum Serum PTH UCa/Cr U.Ca of patients phisms in the index case diagnosis calcium creatinine (pg/mL)* (mg/mg)* (mg/24 h)* index case (years)* (mg/dL)* (mg/dL)* (mg/dL)* Six families with hyperparathyroidism CA02 1 p.Arg185Gln; c.554G>A – Neonatal hyperparathyroidism M 0 13.7 4.8 0.4 58 0.001 NA CA34 2 p.Arg185Gln; c.554G>A p.Ala986Ser Neonatal hyperparathyroidism F 0.5 NA NA NA NA NA NA CA07 3 p.Phe351Ile; c.1051T>A+ – Parathyroid hyperplasia F 56 11.3 3.5 0.9 54 NA 160 CA42 3 p.Arg465Leufs*9; – Neonatal M 0.06 23.2 1.3 0.16 518 0.5 NA c.1392_1404del13‡ hyperparathyroidism, hypercalcemia, severe hypotonia, failure to thrive, dehydration, lethargy,

constipation L Madariagaandothers A García-Castaño, CA19 8 p.Phe789del; p.Ala986Ser Hyperparathyroidism, F 64 11.5 3.3 0.85 75 NA 162 c.2367_2369delCTT parathyroid hyperplasia ME133 2 p.Pro935_Gln945dup; p.Gln1011Glu Nephrolithiasis and M 52 10.6 1.8 0.99 154 0.21 NA c.2803_2835dup33+ hyperparathyroidism, hyperplasia of both left parathyroids. Insufficiency of , normocalcemia Eighteen families with hypercalcemic hypocalciuria CA58 2 p.Arg25*; c.73C>T p.Arg990Gly Hypocalciuric hypercalcemia F 14 NA NA NA NA NA NA CA03 2 p.Pro55Leu; c.164C>T NA Hypocalciuric hypercalcemia F 28 10.7 2.7 NA 50 0.004 NA CA76 3 p.Pro55Leu; c.164C>T – Hypocalciuric hypercalcemia M 15 11.3 3.2 0.75 NA 0.03 63 + CA55 2 p.Cys60Arg; c.178T>C p.Ala986Ser Hypocalciuric hypercalcemia M 5 11.5 4.2 NA 83 NA NA receptor gene Novel mutationsinthecalcium CA04 2 p.Arg220Trp; c.658C>T NA Hypocalciuric hypercalcemia F 40 11.6 2.4 NA 34 0.009 NA CA66 1 p.Arg220Trp; c.658C>T p.Ala986Ser Hypocalciuric hypercalcemia M 12 12.2 3.2 0.5 68 0.001 45

CA15 2 p.Gly222Glu; c.665G>A+ p.Ala986Ser Hypocalciuric hypercalcemia M 42 10.7 2.8 0.76 42 0.04 88 CA36 3 p.Arg465Gln; c.1394G>A p.Arg990Gly Hypocalciuric hypercalcemia F NA NA NA NA NA NA NA CA27 3 p.Cys542Tyr; c.1625G>A+ p.Ala986Ser Hypocalciuric hypercalcemia M 72 12 NA NA NA 0.01 NA CA64 2 p.Cys546Gly; c.1636T>G+ p.Ala986Ser Hypocalciuric hypercalcemia F 0.5 12.1 5 0.2 64 0.002 0.41 CA56 4 p.Arg551Gly; c.1651A>G – Hypocalciuric hypercalcemia M 13 11.4 4 0.64 38 0.01 NA CA69 1 p.Ile555Thr; c.1664T>C – Hypocalciuric hypercalcemia F 8 12.8 3.4 0.58 30 0.01 NA CA70 1 p.Cys677Tyr; c.2030G>A+ p.Ala986Ser/ Hypocalciuric hypercalcemia M 35 11.1 NA NA 54 NA NA Downloaded fromBioscientifica.com at09/27/202104:57:07PM p.Arg990Gly CA25 5 p.Val697Met; c.2089G>A p.Ala986Ser Hypocalciuric hypercalcemia F 7 12.3 4.2 0.56 19 0.001 16.5 CA33 2 p.Val697Met; c.2089G>A p.Ala986Ser Hypocalciuric hypercalcemia M 5.8 12.8 3.7 0.8 59 0.005 57 CA89 2 p.Ile816Val; c.2446A>G+ – Hypocalciuric hypercalcemia F 12 11.4 2.8 0.56 113 0.082 134 CA45 2 p.Ile816Thr; c.2447T>C p.Ala986Ser Hypocalciuric hypercalcemia F 25 11.7 3.8 NA 93 NA NA

CA61 4 p.Ala887Asp; c.2660C>A+ p.Ala986Ser Hypocalciuric hypercalcemia M 14.7 11.1 4.5 0.78 49 0.01 NA 180 One family with hypercalcemic normocalciuria :1 CA57 1 p.Ile816Val; c.2446A>G+ – Normocalciuric hypercalcemia M 15 11.2 3.2 0.5 52 0.15 134.7 One family with hypercalcemic hypercalciuria CA73 2 p.Ala26Ser; c.76G>T+ – Hypercalciuric hypercalcemia M 21 11.4 3.7 0.88 28 NA NA

Reference ranges: total serum calcium (8.5–10.2 mg/dL); serum phosphate (3.4–4.5 mg/dL); serum creatinine (0.7–1.3 mg/dL); PTH (8–51 pg/mL); UCa/Cr (<0.20 mg/mg); U.Ca (100–300 mg/24 h). + † †† ‡

These mutations (also marked in bold) have not been reported to date; Numbering is according to DNA sequence (Ensembl: ENST00000490131); Homozygous polymorphism; Homozygous 62 via freeaccess mutation in the index case; *Laboratory findings of index case. NA, not available; PTH, ; U, urinary. European Journal of Endocrinology in these27patients.Finally, nomutationsweredetected other alterationsinthe and risk ofhypercalcemia or colorectal adenoma ( linked toabnormallyhigh blood ionizedcalciumlevel, polymorphisms thathavebeen previouslyreportedtobe addition, in27patients(27families),wefoundcommon unrelated familiesthatexplainedpatientphenotype.In gene variantsin82outof130patients(63%),from34 inthisgroupofpatients. PTH andagewereobserved No significant associationsbetweentotalserumcalcium, showed intracranialcalcificationsandhypothyroidism. nephrolithiasis. Finally, theindexcaseof familyCA01 adrenal hyperplasia. She also had diabetes mellitus and diagnosed with21-hydroxylasedeficiencyandcongenital (a 41-year-oldwoman).Thelatterpatienthadearlierbeen hypoplasia, in the index case of family CA23 mammary colic, depression,obesity, acanthosis,hirsutismand case offamilyCA12(an11-year-oldgirl),andrenal findings weretetanyandnephrocalcinosis,intheindex UCa/Cr 0.16 these patientspresentedmarkedhypercalciuria (median borderline low(median1.6 while themeanserummagnesiumlevelwaslowor serum phosphate level was high (median 6.6 (median PTH13 calcium levelof7.4 finding,withamedianserum was anincidentallaboratory ofage.Inmostcases,hypocalcemia a medianof12 years ( clinical diagnosis of autosomal dominant hypocalcemia mutations in8outof18families(17patients)witha serum calciumandPTH( between the age at diagnosis and PTH ( ( serum calciumandageatdiagnosis( mutations revealedsignificantassociationsbetweentotal (proband CA25). dizziness andbonelesions(probandCA61)epilepsy in onecaseeach:asthenia(indexoffamilyCA33), in two cases (index cases of families CA03 and CA04), and inthissubgroupwereosteopenia manifestations observed nephrolithiasis (index case of family ME133). Other CaSR-inactivating mutations,wefoundonecasewith (549 U/L). levels two-tofour-foldhigherthanthenormallimit serum phosphate (1.3 Table 2 Fig. 3 Clinical Study In summary, presumedpathogenic weobserved On theotherhand,wefoundpresumedCaSR-activating Statistical analysisofpatientswithCaSR-inactivating In addition,withinthesubgroupofpatientswith ). In contrast, there wasno significant association ). In these patients, the disease was diagnosed at gm)(upeetr al 1).Other Table mg/mg) (Supplementary pg/mL) ( mg/mL) and alkaline phosphatase mg/dL andhypoparathyroidism P CASR

> i. 1 Fig. 0.05). mg/dL). Characteristically, genehavebeendetected L Madariagaandothers A García-Castaño, ). Moreover, themean P P

>

<

0.05) and total 0.01, mg/dL), r 12 =0.38) CASR ). No

( We moreinactivating( observed different mutations,9ofthemwerenovelmutations. for themutation. index caseoffamilyCA42wasfoundtobehomozygous pathogenic and 1duplicationof33nucleotides.Ofthese,14the frameshift mutation,1smalldeletionof3nucleotides 1 grossdeletionof13nucleotidesresultingina mutations in 34 families: 25 missense, 2 nonsense, 24 was found(median6.7 disease of35 years), whereasinfivepatients,hypocalcemia mL) in16patients(withamedianageatdiagnosisofthe 11.4 features includedelevatedserumcalciumlevels(median in theother21patients(21families).Theirlaboratory patients withinactivating mutations ofthe we consider‘likelydisease-causing mutations’. patients, especiallyinthecase ofnovelmutationswhich these changesareresponsible forthephenotypesof analysis; therefore,weareunabletodeterminewhether one ofthelimitsourstudywaslackfunctional and theintracellularcarboxyl-terminaltail.Unfortunately, extracellular domain,theseventransmembranedomains in thethreemajorregionsofreceptor, namely, the previous studiesofthe p.Gln934* andp.Pro935_Gln945dup).Inaccordancewith p.Cys546Gly, p.Cys677Tyr, p.Ile816Val, p.Ala887Asp, p.Glu133Val, p.Gly222Glu, p.Phe351Ile, p.Cys542Tyr, (p.Ala26Ser, p.Cys60Arg, p.Lys119Ile, p.Leu123Met, revealed 14mutations(47%)notpreviouslyreported: detected and,importantly, thecompletegeneticstudy in ourcohort,30different 17 patients from 8 families activating mutations. Overall, patients from26familieshadinactivatingmutationsand suspected alterations in the In thisstudy, weanalyzedacohortof130patientswith Discussion terminal tail). three mutationsintheintracellulardomain(carboxyl- mutations werefoundinthetransmembranedomainand the cysteine-richregionand1inpeptidelinker. Eight mutations, 13wereinthevenusflytrap-likedomain,5 receptor gene Novel mutationsinthecalcium 5 ) mutationsinthispartofthereceptor. Ofthese19 pg/mL (Supplementary Table 1).pg/mL (Supplementary In theextracellulardomain,wefound19(63%) 30 Regarding moleculardiagnosis,weobserved Clinical andbiochemicalfindings inthegroupof mg/dL) andhighintactPTHlevels(median92 CASR genevariantswerenovel.Onlythe mg/dL) withamedianPTHlevelof CASR Downloaded fromBioscientifica.com at09/27/202104:57:07PM CASR CASR gene,wefoundmutations https://eje.bioscientifica.com gene. We found that 65 13 genemutationswere , 180 14 ) thanactivating :1 CASR gene 63 pg/ via freeaccess European Journal of Endocrinology https://eje.bioscientifica.com for activatingmutations( Pro136 regionwasdescribed asanimportanthotspot Fig. 1). depended onthemutationtype (Supplementary calcium-to-creatinineratio calcium levelsandtheurinary be mutation dependent ( profile variesconsiderablyandthisvariabilityisthought to 1).Furthermore,thebiochemical Fig. (Supplementary calciumlevelsarenormal orevenhigh cases, urinary with hypercalcemia andhypocalciuria;however, insome with inactivatingmutationshavetheclassicphenotype by Marx hypocalciuric hypercalcemia undertaken in the 1980s are inconcordancewiththeclinicalstudiesoffamilial represented byadifferentsymbol. serum calciumand8–51 pg/mLforPTH.Eachpatientis reference rangesconsideredwere8.5–10.2 mg/dLfortotal (PTH) levelsforeachphenotypeobservedinourcohort.The Scatter plotsoftotalserumcalciumandparathyroidhormone Figure 1

PTH (pg/mL) Total Serum Calcium (mg/dL) Clinical Study 100 150 200 50 24 10 12 14 16 18 20 22 Specifically, intheextracellular domain,theAla116- 0 6 4 8 t al. et

Hyperparathyroidism ( 13 ,

14 Hypocalciuric hypercalcemia ) . We that most patients observed 15 ). We that the PTH and observed

16 Normocalciuric hypercalcemia ). We found threenovel L Madariagaandothers A García-Castaño,

Hypercalciuric hypercalcemia

Hypercalciuric hypocalcemia

Normocalciuric hypocalcemia Gln945dup mutation. only theprobandandhisfathercarriedp.Pro935_ family membershadnormocalcemichyperparathyroidismbut Pedigree ofafamily(ME133)withhyperparathyroidism.Allfive Figure 2 correlation issignificant( patients withaninactivatingmutation inthe Linear regressioncalciumand age atdiagnosis(b)basedon65 Figure 3 region. Again,allthepatientshadfeaturescompatible p.Cys546Gly, p.Arg551Glyandp.Ile555Thr)inthis Wehighly conserved. foundfourmutations(p.Cys542Tyr, the cysteine-richregion,closetomembrane,thatis Another importantregionintheextracellulardomainis compatible with inactivating mutations ( had hypercalcemia and hypocalciuria,clinicalfeatures and p.Gly222Glu).Thepatientswiththesemutations We foundtwomutationsinthisregion(p.Arg220Trp in theAsp215-Arg227regionthatwasreportedasan hand, severalinactivatingmutationshavebeendescribed On the other Fig. 1). had hypocalcemia (Supplementary p.Glu133Val), andallthepatientswiththesemutations mutations in this region(p.Lys119Ile, p.Leu123Met and important regionforligand–receptorinteraction( receptor gene Novel mutationsinthecalcium p.Pro935_Gln945dup p.Pro935_Gln945dup No Mutation P

Downloaded fromBioscientifica.com at09/27/202104:57:07PM < 0.01, No Mutation r = 0.38). No Mutation 180 :1 CASR gene.The Affected female Affected male al 1 Table 64 17 via freeaccess ). ). European Journal of Endocrinology Clinical Study

Table 2 Clinical characteristics, laboratory findings and molecular results for presumed CaSR-activating mutations in the studied cohort.

Family Number of CASR mutation† Polymorphisms Clinical phenotype of Gender * Age at Total serum Serum PTH UCa/Cr U.Ca patients in the index the index case diagnosis calcium phosphate (pg/mL)* (mg/mg)* (mg/24h)* case (years)* (mg/dL)* (mg/dL)* Six families with hypocalcemic hypercalciuria CA12 1 p.Lys119Ile; – Nephrocalcinosis, hypercalciuria, F 11 6.9 6.4 NA NA NA c.356A>T+ CA23 4 p.Leu123Met; p.Ala986Ser†† Hypocalcemia, congenital F 41 7.8 4.3 24.7 0.36 419 c.367T>A+ adrenal hyperplasia due to L Madariagaandothers A García-Castaño, 21-hydroxylase deficiency, hypertension, nephrolithiasis, diabetes mellitus CA01 3 p.Glu133Val; – , F 0 6.9 8.6 4 1.6 NA c.398A>T+ hypocalcemia CA79 1 p.Glu604Lys; p.Ala986Ser Hypocalcemic infant F 4 7.2 8.5 NA NA NA c.1810G>A OT362* 1 p.Phe788Cys; p.Ala986Ser Hypoparathyroidism, F 33 6.6 6.1 3.9 NA 506 c.2363T>G& hypocalcemia, hypercalciuria, renal lithiasis, fibromyalgia, osteoarthritis, hyperlipidemia,

hypercholesterolemia, basal receptor gene Novel mutationsinthecalcium ganglia CA82 3 p.Ser820Phe; NA Hypoparathyroidism M 9 NA NA NA NA NA c.2459C>T OT362* 1 p.Gln934*; p.Ala986Ser Hypoparathyroidism, F 33 6.6 6.1 3.9 NA 506 c.2800C>T+,& hypocalcemia, hypercalciuria, renal lithiasis, fibromyalgia, osteoarthritis, hyperlipidemia, hypercholesterolemia. basal ganglia calcification

Downloaded fromBioscientifica.com at09/27/202104:57:07PM Two families with hypocalcemic normocalciuria CA60 2 p.Tyr514Cys; p.Ala986Ser Hypoparathyroidism F 0 5.1 10.3 17 0.1 NA c.1541A>G CA72 2 p.Gly830Ser; – Failure to thrive M 13 7.4 6.8 12 0.16 NA https://eje.bioscientifica.com c.2488G>A 180 Reference ranges: total serum calcium (8.5–10.2 mg/dL); serum phosphate (3.4–4.5 mg/dL); serum creatinine (0.7–1.3 mg/dL); PTH (8–51 pg/mL); UCa/Cr (<0.20 mg/mg); U.Ca (100–300 mg/24 h). *Same family; +These mutations (also marked in bold) have not been reported to date; †Numbering is according to DNA sequence (Ensembl: ENST00000490131); ††Homozygous polymorphism; :1 ‡Homozygous mutation in the index case; &Same patient; *Laboratory findings of index case. NA, not available; PTH, parathyroid hormone; U, urinary. 65 via freeaccess European Journal of Endocrinology https://eje.bioscientifica.com more than10%ofitsamino acids),and,asfarweknow, mutation producesastrongly truncatedprotein(missing pathway ( calcineurin-nuclear factorof activatedTcellsignaling mutation increasesintracellular [Ca functional studieshadpreviouslydemonstratedthatthis with hypocalcemiaandhypoparathyroidism( The p.Phe788Cysmutationwasdescribedtobeassociated whether thosemutationswerepresentindifferentalleles. not abletoperformgenetictestinginthefamilyorcheck behave asanactivatingmutation.Unfortunately, wewere and therefore,wesupposethatthesetwomutationstogether hypocalcemia, hypercalciuria andhypoparathyroidism, mutation (intheintracellulardomain).Thispatienthad transmembrane domain)andthenovelp.Gln934* two mutations:thep.Phe788Cysmutation(in localization. probably destabilizestheproteinandaltersitssubcellular the normalCaSRsequenceof1078aminoacids,which both mutationsyieldatruncatedproteinwithlossof of thereceptor( codon 877within the predictedcarboxyl-terminal tail who had an insertion of an A similarphenotypewasdescribedinanotherpatient hypercalcemia, hypocalciuria andhyperparathyroidism. Gln945dup duplicationintheintracellulardomainhad ( sites mightcausecontinuousactivationofthereceptor and the elimination of such important phosphorylation), this region (CaSR being regulated by protein kinase C sites are located in the other hand, phosphorylation interaction withtheGproteinbeingdisrupted.On results inanon-functionalreceptor, likelyduetoits membrane domain( p.Ser820Phe andp.Gly830Ser)mutationsinthetrans­ p.Ile816Val and p.Ile816Thr) activating (p.Phe788Cys, inactivating (p.Cys677Tyr, p.Val697Met, p.Phe789del, inactivating mutations( et al mutation hasapathogeniceffectasreportedbyNissen pathogenic effect.Ourresultssupporttheviewthatthis however, inthatcase, between thosedescribedinourstudy(p.Arg544Gln); previous study, Nissen to beassociatedwithaninactivatingeffect( between aminoacids544and555havebeendescribed with inactivatingmutations.Moreover, manymutations 21 Clinical Study ). In our cohort, patient ME133 with the p.Pro935_ . andconfirmthisregionasanimportanthotspotfor Interestingly, wefoundonepatient(OT362)with Loss ofparttheintracellulardomainpresumably In concordancewithotherstudies,wefoundboth 24 ). Ontheotherhand, novel p.Gln934* 22 19 ). Therefore,wehypothesizethat , t al et in silico 18 20 ). ). . reportedanothermutation Alu analysispredictsanon- -repetitive element after L Madariagaandothers A García-Castaño, 2+ ] andactivatesthe 17 23 , 18 ), while ). Ina is whatexplainswhythemutantdoesnotexerta that theabsenceofadefectivereceptorinmembrane only WTreceptorsareexpressedinthemembraneand less severephenotype.We hypothesizethatinthispatient p.Arg25* mutationintheheterozygousstate,exhibitsa biochemical phenotype( receptor signaland,thereforeproducingamoresevere in the heterozygous state, interfering with the WT point mutationsexertadominant-negativeeffect heterozygous patients( on the membrane in CaSRs had been observed Further, thatdeletionthanWT moreCASRscarrying exerts adominant-positiveeffectinthemembrane. authors suggested that CaSR with this large deletion terminal tail(Ser895-V1075)hasbeenreported,andthe of alargedeletion181aminoacidsinthecarboxyl- hypocalcemia. A similar activating mutation consisting it isthefirstdescribednonsensemutationassociatedwith compound heterozygousstate withthepolymorphisms in 32cases,thehomozygous statein3andthe polymorphism in37patients (intheheterozygousstate be higherinsomepatients( hyperparathyroidismwasreportedto the riskofprimary than those without thispolymorphism ( p.Ala986Ser, especially wheninthehomozygousstate, in individuals with mean serumcalciumbeingobserved has beendescribed associated withhypercalcemia, higher populations (allelefrequencyof0.03).Thispolymorphism rareinAfrican andEastAsian in Caucasians,butvery ExAC, the p.Ala986Ser polymorphism is 0.1269 (according to and c.3031C gene (c.2956G previously described as polymorphisms in the sorption rate( hyperaldosteronism oralowfractionalchloridereab­ of Barttersyndrome,suchashypokalemia,secondary of 17patientswithactivatingmutationshadsymptoms had amildphenotype,similartothatofCA58. parents (whocarriedthemutationinheterozygousstate) This hypothesisissupportedbythefactthatpatient’s that thereceptorwasnotexpressedinmembrane. hyperparathyroidism. In thispatient,weconsideritlikely phenotype andhehadbeendiagnosedwithneonatal mutation inthehomozygousstate,hadamoresevere case offamilyCA42,whopresentedthep.Arg465Leufs*9 dominant-negative effect.Ontheotherhand,index receptor gene Novel mutationsinthecalcium On the other hand, it was hypothesized that some In ourcohort,wefoundthreecommonvariants As farasweknow, noneofthepatientsinourgroup http://exac.broadinstitute.org > 27 G; p.Gln1011Glu).Theallelefrequencyof > T; p.Ala986Ser, c.2968A ). 25 Downloaded fromBioscientifica.com at09/27/202104:57:07PM 26 ). 29 ). PatientCA58,whohadthe ). We foundthep.Ala986Ser ), being around 0.15 180 28 > :1 G; p.Arg990Gly ). Furthermore, CASR 66 via freeaccess

European Journal of Endocrinology without anyotheralteration inthe hyperparathyroidism inthese27patients of primary calcium homeostasisorarerisk factorsforthedevelopment these polymorphismscause significantvariationsin polymorphisms andcalciumhomeostasis( studies havereportednorelationbetweenthesethree with hypocalciuria.Asmentionedpreviously, other gene ( polymorphism: onehadanothermutationinthe ( associated withhigherbloodionizedcalciumlevels studied hasanallelefrequencyof0.0554,anditwas function, buttheotherthreehadhypercalcemia. hypercalciuria, inaccordance withreceptorgainof that twohadhypocalcemia,hypoparathyroidismand ( of themalsohadinactivatingmutationsinthe with thep.Arg990Glypolymorphisminourcohort:three PTH andionizedcalcium( hyperparathyroidism had lower levels of serum secondary or that individualswiththispolymorphismandprimary stone formers( ( polymorphism producesagainoffunctioninthereceptor ( specifically focusingonthep.Arg990Glypolymorphism on single-nucleotidepolymorphismassociations,not colorectal adenoma,butallpreviousstudiesarebased associated withtheriskofchronicpancreatitis( commoninEastAsia(0.50).Ithasbeen (0.04), butvery population andisrareinEurope(0.10)Africa has anallelefrequencyof0.1451inthegeneral homozygous state)whohadhypocalcemia. it in the with this polymorphism (including one carrying calcium ( p.Ala986Ser polymorphism and high levelsofserum have reportednorelationbetweenthepresenceof hand, otherstudies(whichincludefunctionalanalysis) to what has been described previously. On the other initially diagnosedwithhyperparathyroidism,similar patients had hypercalcemia, including four patients gene ( mutations (activatingand/orinactivating)inthe besides thispolymorphism,otherlikelydisease-causing in 20ofthesepatients,while17(indexcases)alsocarried, change wastheonlyalterationfoundin p.Arg990Gly or p.Gln1011Glu in 2);thispolymorphism 12 34 1 Table 33 Clinical Study ). Inourcohort,wefoundthreepatientswiththis ) withincreasedhypercalciuria inpeoplewhoare ). Functionalanalysisshowedthatthep.Arg990Gly The second polymorphism observed (p.Arg990Gly) The secondpolymorphismobserved Further work would be necessary toclarifywhether Further workwouldbenecessary The lastpolymorphism(c.3031C Table 1 als 1 Tables ), while in the other five patients, we observed ), whileintheotherfivepatients,weobserved 30 , ), whereastheothertwohadhypercalcemia 31 and 34 ). Indeed, we observed eightpatients ). Indeed,weobserved ). Inaddition,someauthorsreported 2 ). We that 29 ofthese37 observed 35 ). We foundeightpatients L Madariagaandothers A García-Castaño, CASR > G; p.Gln1011Glu) gene. 30 , 31 CASR CASR ). 32 gene ) and CASR CASR gene

it fromotherentities,butalsoessentialfordetecting important for confirming the diagnosis and distinguish proteins ( intakeofsaltandanimal many factors,includingdietary 1). Variations in calcium excretion can be due to Fig. calciumlevelsmaybenormal(Supplementary urinary hypercalcemia withhypocalciuria,andinfact,their of the cotransporter family( encoding amemberofthetypeIIsodium-phosphate the cytochromeP450superfamilyand subunit ( the The authors declare that there is no conflict of interest that could be could that interest of conflict perceived asprejudicingtheimpartiality ofthisstudy. no is there that declare authors The Declaration ofinterest EJE-18-0129 This islinkedtotheonline version ofthepaperat Supplementary data pathogenicity ofournovelmutations. the needtoconductfunctionalanalysisconfirm structures intheCASR.Nevertheless,werecognized help improveourunderstandingofthefunctiongiven presumably activatingorinactivatingmutationswill clinical descriptionofacohort82patientswith non-pathogenic effect. of someothermutationsdespite mutations intheheterozygousstateandpathogenicity by other groups concerning the dominant effect of some future. Moreover, ourstudysupportstheresultsobserved facilitate individualized treatment of patients in the is criticalforguidingpharmacologicalresearch, andcould functional impactofthemutationsatcellmembrane biochemical features.Inaddition,understandingthe from idiopathic hypoparathyroidism, because of common be performed)andautosomaldominanthypocalcemia maywell hyperparathyroidism(forwhichsurgery primary to differentiate hypocalciuric hypercalcemia type I from normocalcemic. Intheclinicalsetting,itcanbedifficult carriers, assomeindividualswithmutationsmayremain GNA11 associated withcalciumhomeostasisdisorders:the analysis ofthesepatientswillbetostudyfourgenes sequencing the receptor gene Novel mutationsinthecalcium In conclusion,patientswithinactivatingmutations We believethatthiscomprehensivegeneticand Finally, in21patients,noalterationswerefoundby AP2S1 gene,encodingthealphasubunitofG CASR . 37 40 gene,encodingtheadaptor-protein2sigma ); the ). Giventhis,molecularstudiesarenotonly genemaynotpresentaclassicpictureof CASR CYP24A1 gene.Hence,thenextstepinour 38 , Downloaded fromBioscientifica.com at09/27/202104:57:07PM 39 gene,encodingamemberof ). https://eje.bioscientifica.com in silico 180 https://doi.org/10.1530/ :1 predictionsofa SLC34A1 α 11 ( gene, 67 36 via freeaccess ); European Journal of Endocrinology https://eje.bioscientifica.com References d’Hebron –Barcelona). Gema Ariceta,Anna Meseguer (Hospital UniversitarioMaterno-Infantil Vall María Herrero, Aníbal Aguayo (Hospital Universitario Cruces – Barakaldo). Gustavo PérezdeNanclares,AlejandroGarcía-Castaño,MireiaAguirre, Nuestra Señorade Candelaria –Tenerife).Luis Castaño, LeireMadariaga, Universitario (Hospital Lanús Córdoba Elizabeth Yanes, Luis Isabel María Trujillo, Ramos Elena Acosta, González Hilaria Claramunt Martín, Claverie Félix Nieto, Débora Fuente, Rocío Hernández, (Hospital Universitario CentraldeAsturias–Oviedo). Víctor M.García Olaya Riera, Braga Eva Rodríguez, Gil- Julián Ordóñez, Ángel Flor Loredo, Helena Vanessa Coto, Eliecer Peña, Santos, Fernando Group: RenalTube Barakaldo). – Institute LaPiscina (Hospital Universitario Cruces,BiocrucesBizkaiaHealthResearch de Sagunto), ItxasoRica,RosaMartínez,Inés Urrutia, Idoia Martínez de Universitario (Hospital Universitario Valld’Hebron – Barcelona),LeonorGarcía(Hospital (Hospital Fernández-Ramos Basurto –Bilbao),JuanMartí(HospitaldeZumárraga),MaríaClemente Concepción Galdakao), de (Hospital Universitario 12 de Octubre – Madrid), Jorge Hernández (Hospital Lourdes ReyCordo(Complejo Hospitalario Universitario–Vigo),JaimeCruz Madrid), CarmenMaríaGonzález(HospitalRafaelMéndez Carmen – Lorca), Infantil – Badajoz),RaquelBarrio(Hospital Universitario RamónyCajal– Virgen Macarena–Sevilla),EmiliaHidalgo-Barquero (Hospital Materno (Hospital Juan Ramón Jiménez – Huelva), Elena Pérez(Hospital Universitario Universitario Severo Ochoa – Leganés), Belén Jiménez, Pilar Rodríguez Conde (Hospital de Barbastro – Huesca), Beatriz García-Cuartero (Hospital – Getafe),Ismene Bilbao (Hospital Universitario –Donostia), Santiago – Las Palmas de Gran Canaria), Cristina Aparicio (Hospital Universitario Boronat (Complejo Hospitalario Universitario Insular Materno-Infantil – Albacete),CatalinaSocias(Hospital Bidasoa –Hondarribia),Mauro RafaelRuiz (Complejo Sierrallana –Torrelavega), Hospitalario Universitario Universitario PríncipedeAsturias–Madrid),CoralMontalbán (Hospital Spanish Endocrinology Group: Joaquín Ramírez, MercedesGómez(Hospital grateful to the RenalTube group for contributing samples forthis study. and endocrinologists who collaborated with the genetic study. They are The authorsthankpatientsandfamiliestheirpediatricnephrologists Acknowledgements E CL,MA,H,AM. Y, L I M T, R E A, G H M, C F N, G M V C, D F, R H, O R, B E R, J O, Á F L, V C, E B, C M G, C L R C, J C, J H, C F-R, J M, M C, L G, I R, R M, I U, I M d L, F S, H G-P, L C, G A, J R, M G, C M, R R, C S, M B, C A, I B, S C, B G-C, B J, P R, E P, E H-B, R A G-C, L M, L C, G A, S G. Clinical diagnostic and validation: A G-C, L M, S G, Project administration:LM,C,GA.Resources:Visualization: A. G C, L M, Investigation: A G-C, L M,GPd N, LC,GA,S G. Methodology: A G-C, G P,LC. L acquisition: Funding N. d P G G-C, A analysis: Formal G. S Writing: A G-C, L M,C,GA,S G. Conceptualization: A G-C, LM,C,GA, Author contributionstatement to publishorpreparationofthemanuscript. funders had noroleinstudy design, data collectionandanalysis,decision (2014111064) and of Education (IT795-13) of the Basque Government. 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. Accepted 6September2018 Revised versionreceived3August2018 Received 11February2018

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