Review: Understanding Rare Genetic Diseases in Low ...

Rare diseases are caused by function-altering variation(s) in a single gene, and hence are referred to as “single gene disorders” or “monogenic ... Articles HaranathaR.Potteti UniversityofIllinoisatChicago,UnitedStates SajidMalik Quaid-i-AzamUniversity,Pakistan SheikhRiazuddin AllamaIqbalMedicalCollege,Pakistan LiliaRomdhane FacultyofSciencesofBizerte,UniversityofCarthage,Tunisia Theeditorandreviewers'affiliationsarethelatestprovidedontheirLoopresearchprofilesandmaynotreflecttheirsituationatthetimeofreview. Abstract Introduction RareDiseases:SomeFacts ChallengesAssociatedWithRDsandRelatedProgress UnderstandingRareGeneticDisorders:MethodologiesforStudyingMolecularEtiology BurdenofGeneticDisordersinIndia JammuandKashmir:aModelPopulationforStudyingRareGeneticDisorders Bottom-UpApproach Conclusion FuturePerspectives AuthorContributions ConflictofInterest Acknowledgments Footnotes References SuggestaResearchTopic> DownloadArticle DownloadPDF ReadCube EPUB XML(NLM) Supplementary Material Exportcitation EndNote ReferenceManager SimpleTEXTfile BibTex totalviews ViewArticleImpact SuggestaResearchTopic> SHAREON OpenSupplementalData REVIEWarticle Front.Genet.,30April2020 |https://doi.org/10.3389/fgene.2020.00415 Review:UnderstandingRareGeneticDiseasesinLowResourceRegionsLikeJammuandKashmir–India ArshiaAngural1†,AkshiSpolia1†,AnkitMahajan1,VijeshwarVerma2,AnkushSharma3,ParvinderKumar4,ManojKumarDhar4,KamalKishorePandita3,5,EktaRai1*andSwarkarSharma1* 1HumanGeneticsResearchGroup,SchoolofBiotechnology,ShriMataVaishnoDeviUniversity,Katra,India 2BioinformaticsInfrastructureFacility,SchoolofBiotechnology,ShriMataVaishnoDeviUniversity,Katra,India 3ShriMataVaishnoDeviNarayanaSuperspecialityHospital,Katra,India 4InstituteofHumanGenetics,UniversityofJammu,Jammu,India 5IndependentResearcher,HealthClinic,Jammu,India Rarediseases(RDs)aretheclinicalconditionsaffectingafewpercentageofindividualsinageneralpopulationcomparedtootherdiseases.Limitedclinicalinformationandalackofreliableepidemiologicaldatamaketheirtimelydiagnosisandtherapeuticmanagementdifficult.EmergingNext-GenerationDNASequencingtechnologieshaveenhancedourhorizonsonpatho-physiologicalunderstandingofmanyoftheRDsandusheredusintoaneraofdiagnosticandtherapeuticresearchrelatedtothisignoredhealthchallenge.Unfortunately,relevantresearchismeagerindevelopingcountrieswhichlackareliableestimateoftheexactburdenofmostoftheRDs.Indiaistobeconsideredasthe“Pandora’sBoxofgeneticdisorders.”Owingtoitshugepopulationheterogeneityandhighinbreedingorendogamyrates,ahigherburdenofrarerecessivegeneticdiseasesisexpectedandsupportedbytheliteraturefindingsthatendogamyishighlydetrimentaltohealthasitenhancesthedegreeofhomozygosityofrecessiveallelesinthegeneralpopulation.ThepopulationofalowresourceregionJammuandKashmir(J&K)–India,ishighlyinbred.Someofitspopulationgroupsvariablypracticeconsanguinity.Incontextwiththeregion’stypicalgeographicaltopography,highlyinbredpopulationstructureanduniquebutheterogeneousgenepool,ahugeburdenofknownanduncharacterizedgeneticdisordersisexpected.Unfortunately,manysuspectedcasesofgeneticdisordersremainundiagnosedormisdiagnosedduetolackofappropriateclinicalaswellasdiagnosticresourcesintheregion,causingpatientstofaceahugepsycho-socio-economiccrisisandmanyatimesufferlife-longwiththeirailment.Inthisreview,themajorchallengesassociatedwithRDsarehighlightedingeneralandanaccountonthemethodsthatcanbeadoptedforconductingfruitfulmoleculargeneticstudiesingeneticallyvulnerableandlowresourceregionsisalsoprovided,withanexampleofaregionlikeJ&K–India. Introduction Rarediseases(RDs)areprogressive,chronicallydebilitatingand/orlife-threateningheterogeneousclinicalconditionsthataffectalimitedfractionofindividualsfromthegeneralpopulationincomparisontootherprevalentdiseases(Schieppatietal.,2008;Richteretal.,2015;DaneseandLippi,2018).However,thesehavebeenrecentlyrecognizedasoneofthemajorpublichealthconcernsprevalentacrosstheglobe.IthasbeenestimatedthatRDsarealtogetherafflictingasignificantlylargerproportionoftheglobalpopulation(translatingtobillionindividuals)(Schieppatietal.,2008).ThereareseveralchallengesthatmakediagnosisandtherapeuticmanagementofRDscumbersomeandimpedeRDs-relatedresearch.DespiteseveralglobalinitiativestoaddresstheRDs-associatedchallenges,alotofworkneedstobecarriedoutinordertodealwiththisignoredhealthsector.InadevelopingcountrylikeIndia,theRD-relatedresearchhasbeenhamperedbylimitedadvancedclinicalresourcesandfar-to-approach,sporadicallylocalizedgeneticservicescenters(Pradhanetal.,2011;AggarwalandPhadke,2015;Kasthuri,2018). India’sprimitivemigrationhistoryandhighlydiversepopulationarchitecturewhichisepitomizedbysocio-cultural,geographical,linguistic,andreligiousisolationhavebeensuggestedasthemaincontributortothecountry’sgeneticdiversity(Basuetal.,2016).However,biologicalisolationofseveralendogamouspopulationgroupsmighthaveresultedinarelativelyhigherprevalenceofgeneticdisordersinIndia(Pradhanetal.,2011).OneofIndia’shighlydiverseandconglomerationofvariousinbredpopulationgroups,thepopulationofJammuandKashmir(J&K)region,isexpectedtobeanunexploredreservoirofgeneticdisorders.TheJ&Kpopulationischaracterizedbyseveralendogamousgroupswithspecificmaritalaffinities,includingthepracticeofconsanguinity.Inaddition,thegeographicalisolationofvariousgroupsduetocomplexterrainmayprovideJ&Kauniquegeneticarchitectureanddiseaseprofile.Unfortunately,theregionhaslargelyremainedunexploredincontextofgenetic-basedresearch.Thishaspotentiallycontributedtotheignoranceofalargenumberofhereditarydiseaseswhicharelikelyrestrictedtospecificextendedfamiliesorcommunitiesonawhole.Advancedhigh-throughputgenomics-basedapproacheshave,however,outpacedgeneticresearchinJ&Kandprecisediagnosisofsomemonogenicdisorders,whichareotherwiserareinprevalenceacrosstheglobebutarelikelytohaveattainedhigherincidenceintheregionduetohigherinbreedingrates(Raietal.,2016;Kuchayetal.,2019).WeproposethatthepopulationofJ&K,thus,offersaspecialnichetounderstandyet-to-be-exploredunderpinningmolecularetiologyofgeneticdiseases.WithrespecttothepopulationofJ&K,geneticresearchholdsahugepotentialindesigningdiagnosticprotocolsforprevailinggeneticdiseasesanddevelopmentoftherapeutics. ThisreviewprovidesabriefaccountonRDsandtheirprevalence,followedbyadiscussiononthemajorRDs-associatedchallengesingeneral,anaccountonthemethodsthatcanbeadoptedforconductingfruitfulmoleculargeneticstudiesofmonogenicdiseases,andtheexperiencesofgeneticresearchinIndiancontextwithaspecialreferencetoageneticallyvulnerableandlowresourceregionlikeJ&K. RareDiseases:SomeFacts Rarediseasesarecausedbyfunction-alteringvariation(s)inasinglegene,andhencearereferredtoas“singlegenedisorders”or“monogenicdisorders”(Boycottetal.,2017).ThereisnosingleuniversaldefinitionforprevalenceofknownRDs.ThebaseprevalencerateofRDssetbytheWorldHealthOrganization(WHO)isapproximately1in2,000people(Lopesetal.,2018).However,differentnationshavetheirowndefinitionsfortheprevalenceofRDswhichismostlybasedontheprevalenceofadiseaseintheirownpopulation,statusofhealthcaresystemandavailabilityofresources.AgeneticdisorderprevalentintheEuropeanUnion(EU)isconsideredrareonlyifitaffects5orlessper10,000cases,whereastheincidencerateforRDsintheUnitedStatesis7orlessper10,000individuals(LangandWood,1999;Hughesetal.,2005).Thesenumberstranslatetonearly30millionEuropeansand25millionNorthAmericans(approximately1inevery10)affectedbyanyoftheknownRDs(Haffneretal.,2002;Wastfeltetal.,2006).Theincidencerateisestimatedtobe≤2.5casesin10,000-and1in10,000individualsforJapanandAustralia,respectively(LangandWood,1999;Hughesetal.,2005;Zurynskietal.,2008). Nearly7,000distinctRDshavebeendelineatedandnewonesarebeingconsistentlyreportedintheliterature(Ngetal.,2010b).ItisbelievedthatamajorityofRDs(80%ormore)aregeneticinorigin,whereasdistinctunderpinningcausesfortheremainingdisordersarenotwellunderstood(Mckusick,2007;Songetal.,2012a).TheremainingRDsmaybecausedbyenvironmental(forinstance,Jamaicanvomitingsickness,mesothelioma),infectious(forinstance,maternofetalmeasles)orimmunological(forinstance,juvenilechronicarthritis)factors(Guillemetal.,2008;Lopesetal.,2018).ThereisawidevariabilityinseverityandexpressionofdistinctRDs.Manyofthesearecongenitalononsetandcontinuetoexistwithpoorprognosis(lifelong-disabilityand/orearlydeath)overthelifetimeoftheafflictedindividual,whileinsomeindividualsthesymptomsofthedisease(whethersameorother)mayappearlaterinlife,thus,presentingdifficultiesintheirdiagnosis.Ithasbeenestimatedthatnearly50%ofreportedRDsoccurinchildren,30%ofRDspatientsdieduringinfancy(beforetheageof5),and12%ofthemdiebetween5and15yearsofage(Songetal.,2012b). AlthoughRDsaredistinctlydefinedasrareonthebasisoftheirlowprevalence,yettheircumulativeburdenonthepublichealthishuge.TheknownRDsaltogetheraffectasubstantialnumberofestimated350millionindividualsacrosstheglobewhichtranslatestoapproximately10%oftheglobalpopulation(Songetal.,2012b;Boycottetal.,2013).About80%ofthemarecumulativelyaffectedbymerelylesserthan100knownRDs(Luzzattoetal.,2015).Nonetheless,theavailablefiguresfortheirglobalprevalencearealarmingindicatingthatthesealtogetheraffectmoreindividualsthanthosesufferingwithcommondiseases;forinstancediabeteshasanestimatedincidenceof20.8millionand1.4millionamongAmericansandAustralians,respectively(Dunstanetal.,2002;Zurynskietal.,2008). ChallengesAssociatedWithRDsandRelatedProgress DifferenttypesofRDsaltogetherconstituteamajorexigentglobalpublic-healthissueandpresentseveralformidablechallengeswhicharelargelyrelatedtodiagnosis,accesstohealthcareservicesanddisease-specificinterventions,lackofspecializedclinicalpersonnelandspecificinfrastructure,challengesfacedbytheRDpatientsandtheirfamilies,andlackofampleresourcesforRD-associatedresearchanddevelopment(R&D).Furtherthesechallengesinevitablyresultinunreliabilityoftheavailablepatient-registriesand,therefore,vagueepidemiologicaldata.Toaddressthesechallengingissues,adrivetowarduniversalhealthcoverageisrequiredsothattheneedsofRDpatientsgetfulfilledandpublicandgovernment(nationalorinternational)agenciesinvesttheirfundsintofundamentalbiomedicalresearchforunderstandingtheetiologyofdiseasesanddiscoveringtheirnoveltherapeutictargetsandstrategies.However,asignificantscientificandtechnologicalprogresshasbeenwitnessedoverthelastfewdecadesthathavenotablyfilledourknowledgegapsontheunderstandingofseveralRDsandmadetheirdiagnosisandmanagementabitsimpletask.AbriefaccountonmajorRDs-associatedchallengesandtheirrelatedprogresshasbeenprovidedinthissection. ClinicalChallenges,LackofReliablePatient-RegistriesandInaccurateEpidemiologicalData Clinicalchallengesincludelimitedornoavailabilityofappropriateclinicalresources,lackofspecificliteratureandevidence-basedknowledgeordifficultyinassessingknowledgesources,andhasrenderedRDsdiagnosisandmanagementtroublesome(Schieppatietal.,2008).Inadequateclinicalresourcesincludelackofclinicians/expertshavingasoundknowledgeandexperienceinClinicalGenetics,lackofstandardizedclinicalguidelinesandspecificclinicalinfrastructurewhich,thereby,usuallyrequiretheneedypatientstoundergounavoidableclinicalinvestigationsthatendupgettingthemmultipleindefinitediagnoses(KnightandSenior,2006;Zurynskietal.,2008).Itisobviousthataclinician’sdiseasemanagementexpertiseisproportionaltothefrequencyofclinician-patientencounters.SinceRDshavearelativelylessfrequency,suchclinicalencountersareassumedtobenegligiblewhichsubsequentlycontributestoahugeclinicalknowledgegap.Thisknowledgegapisalsocontributedbyinsufficientavailabilityofsourcesofclinicalinformationontheunderpinningcause,patho-physiologyandnaturalcourseofmostoftheRDs,andlimitedavailabilityandendorsementofrelevantguidelinesofthealliedclinicalsocieties.ClinicalheterogeneityofdistinctRDsresultsinmanysubtypesleadingtodifferentclinicalmanifestationsandcoursewhichalongwithlackofrelevantclinicalinformationusuallymakethediagnosisofseveralRDstroublesome.Nodiagnosisormisdiagnosistothepatientsultimatelyresultinnoorlackofreliablepatient-registrieswhichsubsequentlyendsupinthelackofanaccurateepidemiologicaldataonRDs(Zurynskietal.,2008).Unfortunately,oftheestimated≥7,000disordersdefinedas“singlegenedisorders,”adetailedphenotypicinformationonabout5,551ofthesehavebeencurrentlyreportedintheOnlineMendelianInheritanceinMan®database(OMIM®),1whilephenotypicinformationontheremainingdisordersisscantytillnow.AccordingtothedataavailablefromtheOrphanetdatabase,2epidemiologyofonly29%of≥7,000RDshasbeenreported. ProblemsFacedbythePatientsandTheirFamilies RDspatientsandtheirfamiliesfacehugepsycho-socio-economicburdenduetosocialisolation,difficultyinaccessingappropriatehealthcareservices,delayindiagnosis,anduncertaintyabouttheirfutureandfinancialhardships.MostoftheRDsareoftenseverelydisabling,impairtheoverallabilitiesofthepatients,andsubstantiallyreducethequalityoftheirlifeandlifeexpectancy.AbouthalfoftheRDsappearinearlychildhoodwhichmakesithardorimpossiblefortheyoungpatientstoattaintheireducationintheschoolsorcolleges(Zurynskietal.,2008).Thepatientsandtheirfamiliesalsohavetoexperiencesocialstigmaintheformofsocialisolationandoveralldiscrimination.Duetofearofsocialstigmacompoundedbylackofawarenessontheirhealthcondition(whetherbeinganinheritedorgeneticdisorder),manyatimespatientsintentionallydonotgetaclinicalconsultation.This,inturn,directlyimpactsthereliabilityofthepatient-registriessincethesepatientsdonotgetregisteredinthehospitals.PatientsgenerallystruggletofindspecializedclinicianshavingsoundknowledgeandexperienceinClinicalGenetics.ClinicianshavingdeepknowledgeandexpertiseinmanagementofRDsareusuallyconcentratedingeographicallydispersedspecializedcenterswhichmayremainbeyondthepatient’saccessorrequiremostofthepatientstotravellongdistancesortoshifttheirresidencetoanewplaceforgettingadiagnosis(Zurynskietal.,2008).Thesefactorsaltogetherresultindiagnosticdelays,misdiagnosisornodiagnosisandultimatelynoeffectivetreatmentstothepatients.Allthiscomplicatesthemedicalconditionofthepatientsastheyareonlyleftwithanoptionofsufferingwiththeprimaryorsecondaryconsequencesoftheirdiseaseanditslatesequelae(Yangetal.,2013).Besides,along-termsearchforanaccuratediagnosisofRDs,referredtoasthe“diagnosticodyssey,”usuallyincurahugemedicalexpenditurewithunsuccessfulattemptsandconsumptionoflimitedresourceswhichhasitsownfinancialimplicationsonthepatient’sfamilyasraisingadisabledchildisrelativelyexpensivethanforanormalchild(Zurynskietal.,2008;Yangetal.,2013). DiagnosticChallengesofRareDiseases EstablishingtheprecisediagnosesforRDsisusuallydifficult.Theirdiagnosisishighlydependentontheaccesstodiagnostictestingandrequiresdeterminationoftheunderpinninggeneticcause(BoycottandArdigo,2018).Factorsincludingclinicalheterogeneity,co-morbidityandvaryingdiseasecourseamongdifferentRDspatientshighlydemandadifferentialdiagnosisofthediseasewithwhichtheysufferlife-long(Romdhaneetal.,2016;Benjaminetal.,2017).However,establishingdifferentialdiagnosisisameticulousandtime-consumingtaskincurringadiagnosticodysseythatusuallyreliesontheskillsofconcernedclinicianandthediagnosticteststhatapatienthastoundergo,asdiscussedearlier. Since2010,Next-GenerationSequencing(NGS)hasacceleratedtherateofRDsdiagnosis.AlthoughNGShassignificantlyacceleratedtherateofprecisiondiagnosesinRDspatients,butwithadiagnosticyieldofonly25–50%(Lietal.,2018).Fortheremainingsignificantfractionofpatientscomprisingoftheonespresentingcomplexphenotypes,itfailstoyieldanyconfirmeddiagnosisduetoseveraltechnicallimitations(Wengeretal.,2017).However,manyapproachespertainingtogeneticdiagnosishaverecentlyemerged.Anamalgamationofcomparativereanalysisofclinicalaswellasnon-clinicalNGSdatausingvariousnewlyemergeddataanalysispipelinesandsoftwareinconsiderationwithupdatedscientificliteraturecanbeemployedforimprovingthediagnosticyieldthroughNGS.Forinstance,arecentstudyonreanalysisof40unsolvedexomereportslaterledtoaprecisediagnosisinabout10%ofcases(Wengeretal.,2017). ChallengesFacedinRD-RelatedR&DandTherapeutics TheRDs-relatedR&DishighlycomplicatedandimpededbyseveralchallengingissuesincludingahugeknowledgegapabouttheunderpinningcausesofdistinctRDs,lackofaninternationalstandardcodefortheirclassification,assemblingcohortsofpatientsforconductingaresearchstudyowingtotheirdistinctrareprevalence,andinsufficientfundingopportunitiesonRDs-research.Thesechallenges,further,compoundthedeterminationofsuitabletherapeuticinterventionsanddevelopmentofparticulardrugmoleculesfortargetingaspecificclinicalcondition.Nosingleinstitutionand/orcountryhaveasufficientfigureonthenumberofaffectedindividualsforcarryingoutageneralizedclinicalandtranslationalresearch.ThiscouldbemainlyattributedtotheInternationalClassificationofDiseases(ICD)systemusedinmanycountriesfordiseaseclassification.TheICDisnotsuitableformostoftheRDswhichfurtherhampersinclusionofnationalandinternationalpatients’registriesintoreliableepidemiologicaldatabasesandleadtonon-reliableassessmentoftheireconomicandsocialburden(Schieppatietal.,2008).TheothermajorreasonisthatsomeRDsoccursoinfrequently(<1in1,000,000population)thatonlybyconductinginternationalpopulation-basedstudycansufficientnumbersofgeographicallydispersedpatientsbeaccruedforaclinicalinvestigationsothatahigherpowerstudycouldbeyielded.Recruitmentofsuchanumberofpatientsintoaresearchstudyisfurtherimpededbythelackofreliablepatientregistrieswhichsubsequentlyleadtonon-reliableassessmentofdiseaseburden,imprecisecostestimationsofresourceconsumptioninvolvedinthewholeprocessofresearch,drugdevelopmentandclinicaltrialsfordevelopingasuitablediseasemanagementortherapeuticstrategy,andmissingoutapotentialfundingopportunity(Schieppatietal.,2008).Fundingandpolicy-makinghasalsobeenamajorobstacleinestablishinginfrastructureformaintainingregistriesofthepatients(Forrestetal.,2011).Althoughforsomeofthedisorders,nationaland/orinternationalpatientregistrieshavebeenregularlymaintainedbydifferentassociations,yetthereisnorecognitionformostoftheseattheGovernmentlevelduetolackoforlimiteddocumentationofRDspatientsinthelocalhospitals(Schieppatietal.,2008). TakingintoconsiderationofadireneedfortheformulationofauniversalRDsclassificationsystemthatwouldprovidecomprehensiveinformationonknownRDs,theEuropeanRareDiseaseTaskForceoftheHealthandConsumersProtectionDirectorateGeneraloftheEuropeanCommissionincollaborationwithWHOhassetuptheICD-10.Besides,severalotherclassificationsystemsliketheOrphanet,theOMIM®,theSystemizedNomenclatureofMedicine–ClinicalTerms(SNOMED-CT)areavailableforcodingofthesediseases,witheachofthesehavetheirownadvantagesanddisadvantages(Aymeetal.,1998;Mckusick,2007;DeSilvaetal.,2011).Anotherinternationalgroupcalledthe“RareDiseaseTerminologyandDefinitionsUsedinOutcomesResearchWorkingGroup”underaegisofthe“InternationalSocietyforPharmacogenomicsandOutcomesResearch(ISPOR)RareDiseaseSpecialInterestGroup”hasbeenestablishedforthedevelopmentofauniversalRDsdefinition(Richteretal.,2015).Withregularup-gradationofclinicalinfrastructureandupdatingofclinicaldatabasesviacollaborativeefforts,knowledgeofthesediseasesisalsoimproving.SuchanincreaseinfocusoverRDshasbeenmainlyfacilitatedbytherelentlessworkofasignificantnumberoflegislations,NGOcommitteesandpatients’organizationswhichhavehighlightedtheplightofRDspatientsandstressedovertheirtimelytherapeuticmanagement.ThishasfurtherpavedtheimpetustoRDsresearchbyincentivizingpharmaceuticalandbiotechnologycompanies,andinturn,hasproveninstrumentalinfiguringoutdiagnostic,effectivetherapeuticsandpreventativemodalitiesforavarietyofRDs(Richteretal.,2015).UnlessbeforetheUSOrphanDrugAct(1983)andtheEuropeanUnion(EU)Regulation142/2000(2000)onmedicinalproductscameintoeffect,thepharmaceuticalindustrywasignoranttothedevelopmentof“orphandrugs”ordrugsforthetreatmentofdistinctRDs(Haffneretal.,2002;Richteretal.,2015;Auvinetal.,2018).Withtheirenactment,incentiveswereprovidedtopharmaceuticalcompaniesforthedevelopmentofRDsdiagnosticsandtherapeuticstrategiesintheUnitedStatesandtheEU,andsincethen,theirsuccesscouldbedefinedbytheUnitedStatesFoodandDrugAdministration(FDA)andtheEuropeanMedicineAgency(EMA)approvalformarketingofseveralhundredsoftherapeuticdrugsandbiologicalproductsforthetreatmentofmerely5%RDsmainlyincludingrareformsofcancers(Haffneretal.,2002;Haffner,2006;Braunetal.,2010;Richteretal.,2015;Auvinetal.,2018).Althoughtheseapproveddrugshavesignificantlytransformedthetreatmentofonly5%ofthesediseasesforwhichfewerornotreatmentoptionswereavailableearlier,yetthereisalackofavailabilityoftreatmentoptionsforasignificantlyhigherpercentage(95%)ofRDs.Since,theavailabledrugsareextremelyexpensive,theyposeahugefinancialstressontheaffectedfamilies,health-caresystemsanddonoragencies. However,forcreatinganadvancedintegratedresearchpipelineforthedevelopmentofnoveltherapeuticsforRDs,severalgovernmentalandnon-governmentalorganizationsandtheirprogramsincludingtheUnitedStatesNationalInstitutesofHealth’s(NIH)“TherapeuticsforRareandNeglectedDiseases(TRND)”program,the“GeneticandRareDiseases(GARD)InformationCenter”(acollaborativeeffortoftwoNIHcentersnamelytheNationalHumanGenomeResearchInstitute(NHGRI)andtheNationalCenterforAdvancingTranslationalSciences(NCATS)),theNIH’s“OfficeofRareDiseasesResearch”(ORDR),the“NationalOrganizationforRareDiseases”(NARD),the“EuropeanOrganizationforRareDiseases”(EURORDIS),the“InternationalRareDiseasesResearchConsortium”(IRDiRC),the“GeneticAlliance,”the“VerenigingSpierziektenNederland”(VSN),etc.,havebeeninitiatedduringthepastfewyearsforadvocatingRDspatients’needfornational/internationalRDspolicies.Theseinitiativesareprovidingaplatformforpatients’advocacy,researchfundinganddevelopmentofnewadvancedamenitiesforaddressingthechallengespertainingtoignoredRDswithacommonaimofdisseminationoftherelateddataandinformationtothescientificcommunityanddemonstrationoftheiroverallusefulnessinRDsdiagnosticsandtherapeutics.Despite,itisassumedthatthecurrentrateofR&DwouldnotbeabletogeneratetherapeuticsformostoftheRDsforthenextseveralyears.ThisuniversalRD-challengewouldonlybeaddressedbyanunprecedented,largescaleinternationalcooperationbetweendifferentgeographicallyscatteredgovernmentandnon-governmentagenciesandR&Dunitsofdifferentpharmaceuticalcompanies. UnderstandingRareGeneticDisorders:MethodologiesforStudyingMolecularEtiology Ingeneral,thefirst-linediagnosticsincludesdetectionofpathognomonicphenotypicchanges,disease-phenotypecorrelationandbiochemicalanalysisoftheknowndiseasebiomarkersthroughnewborndiseasescreeningmethods,hematologicalevaluation,metabolictesting,andradiographicexaminations.Onceapreliminarydiagnosisisestablished,itsauthenticityprimarilyreliesonthedeterminationofmolecularetiologyofquerydiseasethroughgeneticscreeningofthepatients.Differentgeneticscreeningmethodsincludetraditionalaswellasadvancedcytogenetictechniques,single-genesequencing,andsequencingofapanelofgenesassociatedwithspecificdiseasetypes.Thougheachofthesehavetheirownlimitations,yetarewidelypracticedforthediagnosisofgeneticdiseasesinmanycountriesandarehelpingriskedfamiliesinearlydetectionofapossibledisease,itsearlyinterventionandpreventative/palliativecare. Formanypreviousyears,underpinninggeneticreasonsforseveralhumandiseaseshavebeenprimarilydecipheredthroughlinkageandassociation-basedgeneticepidemiologicalstudies.However,withfurtheradvancementsinseveralbiologicaltechniques,geneticepidemiologicalstudieshaveseenadramaticshiftfromconventionalstudyapproaches(suchastwinstudies,family-basedlinkagestudies)topopulation-basedgenome-wideassociationstudies(GWAS)tothestudiesbasedonthemost-advancedNext-GenerationDNASequencing(NGS)technologies.Ononehandwherepopulation-basedstudydesignslikeGWASandTwin-basedEpidemiologicalStudiesremainuninformativeinunderstandingRDs,specificmethodologiestofacilitatedeterminationoftheirdistinctmolecularetiologyalsoexistbutarecumbersome.Withadventofnew,high-throughputtechnologies,theseeffortshaveimprovedsignificantly.Briefaccountsofthesestudydesignsareprovidedbelow: GeneticLinkageStudies Geneticlinkagestudiesaregenerallyusedformapping/determiningthemostprobablechromosomal/genomiclocico-segregatingwithadiseasephenotypethroughstudieseitherbasedontheinformationofdisease’smodeofinheritance(dominantorrecessive)inthefamilypedigrees(parametricormodel-basedlinkageanalysis)oronsomegenome-widepolymorphicgeneticmarkerssuchasmicrosatellitesintheselectedfamilies(non-parametricormodel-freelinkageanalysis)(DawnTeareandBarrett,2005;TeareandSantibanezKoref,2014).Themainprincipleofthesefamily-basedstudiesisthatphysicallyclosegeneticlocionachromosomeremainhighlylinkedduringmeiosis(thatis,theprobabilityofrecombinationbetweenthemis<50%)andareinheritedindependentlyfromparentstooffspring(Pulst,1999;DawnTeareandBarrett,2005).Ifasetofvariationsarelinkedtogetherinasamehaplotypeinaparticularpopulation,theyareconsideredtobein“linkagedisequilibrium”(LD)andthegeneticlociinLDareconsideredtobelinked(DawnTeareandBarrett,2005).Studiesbasedonlinkagemappinginaffectedandunaffectedsiblingsoraffectedindividualsandtheirparents(child-parenttrio)inmulti-caseextendedfamilies(loadedfamilies)orpedigreesprovideinformationontheco-segregationofgeneticvariationswiththediseasephenotype,andminimizegenotypingerrorandmaximizepowerforassertion(WhittemoreandNelson,1999;Bailey-WilsonandWilson,2011;Barrett,2014).Inthefirstcourseofageneticcharacterizationofaclinicalphenotypeortraitforwhichexactcausalgeneticcomponentisunknown,linkagestudiesareextremelyusefulintheidentificationofageneticlocusthatmightbeharboringadisease-causinggenebeingsharedbetweenaffectedindividualsandneedsjustaveryfewmarkerstocarryoutthesame.However,thisdoesnotmeanthatlinkagestudiescouldmapthecausativegenealone.Forthispurpose,thesemostlyrelyon“positionalcloning”workflows(TeareandSantibanezKoref,2014).Theotherlimitationoflinkagestudiesisthatthepowerofalinkagestudymaygethighlyreducedwhenbothincompletepenetranceandlocusheterogeneityexistsinthestudysubjects(TeareandSantibanezKoref,2014). Linkagestudiesarethemostpowerfultoolintheidentificationofhighlypenetrant,rarevariationsunderpinningraremonogenicMendeliandisordersandbirthdefects(Bailey-WilsonandWilson,2011).Previously,severallinkageworkflowshavehelpedindelineating/mappingtheunderpinninggenesforanumberofRDsinlargemulti-casefamilies(Barrett,2014).Itisworthmentioningthatmolecularetiologyofnearly3,500knownRDshavebeendelineatedprimarilythroughconventionalpositionalcloningmethodsbasedonlinkageanalysisand“homozygositymapping”inwhichinheritancepatternofspecificDNAmarkerssuchassinglenucleotidevariations(SNVs)andmicrosatelliterepeatswasusedforascertainingrecombinationeventsinextendedmulti-casepedigrees.Nevertheless,theremainingdisordershavebeenrefractorytotheseclassicalgeneticscreeningmethodsforseveralreasons:locusheterogeneity,phenotypicheterogeneity,reducedpenetrance,availabilityofonlyasmallnumberofpatientsorfamilieswhichmaynotbesufficientenoughtoattainahigh-powerstudy,andsubstantiallyreducedreproductivefitnessinthepatientsduetoearlydiseaseonsetandsevereeffect(LanderandBotstein,1987;VinkandBoomsma,2002;BotsteinandRisch,2003;Ngetal.,2010c;Boycottetal.,2013).Theseclassicalapproachesremainuninformativeincaseofspontaneousandnon-inheriteddisordersandareexpensive,laborintensiveandtimeconsuming. Next-GenerationDNASequencing Severalrecentlyemergedstate-of-the-artmolecularbiologicaltechniquessuchaschip-basedDNAarraysandseveral“massivelyparallel”orhighthroughputNGStechnologieshashelpedovercometheshortcomingsofpreviouslydescribedstudies.TheinitialinformationonthehumangenomecameintolimelightwithcompletionoftheHumanGenomeProjectthatmostlyreliedonthe“hierarchicalshotgunstrategy”carriedoutthroughtheclassicSangerbiochemistry(Landeretal.,2001;Shendureetal.,2017).After2–3decadesofgradualimprovementsinsequencingbiochemistryandtechnology,NGStechnologieshavevastlyoutpacedourabilityinawaythatpresentlyseveralsamplescanbesequencedsimultaneouslyonasingleplatform(multiplexing)withahigheraccuracyandlessercost(Shendureetal.,2017).Thishassubsequentlyleadtoaneraofunprecedentedproductivity–thatis,progressiveaccumulationandavailabilityofgenomicdataofindividuals(personalgenomesorexomes)ofgeneticallyvariedpopulationsthathasfacilitatedcomprehensiveunderstandingofseveralhumandiseasesandtheirsusceptibilityamongdifferentpopulationgroups(Wangetal.,2013;Middhaetal.,2015).Overthepastdecade,NGShasemergedasaleadingplayerintheodysseyoffindingtheunderpinningcausesofseveralRDs.IthascreatedaparadigmshiftinclinicalgeneticsthrougharelativelyeasierdiscoveryoftheunderpinninggeneticcausesofanumberofRDsatamuchlessercost,thereby,obtainingaprecisedelineationformanyuncharacterizedRDscases(Sawyeretal.,2016;Nambotetal.,2018).Discoveryofnewdisease-associatedgenesandnovelgeneticvariationsthroughdifferentNGSplatformshasefficientlyreinvigoratedourunderstandingontheetiologyofseveralgeneticdisorders,especiallythoseofrareMendeliandisorders,whichhasledtothedevelopmentofdisease-specificdiagnosticproceduresandtherapeutics(Shendureetal.,2017). Sincethepreviousdecade,NGShelpedovercomechallengesassociatedwithtraditionalgenediscoveryapproaches,madeiteasiertoidentifytheunderpinninggeneticetiologyofseveralRDsandprovidedinsightsintotheirdistinctunderpinningbiologicalmechanisms(Shendureetal.,2017).Duetothewiderspan,intrinsiccomplexityandgreatercostofwholegenomesequencing(WGS),wholeexomesequencing(WES)hasrelativelygainedmorepopularityintheidentificationofRDsgeneticssince2010.Itisbasedonthesequenceanalysisofexome(thetotalprotein-codingcontentofthegenome)whichrepresentsabout1%ofthehumangenomeandisknowntoharbornearly85%ofthegeneticvariationsthathavelargeeffectsonthehumanphysiologyandcausedisease-relatedphenotype(Choietal.,2009).Withcontinuousrefinementsinthesequencingtechnologyandhavingaidedindiscoveryofmorethan550distinctnoveldisease-associatedgenes,WEShasemergedasanamazingtechnologicaladvancementthathasenhancedourunderstandingofthestructureandfunctionofthehumangenomeandthemolecularpathwaysunderpinninghumandevelopmentanddiseasebiology(Boycottetal.,2013). Theinitialproof-ofconceptforthepotentialroleofWESinRDsdiagnosticsandresearchcameintolimelightwiththeidentificationofgenesresponsibleforKabukiSyndrome(OMIM:147920)andMillersyndrome(OMIM:263750).UsingWESandtargetedsequencing,theresearchersatUniversityofWashingtonhadidentifiedandreporteddistinctMLL2gene(currentlyknownasKMT2Dgene)andDHODHgenevariantsresponsibleforKabukiSyndromeandMillersyndrome,respectively,inmajorityoftheafflictedindividuals(Ngetal.,2010a,b).Later,proteintruncatingPRRT2variationswereidentifiedinafamilialcaseofParoxysmalKinsesigenicDyskinesiathroughWES(Chenetal.,2011).VariationsinSMARCB1,SMARCA4,SMARCA2,SMARCE1,ARID1A,andARID1BgenesencodingproteinsbelongingtoSWI/SWFchromatin-remodelingcomplexwereidentifiedusingWESandimplicatedinararecongenitalanomalycalledCoffin-Sirissyndrome(OMIM:135900)(Tsurusakietal.,2012).AcaseofectrodactylyandlethalpulmonaryacinardysplasiaassociatedwithFGFR2variationsina5dayoldbabyborntonormalconsanguineouswasidentifiedusingWES(Barnettetal.,2016).Evidently,therehasbeenaproportionalflurryofreportsatanacceleratingrateondiscoveryofRDs-associatedgenesandnovelvariationswhich,inturn,hasacceleratedprecisediagnosisofanumberofsuspectedcharacterizedaswellasuncharacterizedRDssincetheadventofWESin2010(Yangetal.,2014;Shendureetal.,2017).WEShasbeensuccessfulinleadingtoprecisediagnosesinanestimated30–50%ofRDscasesinclinicalsettings(FresardandMontgomery,2018).Despite,themolecularetiologyofnearlyone-thirdofRDsisstillunknownandremainstobediscovered(BoycottandArdigo,2018).WESisstillnotthegold-standarddiagnosticapproachforclinicallyuncharacterizeddiseasesowingtoitstechnicallimitationsthatrestrictthedeterminationofvariationsinnon-codingand/orregulatorygenomicregions,structuralvariationsandcomplexgeneticmechanismssuchassomaticmosaicismandgeneimprintingunderlyingasubstantialfractionofRDs(FresardandMontgomery,2018).Here,comingtorescuefromthelimitationsofWESisanotherNGStechnology,thatis,theWGS.BesidestheidentificationofSNVs,WGSisalsocapableofdeterminingstructuralaswellasepigeneticchangesinthegenome(Wangetal.,2015). SuccessofNGSmostlyreliesontheaccuracyofdataminingtoolsusedfortheanalysisofsequencingdata.ThereisampleavailabilityofrawNGSdataprocessingandvariantcallingtools.Initially,processingofrawNGSdataincludesseveralstepssuchasqualitycheck,adaptertrimming,post-trimmingqualitycheck,PCRduplicateremoval,alignmentofsequencingreadstothereferencegenomeandvariantcalling.FastQCisatoolusedforcheckingqualityreportsofpre-andpost-trimmingsequencingdata(Andrews,2010).Trimmomaticisusedfortheinitialqualitytrimmingofthereads(Bolgeretal.,2014).MarkDuplicatesutilityofPicardtoolisusedforPCRduplicatesremoval(Wysokeretal.,2013).Bowtie(Langmeadetal.,2009)andBurrows-WheelerAlignment(BWA)tool(LiandDurbin,2009)areusedforalignmentofthereadstothereferencegenomes.ToolsthatareusedforvariantcallingfromNGSdataincludeDISCOVAR(Weisenfeldetal.,2014),genomeanalysistoolkit(GATK)(Mckennaetal.,2010)forSNVs,DELLY(Rauschetal.,2012),GASV(Sindietal.,2009),LUMPY(Layeretal.,2014)forStructuralVariations,CoNIFER(Krummetal.,2013),CONTRA(Lietal.,2012),XHMM(Fromeretal.,2012)forCopyNumberVariations,Platypus(Rimmeretal.,2014)forSNVs,indels,repeatelements,denovovariations,andSAMtools(Lietal.,2009)forSNVsandshortindels.ThesetoolsarecapableofminingdifferenttypesofvariationsfromNGSdatawithahigheraccuracy. BurdenofGeneticDisordersinIndia PopulationstratificationinIndiahasaddedtothecountry’spopulationdiversityandgenepool.AhighinbreedingrateinsomespecificIndianpopulationclustershinttowardarelativelyhigherburdenofspecificgeneticdiseasesandfoundervariations.Indiais,thus,consideredasauniquehotspotofinheritedgeneticdisordersandvariations.WithIndia’srecentacceleratingclinicaldemographicswitchtonon-communicablediseases,congenitalmalformations/birthdefectsandgeneticdisordershaveemergedasthemajorcausesofmortalityintheperinatalperiod(VermaandBijarnia,2002).AhigherburdenofinheritedgeneticdisordersandvariationshighlightstheimportanceofdissectingthegeneticetiologyandpathogenesisofseveralrecessivedisordersandcomplexdiseasesinIndia. Briefaccountsonthepopulationstratification,inbreeding,geneticdisorders,andgeneticservicesinIndiaareasfollow: PopulationArchitectureofIndia India,theworld’ssecondmostpopulouscountry,holdsthedistinctionofbeingthesixthlargesthometomorethanone-sixthoftheglobalhumanpopulation(AggarwalandPhadke,2015).Duringprehistoricandhistorictimes,thecountryhasservedasamajorcorridorfordifferentmigratorywavesofanatomicallymodernhumans(MajumderandBasu,2014).ThesemigratoryeventshavesignificantlycontributedtohighheterogenicpopulationstratificationintheinhabitingIndianpopulationgroupsintermsoftheirreligious,socio-cultural,linguisticandracialbackgrounds.Inanevolutionarycontext,thepopulationdiversityinIndiahasbeenconsideredasaresultofadmixtureofmultiplemigratorypopulationsandinvadersbelongingtothenorthwesternandeasterncornersoftheglobethathadenteredintothecountrybyfollowinglandandcoastalroutes(Basuetal.,2016).GeneticstudieshaveindicatedthatthemodernIndianpopulationisasanadmixtureoffivelargeancestral,geneticallydivergent,heterogeneouspopulationgroups.TheancestralgroupscomprisingtheIndianmainlandersincludethe“AncestralNorthIndian(ANI),”“AncestralSouthIndian(ASI),”“AncestralAustro-Asiatic(AAA)”and“AncestralTibeto-Burman(ATB),”andaseparateancestralgroupnamedthe“AncientAncestralSouthIndian(AASI)–related”forthepeopleoftheAndamanarchipelago(Basuetal.,2016;Narasimhanetal.,2018).However,thecurrentIndianpopulationcanalsobecategorizedintofourethno-racialgroupsnamelythe“Australoids,”“Caucasoids,”“Mongoloids,”and“Negritos,”stratifiedintomorethan4,000anthropologicallydistinctpopulationgroupshavingtheirindividuallinguisticprofiles(AggarwalandPhadke,2015).Basedonreligious-socio-culturalbackgrounds,theIndianpopulationisfurthersub-classifiedintodifferentreligiousgroups,castesandtribes.Avastmajority(∼80%)oftheIndianpopulationcomprisesoftheHindupopulationgroupswhichisfurthersub-dividedintocastesandsub-castes,about8%isrepresentedbythetribalpopulationswhiletherestofthepopulationiscomprisedofotherreligiousgroupssuchasMuslims,Christians,Buddhists,Jews,Sikhs,andothers(IndianGenomeVariationConsortium,2005). InbreedinginIndia ThecontemporaryIndianpopulationgroupsisanagglomerationofseveralthousandsofseparateendogamousgroups(>50,000)residingintopographicallyalienatedpockets,manyofwhichhavebeeninexistenceforatleast100generations(Mcelreaveyetal.,2005).Thesegroupsrepresentdistinctconservativebreedingpoolsinwhichmarriagesareusuallyrestrictedwithinsamereligion,casteandbiraderiaccordingtothecustomsdatingbacktosome3,000years(Bittlesetal.,2002).InIndia,theestablishmentofmaritalrelationshipsamongindividualpopulationgroupsisguidedbydifferentregulationswhichareusuallybasedontheirdistinctreligious-socio-culturalnorms.Forinstance,theHindureligiousgroupisstructuredintoseveralhierarchicalsocio-culturalgroupscalledvarnas(Brahmins,Kshatriyas,Vaishyas,Shudras)whicharesub-dividedintocastes(orjatis)(IndianGenomeVariationConsortium,2005).Thepopulationgroupsbasedonvarnasandcastesareusuallyendogamous.Eachcastegroupissub-dividedintopatrilinealgroupsorsub-castesknownasgotras,eachrepresentinganexogamousgroup.Thetribalsectionsortheancestor-worshippersaremainlyendogamous(IndianGenomeVariationConsortium,2005). Nevertheless,consanguinityisalsopracticedasacustominsomespecificIndianpopulationgroups,withrateofconsanguinityrangingbetween20and30%(Bittles,2002a).AmongtheIndianHindus,non-uniformviewspertainingtoconsanguinitysubsistwithmorecomplexmarriageregulations(BittlesandBlack,2010a).Accordingtoageneralprohibitiondatingbackto200BC,themajorityHindupopulationinthenorthern,eastern,andnorth-easternstatesrigorouslyforbidconsanguinitybyavoidingsame“gotra”marriageincludingthosebetweenkinsandbetweenamanandhisfather’ssister’sormother’ssister’sormother’sbrother’sdaughter;thoughalongtraditionoffirst-cousinmaritalunion,uncle-niecemarriageandmarriagebetweenamanandhismaternaluncle’sdaughterisprevalentamongDravidianHindusbelongingtosouthernIndiaandinmostChristiandenominations,mostlyreportedfromruralcommunitiesandamongtheunderprivileged(includingthepoorest,illiterate,andleasteducated)groups(HussainandBittles,2000;Bittles,2002b).First-cousinmarriage,particularlybetweenamanandhismaternaluncle’sdaughter,isgenerallypreferredinAndhraPradesh,Karnataka,andTamilNaduandinKerala,Goa,andsouthernMaharashtratoalesserextent(Bittles,2002a).TheMuslimreligiousgrouppracticeconsanguinityatahigherratewithnocomparablenorth-southdistinctioninconsanguinity,asindicatedinTable1(Bittles,2002b).AlthoughconsanguineousmarriagesareforbiddenintheSikhreligion,someminoritySikhgroupsinIndiaappeartoexerciseflexibilityintheobservanceofthisproscriptionbyallowingfirst-orsecond-cousinmarriages(Table1)(Bittles,2002b). TABLE1 Table1.RateofconsanguineousmarriagesamongvariousreligiousgroupsinIndiaduring1992–1993(Bittles,2002b). IndiaasaTroveofGeneticDisorders Ofthe350millionglobalestimateofRDspatients,Indiaaloneisahometoapproximately70million(equatingto1in20)patientswithanyoftheknownprogressive,life-threateningandchronicallydebilitatingrarehealthanomalieswhichencompassawiderangeofsystemicdisordersincludingimmunodeficiencysyndromes,blooddisorders,skeletaldisorders,neurologicaldisorders,andmanymore(KumarH.etal.,2017).ThisindicatesthatthecumulativeburdenofRDsisquitesignificantinIndiaincomparisontotheworldaverageowingtoitshighlyinbredpopulationstructure(KumarH.etal.,2017).Unfortunately,thereisnostandarddefinitiontodescribetheprevalenceofaRDinIndia;thoughOrganizationforRareDiseasesIndia(ORDI)hassuggestedathresholdfordefiningadiseaseasrareifitafflicts1in5,000individualsinIndia(Rajasimhaetal.,2014). ReligiousrestrictionscompoundedbythegeographicalisolationofsomeIndianhabitatsduetothecountry’sdiversetopographyhavecontributedsignificantlytoarelativelyhigherrateofinbreeding(population-inbreedingcoefficientofIndia=0.00–0.20)and,thus,servedasbarrierstorandommatingandfreegeneflowleadingtothedistinctgenepooloftheIndiansub-populations(Bittlesetal.,2002).Inparallel,thereisalsoarelativelyhigherburdenofspecificdiseasesusuallyrestrictedand/oruniquetospecificIndianethnicgroups,sub-castes,tribesorclansundertheinfluenceoffoundereventsthathaveoccurred50–100generationsback(Bittles,2002a;Mcelreaveyetal.,2005;Pradhanetal.,2011;Dixitetal.,2015).Alistofsomeofthedisease-associatedfoundervariationsdeterminedfromtheIndianpopulationhasbeenindicatedinTable2.Oneofthebiggestexamplessupportingtheassociationofendogamyandcommunity-specificdiseaseburdeninIndiaisahighlyendogamousAgarwalcommunity.GeneticdiseasessuchasMegalencephalicLeukodystrophywithsub-corticalcysts(OMIM#604004),PanthothenateKinase-AssociatedNeurodegenerationwithBrainIronAccumulation(PKAN;OMIM#234200)andSpinocerebellarataxiatype12(OMIM#604326)associatedwithanumberoffoundervariationsinCAPN3(OMIM#114240),MLC1(OMIM#605908),PANK2(OMIM#606157),PPP2R2B(OMIM#604325)genes,respectively,arefrequentlyobservedinthisIndiancommunity(Table3).GeneticdisordersreportedfromIndiaareenlistedinTable4.BesidesahigherrateofinbreedingandfoundereffectsinsomeIndiansub-populationgroups,otherfactorsincludinglackofdiagnostic,diseasemanagementandrehabilitationinfrastructureinthecountry,itslargepopulationsizeandhighbirthratehavecontributedsignificantlytoarelativelyhigherincidenceofrecessivedisordersinIndia(Verma,2000;VermaandBijarnia,2002). TABLE2 Table2.Alistofsomeofthedisease-associatedfoundervariationsprevalentinIndia. TABLE3 Table3.AtablerepresentingsomeofthefoundervariationsprevalentinahighlyendogamousIndianAgrawalcommunity. TABLE4 Table4.TheestimatedrateofincidenceofcommongeneticdisordersinIndia(Ankalaetal.,2015). StatusofRDs-GeneticServicesinIndia Likeotherdevelopingcountries,IndiatoofacesanumberofchallengesindealingwithRDsasamajorpublichealthissue.Thereisalackoftrue,reliablequantitativedataonindividualaswellascumulativeprevalenceofRDsatthenationalandregionallevelandtheepidemiologyofassociatedmorbidityandmortality.ThesefactorshavefurtherimpededtheRDs-relatedreliablecostestimationsandtheimplementationofrelevantresearchanddevelopmentprogramsforRDstherapeuticmanagementinthecountry.Otherchallengesincludelackofavailabilityofspecializedmedicalpersonnel,moleculardiagnosticinfrastructure,andstandardtherapeuticdrugsandprotocolscommittedtounderstandingandmanagementofRDs.ThegeneticbackgroundoftheIndianpopulationisalsonotwellunderstoodduetoitsunder-representationinthemajorglobalgenomicstudies. Severalindividualresearchgroups,pharmaceuticalcompaniesandpatientassistanceorganizationsandtheircharitableprogramsareconsistentlyworkingonadvocacyoftheimportanceofRDsdiagnosis,researchanddrugdevelopmentandframingofanationalRDspolicyinIndia.TheseincludeORDI,IndianOrganizationforRareDiseases(I-ORD),FoundationforResearchonRareDiseasesandDisorders-RareDiseasesIndia(FRRDD-RDI),OpenPlatformforRareDiseases(OPFORD),GenomicsforUnderstandingRareDiseasesIndiaAllianceNetwork(GUaRDIAN)andmanymore(Rajasimhaetal.,2014).SeveralcasereportsfordistinctRDsfromIndiahavebeenpublishedduringtherecentyears.TheseincludeaseriesofreportsonaccuratediagnosisofRDscasesincludingAcidSphingomyelinase(ASM)-DeficientNiemann-PickDisease,AllgroveorTripleA(AAA)syndrome,EthylmalonicEncephalopathy,Fanconi-BickelSyndrome,Fructose-1,6-biphophataseDeficiency,Homozygousfamilialhypercholesterolemia,MucopolysaccharidosestypeIandtypeII,Rhizomelicchondroplasiapunctatatype1,progressivepseudorheumatoiddysplasiaandmanymoreusingSangerSequencingofdisease-associatedgenes(Phadkeetal.,2010;Dalaletal.,2012;Keharetal.,2014;Bijarnia-Mahayetal.,2016;Ranganathetal.,2016;Setiaetal.,2016;Uttarillietal.,2016;Anguraletal.,2018;Bhaietal.,2018).Besides,thereareseveralcasereportsindicatingthemajorapplicationofNGStechnologyinRDsdiagnosticsincludingthatofichthyosis,raresyndromesofmineralocorticoidexcess,dystrophicepidermolysisbullosa,conedystrophy,sporadicacrokeratosisverruciformis,Dowling-Degosdisease,SpasticParaplegia79andmanymore,(Guptaetal.,2015,2016,2017;KaruthedathVellarikkaletal.,2016;Narayananetal.,2016;DasBhowmiketal.,2018;Virmanietal.,2018).ManyofthesediagnosedRDsaresorarethattheyhavebeenreportedeitherdistinctlyorasspectrumdisordersforthefirsttimefromIndia.Inthiscontext,thereisadireneedofconductinggeneticscreeningoftheuncharacterizedRDspatientsandtheirfamiliesandpopulationgeneticstudiesforelucidatingtheloadofpathogenicvariationsharboredbytheIndianpopulationandestablishingitsownpopulation-basedgeneticvariationdatabase. JammuandKashmir:aModelPopulationforStudyingRareGeneticDisorders Despitecontinuousglobalefforts,averylittleattentionhasbeenpaidtothemostchallenginghealthissueofRDsinIndiawithspecialreferencetoregionlikeJ&K.J&KislocatedinthenorthernpartoftheIndiansub-continentinthevicinityoftheKarakoramandwesternmostHimalayanmountainranges(outerhills,middleHimalayas,andinnerHimalayas)(BhasinandNag,2002b).TheregionisbeingborderedbycountriessuchasPakistaninthewest,Afghanistaninthenorth-westandChinainthenorth-east(BhasinandNag,2002a).Topographically,J&Kisdividedintothreemainisolateddivisions,namelyJammu,Kashmir,andLadakh(Sharmaetal.,2018).Theregionischaracterizedbytoughmountainousterrainsthathaverenderedgeographicalisolationofitsheterogeneouspopulation.J&Khasbeenconsistentlyundergeo-politicalturbulencesandterrorismand,ingeneral,haslowresourcesincludingbasicfacilitiesofeducation,feeding,healthcare,electricity,sanitation,andtransportation.Owingtogeographicalisolationandreligioussocio-culturalnorms,majorityoftheinhabitingpopulationishighlyendogamousandconsanguineous. AhugeburdenofRDshasbeensuspectedinJ&K.AccordingtotheinformationavailablefromRareDiseasesIndiaOrganization,morethan0.7millionindividualsfromJ&KarelikelysufferingfromRDs.Nevertheless,thisinformationappearstobevagueduetolackofacentralizedpatient-registrywhichhasresultedinanunfortunatelackofaccurateepidemiologicaldataondistinctRDsprevalentintheregion.Addingfurthertothisisthelackofappropriateclinicalinformationonthesediseases,lackofawarenessamongthegeneralpopulationandbasicfacilitiessuchastertiarycarehospitals,medicalpersonnel,diagnosticfacilities,andscantyR&Dcenters.AlthoughthereisamagnanimousquantitativeburdenofRDsinJ&K,butthesehavemostlyremainedclinicallyignoredowingtoaforementionedissues.Unfortunately,thepopulationgroupsofJ&Khavealsolargelyremainedunder-representedinthepreviousIndiangeneticstudiesuntilrecently,resultinginalackofinformationonthegeneticmake-upoftheJ&Kpopulationstrataandtheirdiseaseheritage.Incontextwiththeregion’sgeographicaltopographyandhigherinbreedingrate,theunexploredcasesofgeneticdisordersarelikelytobeassociatedwithfoundereventsuniquetodifferentpopulationgroupsorfamilieswhichmayalsoholdcluesfortheirevolutionaryperspectives.Thegeneticassessmentoftheaffectedindividualsfromtheregionis,thus,aneed-of-the-hourandessentialforthedevelopmentofappropriatetherapeuticinterventions.Thiswouldaidingeneticcounselingandmanagementofthereportedgeneticdiseases. Inthissectionofthereview,briefaccountsonthepopulationarchitectureofJ&K,burdenofgeneticdisordersandgeneticstudiesconductedintheregionhavebeenprovided. PopulationArchitectureofJ&K ThepopulationofJ&Kistypicallyheterogeneouswithmanyanthropologicallywell-defineddistinctethnicandreligiousgroups.TheethnicgroupsresidinginJ&KmainlyincludeArghuns,Bakerwals,Baltis,Bedas,Bodhs,Brokpas,Changpas,Dogras,Garras,Gujjars,Harijans,Kashmiris(PanditsandMuslims),Khatris,Kishtwaris,Ladakhis,Mahajans,Mons,PaharisandPurigpas(BhasinandNag,2002a).ThemajorreligiousgroupsofJ&KincludetheBuddhists,Christians,Hindus,Jains,Muslims,Sikhsandothers.AccordingtothePopulationCensusofIndia–2011,Muslims(68.31%)constitutethemajorinhabitingpopulationstratainJ&K,followedbyHindus(28.44%),Sikhs(1.87%),Christians(0.28%),Jains(0.02%)andothers(0.17%).ThecontemporarypopulationofJ&Kspeakslanguagesbelongingtothreedifferentlinguisticfamilies–theIndo-European,Tibeto-Burman(inLadakh)andvariousdialectofDardic(inKashmir)fromIndo-Aryanlanguagegroup(Sharmaetal.,2018).Thesepopulationgroupsusuallyresideinsmall,geographicallyandsociallyisolatedpocketssincemanycenturiesandarevariablyreinforcedbytheirsocietalorreligiouscustomstopracticeendogamyorconsanguinity.EndogamyispreferredbyalmostallthepopulationgroupsofJ&K,whereastheconsanguinityismostlypreferredbytheMuslimpopulationgroups(BhasinandNag,2002b).AstudyfromtheRajouriandPoonchareasofJ&KhasindicatedthattherateofconsanguinityisquitehighamongthenativeMuslimpopulationswhichaccountnearly35–50%withnearly70–80%oftheinbreedingoccurringamongfirstcousins(FareedandAfzal,2014a,2017).However,thereportedfigureswerelimitedtotheMuslimpopulationofonlytwoareasinJ&Kandmightvarybetweendifferentregionsandcommunities. BurdenofGeneticDiseasesinJ&K ThepopulationstratificationofJ&Kholdsahighsignificanceinthehistorical,religious,socio-culturalandlinguisticdiversificationoftheIndianpopulation.Ithasbeensuggestedthatvariouspre-historicandhistoriceventsofmigrationsandimmigrationstowardtheIndiansub-continenthaveoccurredthroughJ&Kalongthenorth-easternandnorth-westernroutes(BhasinandNag,2002a;Pandithetal.,2015;Sharmaetal.,2018).IntenseendogamywithinthepopulationgroupsofJ&Khasresultedinrestrictedgeneflowandgeneticisolationforseveralcenturies,thus,makingthemuniqueintermsoftheirgenepoolanddiseaseheritage.Itisknownthatgeneticallyisolatedandhighlyendogamouspopulationgroupshavehigherlevelsofgenetichomozygosityand,thus,arerelativelymorepronetohaveahigherburdenofgeneticdisorders,especiallyrecessiveRDs(Woodsetal.,2006;BittlesandBlack,2010).IncontexttoJ&K,thisfactcouldbesupportedbyastudyconductedon995individualsbelongingtosixMuslimpopulationgroups(includingGujjarsandBakerwals,Khans,Maliks,Mirs,Mughals,andSyed)fromRajouriandPoochareasinJ&KwhichhasindicatedarelativelyhigherlevelofhomozygosityforRhesusfactoralleles(Fareedetal.,2014). Sincepastfewyears,anumberofsuspectedcasesofgeneticdisorders(includingnew/knownmonogenicdiseasesandotherknowngeneticdiseaseswithatypicalclinicalfeatures)havebeenreportedfromJ&Kregion.Casesofchromosomalgeneticdisorders(suchasDownSyndrome,TurnerSyndrome,KlinefelterSyndrome,PatauSyndrome;Table5),anemias,blooddisorders(includingThalassemia),congenitalanomalies,disordersofsexdevelopment,metabolicdisorderslikeG6PDdeficiency,neurologicaldisorders(Table6)andothersarefrequentlyreportedinJ&K(Razdanetal.,1994;Kumaretal.,2010;Upmaetal.,2010;VasudevandSawhney,2014;Araetal.,2018;Daretal.,2018;Hockhametal.,2018).Thedetrimentaleffectsofconsanguinityandinbreedingdepressiononchildhealthandmortality,cognitivebehaviorandfertilityandanincreasedriskofcardiovasculardiseasesinsmallpopulationgroupsfromJ&Khasalsobeenreported(BhasinandNag,2002b;FareedandAfzal,2014a,b,2016;Fareedetal.,2017).However,theJ&Kpopulationhasbeenlargelyunder-representedinthesurveys/screeningstudiesconductedincontextwithdiseaseincidence.ThesestudieswereusuallyrestrictedtosmallregionalpocketsindifferentareasofJ&Kand,therefore,havecreatedahugegapintheliteratureonincidenceoftheprevalentdiseases. TABLE5 Table5.FiguresonthereportedcasesofchromosomalanomaliesinJ&K(Kumaretal.,2010). TABLE6 Table6.Prevalenceofvariousneurologicaldiseases,asreportedinruralKashmirduring1986(Razdanetal.,1994). TherearesomecuriouscasesofcertainclinicalconditionsthatarehighlyprevalentindifferenthotspotsorvillageslocatedinisolatedremoteareasofJ&K.Thesereportsindicateahigherburdenofsomediseasesintheregionrestrictedtoindividualvillagesorfamilies.Informationonthesecaseshasbeenmostlyportrayedovernarrowapproachesintheformofgeneralizedorjournalisticinformation.Summarizedreportsonsomeofthesehavebeenprovidedasfollows: Report1–TheVillageofSilence,J&K:DadhkaivillageofBhalessa,oftenknownasthe“VillageofSilence,”inDodaareahasbeenreportedtohaveahighincidenceofhearingloss.Asperthereports(bothjournalisticaswellasliterary),thishamletisinhabitedbymorethan2,500individualswithover95membersbeingdeafandmute.Atleastthereisonememberineachfamilywhocanneitherhearorismute.However,ithasbeenreportedthatthereisanincreaseinnumberofaffectedindividualsintheregionduringthepastdecades.ThevillageislocatedinaremoteisolatedareacharacterizedbyaverytoughterrainintheHimalayanMountains.Itlacksbasicfacilitiesofnutrition,healthcare,immunizationandrehabilitation,education,electricity,roads,andtransportation.Itisconnectedtothemainlandthroughafoot-bridge.Someoftheindividualshavebeenfoundtobeiodine-andsalt-deficientbytheclinicians.Theresidentsofthevillageareoftheopinionthatthecommunitymighthavebeenundersomecurse.ThemajorityofthepopulationinDadhkaivillagebelongstoascheduledtribe“Gujjar”communityofMuslimreligion.However,givenimpetustoprolongedendogamouspracticesamongthiscommunity,theclinicalconditionmusthavegeneticoriginand,thus,attainedarelativelyhighincidenceinthevillage.PedigreesoffourfamiliesfromDadhkaiarealreadyavailableintheliterature(Rainaetal.,2017). Report2–AraivillageinPoonch,J&K:TherearereportsonamysteriousskeletaldiseasehighlyprevalentinArai,aclusterofthreevillages,inMandiareaofPoonch–J&K.Thesymptomsofthediseaseusuallyappearbetween4and8yearsofageandprogressinseveritywithadvancingage.Theaffectedindividualsdevelopenlargedjointsthatlookknobbyinappearance,paininjoints,gaitdisturbance,abnormalposture,andshortstature.Morethan100individualsovertwoextendedfamiliesinthevillagehavebeenrenderedcrippledfortheirlifewiththisdisease.AraiislocatedintheremotemountainousterrainsoftheHimalayasandhasremainedisolatedduetolackoftransportfacilitiesuntiltheroadswerelaidrecentlyinthevillagearea.Theregionalsolacksbasicfacilitiesofeducation,electricity,healthcare,andsanitation.TheresidentsofAraiareeconomicallypoor.Owingtotheirincurableailmentandlackofmedicalawareness,localbeliefofacurseresultingintheskeletaldisorderinfamilieshasremainedfordecadesand,thus,familieshavegivenuponmedicalconsultations.RecentextensiveeffortsandastudyreportedfromAraivillagecharacterizedthedisorderasprogressivepseudorheumatoiddysplasia(PPD),anautosomalrecessivegeneticdiseasewithvariantsingeneWISP3asthecause(Raietal.,2016). Report3–VillageofdeafandmuteinParalkot,J&K:InaremotevillagenamedParalkot,about80%ofthepopulationhasbeenreportedtobedeafandmute.ThevillagefallsinSawjianareaofMandisub-division,Poonch,J&KneartheLineofControl(LoC),andlacksbasicfacilitiesofeducation,health-careandtransportation.Majorityoftheinhabitingpopulationispoorandarelaborers.Thevillagershavedifferenttelltalesregardingtheirclinicalcondition.Somebelievethatthepersonwhoseesthefairiesresidinginanearbymountainbecomedeafandmute,whileothersbelievethereareevilspiritsintheareaorsomecurseshadowedovertheirfamilies.Ithasbeenreportedthatowingtotheirclinicalcondition,about30familiesfromthisvillagehadmigratedtoPakistanoccupiedKashmir(PoK)during1990–1991.However,thevillagersmarrytheircloserelativeswithinthesamevillage.Owingtotheirendogamousbackground,therecouldbealikelygeneticcausefortheirhearingdisabilityasindicatedbyourpreliminary(unpublished)findings. TherearefurtherseriesofcasereportsonRDsincludingEllis-vanCreveldSyndrome,EpidermolysisBullosa,Fabry’sdisease,Fahr’sdisease,HereditaryStomatocytosis,Holt-OramSyndrome,Ollier’sdisease,differenttypesofPorphyria,RogersSyndrome,Wolframsyndrome,congenitalanomalies,andmanymorefromdifferentregionsofJ&K(BhatJ.I.etal.,2010;BhatY.J.etal.,2010;Bhatetal.,2015;Qayoometal.,2010;Ganieetal.,2011,2012;Majidetal.,2012;Shoibetal.,2012;HassanandKeen,2013;Rasooletal.,2015;Wanietal.,2016;KumarS.etal.,2017;NazirandChalkoo,2017;Ilyasetal.,2019).MostofthesearemerelypresentedascasereportsandtheunderpinningmolecularetiologyofmanyofthereportedaswellasunreportedRDscasesfromJ&Khavebeendueforyears.Furtherthereisalikelihoodofmoreunder-representedoryettobeidentifiedRDscasesfromJ&K. RDs-AssociatedGeneticStudiesConductedintheRegionsoFar Withadvancesinthegenomeresearchtechnologies,researchershavebeenrecentlysuccessfulindelineatingsomecasesofRDsprevalentinJ&K.Thefindingsofthesestudieshaveimmensecontributioninexpandingthegenotype-phenotypeandgeographicalspectrumofthereportedRDs.Interestingly,thesereportshavebeenvariablyreportedfromdifferentregionsacrosstheglobeinhighnumbersfromconsanguineous/endogamouspopulationgroupsinassociationwithspecificfounderevents.Sofar,thesereportedRDscaseshavebeenreportedforthefirsttimefromJ&Kinassociationwithvariationsrestrictedtoindividualfamiliesorpopulationgroupsofwhichafewindicatesthepresenceoflikelyfounderevents. TwostudiesbasedonCFTR(OMIM#602421)variationanalysisbySher-e-KashmirInstituteofMedicalSciences,KashmirintheKashmiripopulationsuspectedwithCysticFibrosis(OMIM#219700)hasrevealeddisease’sassociationwithhighlyfrequentCFTR.ΔF508(c.1521_1523delCTT;p.Phe508del)andCFTR.3,849+10kbC>Tvariations(Kawoosaetal.,2014;Pandithetal.,2015).ThemajorlimitationofthesestudieswasthatthesewerebasedonscreeningofonlytwoCFTRvariationsinlimitedsamplesize.ConsideringtheheterogeneityoftheJ&Kpopulation,itwouldhavebeenhighlyinformativeifthesestudiescouldhavescreenedthewholeCFTRgene.Nevertheless,itispertinenttomentionthatuntilthesemolecularstudieshadbeenconducted,CysticFibrosiswasearlierconsideredtobeuncommoninJ&K.AteamofresearchersfromJammuhadconductedapilotstudyonscreeningofcodingregionexon2ofGJB2gene(OMIM#121011)inrandomlyselected17affectedindividualsfromthepreviouslymentionedDadhkaivillage(VillageofSilence)inDoda–J&K(Razdanetal.,2012).Throughthisstudy,GJB2variationsinonly4out17subjectsweredetectedwhichincludedp.G12V,p.L6L,p.R165W,p.L214P,andDelTatnt636variations.Thestudycouldhavebenefittedandinformativeifscreeningofothergenesassociatedwithhearinglossbeenperformed.Afurtherinterestingstudyoneightfamilies(onelargeandsevensmallfamilies)representingabout50%oftheaffectedindividualsfromthesamevillagewasconductedjointlybytheDepartmentofBiotechnologyinUniversityofKashmir(Kashmir),MolecularBiologyandGeneticsUnitinJawaharlalNehruCentreforAdvancedScientificResearch(Bengaluru)andDepartmentofAudiologyinAliYavarJungNationalInstitutefortheHearingHandicapped(Mumbai)(Pandeyetal.,2017).ThefamilieswerescreenedusingmethodslikeGenome-widescanandlinkageanalysis,mutationanalysisofOTOF(OMIM#603681),Cx26orGJB2,TMIE(OMIM#607237),CLDN14(OMIM#605608),SLC26A4(OMIM#605646),TMPRSS3(OMIM#605511),TMC1(OMIM#606706),andUSH1C(OMIM#605242)genes.ThefindingsofthisstudysuggestedgeneticheterogeneityunderpinninghearinglossamonginhabitantsofthehighlyendogamousDadhkaivillage.Genome-widescanandlinkageanalysisofthelargefamilyextendinguptosixgenerationsmappeddeafnesstoachromosomeloci2p24-p22.Thefindingsofthemutationanalysisindicatedanovel,foundervariationNM_194248.2:c.2122C>T(NP_919224.1:p.R708∗)inexon18ofOTOFgene,NM_144492.2:c.254T>A(NP_652763.1:p.V85D)inexon7ofCLDN14gene(afoundervariationwithorigininPakistan)andanovelNM_00441.1:c.1668T>A(NP_000432.1:p.Y556∗)inexon16ofSLC26A4genecausinghearinglossin94%ofthecasesgeneticallyscreenedthroughthisstudy.Furthermore,anunidentifiedunderpinninggeneticcauseinoneofthefamilieswassuspectedtobethefourthcause. AcaseofAutosomalRecessiveSpasticAtaxiaofCharlevoix-Saguenay(ARSACS;OMIM#270550)inindividualborntoconsanguineousparentsoftribaloriginfromRajouriareaofJ&KhasbeenrecentlydelineatedthroughWESandreportedinareputedpeer-reviewedjournal(Kuchayetal.,2019).Thestudyidentifiedanovel,frame-shiftvariationNM_014363:c.8605delT(p.Cys2869ValfsTer15)inexon10ofSACSgene(OMIM#604490),awellknowncausativegeneofARSACS.Theprobandwasfoundtobehomozygousforthereportedvariation,whereashisconsanguineousparentswerecarriers. Ourresearchgroup(HumanGeneticsResearchGroup)atShriMataVaishnoDeviUniversity,Katra,J&K–Indiaisactivelyengagedinelucidatingtheunderlyinggeneticcausesforvariouscomplexdisordersincludingtype2Diabetes,ScoliosisaswellasRDsandunderstandingtheevolutionaryperspectivesofthepopulationofJ&K.IncontextwithRDs,wehavecollectedclinicalinformationandsamplesfrom60distinctextendedfamiliessuspectedwithuncharacterizedgeneticdisordersfromdifferentregionsofJ&K.Oftheserecruitedfamilies,theexactgeneticetiologyofthreedistinctRDshavebeendeterminedandreportedrecently.TheAraivillagewithahighincidenceofamysteriousskeletaldisorder(asmentionedearlier)wasidentifiedasarareskeletaldisorder“ProgressivePseudorheumatoidDysplasia”(PPD;OMIM#208230)usingWESbyourresearchgroup(Raietal.,2016).BothaffectedaswellasunaffectedmembersoftwohighlyextendedconsanguineousfamiliesinAraivillagewererecruitedfortheirgeneticscreening.ThroughWESinthreemembers(affectedsiblingsandtheirdistantuncle)belongingtooneofthefamiliesrevealedtwoco-segregating,highlyautosomalrecessivevariationsNM_003880.3:c.156C>A(NP_003871.1:p.Cys52∗;rs121908901)andNM_003880.3:c.248G>A(NP_003871.1:p.Gly83Glu;rs147337485)inexon3ofWISP3gene(OMIM#603400).Thesevariationsarealreadyknowntoco-segregatewiththediseasephenotypeinsomefamiliesbelongingtodifferentpartsoftheworldandmightbeanoutcomeinaregionasafounderevent,mostlikelyofamiddle-easternorigin(Delagueetal.,2005).BothofthesevariationshavealsobeenreportedinsomeSouthIndianfamilies(Dalaletal.,2012).However,thesevariationswerenotfoundintheotherrecruitedfamilyforwhichsequencingofwholeWISP3genewascarriedoutanditwasfoundthatthesecondfamilyisharboringanovel,autosomalrecessive,splice-sitevariationNM_003880.3:c.643+1G>A(rs879255273)attheWISP3exon4–intron4junction.Interestingly,PPDisararedisorderhavingaprevalenceof1in1millionindividualsintheUnitedKingdom,buthaveattainedahigherprevalenceinAraivillage(upto1000times)duetotheresidingcommunity’sconsanguineousmaritalpractices(Wynne-Daviesetal.,1982).ThiswasthefirsteverstudyfromtheregionthatexploitedaNGStechnique(WES)fortheidentificationandcharacterizationofanunknowndisease. InanindependentstudyusingaPCR-basedDirectSangerSequencingStrategy,ourresearchgrouphasidentifiedanautosomalrecessivevariantNM_153638.3:c.1069C>T(NP_705902.2:p.Arg357Trp;rs753376100)locatedinexon3ofPANK2geneinaclinicallysuspectedfamilialcaseofneurodegenerativedisorder“PantothenateKinase-AssociatedNeurodegeneration”(PKAN;OMIM#234000)(Anguraletal.,2017).PKANisaprogressiveneurodegenerativedisordercharacterizedbyanabnormalaccumulationofironinthebasalgangliainbrainandextra-pyramidalmanifestation(Hayflicketal.,2003).Thus,throughthisstudythesuspectedPKANdiagnosisamongtwoaffectedsiblings,belongingtoaremotevillageinDoda–J&K,wasconfirmednearlywithinadecadeoftheirpreliminarydiagnosis.Thevariationidentifiedislocatedatahighlyconservedregionincodon357ofhPank2proteinandpredictedtobepathogenicthroughinsilicopathogenicitypredictiontools.Throughacomparativemoleculardynamicsstudyofthewild-typeandvarianthPank2proteinmodels,itwasobservedthatthereportedvariationhasrenderedrigiditytotheotherhighlydynamicproteinstructurewhichmighthavecausedafunctionalcompromiseinhPank2molecules(Anguraletal.,2017). Recently,ourgrouphasalsosuccessfullyidentifiedanuncharacterizedneurologicalcaseina9yearsoldboyasanatypicalformofLeighSyndrome(LS;OMIM#256000)throughWholeMitochondrialGenomeSequencing,andwasfoundtobeassociatedwithanovelheteroplasmicMTP6gene(OMIM#516060)variantm.8936T>A(Anguraletal.,2019).LSisaprogressiveneurodegenerativedisorderofinfancyorearlychildhoodwithaclinicalandgeneticallyheterogeneousbackground(Sofouetal.,2014).Theprobandinthisstudydepictedanatypicalfeatureofcalcificationinbasalgangliawhichwasreportedforthefirsttimethroughthisstudy. Bottom-UpApproach Forcarryingoutgeneticscreeningofcasesofsuspectedgeneticdisorders,ourresearchgrouphasadoptedthe“Bottom-upApproach.”Thisapproachishighlyefficientindelineatingthegeneticetiologyofvariousgeneticdisorders,especiallyRDs,prevalentindifferentglobalpopulationgroups.AnoutlineofthedesignedBottom-upApproachhasbeenprovidedinFigure1.Thestrategybeginswiththecollectionofclinicalinformationofthepatientandhis/herfamily’sclinicalhistoryintheformofapedigree.Thisisfollowedbyastrategiccollectionofsamples(blood,saliva,andtissue)fromthepatientsandsomeoftheirunaffectedfamilymembers,sothatacomparativegeneticscreeningisconducted.Basedontheclinicalsuspicionandpriorknowledgeonthemolecularetiologyofthesuspecteddisorders,twodistinctapproachesareconsideredforthegeneticscreening.IncaseofclinicallysuspectedbutgeneticallycharacterizedRDs,targetedscreeningofknowndisease-associatedcandidategenesthroughaPCR-basedDirectSangerSequencingapproach(candidategenescreening)issuggested.Ontheotherhand,WESapproachisadoptedincaseofclinicallysuspectedbutgeneticallyuncharacterizedRDs,theresultsofwhicharefurthervalidatedthroughatargetedPCR-basedDirectSangerSequencingapproachtoscreentheidentifiedvariationsintherecruitedsubjects.Afterrigorousanalysesandinterpretationoftherawsequencingdatathroughvarioustools,theidentifiedvariationsshouldbefurtheranalyzedfortheirplausiblepathogenicitythroughvariousonlineinsilicopredictiontools,thebriefdetailsofwhichhavebeenprovidedinTable1.Theplausiblepathogenicvariationsarethenmarkedforestablishingtheircorrelationwiththeclinicalphenotypeofthepatientsfollowedbyanalysesoftheirco-segregationwiththesuspecteddisorderintherecruitedfamily.However,thefinalproofforvariant-diseasephenotypecorrelationisobtainedthroughmolecularfunctionalstudiesbasedonDNA-RNA,DNA-Protein,RNA–RNA,andprotein–proteininteractionsandexpressionstudiesdealingwiththeelucidationofqueryvariationinageneinmodelorganisms.Onceavariationisidentifiedaspathogenic,a“Bottom-upApproach”isthenfollowedwhichincludescollectionofmoreclinicalinformationofthepatientandhis/heraffectedfamilymembers,andacomprehensiveclinicalevaluationinordertoestablishadifferentialdiagnosisofthedisease,followedbyrecruitmentandgeneticscreeningofotherfamilyindividuals(includingbothaffectedaswellasunaffected)forassessingthecarrierfrequencyoftheidentifiedgeneticvariation. FIGURE1 Figure1.Anillustrationoutliningtheproposed“Bottom-upApproach”forthecharacterizationofgeneticdisorders. Conclusion RDsareanimportantpublichealthissuewhichneedstobeovercome.ToaddressRDs-associatedchallenges,adrivetowarduniversalhealthcoveragetofulfillRDspatients’needsisrequiredalongwithinvestmentofpublicandgovernment(nationalorinternational)fundinginfundamentalbiomedicalresearchforunderstandingthediseaseetiology,discoveryofnoveldiagnosticbiomarkersandtherapeutictargets,anddevelopmentofpersonalizedinterventionstrategiesforindividualRDspatients.InpromotingRDs-relatedR&D,significantprogresshasbeenmadeacrosstheglobeinrecentyearsandmanyopportunitieshavebeendevelopedtobuildonthesuccessfulprograms,projectsandcollaborations.ForseveralRDs,remarkablefundamentalresearchintothediseaseprocesshasincreasedourunderstandingofRDspatho-physiologyandledtodevelopmentofsuitableorphandrugs,healthcareinnovation,andtherapeuticinterventions.Despite,therearestillseveralhurdlesinRDsresearchandhealthcareandmoreemphasisisrequiredtosupportappropriateRDs-relatedR&Dandpolicyprogramswithinindividualcountriessothatallglobalpatientswouldhaveequalaccesstotherapeuticinterventions.Itisanironythatunlikeotherdevelopingcountries,IndiaislaggingbehindincontexttoregulationofRDs-basedR&Dduetoseveralkeyissuesthatneedstobeurgentlyaddressed.TheprecisedelineationofdistinctRDsispossiblebyameticulousclinicalevaluationofthepatientsandtheirgeneticscreening.Recognitionofcarriersharboringclinicallypathogenicgeneticvariationsisimportantastoprovidepropergeneticcounselingtothesuspectedindividuals/familiesandanappropriatemanagementofthedisorderinanaffectedindividualinatimelymanner.Forthispurpose,weproposeahighlypotentialworkflownamed“Bottom-upApproach”whichcouldpossiblyaidinaddressingthischallengenotonlyfortheIndianpopulation,butforseveralotherendogamous/consanguineouspopulationgroupsexistingacrosstheglobe.WefurtherproposethattheheterogeneouspopulationofJ&Kcouldinspirefuturegeneticstudiesandcouldserveasaninterestingpopulation-modelforthesamepurpose.Besides,stronglegislativepoliciesandinitiativesarealsorequiredfromgovernmentandotherinstitutionsforcarryingoutRDs-relatedresearch. FuturePerspectives AlthoughthemainsourceofinformationonRDssupportandresearchgroupsforthepatientsandtheirfamiliesremainstheinternet,yetnationalRDssupportwebsitesarestillneededinmanycountries(Cullen,2002;Laskeretal.,2005;Zurynskietal.,2008).ToaddresstheclinicalchallengesassociatedwithRDs,itbecomesimperativethatdifferentsourcesofclinicalinformationandtheclinicalinfrastructureshouldbeupdatedregularly.AseparatecourseonClinicalGeneticsmustbeincludedintheacademiccurriculumofmedicalstudentsinordertoprovidethemknowledgeonthebasicconceptsofGeneticsanditsapplicationsinhumanhealth. IncontexttothelimitationsofWES,itbecomesimperativetouseWGSandother“omics”platformasanalternativefordeterminingtheunderpinningcomplexmolecularetiologyofRDs.Thisshouldalsobeaccompaniedwiththedevelopmentofinnovativeapproachesthatcouldpossiblymaintainandacceleratethecurrentpaceofclinicalaswellasgeneticdiscoveriesandinformfuturetherapeuticdevelopments. WealsoemphasizethatgeneticscreeningofsuspectedpopulationgroupsinJ&Kthroughthe“Bottom-upApproach”basedonthestate-of-the-artbiologicaltechniquesinamalgamationwithclinicalexpertiseshouldbecarriedout.InordertoascertainthegeneticprofileoftheJ&Kpopulationandtheburdenofcarriersharboringpathogenicvariations,abaselinedatabasetargetingeachandeveryendogamousgroupfromtheregionneedstobecreated,adauntingtaskundertakenbyourHumanGeneticsResearchGroupatSMVDUbutcomingoutwithpromisingoutcomes. AuthorContributions AAandASpprimarilywrotethemanuscriptandpreparedthefigureandtables.SSandERcriticallyeditedthemanuscript,plannedvariousstudies,anddevelopedproceduresandexecutionpipelines.AA,ASp,AM,VV,ASh,PK,MD,KP,ER,andSSwereinvolvedincarryingoutvariousstudies.MDfacilitatedSangerSequencingatDNASequencingFacility,UniversityofJammu. ConflictofInterest SShasrecentlyfoundedasaChiefScientificAdvisorastart-up“BiodroidInnovationsPvt.Ltd.,”involvedindevelopinggeneticandMedtechsolutions. Theremainingauthorsdeclarethattheresearchwasconductedintheabsenceofanycommercialorfinancialrelationshipsthatcouldbeconstruedasapotentialconflictofinterest. Acknowledgments WethankpatientsandtheirfamilieswhohavebeenactivelyparticipatinginthegeneticstudiesbeingcarriedoutatHumanGeneticsResearchGroup,SchoolofBiotechnology,SMVDU.AA,ASp,andSSthankSMVDUforprovidingdoctoralfellowshiptoAAandASp.AMandSSthankDST-SERBforNPDFtoAM. Footnotes ^https://www.omim.org/ ^https://www.orpha.net/consor/cgi-bin/index.php References Aggarwal,S.,andPhadke,S.R.(2015).MedicalgeneticsandgenomicmedicineinIndia:currentstatusandopportunitiesahead.Mol.Genet.GenomicMed.3,160–171.doi:10.1002/mgg3.150 PubMedAbstract|CrossRefFullText|GoogleScholar Andrews,S.(2010).FastQC:AQualityControlToolforHighThroughputSequenceData.Availableat:http://www.bioinformatics.babraham.ac.uk/projects/fastqc/(accessedOctober6,2011). GoogleScholar Angural,A.,Sharma,I.,Pandoh,P.,Sharma,V.,Spolia,A.,Rai,E.,etal.(2018).AcasereportonanovelMT-ATP6genevariationinatypicalmitochondrialLeighsyndromeassociatedwithbilateralbasalgangliacalcifications.Mitochondrion46,209–213.doi:10.1016/j.mito.2018.06.005 PubMedAbstract|CrossRefFullText|GoogleScholar Angural,A.,Sharma,I.,Pandoh,P.,Sharma,V.,Spolia,A.,Rai,E.,etal.(2019).AcasereportonanovelMT-ATP6genevariationinatypicalmitochondrialLeighsyndromeassociatedwithbilateralbasalgangliacalcifications.Mitochondrion46,209–213.doi:10.1016/j.mito.2018.06.005 PubMedAbstract|CrossRefFullText|GoogleScholar Angural,A.,Singh,I.,Mahajan,A.,Pandoh,P.,Dhar,M.K.,Kaul,S.,etal.(2017).AvariationinPANK2geneiscausingPantothenatekinase-associatedNeurodegenerationinafamilyfromJammuandKashmir-India.Sci.Rep.7:4834.doi:10.1038/s41598-017-05388-9 PubMedAbstract|CrossRefFullText|GoogleScholar Ankala,A.,Kohn,J.N.,Dastur,R.,Gaitonde,P.,Khadilkar,S.V.,andHegde,M.R.(2013).Ancestralfoundermutationsincalpain-3intheIndianAgarwalcommunity:historical,clinical,andmolecularperspective.MuscleNerve47,931–937.doi:10.1002/mus.23763 PubMedAbstract|CrossRefFullText|GoogleScholar Ankala,A.,Tamhankar,P.M.,Valencia,C.A.,Rayam,K.K.,Kumar,M.M.,andHegde,M.R.(2015).ClinicalapplicationsandimplicationsofcommonandfoundermutationsinIndiansubpopulations.Hum.Mutat.36,1–10.doi:10.1002/humu.22704 PubMedAbstract|CrossRefFullText|GoogleScholar Ara,A.,Kumar,D.,Dewan,D.,andDigra,N.(2018).IncidenceofcongenitalanomaliesinaruralpopulationofJammu-Aprospectivestudy.IndianJ.PublicHealth62,188–192.doi:10.4103/ijph.IJPH_77_17 PubMedAbstract|CrossRefFullText|GoogleScholar Auvin,S.,Irwin,J.,Abi-Aad,P.,andBattersby,A.(2018).Theproblemofrarity:estimationofprevalenceinraredisease.ValueHealth21,501–507.doi:10.1016/j.jval.2018.03.002 PubMedAbstract|CrossRefFullText|GoogleScholar Ayme,S.,Urbero,B.,Oziel,D.,Lecouturier,E.,andBiscarat,A.C.(1998).Informationonrarediseases:theOrphanetproject.Rev.Med.Interne19(Suppl.3),376S–377S. GoogleScholar Bailey-Wilson,J.E.,andWilson,A.F.(2011).Linkageanalysisinthenext-generationsequencingera.Hum.Hered.72,228–236.doi:10.1159/000334381 PubMedAbstract|CrossRefFullText|GoogleScholar Barnett,C.P.,Nataren,N.J.,Klingler-Hoffmann,M.,Schwarz,Q.,Chong,C.E.,Lee,Y.K.,etal.(2016).EctrodactylyandlethalpulmonaryacinardysplasiaassociatedwithhomozygousFGFR2mutationsidentifiedbyexomesequencing.Hum.Mutat.37,955–963.doi:10.1002/humu.23032 PubMedAbstract|CrossRefFullText|GoogleScholar Barrett,J.H.(2014).Rarevariantsanddisease.Brief.Funct.Genomics13,351–352. GoogleScholar Basu,A.,Sarkar-Roy,N.,andMajumder,P.P.(2016).GenomicreconstructionofthehistoryofextantpopulationsofIndiarevealsfivedistinctancestralcomponentsandacomplexstructure.Proc.Natl.Acad.Sci.U.S.A.113,1594–1599.doi:10.1073/pnas.1513197113 PubMedAbstract|CrossRefFullText|GoogleScholar Benjamin,K.,Vernon,M.K.,Patrick,D.L.,Perfetto,E.,Nestler-Parr,S.,andBurke,L.(2017).Patient-reportedoutcomeandobserver-reportedoutcomeassessmentinrarediseaseclinicaltrials:anISPORCOAemerginggoodpracticestaskforcereport.ValueHealth20,838–855.doi:10.1016/j.jval.2017.05.015 PubMedAbstract|CrossRefFullText|GoogleScholar Bhai,P.,Bijarnia-Mahay,S.,Puri,R.D.,Saxena,R.,Gupta,D.,Kotecha,U.,etal.(2018).ClinicalandmolecularcharacterizationofIndianpatientswithfructose-1,6-bisphosphatasedeficiency:identificationofafrequentvariant(E281K).Ann.Hum.Genet.doi:10.1111/ahg.12256[Epubaheadofprint]. CrossRefFullText|PubMedAbstract|GoogleScholar Bhasin,M.,andNag,S.(2002a).AdemographicprofileofthepeopleofJammuandKashmir1.PopulationStructure.J.Hum.Ecol.13,1–55.doi:10.1080/09709274.2002.11905524 CrossRefFullText|GoogleScholar Bhasin,M.K.,andNag,S.(2002b).ConsanguinityanditseffectsonfertilityandchildsurvivalamongMuslimsofLadakhinJammuandKashmir.Anthropologist4,131–140.doi:10.1080/09720073.2002.11890739 CrossRefFullText|GoogleScholar Bhat,J.I.,Qureeshi,U.A.,andBhat,M.A.(2010).Acuteintermittentporphyriawithtransientcorticalblindness.IndianPediatr.47,977–978.doi:10.1007/s13312-010-0152-9 PubMedAbstract|CrossRefFullText|GoogleScholar Bhat,Y.,Hassan,I.,Sajad,P.,andWani,R.(2015).PretibialepidermolysisbullosainaKashmirigirl.IndianJ.Paediat.Dermatol.16,203–206. GoogleScholar Bhat,Y.J.,Baba,A.N.,Manzoor,S.,Qayoom,S.,Javed,S.,andAjaz,H.(2010).Ellis-vanCreveldsyndromewithfacialhemiatrophy.IndianJ.Dermatol.Venereol.Leprol.76,266–269.doi:10.4103/0378-6323.62968 PubMedAbstract|CrossRefFullText|GoogleScholar Bijarnia-Mahay,S.,Gupta,D.,Shigematsu,Y.,Yamaguchi,S.,Saxena,R.,andVerma,I.C.(2016).EthylmalonicEncephalopathyinanIndianBoy.IndianPediatr.53,914–916.doi:10.1007/s13312-016-0959-0 PubMedAbstract|CrossRefFullText|GoogleScholar Bittles,A.H.(2002a).Endogamy,consanguinityandcommunitygenetics.J.Genet.81,91–98.doi:10.1007/bf02715905 PubMedAbstract|CrossRefFullText|GoogleScholar Bittles,A.H.(2002b).TheimpactofconsanguinityontheIndianpopulation.IndianJ.Hum.Genet.8,45–51. GoogleScholar Bittles,A.H.,andBlack,M.L.(2010).Theimpactofconsanguinityonneonatalandinfanthealth.EarlyHum.Dev.86,737–741.doi:10.1016/j.earlhumdev.2010.08.003 PubMedAbstract|CrossRefFullText|GoogleScholar Bittles,A.H.,andBlack,M.L.(2010a).Consanguinity,humanevolution,andcomplexdiseases.Proc.Natl.Acad.Sci.U.S.A.107,1779–1786.doi:10.1073/pnas.0906079106 PubMedAbstract|CrossRefFullText|GoogleScholar Bittles,A.H.,Savithri,H.S.,andAppajiRao,N.(2002).Communitygeneticsindevelopingcountries.CommunityGenet.5,151–152.doi:10.1159/000066327 PubMedAbstract|CrossRefFullText|GoogleScholar Bolger,A.M.,Lohse,M.,andUsadel,B.(2014).Trimmomatic:aflexibletrimmerforIlluminasequencedata.Bioinformatics30,2114–2120.doi:10.1093/bioinformatics/btu170 PubMedAbstract|CrossRefFullText|GoogleScholar Botstein,D.,andRisch,N.(2003).Discoveringgenotypesunderlyinghumanphenotypes:pastsuccessesformendeliandisease,futureapproachesforcomplexdisease.Nat.Genet.33(Suppl.),228–237.doi:10.1038/ng1090 PubMedAbstract|CrossRefFullText|GoogleScholar Boycott,K.M.,andArdigo,D.(2018).Addressingchallengesinthediagnosisandtreatmentofraregeneticdiseases.Nat.Rev.DrugDiscov.17,151–152.doi:10.1038/nrd.2017.246 PubMedAbstract|CrossRefFullText|GoogleScholar Boycott,K.M.,Rath,A.,Chong,J.X.,Hartley,T.,Alkuraya,F.S.,Baynam,G.,etal.(2017).Internationalcooperationtoenablethediagnosisofallraregeneticdiseases.Am.J.Hum.Genet.100,695–705.doi:10.1016/j.ajhg.2017.04.003 PubMedAbstract|CrossRefFullText|GoogleScholar Boycott,K.M.,Vanstone,M.R.,Bulman,D.E.,andMackenzie,A.E.(2013).Rare-diseasegeneticsintheeraofnext-generationsequencing:discoverytotranslation.Nat.Rev.Genet.14,681–691.doi:10.1038/nrg3555 PubMedAbstract|CrossRefFullText|GoogleScholar Braun,M.M.,Farag-El-Massah,S.,Xu,K.,andCote,T.R.(2010).EmergenceoforphandrugsintheUnitedStates:aquantitativeassessmentofthefirst25years.Nat.Rev.DrugDiscov.9,519–522.doi:10.1038/nrd3160 PubMedAbstract|CrossRefFullText|GoogleScholar Chaki,M.,Mukhopadhyay,A.,Chatterjee,S.,Das,M.,Samanta,S.,andRay,K.(2005).HigherprevalenceofOCA1inanethnicgroupofeasternIndiaisduetoafoundermutationinthetyrosinasegene.Mol.Vis.11,531–534. PubMedAbstract|GoogleScholar Chen,W.J.,Lin,Y.,Xiong,Z.Q.,Wei,W.,Ni,W.,Tan,G.H.,etal.(2011).ExomesequencingidentifiestruncatingmutationsinPRRT2thatcauseparoxysmalkinesigenicdyskinesia.Nat.Genet.43,1252–1255.doi:10.1038/ng.1008 PubMedAbstract|CrossRefFullText|GoogleScholar Choi,M.,Scholl,U.I.,Ji,W.,Liu,T.,Tikhonova,I.R.,Zumbo,P.,etal.(2009).GeneticdiagnosisbywholeexomecaptureandmassivelyparallelDNAsequencing.Proc.Natl.Acad.Sci.U.S.A.106,19096–19101.doi:10.1073/pnas.0910672106 PubMedAbstract|CrossRefFullText|GoogleScholar Cullen,R.J.(2002).Insearchofevidence:familypractitioners’useoftheInternetforclinicalinformation.J.Med.Libr.Assoc.90,370–379. PubMedAbstract|GoogleScholar Dalal,A.,Bhavani,G.S.,Togarrati,P.P.,Bierhals,T.,Nandineni,M.R.,Danda,S.,etal.(2012).AnalysisoftheWISP3geneinIndianfamilieswithprogressivepseudorheumatoiddysplasia.Am.J.Med.Genet.A158A,2820–2828.doi:10.1002/ajmg.a.35620 PubMedAbstract|CrossRefFullText|GoogleScholar Danese,E.,andLippi,G.(2018).Rarediseases:theparadoxofanemergingchallenge.Ann.Transl.Med.6:329.doi:10.21037/atm.2018.09.04 PubMedAbstract|CrossRefFullText|GoogleScholar Dar,S.,Nazir,M.,Lone,R.,Sameen,D.,Ahmad,I.,Wani,W.,etal.(2018).Clinicalspectrumofdisordersofsexdevelopment:across-sectionalobservationalstudy.IndianJ.Endocr.Metab.22,774–779.doi:10.4103/ijem.IJEM_159_18 PubMedAbstract|CrossRefFullText|GoogleScholar DasBhowmik,A.,Patil,S.J.,Deshpande,D.V.,Bhat,V.,andDalal,A.(2018).Novelsplice-sitevariantofUCHL1inanIndianfamilywithautosomalrecessivespasticparaplegia-79.J.Hum.Genet.63,927–933.doi:10.1038/s10038-018-0463-6 PubMedAbstract|CrossRefFullText|GoogleScholar DawnTeare,M.,andBarrett,J.H.(2005).Geneticlinkagestudies.Lancet366,1036–1044. PubMedAbstract|GoogleScholar DeSilva,T.S.,Macdonald,D.,Paterson,G.,Sikdar,K.C.,andCochrane,B.(2011).Systematizednomenclatureofmedicineclinicalterms(SNOMEDCT)torepresentcomputedtomographyprocedures.Comput.MethodsProgramsBiomed.101,324–329.doi:10.1016/j.cmpb.2011.01.002 PubMedAbstract|CrossRefFullText|GoogleScholar Delague,V.,Chouery,E.,Corbani,S.,Ghanem,I.,Aamar,S.,Fischer,J.,etal.(2005).MolecularstudyofWISP3inninefamiliesoriginatingfromtheMiddle-Eastandpresentingwithprogressivepseudorheumatoiddysplasia:identificationoftwonovelmutations,anddescriptionofafoundereffect.Am.J.Med.Genet.A138A,118–126.doi:10.1002/ajmg.a.30906 PubMedAbstract|CrossRefFullText|GoogleScholar Dixit,S.,Sahu,P.,Kar,S.K.,andNegi,S.(2015).Identificationofthehot-spotareasforsicklecelldiseaseusingcordbloodscreeningatadistricthospital:anIndianperspective.J.CommunityGenet.6,383–387.doi:10.1007/s12687-015-0223-7 PubMedAbstract|CrossRefFullText|GoogleScholar Dunstan,D.W.,Zimmet,P.Z.,Welborn,T.A.,DeCourten,M.P.,Cameron,A.J.,Sicree,R.A.,etal.(2002).Therisingprevalenceofdiabetesandimpairedglucosetolerance:theAustralianDiabetes.ObesityandLifestyleStudy.DiabetesCare25,829–834.doi:10.2337/diacare.25.5.829 PubMedAbstract|CrossRefFullText|GoogleScholar Fareed,M.,andAfzal,M.(2014a).Estimatingtheinbreedingdepressiononcognitivebehavior:apopulationbasedstudyofchildcohort.PLoSOne9:e109585.doi:10.1371/journal.pone.0109585 PubMedAbstract|CrossRefFullText|GoogleScholar Fareed,M.,andAfzal,M.(2014b).Evidenceofinbreedingdepressiononheight,weight,andbodymassindex:apopulation-basedchildcohortstudy.Am.J.Hum.Biol.26,784–795.doi:10.1002/ajhb.22599 PubMedAbstract|CrossRefFullText|GoogleScholar Fareed,M.,andAfzal,M.(2016).Increasedcardiovascularrisksassociatedwithfamilialinbreeding:apopulation-basedstudyofadolescentcohort.Ann.Epidemiol.26,283–292.doi:10.1016/j.annepidem.2016.03.001 PubMedAbstract|CrossRefFullText|GoogleScholar Fareed,M.,andAfzal,M.(2017).Geneticsofconsanguinityandinbreedinginhealthanddisease.Ann.Hum.Biol.44,99–107.doi:10.1080/03014460.2016.1265148 PubMedAbstract|CrossRefFullText|GoogleScholar Fareed,M.,Hussain,R.,Shah,A.,andAfzal,M.(2014).A1A2BOandRhgenefrequenciesamongsixpopulationsofJammuandKashmir,India.Transfus.Apher.Sci.50,247–252.doi:10.1016/j.transci.2014.01.014 PubMedAbstract|CrossRefFullText|GoogleScholar Fareed,M.,KaisarAhmad,M.,AzeemAnwar,M.,andAfzal,M.(2017).Impactofconsanguineousmarriagesanddegreesofinbreedingonfertility,childmortality,secondarysexratio,selectionintensity,andgeneticload:across-sectionalstudyfromNorthernIndia.Pediatr.Res.81,18–26.doi:10.1038/pr.2016.177 PubMedAbstract|CrossRefFullText|GoogleScholar Forrest,C.B.,Bartek,R.J.,Rubinstein,Y.,andGroft,S.C.(2011).Thecaseforaglobalrare-diseasesregistry.Lancet377,1057–1059.doi:10.1016/s0140-6736(10)60680-0 CrossRefFullText|GoogleScholar Fresard,L.,andMontgomery,S.B.(2018).Diagnosingrarediseasesaftertheexome.ColdSpringHarb.Mol.CaseStud.4:a003392.doi:10.1101/mcs.a003392 PubMedAbstract|CrossRefFullText|GoogleScholar Fromer,M.,Moran,J.L.,Chambert,K.,Banks,E.,Bergen,S.E.,Ruderfer,D.M.,etal.(2012).Discoveryandstatisticalgenotypingofcopy-numbervariationfromwhole-exomesequencingdepth.Am.J.Hum.Genet.91,597–607.doi:10.1016/j.ajhg.2012.08.005 PubMedAbstract|CrossRefFullText|GoogleScholar Ganie,M.A.,Ali,I.,Ahangar,A.G.,Wani,M.M.,Ahmed,S.,Bhat,M.A.,etal.(2012).Thiamineresponsivemegaloblasticanemiasyndromeassociatedwithpatentductusarteriosus:firstcasereportfromKashmirValleyoftheIndiansubcontinent.IndianJ.Endocrinol.Metab.16,646–650.doi:10.4103/2230-8210.98033 PubMedAbstract|CrossRefFullText|GoogleScholar Ganie,M.A.,Laway,B.A.,Nisar,S.,Wani,M.M.,Khurana,M.L.,Ahmad,F.,etal.(2011).PresentationandclinicalcourseofWolfram(DIDMOAD)syndromefromNorthIndia.Diabet.Med.28,1337–1342.doi:10.1111/j.1464-5491.2011.03377.x PubMedAbstract|CrossRefFullText|GoogleScholar Gorospe,J.R.,Singhal,B.S.,Kainu,T.,Wu,F.,Stephan,D.,Trent,J.,etal.(2004).IndianAgarwalmegalencephalicleukodystrophywithcystsiscausedbyacommonMLC1mutation.Neurology62,878–882.doi:10.1212/01.wnl.0000115106.88813.5b PubMedAbstract|CrossRefFullText|GoogleScholar Guillem,P.,Cans,C.,Robert-Gnansia,E.,Ayme,S.,andJouk,P.S.(2008).Rarediseasesindisabledchildren:anepidemiologicalsurvey.Arch.Dis.Child93,115–118.doi:10.1136/adc.2006.104455 PubMedAbstract|CrossRefFullText|GoogleScholar Gupta,A.,Sharma,Y.,Deo,K.,Vellarikkal,S.,Jayarajan,R.,Dixit,V.,etal.(2015).CaseReport:Wholeexomesequencinghelpsinaccuratemoleculardiagnosisinsiblingswitharareco-occurrenceofpaternallyinherited22q12duplicationandautosomalrecessivenon-syndromicichthyosis.F1000Res.4:446.doi:10.12688/f1000research.6779.1 PubMedAbstract|CrossRefFullText|GoogleScholar Gupta,A.,Sharma,Y.K.,Vellarikkal,S.K.,Jayarajan,R.,Dixit,V.,Verma,A.,etal.(2016).Whole-exomesequencingsolvesdiagnosticdilemmainararecaseofsporadicacrokeratosisverruciformis.J.Eur.Acad.Dermatol.Venereol.30,695–697.doi:10.1111/jdv.12983 PubMedAbstract|CrossRefFullText|GoogleScholar Gupta,S.,Chaurasia,A.,Pathak,E.,Mishra,R.,Chaudhry,V.N.,Chaudhry,P.,etal.(2017).WholeexomesequencingunveilsaframeshiftmutationinCNGB3forconedystrophy:acasereportofanIndianfamily.Medicine96:e7490.doi:10.1097/MD.0000000000007490 PubMedAbstract|CrossRefFullText|GoogleScholar Haffner,M.E.(2006).Adoptingorphandrugs–twodozenyearsoftreatingrarediseases.N.Engl.J.Med.354,445–447.doi:10.1056/nejmp058317 PubMedAbstract|CrossRefFullText|GoogleScholar Haffner,M.E.,Whitley,J.,andMoses,M.(2002).Twodecadesoforphanproductdevelopment.Nat.Rev.DrugDiscov.1,821–825.doi:10.1038/nrd919 PubMedAbstract|CrossRefFullText|GoogleScholar Hart,T.C.,Hart,P.S.,Michalec,M.D.,Zhang,Y.,Firatli,E.,VanDyke,T.E.,etal.(2000).Haim-MunksyndromeandPapillon-LefevresyndromeareallelicmutationsincathepsinC.J.Med.Genet.37,88–94.doi:10.1136/jmg.37.2.88 PubMedAbstract|CrossRefFullText|GoogleScholar Hassan,I.,andKeen,M.A.(2013).Severegeneralizedrecessivedystrophicepidermolysisbullosa(Hallopeau-Siemen’s):acasereport.J.Pediatr.Sci.5:e190. GoogleScholar Hayflick,S.J.,Westaway,S.K.,Levinson,B.,Zhou,B.,Johnson,M.A.,Ching,K.H.L.,etal.(2003).Genetic,clinical,andradiographicdelineationofHallervorden–Spatzsyndrome.N.Engl.J.Med.348,33–40.doi:10.1056/nejmoa020817 PubMedAbstract|CrossRefFullText|GoogleScholar Hockham,C.,Bhatt,S.,Colah,R.,Mukherjee,M.B.,Penman,B.S.,Gupta,S.,etal.(2018).Thespatialepidemiologyofsickle-cellanaemiainIndia.Sci.Rep.8,17685.doi:10.1038/s41598-018-36077-w PubMedAbstract|CrossRefFullText|GoogleScholar Hughes,D.A.,Tunnage,B.,andYeo,S.T.(2005).Drugsforexceptionallyrarediseases:dotheydeservespecialstatusforfunding?QJM98,829–836.doi:10.1093/qjmed/hci128 PubMedAbstract|CrossRefFullText|GoogleScholar Hussain,R.,andBittles,A.H.(2000).SociodemographiccorrelatesofconsanguineousmarriageintheMuslimpopulationofIndia.J.Biosoc.Sci.32,433–442.doi:10.1017/s0021932000004338 PubMedAbstract|CrossRefFullText|GoogleScholar Ilyas,M.,Wani,A.,Ahmad,Z.,AhmadKazimi,M.,andChoh,N.(2019).Holt-oramsyndrome-caseseriesoftworeports.CHRISMEDJ.HealthRes.6,119–122.doi:10.1002/ajmg.a.36783 PubMedAbstract|CrossRefFullText|GoogleScholar IndianGenomeVariationConsortium(2005).TheIndianGenomeVariationdatabase(IGVdb):aprojectoverview.Hum.Genet.118,1–11.doi:10.1007/s00439-005-0009-9 PubMedAbstract|CrossRefFullText|GoogleScholar Lander,E.S.,Linton,L.M.,Birren,B.,Nusbaum,C.,Zody,M.C.,Baldwin,J.,etal.(2001).Initialsequencingandanalysisofthehumangenome.Nature409,860–921.doi:10.1038/35057062 PubMedAbstract|CrossRefFullText|GoogleScholar KaruthedathVellarikkal,S.,Jayarajan,R.,Verma,A.,Nair,S.,Ravi,R.,Senthivel,V.,etal.(2016).CaseReport:wholeexomesequencingrevealsanovelframeshiftdeletionmutationp.G2254fsinCOL7A1associatedwithautosomalrecessivedystrophicepidermolysisbullosa.F1000Res.5:900.doi:10.12688/f1000research.8380.2 PubMedAbstract|CrossRefFullText|GoogleScholar Kasatkar,P.,Ghosh,K.,andShetty,S.(2013).Acommonfoundermutationp.P2063Sinexon36ofVWFin11unrelatedIndianvonWillebranddisease(VWD)families.Ann.Hematol.92,1147–1148.doi:10.1007/s00277-013-1680-x PubMedAbstract|CrossRefFullText|GoogleScholar Kasthuri,A.(2018).ChallengestoHealthcareinIndia-TheFiveA’s.IndianJ.CommunityMed.43,141–143. GoogleScholar Kawoosa,M.S.,Bhat,M.A.,Ali,S.W.,Hafeez,I.,andShastri,S.(2014).ClinicalandmutationprofileofchildrenwithcysticfibrosisinJammuandKashmir.IndianPediatr.51,185–189.doi:10.1007/s13312-014-0381-4 PubMedAbstract|CrossRefFullText|GoogleScholar Kehar,M.,Bijarnia,S.,Ellard,S.,Houghton,J.,Saxena,R.,Verma,I.C.,etal.(2014).Fanconi-Bickelsyndrome-mutationinSLC2A2gene.IndianJ.Pediatr.81,1237–1239.doi:10.1007/s12098-014-1487-3 PubMedAbstract|CrossRefFullText|GoogleScholar Knight,A.W.,andSenior,T.P.(2006).Thecommonproblemofrarediseaseingeneralpractice.Med.J.Aust.185,82–83.doi:10.5694/j.1326-5377.2006.tb00477.x PubMedAbstract|CrossRefFullText|GoogleScholar Kotecha,U.H.,Movva,S.,Puri,R.D.,andVerma,I.C.(2012).Trichohepatoentericsyndrome:foundermutationinasianindians.Mol.Syndromol.3,89–93.doi:10.1159/000339896 PubMedAbstract|CrossRefFullText|GoogleScholar Krumm,N.,O’roak,B.J.,Karakoc,E.,Mohajeri,K.,Nelson,B.,Vives,L.,etal.(2013).TransmissiondisequilibriumofsmallCNVsinsimplexautism.Am.J.Hum.Genet.93,595–606.doi:10.1016/j.ajhg.2013.07.024 PubMedAbstract|CrossRefFullText|GoogleScholar Kuchay,R.A.H.,Mir,Y.R.,Zeng,X.,Hassan,A.,Musarrat,J.,Parwez,I.,etal.(2019).ARSACSasaworldwidedisease:novelSACSmutationsidentifiedinaconsanguineousfamilyfromtheremotetribalJammuandKashmirRegioninIndia.Cerebellum18,807–812.doi:10.1007/s12311-019-01028-2 PubMedAbstract|CrossRefFullText|GoogleScholar Kumar,H.,Sarma,P.,andMedhi,B.(2017).Orphandrugs:Indianperspective.IndianJ.Pharmacol.49,267–269. GoogleScholar Kumar,P.,Gupta,A.,Fotra,R.,Upma,Raina,S.,Sethi,S.,etal.(2010).Chromosomalanomaliesinreferredcaseswithsuspectedgeneticdisorders:firstreportfromJammuandKashmir.Int.J.Hum.Genet.10,41–47.doi:10.1080/09723757.2010.11886083 CrossRefFullText|GoogleScholar Kumar,S.,Bhalla,A.,Sharma,N.,Dhibar,D.P.,Kumari,S.,andVarma,S.(2017).Clinical,biochemicalcharacteristicsandhospitaloutcomeofacuteintermittentporphyriapatients:adescriptivestudyfromNorthIndia.Ann.IndianAcad.Neurol.20,263–269. GoogleScholar Lander,E.S.,andBotstein,D.(1987).Homozygositymapping:awaytomaphumanrecessivetraitswiththeDNAofinbredchildren.Science236,1567–1570.doi:10.1126/science.2884728 PubMedAbstract|CrossRefFullText|GoogleScholar Lang,J.,andWood,S.C.(1999).Developmentoforphanvaccines:anindustryperspective.Emerg.Infect.Dis.5,749–756.doi:10.3201/eid0506.990602 PubMedAbstract|CrossRefFullText|GoogleScholar Langmead,B.,Trapnell,C.,Pop,M.,andSalzberg,S.L.(2009).Ultrafastandmemory-efficientalignmentofshortDNAsequencestothehumangenome.GenomeBiol.10:R25.doi:10.1186/gb-2009-10-3-r25 PubMedAbstract|CrossRefFullText|GoogleScholar Lasker,J.N.,Sogolow,E.D.,andSharim,R.R.(2005).Theroleofanonlinecommunityforpeoplewithararedisease:contentanalysisofmessagespostedonaprimarybiliarycirrhosismailinglist.J.Med.InternetRes.7:e10.doi:10.2196/jmir.7.1.e10 PubMedAbstract|CrossRefFullText|GoogleScholar Layer,R.M.,Chiang,C.,Quinlan,A.R.,andHall,I.M.(2014).LUMPY:aprobabilisticframeworkforstructuralvariantdiscovery.GenomeBiol.15:R84.doi:10.1186/gb-2014-15-6-r84 PubMedAbstract|CrossRefFullText|GoogleScholar Li,D.,Tian,L.,andHakonarson,H.(2018).IncreasingdiagnosticyieldbyRNA-Sequencinginraredisease-bypasshurdlesofinterpretingintronicorsplice-alteringvariants.Ann.Transl.Med.6:126.doi:10.21037/atm.2018.01.14 PubMedAbstract|CrossRefFullText|GoogleScholar Li,H.,andDurbin,R.(2009).FastandaccurateshortreadalignmentwithBurrows–Wheelertransform.Bioinformatics25,1754–1760.doi:10.1093/bioinformatics/btp324 PubMedAbstract|CrossRefFullText|GoogleScholar Li,H.,Handsaker,B.,Wysoker,A.,Fennell,T.,Ruan,J.,Homer,N.,etal.(2009).ThesequenceAlignment/MapformatandSAMtools.Bioinformatics25,2078–2079.doi:10.1093/bioinformatics/btp352 PubMedAbstract|CrossRefFullText|GoogleScholar Li,J.,Lupat,R.,Amarasinghe,K.C.,Thompson,E.R.,Doyle,M.A.,Ryland,G.L.,etal.(2012).CONTRA:copynumberanalysisfortargetedresequencing.Bioinformatics28,1307–1313.doi:10.1093/bioinformatics/bts146 PubMedAbstract|CrossRefFullText|GoogleScholar Lopes,M.T.,Koch,V.H.,Sarrubbi-Junior,V.,Gallo,P.R.,andCarneiro-Sampaio,M.(2018).Difficultiesinthediagnosisandtreatmentofrarediseasesaccordingtotheperceptionsofpatients,relativesandhealthcareprofessionals.Clinics73:e68.doi:10.6061/clinics/2018/e68 PubMedAbstract|CrossRefFullText|GoogleScholar Luzzatto,L.,Hollak,C.E.,Cox,T.M.,Schieppati,A.,Licht,C.,Kaariainen,H.,etal.(2015).Rarediseasesandeffectivetreatments:arewedelivering?Lancet385,750–752.doi:10.1016/s0140-6736(15)60297-5 CrossRefFullText|GoogleScholar Majid,S.,Ahmad,Q.M.,andShah,I.H.(2012).PorphyriasamongstpatientswithcutaneousphotosensitivityinKashmir:asevenyearexperience.IndianJ.Dermatol.Venereol.Leprol.78:520.doi:10.4103/0378-6323.98097 PubMedAbstract|CrossRefFullText|GoogleScholar Majumder,P.P.,andBasu,A.(2014).AgenomicviewofthepeoplingandpopulationstructureofIndia.ColdSpringHarb.Perspect.Biol.7:a008540.doi:10.1101/cshperspect.a008540 PubMedAbstract|CrossRefFullText|GoogleScholar Mcelreavey,K.,Wang,W.,andBittles,A.H.(2005).FirstAsianworkshopongenomicsandcommunitygenetics.CommunityGenet.8,130–132.doi:10.1159/000084784 PubMedAbstract|CrossRefFullText|GoogleScholar Mckenna,A.,Hanna,M.,Banks,E.,Sivachenko,A.,Cibulskis,K.,Kernytsky,A.,etal.(2010).Thegenomeanalysistoolkit:aMapReduceframeworkforanalyzingnext-generationDNAsequencingdata.GenomeRes.20,1297–1303.doi:10.1101/gr.107524.110 PubMedAbstract|CrossRefFullText|GoogleScholar Mckusick,V.A.(2007).MendelianInheritanceinMananditsonlineversion,OMIM.Am.J.Hum.Genet.80,588–604.doi:10.1086/514346 PubMedAbstract|CrossRefFullText|GoogleScholar Middha,S.,Lindor,N.M.,Mcdonnell,S.K.,Olson,J.E.,Johnson,K.J.,Wieben,E.D.,etal.(2015).Howwelldowholeexomesequencingresultscorrelatewithmedicalfindings?Astudyof89MayoClinicBiobanksamples.Front.Genet.6:244.doi:10.3389/fgene.2015.00244 CrossRefFullText|GoogleScholar Mistri,M.,Tamhankar,P.M.,Sheth,F.,Sanghavi,D.,Kondurkar,P.,Patil,S.,etal.(2012).IdentificationofnovelmutationsinHEXAgeneinchildrenaffectedwithTaySachsdiseasefromIndia.PLoSOne7:e39122.doi:10.1371/journal.pone.0039122 PubMedAbstract|CrossRefFullText|GoogleScholar Nambot,S.,Thevenon,J.,Kuentz,P.,Duffourd,Y.,Tisserant,E.,Bruel,A.L.,etal.(2018).Clinicalwhole-exomesequencingforthediagnosisofraredisorderswithcongenitalanomaliesand/orintellectualdisability:substantialinterestofprospectiveannualreanalysis.Genet.Med.20,645–654.doi:10.1038/gim.2017.162 PubMedAbstract|CrossRefFullText|GoogleScholar Narasimhan,V.M.,Patterson,N.J.,Moorjani,P.,Lazaridis,I.,Mark,L.,Mallick,S.,etal.(2018).ThegenomicformationofSouthandCentralAsia.bioRxiv[Preprint].doi:10.1101/292581 CrossRefFullText|GoogleScholar Narayanan,R.,KaruthedathVellarikkal,S.,Jayarajan,R.,Verma,A.,Dixit,V.,Scaria,V.,etal.(2016).CaseReport:Applicationofwholeexomesequencingforaccuratediagnosisofraresyndromesofmineralocorticoidexcess.F1000Res.5:1592.doi:10.12688/f1000research.8779.2 PubMedAbstract|CrossRefFullText|GoogleScholar Nazir,N.,andChalkoo,A.H.(2017).OralmanifestationsofapatientwithEpidermolysisBullosa.Biomed.J.Sci.Tech.Res.1,1562–1565. GoogleScholar Ng,S.B.,Bigham,A.W.,Buckingham,K.J.,Hannibal,M.C.,Mcmillin,M.J.,Gildersleeve,H.I.,etal.(2010a).ExomesequencingidentifiesMLL2mutationsasacauseofKabukisyndrome.Nat.Genet.42,790–793.doi:10.1038/ng.646 PubMedAbstract|CrossRefFullText|GoogleScholar Ng,S.B.,Buckingham,K.J.,Lee,C.,Bigham,A.W.,Tabor,H.K.,Dent,K.M.,etal.(2010b).Exomesequencingidentifiesthecauseofamendeliandisorder.Nat.Genet.42,30–35.doi:10.1038/ng.499 PubMedAbstract|CrossRefFullText|GoogleScholar Ng,S.B.,Nickerson,D.A.,Bamshad,M.J.,andShendure,J.(2010c).Massivelyparallelsequencingandraredisease.Hum.Mol.Genet.19,R119–R124.doi:10.1093/hmg/ddq390 PubMedAbstract|CrossRefFullText|GoogleScholar OrphanDrugAct(1983).PubL.No.97-414,96Stat.2049. GoogleScholar Pandey,N.,Rashid,T.,Jalvi,R.,Sharma,M.,Rangasayee,R.,Andrabi,K.I.,etal.(2017).MutationsinOTOF,CLDN14&SLC26A4genesasmajorcausesofhearingimpairmentinDhadkaivillage,Jammu&Kashmir,India.IndianJ.Med.Res.146,489–497. GoogleScholar Pandith,A.A.,Sheikh,S.A.,Faheem,S.,Zargar,M.H.,Malla,T.M.,Shah,Z.A.,etal.(2015).IdentificationofuniquepatternofCFTRgenemutationsincysticfibrosisinanethnicKashmiriPopulation(NorthIndia).J.Genet.Syndr.GeneTher.6,2157–7412. GoogleScholar Phadke,S.R.,Gupta,N.,Girisha,K.M.,Kabra,M.,Maeda,M.,Vidal,E.,etal.(2010).Rhizomelicchondrodysplasiapunctatatype1:reportofmutationsin3childrenfromIndia.J.Appl.Genet.51,107–110.doi:10.1007/BF03195717 PubMedAbstract|CrossRefFullText|GoogleScholar Pradhan,S.,Sengupta,M.,Dutta,A.,Bhattacharyya,K.,Bag,S.K.,Dutta,C.,etal.(2011).Indiangeneticdiseasedatabase.NucleicAcidsRes.39,D933–D938.doi:10.1093/nar/gkq1025 PubMedAbstract|CrossRefFullText|GoogleScholar Pulst,S.M.(1999).Geneticlinkageanalysis.Arch.Neurol.56,667–672. PubMedAbstract|GoogleScholar Qayoom,S.,Masood,Q.,Sultan,J.,Hassan,I.,Jehangir,M.,Bhat,Y.J.,etal.(2010).Epidermolysisbullosa:aseriesof12patientsinkashmirvalley.IndianJ.Dermatol.55,229–232.doi:10.4103/0019-5154.70668 PubMedAbstract|CrossRefFullText|GoogleScholar Quadros,L.,Ghosh,K.,andShetty,S.(2009).NovelmutationsinfactorIXgenefromwesternIndiawithreferencetotheirphenotypicandhaplotypicattributes.J.Pediatr.Hematol.Oncol.31,157–160.doi:10.1097/MPH.0b013e31818b3759 PubMedAbstract|CrossRefFullText|GoogleScholar Rai,E.,Mahajan,A.,Kumar,P.,Angural,A.,Dhar,M.K.,Razdan,S.,etal.(2016).WholeexomescreeningidentifiesnovelandrecurrentWISP3mutationscausingprogressivePseudorheumatoidDysplasiainJammuandKashmir-India.Sci.Rep.6:27684.doi:10.1038/srep27684 PubMedAbstract|CrossRefFullText|GoogleScholar Raina,S.,Saroch,M.,Yadav,G.,andBhardwaj,A.(2017).Establishingancestrythroughpedigreeofavillagewithhighprevalenceofhearing-impaired.J.IndianSpeechLang.Hear.Assoc.31,1–4. GoogleScholar Rajasimha,H.K.,Shirol,P.B.,Ramamoorthy,P.,Hegde,M.,Barde,S.,Chandru,V.,etal.(2014).OrganizationforrarediseasesIndia(ORDI)-addressingthechallengesandopportunitiesfortheIndianrarediseases’community.Genet.Res.96:e009.doi:10.1017/S0016672314000111 PubMedAbstract|CrossRefFullText|GoogleScholar Ranganath,P.,Matta,D.,Bhavani,G.S.,Wangnekar,S.,Jain,J.M.,Verma,I.C.,etal.(2016).SpectrumofSMPD1mutationsinAsian-Indianpatientswithacidsphingomyelinase(ASM)-deficientNiemann-Pickdisease.Am.J.Med.Genet.A170,2719–2730.doi:10.1002/ajmg.a.37817 PubMedAbstract|CrossRefFullText|GoogleScholar Rasool,J.,Manzoor,F.,Bhat,S.,Bashir,N.,andGeelani,S.(2015).Hereditarystomatocytosis:FirstcasereportfromValleyofKashmir.Med.J.Dr.DYPatil.Univ.8,347–349. GoogleScholar Rausch,T.,Zichner,T.,Schlattl,A.,Stutz,A.M.,Benes,V.,andKorbel,J.O.(2012).DELLY:structuralvariantdiscoverybyintegratedpaired-endandsplit-readanalysis.Bioinformatics28,i333–i339.doi:10.1093/bioinformatics/bts378 PubMedAbstract|CrossRefFullText|GoogleScholar Razdan,S.,Kaul,R.L.,Motta,A.,Kaul,S.,andBhatt,R.K.(1994).PrevalenceandpatternofmajorneurologicaldisordersinruralKashmir(India)in1986.Neuroepidemiology13,113–119.doi:10.1159/000110368 PubMedAbstract|CrossRefFullText|GoogleScholar Razdan,S.,Raina,S.K.,Pandita,K.K.,Razdan,S.,Nanda,R.,Kaul,R.,etal.(2012).Inbreedingasacausefordeafness:Dadhkaistudy.IndianJ.Hum.Genet.18,71–74.doi:10.4103/0971-6866.96655 PubMedAbstract|CrossRefFullText|GoogleScholar Richter,T.,Nestler-Parr,S.,Babela,R.,Khan,Z.M.,Tesoro,T.,Molsen,E.,etal.(2015).Rarediseaseterminologyanddefinitions-asystematicglobalreview:reportoftheISPORrarediseasespecialinterestgroup.ValueHealth18,906–914.doi:10.1016/j.jval.2015.05.008 PubMedAbstract|CrossRefFullText|GoogleScholar Rimmer,A.,Phan,H.,Mathieson,I.,Iqbal,Z.,Twigg,S.R.F.,Consortium,W.G.S.,etal.(2014).Integratingmapping-,assembly-andhaplotype-basedapproachesforcallingvariantsinclinicalsequencingapplications.Nat.Genet.46,912–918.doi:10.1038/ng.3036 PubMedAbstract|CrossRefFullText|GoogleScholar Romdhane,L.,Messaoud,O.,Bouyacoub,Y.,Kerkeni,E.,Naouali,C.,CherifBenAbdallah,L.,etal.(2016).ComorbidityintheTunisianpopulation.Clin.Genet.89,312–319.doi:10.1111/cge.12616 PubMedAbstract|CrossRefFullText|GoogleScholar Saha,B.,Lessel,D.,Nampoothiri,S.,Rao,A.S.,Hisama,F.M.,Peter,D.,etal.(2013).Ethnic-specificWRNmutationsinsouthAsianWernersyndromepatients:potentialfoundereffectinpatientswithIndianorPakistaniancestry.Mol.Genet.GenomicMed.1,7–14.doi:10.1002/mgg3.1 PubMedAbstract|CrossRefFullText|GoogleScholar Sawyer,S.L.,Hartley,T.,Dyment,D.A.,Beaulieu,C.L.,Schwartzentruber,J.,Smith,A.,etal.(2016).Utilityofwhole-exomesequencingforthoseneartheendofthediagnosticodyssey:timetoaddressgapsincare.Clin.Genet.89,275–284.doi:10.1111/cge.12654 PubMedAbstract|CrossRefFullText|GoogleScholar Schieppati,A.,Henter,J.I.,Daina,E.,andAperia,A.(2008).Whyrarediseasesareanimportantmedicalandsocialissue.Lancet371,2039–2041.doi:10.1016/s0140-6736(08)60872-7 CrossRefFullText|GoogleScholar Setia,N.,Saxena,R.,Arora,A.,andVerma,I.C.(2016).SpectrumofmutationsinhomozygousfamilialhypercholesterolemiainIndia,withfournovelmutations.Atherosclerosis255,31–36.doi:10.1016/j.atherosclerosis.2016.10.028 PubMedAbstract|CrossRefFullText|GoogleScholar Sharma,I.,Sharma,V.,Khan,A.,Kumar,P.,Rai,E.,Bamezai,R.N.K.,etal.(2018).AncienthumanmigrationstoandthroughJammuKashmir-Indiawerenotofmalesexclusively.Sci.Rep.8:851.doi:10.1038/s41598-017-18893-8 PubMedAbstract|CrossRefFullText|GoogleScholar Shendure,J.,Balasubramanian,S.,Church,G.M.,Gilbert,W.,Rogers,J.,Schloss,J.A.,etal.(2017).DNAsequencingat40:past,presentandfuture.Nature550,345–353.doi:10.1038/nature24286 PubMedAbstract|CrossRefFullText|GoogleScholar Shoib,S.,MaqboolDar,M.,Arif,T.,Bashir,H.,andAhmed,J.(2012).Fahr’sdiseasepresentingasmania:acasereport.Iran.J.PsychiatryBehav.Sci.6,102–104. PubMedAbstract|GoogleScholar Sindi,S.,Helman,E.,Bashir,A.,andRaphael,B.J.(2009).Ageometricapproachforclassificationandcomparisonofstructuralvariants.Bioinformatics25,i222–i230.doi:10.1093/bioinformatics/btp208 PubMedAbstract|CrossRefFullText|GoogleScholar Sinha,K.(2005).SpinocerebellarAtaxia12(SCA12).ATremorDominantDisease,TypicallySeeninIndia.Mumbai:AssociationofPhysiciansofIndia,600–602. GoogleScholar Sofou,K.,DeCoo,I.F.,Isohanni,P.,Ostergaard,E.,Naess,K.,DeMeirleir,L.,etal.(2014).AmulticenterstudyonLeighsyndrome:diseasecourseandpredictorsofsurvival.Orphanet.J.RareDis.9:52.doi:10.1186/1750-1172-9-52 PubMedAbstract|CrossRefFullText|GoogleScholar Song,P.,Gao,J.,Inagaki,Y.,Kokudo,N.,andTang,W.(2012a).IntractableandrarediseasesresearchinAsia.BiosciTrends6,48–51. GoogleScholar Song,P.,Gao,J.,Inagaki,Y.,Kokudo,N.,andTang,W.(2012b).Rarediseases,orphandrugs,andtheirregulationinAsia:currentstatusandfutureperspectives.IntractableRareDis.Res.1,3–9. GoogleScholar Teare,M.D.,andSantibanezKoref,M.F.(2014).LinkageanalysisandthestudyofMendeliandiseaseintheeraofwholeexomeandgenomesequencing.Brief.Funct.Genomics13,378–383.doi:10.1093/bfgp/elu024 PubMedAbstract|CrossRefFullText|GoogleScholar Tsurusaki,Y.,Okamoto,N.,Ohashi,H.,Kosho,T.,Imai,Y.,Hibi-Ko,Y.,etal.(2012).MutationsaffectingcomponentsoftheSWI/SNFcomplexcauseCoffin-Sirissyndrome.Nat.Genet.44,376–378.doi:10.1038/ng.2219 PubMedAbstract|CrossRefFullText|GoogleScholar Turton,J.P.,Mehta,A.,Raza,J.,Woods,K.S.,Tiulpakov,A.,Cassar,J.,etal.(2005).MutationswithinthetranscriptionfactorPROP1arerareinacohortofpatientswithsporadiccombinedpituitaryhormonedeficiency(CPHD).Clin.Endocrinol.63,10–18.doi:10.1111/j.1365-2265.2005.02291.x PubMedAbstract|CrossRefFullText|GoogleScholar Udani,V.,Das,S.,andChhabria,R.(2017).PanthotenateKinase-associatedneurodegenerationhasafoundermutation(c.215_216insa)inIndianAgrawalpatients.Mov.Disord.Clin.Pract.4,96–99.doi:10.1002/mdc3.12341 PubMedAbstract|CrossRefFullText|GoogleScholar Upma,Kumar,P.,Raina,T.R.,Sharma,K.,andGupta,S.(2010).ClinicalandcytogeneticanalysisofhumananemiasfromJammuregionofJammuandKashmirstate.AsianJ.Transfus.Sci.4,99–101.doi:10.4103/0973-6247.67031 PubMedAbstract|CrossRefFullText|GoogleScholar Uttarilli,A.,Ranganath,P.,Matta,D.,MdNurulJain,J.,Prasad,K.,Babu,A.S.,etal.(2016).Identificationandcharacterizationof20novelpathogenicvariantsin60unrelatedIndianpatientswithmucopolysaccharidosestypeIandtypeII.Clin.Genet.90,496–508.doi:10.1111/cge.12795 PubMedAbstract|CrossRefFullText|GoogleScholar Vasudev,R.,andSawhney,V.(2014).ThalassemiamajorandIntermediainJammuandKashmir,India:aregionaltransfusioncentreexperience.IndianJ.Hematol.BloodTransfus.30,297–300.doi:10.1007/s12288-013-0301-0 PubMedAbstract|CrossRefFullText|GoogleScholar Verma,I.(2000).BurdenofgeneticdisordersinIndia.IndianJ.Pediatr.67,893–898.doi:10.1007/bf02723953 PubMedAbstract|CrossRefFullText|GoogleScholar Verma,I.,andBijarnia,S.(2002).TheburdenofgeneticdisordersinIndiaandaframeworkforcommunitycontrol.PublicHealthGenomics5,192–196.doi:10.1159/000066335 PubMedAbstract|CrossRefFullText|GoogleScholar Vink,J.M.,andBoomsma,D.I.(2002).Genefindingstrategies.Biol.Psychol.61,53–71.doi:10.1016/s0301-0511(02)00052-2 PubMedAbstract|CrossRefFullText|GoogleScholar Virmani,N.,Vellarikkal,S.K.,Verma,A.,Jayarajan,R.,Sakhiya,J.,Desai,C.,etal.(2018).Wholeexomesequencinginamulti-generationfamilyfromIndiarevealsageneticvariationc.10C>T(p.Gln4Ter)inkeratin5geneassociatedwithDowling-Degosdisease.IndianJ.Dermatol.Venereol.Leprol.84,344–346. GoogleScholar Wajnrajch,M.P.,Gertner,J.M.,Sokoloff,A.S.,Ten,I.,Harbison,M.D.,Netchine,I.,etal.(2003).HaplotypeanalysisofthegrowthhormonereleasinghormonereceptorlocusinthreeapparentlyunrelatedkindredsfromtheIndiansubcontinentwiththeidenticalmutationintheGHRHreceptor.Am.J.Med.Genet.A120A,77–83.doi:10.1002/ajmg.a.10209 PubMedAbstract|CrossRefFullText|GoogleScholar Wang,Q.,Lu,Q.,andZhao,H.(2015).Areviewofstudydesignsandstatisticalmethodsforgenomicepidemiologystudiesusingnextgenerationsequencing.Front.Genet.6:149.doi:10.3389/fgene.2015.00149 PubMedAbstract|CrossRefFullText|GoogleScholar Wang,Z.,Liu,X.,Yang,B.Z.,andGelernter,J.(2013).Theroleandchallengesofexomesequencinginstudiesofhumandiseases.Front.Genet.4:160.doi:10.3389/fgene.2013.00160 PubMedAbstract|CrossRefFullText|GoogleScholar Wani,M.M.,Khan,I.,Bhat,R.A.,andAhmad,M.(2016).Fabry’sdisease:caseseriesandreviewofliterature.Ann.Med.HealthSci.Res.6,193–197.doi:10.4103/2141-9248.183935 PubMedAbstract|CrossRefFullText|GoogleScholar Wastfelt,M.,Fadeel,B.,andHenter,J.I.(2006).Ajourneyofhope:lessonslearnedfromstudiesonrarediseasesandorphandrugs.J.Intern.Med.260,1–10.doi:10.1111/j.1365-2796.2006.01666.x PubMedAbstract|CrossRefFullText|GoogleScholar Weisenfeld,N.I.,Yin,S.,Sharpe,T.,Lau,B.,Hegarty,R.,Holmes,L.,etal.(2014).Comprehensivevariationdiscoveryinsinglehumangenomes.Nat.Genet.46,1350–1355.doi:10.1038/ng.3121 PubMedAbstract|CrossRefFullText|GoogleScholar Wenger,A.M.,Guturu,H.,Bernstein,J.A.,andBejerano,G.(2017).Systematicreanalysisofclinicalexomedatayieldsadditionaldiagnoses:implicationsforproviders.Genet.Med.19,209–214.doi:10.1038/gim.2016.88 PubMedAbstract|CrossRefFullText|GoogleScholar Whittemore,A.S.,andNelson,L.M.(1999).Studydesigningeneticepidemiology:theoreticalandpracticalconsiderations.J.Natl.CancerInst.Monogr.61–69.doi:10.1093/oxfordjournals.jncimonographs.a024228 PubMedAbstract|CrossRefFullText|GoogleScholar Woods,C.G.,Cox,J.,Springell,K.,Hampshire,D.J.,Mohamed,M.D.,Mckibbin,M.,etal.(2006).Quantificationofhomozygosityinconsanguineousindividualswithautosomalrecessivedisease.Am.J.Hum.Genet.78,889–896.doi:10.1086/503875 PubMedAbstract|CrossRefFullText|GoogleScholar Wynne-Davies,R.,Hall,C.,andAnsell,B.M.(1982).Spondylo-epiphysialdysplasiatardawithprogressivearthropathy.A“new”disorderofautosomalrecessiveinheritance.J.BoneJointSurg.Br.64,442–445.doi:10.1302/0301-620x.64b4.6807993 CrossRefFullText|GoogleScholar Wysoker,A.,Tibbetts,K.,andFennell,T.(2013).PicardToolsversion1.90.Availableonlineat:http://picard.sourceforge.net(accessedMarch10,2020). GoogleScholar Yang,Y.,Muzny,D.M.,Reid,J.G.,Bainbridge,M.N.,Willis,A.,Ward,P.A.,etal.(2013).Clinicalwhole-exomesequencingforthediagnosisofmendeliandisorders.N.Engl.J.Med.369,1502–1511.doi:10.1056/NEJMoa1306555 PubMedAbstract|CrossRefFullText|GoogleScholar Yang,Y.,Muzny,D.M.,Xia,F.,Niu,Z.,Person,R.,Ding,Y.,etal.(2014).Molecularfindingsamongpatientsreferredforclinicalwhole-exomesequencing.JAMA312,1870–1879.doi:10.1001/jama.2014.14601 PubMedAbstract|CrossRefFullText|GoogleScholar Zurynski,Y.,Frith,K.,Leonard,H.,andElliott,E.(2008).Rarechildhooddiseases:howshouldwerespond?Arch.Dis.Child93,1071–1074.doi:10.1136/adc.2007.134940 PubMedAbstract|CrossRefFullText|GoogleScholar Keywords:rarediseases,Next-GenerationDNASequencing,endogamy,consanguinity,India,JammuandKashmir,Bottom-upApproach Citation:AnguralA,SpoliaA,MahajanA,VermaV,SharmaA,KumarP,DharMK,PanditaKK,RaiEandSharmaS(2020)Review:UnderstandingRareGeneticDiseasesinLowResourceRegionsLikeJammuandKashmir–India.Front.Genet.11:415.doi:10.3389/fgene.2020.00415 Received:03August2019;Accepted:01April2020;Published:30April2020. Editedby: HaranathaR.Potteti,UniversityofIllinoisatChicago,UnitedStates Reviewedby: SheikhRiazuddin,AllamaIqbalMedicalCollege,Pakistan SajidMalik,Quaid-i-AzamUniversity,Pakistan LiliaRomdhane,UniversityofCarthage,Tunisia Copyright©2020Angural,Spolia,Mahajan,Verma,Sharma,Kumar,Dhar,Pandita,RaiandSharma.Thisisanopen-accessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense(CCBY).Theuse,distributionorreproductioninotherforumsispermitted,providedtheoriginalauthor(s)andthecopyrightowner(s)arecreditedandthattheoriginalpublicationinthisjournaliscited,inaccordancewithacceptedacademicpractice.Nouse,distributionorreproductionispermittedwhichdoesnotcomplywiththeseterms. *Correspondence:EktaRai,[email protected];SwarkarSharma,[email protected] †Theseauthorshavecontributedequallytothiswork COMMENTARY ORIGINALARTICLE Peoplealsolookedat SuggestaResearchTopic>