Understanding Parkinson disease in Spain: Genetic and clinical insights

Eur J Neurol. 2025;32:e16499.   | 1 of 9 https://doi.org/10.1111/ene.16499 wileyonlinelibrary.com/journal/ene Received:7May2024 | Accepted:16September2024 DOI: 10.1111/ene.16499 ORIGINAL ARTICLE Understanding Parkinson disease in Spain: Genetic and clinical insights Pilar GómezGarre1,2 | Miguel MartínBórnez1 | Laura MuñozDelgado1,2 | Rafael DíazBelloso1,2,3 | María Teresa Periñán1,2,4 | Marta BonillaToribio1,2 | Dolores BuizaRueda1,2 | Daniel MacíasGarcía1,2 | Silvia Jesús1,2 | Astrid AdarmesGómez1,2 | Elena Ojeda1,2 | Antonio LuqueAmbrosiani1 | Sergio GarcíaDíaz1 | Rocío Pineda Sánchez1,2 | Fátima Carrillo1,2,3 | Pablo Mir1,2,3 1UnidaddeTrastornosdelMovimiento,ServiciodeNeurología,InstitutodeBiomedicinadeSevilla,IBiS/HospitalUniversitarioVirgendelRocío/CSIC/ UniversidaddeSevilla,Seville,Spain 2CentrodeInvestigaciónBiomédicaenRedsobreEnfermedadesNeurodegenerativas,InstitutodeSaludCarlosIII,Madrid,Spain 3DepartamentodeMedicina,FacultaddeMedicina,UniversidaddeSevilla,Seville,Spain 4CentreforPreventiveNeurologyUnit,WolfsonInstituteofPopulationHealth,QueenMaryUniversityofLondon,London,UK ThisisanopenaccessarticleunderthetermsoftheCreativeCommonsAttribution-NonCommercialLicense,whichpermitsuse,distributionandreproduction inanymedium,providedtheoriginalworkisproperlycitedandisnotusedforcommercialpurposes. ©2024TheAuthor(s).European Journal of NeurologypublishedbyJohnWiley&SonsLtdonbehalfofEuropeanAcademyofNeurology. Correspondence PilarGómez-GarreandPabloMir,Unidad de Trastornos del Movimiento, Instituto deBiomedicinadeSevilla(IBiS),Hospital UniversitarioVirgendelRocío/CSIC/ UniversidaddeSevilla,AvanzadaManuel Siurots/n,Seville41013,Spain. Email:mgomez-[email protected] and [email protected] Funding information ConsejeríadeEconomía,Innovación, CienciayEmpleodelaJuntadeAndalucía, Grant/AwardNumber:CTS-7685,CVI- 02526andPY20_00896;Consejeríade SaludyBienestarSocialdelaJuntade Andalucía,Grant/AwardNumber:PE- 0 1 8 6 - 2 0 1 9 ,  P E - 0 2 1 0 - 2 0 1 8 ,  P I - 0 4 5 9 - 2 0 1 8  andPI-0471-2013;Grant/AwardNumber: RTC2019-007150-1,InstitutodeSalud CarlosIII(ISCIII)andco-fundedbythe EuropeanUnion,Grant/AwardNumber: PI14/01823,PI16/01575,PI18/01898, PI19/01576andPI21/01875 Abstract Background and purpose: Parkinsondisease(PD)isacomplexandheterogeneousneurodegenerativedisorderwithabroadspectrumofclinicalmanifestations,determinedbya complexinterplayofenvironmentalandgeneticfactors.Thisstudyaimedtoinvestigate geneticvariantsassociatedwithPDandassesstheirimpactonthediseasephenotype through genotype–phenotype correlations. Methods: Weemployedatargetedresequencingpaneltoanalyze27geneslinkedto PD in a cohort of 1185 PD patients from southern Spain. Variants were categorized basedontheAmericanCollegeofMedicalGeneticsandGenomicspathogenicitycriteria. Demographic and clinical data were also collected. Results: Amongthepatientsanalyzed,13.5%carriedpotentialdisease-causingpathogenicorlikelypathogenicvariantsin12differentgenes,indicatingsignificantgenetic heterogeneity.ThemostfrequentlyaffectedgeneswereLRRK2, PRKN, and GBA1(accountingfor 72.1%of positive cases).Sex-specific differenceswere observed, with a higherproportionoffemalepatientscarryingLRRK2variants.Differencesinageatonset andclinicalfeatureswerealsoobservedamongthedifferentmutatedgenes.Notably, variants in genes associated with atypical parkinsonism presented distinct clinical presentations,highlightingtheimportanceofgeneticfactorsinthedifferentialdiagnosis. Conclusions: OurstudyprovidesvaluableinformationonthegeneticlandscapeofPD anditsclinicalmanifestations.Theobservedgenotype–phenotypecorrelations, along withsex-specificdifferences,emphasizethecomplexityofPDpathogenesis,underliningtheimportanceofpersonalizedapproachestoPDdiagnosisandtreatment.Further 2 of 9 | GÓMEZ-GARRE et al. INTRODUCTION Parkinsondisease(PD)isacommonneurodegenerativedisease, clinically characterized by three cardinal motor symptoms: bradykinesia,resting tremor,andmusclerigidity.However,PDisa complex,heterogeneousdisorderwithabroadspectrumofclinical manifestations. Beyond motor symptoms, PD also presents numerousnonmotorsymptoms,oftenappearinglongbeforethe onsetofclassicmotor symptomsandsignificantly affecting the quality of life. Dementia is particularly prevalent, occurring in 83%ofpatientswithPD,withadiseasedurationofapproximately 20 years[1]. The risk of developing PD is determined by a complex interplayofenvironmentalandgeneticriskfactors[2], with age being themostimportantriskfactor.TheincidenceandprevalenceofPD increase almost exponentially with age, peaking at >80 years [3]. Likewise,sexisanestablishedriskfactor,withamale/femaleratio ofapproximately3:2[1]. Withinitsmultifactorialetiopathogenesis,PDisrecognizedas ahighlycomplexgeneticdisease.NumerousstudieshaveinvestigatedriskandprotectivefactorsofPD,withsignificantprogressin geneticresearchduetorapidadvancesinsequencingmethods.The geneticcontributiontoPDisexplainedbyawidespectrumofgeneticvariants,rangingfromcommonvariantsthatconfersusceptibilitytodevelopingPD,withamoderatetoweakeffectsize,torare variantforms,highlypenetrant,wherethepresenceofthevariant issufficienttocausethedisease[4]. To date, pathogenic variants associatedwithmonogenicPDhavebeendescribedin>20 genes, mostofwhicharehighlypenetrantandoftencauseearlyonsetor atypicalPD[4].Althoughithasbeenreportedthatonly5%–10%of PDcasesaremonogenic[5], these data are mainly based on individualsofEuropean/Whiteancestry.Thefrequency,penetrance,and clinicalimpactofgeneticvariantscanvaryacrossethnicities.Spain has a complex demographic history, characterized by continuous occupation since ancient times by various populations with diverse origins,includinganeight-centuryperiodofArabdomination(from MiddleEastandNorthAfrica),withacleargeneticimpact[6]. This study aimed to assess the presence and contribution of variantsinPD-relatedgenesinacohortofPDpatientsfromsouthernSpain,andtoestablishgenotype–phenotypecorrelations.We usedatargetedresequencingpanel,focusingon27genesassociatedwithPD,andanalyzedthedatausinganin-housepipeline.Our findingssignificantlycontributetounderstandingofPD,shedding lightontheeffectofgeneticvariantsondiseasedevelopmentand offeringimportantinsightsintodiagnosisandtreatmentofPDinour population. PATIENTS AND METHODS SeeAppendixS9. Study participants and clinical assessments Atotalof1185PDpatientswereincludedfrom2008.Thediagnoses wereperformedfollowingtheUnitedKingdomParkinson'sDisease SocietyBrainBankcriteria[7]forpatientsrecruitedupto2018and MovementDisorderSocietyclinicaldiagnosticcriteria[8]from2019 onward. Genetic analysis Genomic DNA was isolated from peripheral blood samples from eachparticipant.Acombinationofhigh-resolutionmeltinganalysis, multiplexligation-dependentprobeamplificationanalysis,andnext generation sequencing (NGS)-based targeted resequencing was applied(Figure S1).VariantswerecategorizedaccordingtotheinternationalguidelinesoftheAmericanCollegeofMedicalGenetics andGenomics(ACMG)[9](Figure S2).Onlyvariantsconformingto thegene'sinheritancepatternwereconsideredpotentialdisease- causing variants, and the genetic diagnosis was considered positive. Bioinformatics analysis for NGS results All NGS data were analyzed employing an in-house pipeline (AppendixS1). TobetterdefinetheroleofGBA1variantsinPD,variantsinthis gene were classified using the GBA1-PD browser (ht tps:// pdgen e t i c s . s h i n y a p p s . i o / G B A 1 B r o w s e r / )[10]. Inaddition,copynumbervariations(CNVs)werealsoanalyzed. Lossesweredefinedbyamaximumlog2ratioof−0.55andaminimumlog2ratioof0.4forgains[11]. Statistical analysis Associationanalysesof the variants and PD risk were performed withPLINKsoftware[12].Ararevariantburdenanalysiswasconductedwithconsistentsummarycounts-basedrarevariantburden test[13].Ascontrols,weusedtwosubcohorts:non-neuroandcontrolsintheNon-FinnishEuropean-SouthernEuropean(NFE-SEU) investigationsintogeneticinteractionsandpopulation-specificeffectsarewarrantedto enhanceourunderstandingofPDetiologyandimprovepatientcare. KEYWORDS clinicalinsights,geneticinsights,Parkinsondisease,targetedsequencing 14681331, 2025, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/ene.16499 by Readcube (Labtiva Inc.), Wiley Online Library on [14/04/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License | 3 of 9 PARKINSON DISEASE IN SPAIN cohort.WealsouseddatafromtheCIBERERSpanishVariantServer (CSVS;h t t p s : // c s v s . c l i n b i o i n f o s s p a . e s / )[14]. RESULTS Demographic characteristics Weanalyzed27genesassociatedwithPDinacohortof1185unrelatedPDpatients(Table 1).Amongthem,162(13.7%)reporteda familyhistoryofPD,and387(32.7%)hadearlyonsetPD(EOPD).The ageatonset(AAO)wasunavailableforthreepatients,buttheywere classifiedashavingEOPDbasedontheircurrentage(<50 years). Genetic spectrum Weidentifiedatotalof1014variantsinthecodingsequenceand spliceregionsofthe27analyzedgenes,allwithaminorallelefrequency(MAF)< 5%inthegeneralpopulation.Thesevariantswere categorizedaccordingtoACMGcriteriaasbenign(B),likelybenign (LB),variantofunknownsignificance(VUS),likelypathogenic(LP), andpathogenic(P).Amongtheidentifiedvariants,898werelocated incodingregionsand116inspliceregions(Table S1A and Figure S3A). Specifically,71variantswereclassifiedasP/LP,156asVUS,and788 asB/LB(Table S1B and Figure S3B).TheP/LPvariantswerefoundin 207patients(17.5%),affecting17genes,includingATP13A2, DCTN1, DNAJC6, FBXO7, LRP10, LRRK2, PRKN, PARK7, PINK1, PLA2G6, POLG, SMPD1, SPR, SYNJ1, VPS13A, VPS13C, and GBA1. CNVs were detected in the PRKN and SNCAgenes.Consideringtheinheritance patternofeachaffectedgene,potentiallydisease-causingP/LPvariantswereidentifiedin160patients(13.5%ofthecohort),involving12distinctgenes(DCTN1, DNAJC6, LRP10, LRRK2, PRKN, PINK1, POLG, SMPD1, SNCA, SPR, VPS13A, and GBA1).Amongthese160 patientswithpositivegeneticdiagnosis(PGD),sevencarriedP/LP variantsinmorethanonegene.Nodisease-causingvariantswere identifiedin960patients(80.9%),whowereconsideredtohavea negativegenetic diagnosis (NGD).Additionally, 65patients (5.5%; Figure 1a and Table S2),wereclassifiedashavinganunknowngeneticdiagnosis(UGD),ThisgroupincludedpatientscarryingVUS, those lacking a second variant in recessive genes, and those with GBA1variantsconsideredPDriskfactorsaccordingtotheGBA1-PD browser.Geneticanalysisresults foreachpatientareprovided in Table S3. Variantsinthreegenes(LRRK2, PRKN, and GBA1)accountedfor 72.1%ofthepositivecases.ThemostprevalentgenewasLRRK2, with61patients(37.9%)carryingP/LPvariants(including49with theG2019Svariant),followedbyPRKN(18%,including12patients withtwovariantsbutwithoutsegregationanalysis)andGBA1(18%; Figure 1b). We identified 29 patients (2.45%) carrying P/LP variants in GBA1. Severe variants were present in 10 patients (0.84%), mild variantsin14patients(1.18%),and unknown variantsinfour patients. Moreover, 21 patients (1.76%) carried risk variants. Three patients carried two variants in GBA1butdidnotexhibitsymptoms of Gaucher disease (GD). The most frequent P/LP variants were p.N409Sandp.L483P,andthemostfrequentvariantwastherisk variantp.E365K. ThedistributionofpatientswithPGD,NGD,orUGDacrossdifferentgroupsisshowninFigure 1c. Genotype–phenotype correlations Patients with PGD had a younger mean AAO (50.6 ± 12.4 years) compared to those with NGD (56.7 ± 11.6 years) or UGD (55.0 ± 12.3 years).TheAAOvariedamongdifferentmutatedgenes, withheterogeneityevenamongcarriersofvariantsinthesamegene (Figure 2).TheproportionofpatientswithafamilyhistoryofPDwas similarbetweenthosewithPGDandUGD(18.8%and18.5%,respectively),andhigherthaninthosewithNGD(12.5%;Figure 3a).A higherproportionofwomen(44.4%)wasnotedamongpatientswith PGD,particularlyamongthosecarryingP/LPvariantsintheLRRK2 gene(Figure 3b). Wealsoexploredtheassociationsbetweenthegenotypesand clinical characteristics in PGD patients (Table S4). Despite some missing clinical data, GBA1PDpatientsexhibitedahigherproportionofrapideyemovementsleepbehaviordisorder(RBD)andolfactorydysfunction(88%and68.4%,respectively),whereasalower frequencyofRBDwasobservedinLRRK2PDpatients(39.6%)and a lower frequency of olfactory dysfunction in PRKN PD patients (6.7%; Table S4). Cognitive impairment (CI) was more frequent amongpatientswithPGDrelatedtoGBA1, DCTN1, POLG, and SPR, withahigherprevalenceinmen(Table 2). Cohort Participants, nSex, men, n (%) Age, years, mean ± SD AAO, years, mean ± SD Family PD, n (%) Total 1185 707(59.7) 64.6 ± 11 55.8 ± 11.9 162(13.7) EOPD 387 242(62.5) 53.3 ± 9.2 42.5 ± 7.4 53(13.7) LOPD 798 465(58.3) 69.6 ± 7.8 62.3 ± 7.5 109(13.7) FamilyPD 162 89(54.9) 65.8 ± 10.2 56.0 ± 12.3 162(100) SporadicPD 1023 618(60.4) 64.4 ± 11.1 55.8 ± 11.9 0(0) Abbreviations:AAO,ageatonset;EOPD,earlyonsetPD(ageatonset≤ 50 years);LOPD,lateonset PD;PD,Parkinsondisease. TABLE 1 DemographicdataofthePD cohort. 14681331, 2025, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/ene.16499 by Readcube (Labtiva Inc.), Wiley Online Library on [14/04/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 4 of 9 | GÓMEZ-GARRE et al. Genotype–phenotype correlations in wellestablished PDrelated genes LRRK2 Dystoniawasobservedin68.4%ofLRRK2PDpatients,andresting tremorwasobservedin59.3%.Asubstantialmajorityexperienced motorfluctuation(91.8%)anddyskinesias(83.3%),withCIandRBD presentin31.0%and39.6%,respectively. A familyhistory of PD wasnotedonlyamongcarriersofthepathogenicG2019Svariant (Table S4). In our cohort, LRRK2 PD patients were predominantly women (60.7%).Thishigherproportionwasonlyobservedincarriersofthe G2019S variant, among whom 60% were women. No significant differencesinAAOwereobservedbasedonthespecificvariantcarried among LRRK2PDpatients,although23patients(37.7%)presented EOPD.Amongthese,fivepatientscarriedP/LPvariantsinothergenes. Onepatient(EP-40)carriedP/LPvariantsinboththeLRRK2 and DCTN1genes,withanAAOof51 yearsandabenigndiseasecourse without notable nonmotor symptoms. Twopatients(EP-10andEP-16)werefoundtocarrytheG2019S variantandhomozygousPRKNvariants,exhibitingclinicalfeatures similar to those associated with PRKNPD. Lastly, two patients (EP-215 and EP-223) carried VUSs and were therefore included in the UGD group. Their AAOs were 62 and54 years,respectively,andtheypresentedwithrestingtremor. Despitetheirrelativelybriefclinicalfollow-up,theirclinicaldisease course has been benign. FIGURE 1 Thefrequencyofcausative genes in patients initially diagnosed withParkinsondisease(PD).(a)Overall diagnosticyield.(b)Distributionof disease-causativegenesin160probands. (c)FeaturesoftheanalyzedPDcohort. EOPD,earlyonsetPD(ageatonset ≤ 50 years);LOPD,lateonsetPD(age at onset > 50 years);NGD,patients withanegativegeneticdiagnosis;PGD, patients with a positive genetic diagnosis; UGD,patientswithanunknowngenetic diagnosis. FIGURE 2 Boxplotsofageatonset underspecificgenes.Dotsrepresent the age at onset in each patient carrying putative causative variants in each gene. Horizontallinesrepresentthemedian onset.M,mild;NGD,Parkinsondisease (PD)patientswithanegativegenetic diagnosis;PGD,PDpatientswitha positivegeneticdiagnosis;RF,riskfactor; S,severe;T,totalcohortwithpathogenic or likely pathogenic variants in GBA1 and those with risk variants in GBA1;UGD, PDpatientswithanunknowngenetic diagnosis. 14681331, 2025, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/ene.16499 by Readcube (Labtiva Inc.), Wiley Online Library on [14/04/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License | 5 of 9 PARKINSON DISEASE IN SPAIN SNCA Twopatients(EP-114andEP-135)carriedduplicationsoftheentire SNCAgeneinheterozygosis.However,thecutoffpointscouldnot bedetermined.OnepatientpresentedwithalateAAO,whereasthe otherhadanearlydiseaseonset.ThepatientwiththeearlierAAO also carried a risk variant in the GBA1 gene. PRKN AlthoughmostPRKNPDpatientsexhibitedanearlyAAOof PD,14.3%presentedlateAAO(rangingfrom55to59 years), motor fluctuations and dyskinesias were common (82.1% and77.8%,respectively),andCIandhallucinations/illusions wererelativelyrare(25.9%and18.9%,respectively),with FIGURE 3 Bargraphsshowingfamily historyofParkinsondisease(PD)and genderdistributionacrossspecific genes.NGD,PDpatientswithanegative geneticdiagnosis;PGD,PDpatients withapositivegeneticdiagnosis;UGD, PDpatientswithanunknowngenetic diagnosis. TABLE 2 Cognitiveimpairmentinthepositivegeneticdiagnosiscohort. LRRK2 PRKN GBA1 GBA1, severe GBA1, mild DCTN1 POLG SMPD1 SPR n42 27 25 912 3 9 8 8 CI(%) 31.0 25.9 64 55.6 58.3 66.7 66.7 25 62.5 CI-F(%) 33.3 8.3 28.6 25 0 0 66.7 050 CI-M(%) 20 40.0 77.8 80 70 100 66.7 28.6 66.7 D P - C I 15 ± 7.9 16.6 ± 7.8 7.8 ± 6.0 4.8 ± 1.8 11.1 ± 7.7 7.5 ± 3.5 10.0 ± 5.0 7.5 ± 3.5 9.4 ± 2.9 D P - C I - F 15.8 ± 6.9 15 4.5 ± 3.5 2 0 0 11.5 ± 6.4 010 D P - C I - M 16.5 ± 13.4 16.8 ± 8.5 8.2 ± 6.3 5.1 ± 1.0 11.1 ± 7.7 7.5 ± 3.5 9.3 ± 5.1 7.5 ± 3.5 9.3 ± 3.3 Note:ThedevelopmentofCIwasconsideredthroughoutthefollow-up.Themeanfollow-uptimewas15.1 ± 8.5 years. Abbreviations:CI,cognitiveimpairment;CI(%),percentageofPGD-patientswithCI;CI-F(%),percentageofwomenwithPGDandCI;CI-M(%),percentage ofmenwithPGDandCI;DP-CI,diseaseprogressiontoCI(meanofyears ± SD);DP-CI-F,diseaseprogressiontoCI(meanofyears ± SD)inwomen;DP- CI-M,diseaseprogressiontoCI(meanofyears ± SD)inmen;n,sampleswithpositivegeneticdiagnosis(PGD)andCIdata;PGD,positivegeneticdiagnosis. 14681331, 2025, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/ene.16499 by Readcube (Labtiva Inc.), Wiley Online Library on [14/04/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 6 of 9 | GÓMEZ-GARRE et al. CI more prevalent among men (40%) than women (8.3%; Table 2). PINK1 Onepatient(EP-3)carriedLPmissensevariantsinputativecompound heterozygosityinthePINK1gene.ThispatientpresentedwithearlyAAO (49 years),motorfluctuations,dyskinesias,and dystonia.Additionally, althoughCIwasnotpresent,hallucinations/illusionsoccurred. GBA1 Wedetected13differentvariantsintheGBA1gene.Accordingto theireffectonGDandPD,fiveweredescribedassevere,threeas mild,andtwoasriskvariants,theirfrequencybeing4.9%inEOPD and3.1%inlateonsetPD.TheAAOofthosecarryingtheriskvariantsp.E365Kandp.T408Mwaslaterthantherest. Motorfluctuationsanddyskinesiasweremorefrequentinpatients with mild variants compared with those with severe variants (61.5%vs.50%,and69.2%vs.66.7%,respectively).Although1patient(EP-105)presentedwithtwoseverevariantsinGBA1, no signs of GD have been reported in his clinical history, suggesting that those variants are probably in cis. RBD and olfactory dysfunction were observed with higher frequency in patients carrying severe variants (87.5% and 71.4%, respectively) compared with those with mild variants (84.6% and 66.7%,respectively). CIwaspresentinapproximatelyhalfoftheGBA1patients(affectingahigherproportionofmalepatients),anddiseaseprogressiontoCIwaslowerinthepatientscarryingGBA1 severe variants compared with those with GBA1mildvariants(Table 2). Genotype–phenotype in genes linked with parkinsonism with atypical signs DCTN1 Sixpatients(0.5%ofthePDcohort)harboredanLPvariantinthe DCTN1 gene: c.414 + 1G > A (rs576198476). This variant displayed acombined annotation dependent depletion(CADD) valueof 33 andwasdescribedasLPaccordingtoACMGcriteria.Inourpopulation, DCTN1PDpatientspresentedameanAAOof59.5 ± 6.9 years (Figure 2),goodresponsetolevodopa,andmotorfluctuationsand dyskinesia.Mostofthesepatientsexperiencedparticularlysevere neuropsychiatricsymptoms,andthereforetheydidnottoleratehigh dosesofdopaminergicmedication.Otherfeatures,suchasrespiratory symptoms and weight loss, were absent in their clinical history. Additionally,onepatientcarriedboththeDCTN1c.414 + 1G > Avariant and the LRRK2G2019Svariant,showinganearlierAAOanda lackofneuropsychiatricsymptoms. DNAJC6 Onepatient(EP-47)withalateAAOharboredtwoLPvariantsin DNAJC6, presenting with a typical PD phenotype. Both variants were frameshift changes likely in compound heterozygosity but withoutbiallelicconfirmation. LRP10 Onepatient(EP-29)withnofamilyhistoryofPDcarriedanonsense variant in the LRP10gene.ThispatientpresentedalateAAO anddevelopedCIandhallucinations/illusions5 yearsafterdisease onset. POLG Seven different P/LP variants were identified in 11 patients with a wide range of AAOs. It is known that two ofthesevariants(p.Thr251Ileandp.Pro587Leu)arelinked andin cis. Therefore,all POLG patients were considered monoallelic. Motorfluctuationswerepresentin81.8%ofthePOLGPDpatients. Dyskinesias, CI, RBD, and olfactory dysfunction were also frequentlyobserved(70%,80%,70%,and71.4%,respectively).No patientwasspecificallydescribedashavingataxiaorperipheralneuropathy.Onepatient(EP-54)presentedprogressiveexternalophthalmoplegia(PEO). SMPD1 ElevendifferentP/LPvariantsinSMPD1weredetectedin15PDpatients,whofrequentlyexhibitedmotorfluctuationsandRBD(71.4% and75%,respectively).Dyskinesias(61.5%)andhyposmia(62.5%) were also common, although hallucinations/illusions were rarely reported(23.1%). SPR AnLPvariant(p.Asp69Glu;c.207C > G)intheSPR gene was detected ineightpatientswithPD.Motorfluctuationsanddyskinesiaswere highly prevalent in SPRPDpatients(87.5%and75%,respectively); however,dystoniawasobservedin50%.OnlyoneSPRPDpatient reportedafamilyhistoryofPD. VPS13A OnepatientcarriedanLPvariantinVPS13A;however,clinicalinformationforthisindividualisunavailable. 14681331, 2025, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/ene.16499 by Readcube (Labtiva Inc.), Wiley Online Library on [14/04/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License | 7 of 9 PARKINSON DISEASE IN SPAIN Gene burden analysis Asignificantassociationwithveryrarevariantswasfoundforthe DNAJC13 gene, and PRKNshowedasimilarpatterninthecontextof loss-of-functionvariantswithaCADD> 25.Furthermore,PLA2G6 and POLG showed associations withnoncommonloss-of-function variants(Table S5). DISCUSSION This studyaimedtodefinetheclinicalspectrum and implications ofgeneticvariantsacross27genesinaPDpopulationfromsouthernSpain.Ourfindingsrevealedasignificantgeneticheterogeneity,withpotentiallycausativeP/LPvariantsidentifiedin12genes (DCTN1, DNAJC6, LRP10, LRRK2, PRKN, PINK1, POLG, SMPD1, SNCA, SPR, VPS13A, and GBA1). The analysis of typical and atypical forms of monogenic PD revealed several notable differences. Patients with atypical formsexhibitedahigherAAOandareducedfamilyhistoryofPD. Furthermore,weobservedsex-specificdifferencesintheprevalence ofP/LPvariants,particularlyintheLRRK2gene.Ahigherproportion ofwomenwerefoundtocarryLRRK2 variants, driven particularly bytheG2019Svariant(with60%ofthecarriersbeingwomen)but notamongcarriersofotherLRRK2variants.Thisfindingalignswith previousstudiesindicatingafemalepredominanceamongG2019S variantcarriers[15].Althoughotherstudieshavenotreportedthis gendereffect[16],alargemeta-analysisin2018ofLRRK2-related clinical features reported a similar female predominance among G2019Scarriers[17]. LRRK2 emerged as the most prevalent gene, with a notable proportionofpatientscarryingtheG2019Svariant(4%ofcases), similartootherEuropeans[18] but slightly higher than that reportedinthebroaderSpanishpopulation[19].Clinically,LRRK2- associatedPDpatientsexhibitedfeaturessimilartothosewith sporadicPD.However,therelativelylowerprevalenceofCIand RBDsuggestspotentialdifferencesindiseaseprogressionand underlyingneuropathology.Theidentificationofafamilyhistory ofPDexclusivelyamongcarriersoftheG2019Svariantunderscorestherelevanceofspecificgeneticvariantsinfamilialforms ofPDandemphasizestheimportanceofgeneticcounseling[20, 21]. Inlinewithotherstudies,ourcohortshowsahigherfrequency of the deleterious p.Asn52fs variant in PRKN compared to other populations. This variant has been related with an intronic signal in PRKN,describedasanAAOmodifier,highlightingtheimportanceof studying diverse populations to better understand the genetic contributionstoPD[19]. Interestingly,nosignificantdifferencesinAAOwereobserved among LRRK2 variant carriers, although some patients with LRRK2 and PRKNvariantspresentedwithAAOsoutsidethetypicalrange. Thisreinforcesthenotionthatadditionalgeneticandenvironmental factorslikelymodulatediseasepresentation. Moreover,CIwaslesscommonandtendedtoappearlaterinpatientswithPGDassociatedwithLRRK2 or PRKN compared to those withPGDassociatedwithothergenes,suchasGBA1, DCTN1, POLG, and SPR. AlthoughPINK1-associatedPDhasbeenreportedasthesecond mostcommoncauseofautosomal-recessivePD,itwasveryrarein our cohort. TwopatientswerefoundtocarryduplicationsintheSNCA gene; however,theprecisebreakpointsoftheseduplicationscouldnotbe determinated.Interestingly,oneofthesepatientsalsocarriedthe p.T408MriskvariantinGBA1andhadamuchearlierAAO(38vs. 61 years),suggestingthatgeneticinteractionsbetweenthesegenes mayinfluencediseasecourse. VariantsintheGBA1genehavebeenclassifiedaccordingtotheir effectonGDandPD[10, 22].Contrarytopreviousfindings[23], our studyfoundthatpatientscarryingmildGBA1 variants had an earlier AAOcomparedtothosewithseverevariants.Additionally,twopatientscarryingbothaseverevariantandariskvariant(withoutevidenceofGD)presentedwithalaterAAO,suggestinganinteraction between these two variants. Variantsingenesassociatedwithatypicalparkinsonism,suchas LRP10, DCTN1, DNAJC6, POLG, SPR, and SMPD1, were linked to distinct clinical presentations and disease courses, highlighting the importanceofconsideringgeneticfactorsinthedifferentialdiagnosis ofparkinsoniansyndromes. Therefore, although mutations in POLG gene, inherited in an autosomaldominantorrecessivemanner,havebeenlinkedtoPEO [24],inourcohortonlyonepatientexhibitedit,carryingthep.Tyr955Cysvariation,whichhasbeenpreviouslyassociatedwithPEO [24]. This suggests a genotype–phenotype correlation. The LRP10genehasbeenproposedascausativeinautosomal- dominantPD,withorwithoutdementia[25, 26]. One patient in our cohort presented clinical characteristics consistent with LRP10- related PD,althoughthe absenceofa familyhistoryofPD could suggestlowpenetranceforthisvariant. DCTN1genevariantshavebeenlinkedtoPerrysyndrome(PS) [27], arareautosomal-dominant adultonset neurodegenerative disordercharacterizedbyatypicalparkinsonism,depression/apathy,weightloss,andrespiratorysymptoms[28].PathogenicmutationsinPSaretypicallyfoundwithinthecytoskeleton-associated protein glycine-rich(CAP-Gly) domain[29].However,ourstudy identifiedthec.414 + 1G > Avariantoutsidethisdomain.Although carriersofthisvariantwereclassifiedashavingaPGD,thereremains uncertainty about its actual involvement in PD development.Thisvariantwasalsopresentinhealthycontrolsfromthe CSVSpopulation(MAF = 0.0019),althoughwithslightlylowerfrequencycomparedwithourPDpopulation(MAF = 0.0025).Thus, moreevidenceisneededtoconfirmitspathogenicity.Ourfindings suggest that variants in the DCTN1geneoutsidetheCAP-GlydomaincouldhaveuniqueimplicationsinPD,possiblypresentinga riskfactor. HomozygousvariantsinDNAJC6havebeenlinkedtoautosomal- recessiveearlyonsetPD.Inourcohort,onepatientcarriedtwoLP 14681331, 2025, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/ene.16499 by Readcube (Labtiva Inc.), Wiley Online Library on [14/04/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 8 of 9 | GÓMEZ-GARRE et al. variants,withalaterAAOthanpreviouslyreported.Additionaltests arerequiredtoconfirmtheircausality. EightindividualscarriedanLPvariant(p.Asp69Glu;c.207C > G) in the SPR gene, suggesting a founder effect in our cohort. A population-specificroleforthisgenehasbeensuggested[30]. The SPR gene encodes sepiapterin reductase, a key protein in tetrahydrobiopterin(BH4)synthesis,acofactorcriticalinthesynthesisand secretionofneurotransmitterssuchasdopamine.BH4 deficiency hasbeenassociatedwithconditionscharacterizedbymovementdisordersandmentalretardation,amongothers[31].However,there isconflictingevidenceregardingtheroleofSPRinPDpathogenesis [31].DescribedasaVUSinClinVar,thevariantc.207C > Ghasbeen previously associated with autosomal-dominant dopa-responsive dystonia[32].Inourcohort,62%ofSPRpatientspresentedwithCI, andonly50%presentedwithdystonia,suggestingthatthisvariant maybeaPDriskfactorinourpopulation. HeterozygousvariantsinSMPD1(encodingacidsphingomyelinase)havebeenidentifiedasriskfactorsforPD[33]. In our cohort, 1.26%ofpatientscarriedP/LPvariantsinSMPD1, supporting its role inPD.Notably,halfofthesepatientsexhibitedRBD,underscoring theclinicalimportanceofthisgene. Actually,itiscriticaltoincreaseourknowledgeofgeneticrisks for PD in individuals of varied genetic backgrounds and increase theavailabilityofdetailedclinaldataassociatedwiththem.Inthis context,studyingourPDpopulationfromaregionwithhistorically uniquedemographiccharacteristicsinEuropeaddsvaluabledatato ourunderstandingofPDpathogenesis. Toourknowledge,thisisthelargeststudyofPDtodateconductedinsouthernSpain,providinginformationintothegenetic variants and their associated clinical features. However, we acknowledge several limitations, including the inability to establish a cleargenotype–phenotypecorrelationforsomegenesduetothe limitedsizeofthesampleswithPGD,affectingthosegenesand challengesrelatedtomissingclinicaldata.Inaddition,theuseofa targetedgenepanelrestrictedourabilitytoanalyzerecentlydiscovered genes such as RAB32[34]. Finally, it is important to note thattherelativelylowfrequencyofGBA1 pathogenic variants in our population could be due to technical challenges related to the existenceofahighlysimilarpseudogene.Thismayhaveledtoan underestimation ofthetrue prevalenceofGBA1variantsinPD patients. Despite these limitations, our study provides important insights intothegeneticbasisofPDanditsclinicalmanifestations.Theobserved genotype–phenotype correlations and sex-specific differenceshighlighttheimportanceofpersonalizedapproachestoPD diagnosis and treatment. CONCLUSIONS OurresultshighlightthecomplexityofgeneticcontributionstoPD phenotype and prognosis. Comprehensive genetic profiling and personalizedriskstratificationarecrucialforguidingprognosisand therapeutic decision-making in PD patients. There are numerous mechanismsassociatedwithPDpathogenesisandprogressionthat leadtoPD.Furtherinvestigationsintogeneticinteractionsandthe inclusionoffamilyhistoryandsex-specificdatawillbekeytobetter understandingPDpathogenesis. AUTHOR CONTRIBUTIONS Pilar GómezGarre:Conceptualization;supervision;formalanalysis; fundingacquisition;projectadministration;writing–originaldraft; writing – review and editing. Miguel MartínBórnez: Data curation; formalanalysis;methodology;software;writing–reviewandediting. Laura MuñozDelgado: Data curation; investigation; resources; writing – review and editing. Rafael DíazBelloso: Formal analysis; investigation; writing – review and editing. María Teresa Periñán: Formal analysis; investigation; writing – review and editing. Marta BonillaToribio: Methodology; writing – review and editing. Dolores BuizaRueda: Methodology; writing – review and editing. Daniel MacíasGarcía: Resources; writing – review and editing. Silvia Jesús: Resources; writing – review and editing. Astrid AdarmesGómez: Resources; writing – review and editing. Elena Ojeda: Resources; writing – review and editing. Antonio LuqueAmbrosiani: Resources; writing – review and editing. Sergio GarcíaDíaz:Writing–review and editing. Rocío Pineda Sánchez: Methodology. Fátima Carrillo: Resources. Pablo Mir: Conceptualization; supervision; funding acquisition; project administration; writing – review and editing; resources. ACKNOWLEDGMENTS Theauthorswouldliketothankthedonors,theUniversityHospital Virgen del Rocio, and the Biomedical Institute of Seville Biobank (Andalusian Public Health System Biobank and ISCIII-Red de BiobancosPT20/00069)forthehumanspecimensusedinthisstudy. FUNDING INFORMATION Thisworkwassupportedby theSpanish MinistryofScienceand Innovation (RTC2019-007150-1); the Instituto de Salud Carlos III (ISCIII)cofundedbytheEuropeanUnion(PI14/01823,PI16/01575, PI18/01898,PI19/01576,PI21/01875);theConsejeríadeEconomía, Innovación,CienciayEmpleodelaJuntadeAndalucía(CVI-02526, CTS-7685,PY20_00896);andtheConsejeríadeSaludyBienestar SocialdelaJuntadeAndalucía(PI-0471-2013,PE-0210-2018,PI- 0459-2018,PE-0186-2019).P.G.-G.wassupportedbytheNicolás Monardesprogram(C-0048-2017)ofAndalusianRegionalMinistry of Health. D.M.-G. was supported by the Juan Rodés program (JR22/00073) of ISCIII cofunded by the European Union FSE+ (ISCIII-FEDER).L.M.-D.wassupportedbytheRíoHortegaprogram (CM21/00051) of ISCII cofunded by the European Union FSE+ (ISCIII-FEDER).Thefundershadnoroleinthestudydesign,data collection and analysis, decision to publish, or preparation of the manuscript. CONFLICT OF INTEREST STATEMENT Theauthorsdeclarenoconflictofinterest. 14681331, 2025, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/ene.16499 by Readcube (Labtiva Inc.), Wiley Online Library on [14/04/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License | 9 of 9 PARKINSON DISEASE IN SPAIN DATA AVAILABILITY STATEMENT Thedatathatsupportthefindingsofthisstudyareavailableinthe supplementarymaterialofthisarticle. 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SUPPORTING INFORMATION Additional supporting information can be found online in the SupportingInformationsectionattheendofthisarticle. How to cite this article: Gómez-GarreP,Martín-BórnezM, Muñoz-DelgadoL,etal.UnderstandingParkinsondiseasein Spain:Geneticandclinicalinsights.Eur J Neurol. 2025;32:e16499. doi:10.1111/ene.16499 14681331, 2025, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/ene.16499 by Readcube (Labtiva Inc.), Wiley Online Library on [14/04/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License