ARTICLE OPEN ACCESS

Ethnicity-related DMD Genotype Landscapes inEuropean and Non-European CountriesRita Selvatici PhD Rachele Rossi PhD Fernanda Fortunato MD Cecilia Trabanelli MSc Yamina Sifi PhD

AliceMarguttiMScMarcella NeriMD PhD FrancescaGualandiMD PhD Lena SzaboMD Balint FeketeMD

Lyudmilla Angelova MD Ivan Litvinenko MD Ivan Ivanov MD Yurtsever Vildan MD

Oana Alexandra Iuhas MD Mihaela Vintan MD Carmen Burloiu MD Butnariu Lacramioara MD

Gabriela Visa MD Diana Epure MD Cristina Rusu MD Daniela Vasile MD Magdalena Sandu MD

Dmitry Vlodavets MD Monica Mager MD PhD Theodore Kyriakides MD PhD Sanja Delin MD

Ivan Lehman MD Jadranka Sekelj Fures MD Veneta Bojinova MD Mariela Militaru MD

Velina Guergueltcheva MD Birute Burnyte MD Maria Judith Molnar MD PhD Niculina Butoianu MD

Selma Dounia Bensemmane MD SamiraMakri-Mokrane MD Agnes Herczegfalvi MD Monica Panzaru MD

Adela Chirita Emandi MD Anna Lusakowska MD Anna Potulska-Chromik MD

Anna Kostera-Pruszczyk MD PhD Andriy Shatillo MD Djawed Bouchenak Khelladi MD

Oussama Dendane MD Mingyan Fang PhD Zhiyuan Lu PhD and Alessandra Ferlini MD PhD

Neurol Genet 20217e536 doi101212NXG0000000000000536

Correspondence

Dr Ferlini

flaunifeit

AbstractObjectiveGenetic diagnosis and mutation identification are now compulsory for Duchenne (DMD) andBecker muscular dystrophies (BMD) which are due to dystrophin (DMD) gene mutationseither for disease prevention or personalized therapies To evaluate the ethnic-related geneticassortments of DMD mutations which may impact on DMD genetic diagnosis pipelines westudied 328 patients with DMD and BMD from non-European countries

MethodsWe performed a full DMD mutation detection in 328 patients from 10 Eastern Europeancountries (Poland Hungary Lithuania Romania Serbia Croatia Bosnia Bulgaria Ukraineand Russia) and 2 non-European countries (Cyprus and Algeria) We used both conventionalmethods (multiplex ligation-dependent probe amplification [MLPA] followed by gene-specificsequencing) and whole-exome sequencing (WES) as a pivotal study ran in 28 patients whereDMD mutations were already identified by standard techniques WES output was also in-terrogated for DMD gene modifiers

ResultsWe identified DMD gene mutations in 222 male patients We identified a remarkable alleleheterogeneity among different populations with a mutation landscape often country specificWe also showed thatWES is effective for picking up allDMD deletions and small mutations and

From the Medical Genetics Unit (RS RR FF CT AM MN FG AF) Department of Medical Sciences University of Ferrara Italy Neurologie (YS) CHU de BenbadisConstantine Algerie 2nd Department of Paediatrics Clinic (LS AH) Semmelweis University Institute of GenomicMedicine and Rare Disorders (BF MJM) Semmelweis UniversityBudapest Hungary Department of Medical Genetics (LA) Medical University Varna Bulgaria Department of Pediatrics (I Litvinenko) Medical University Sofia Department of ChildNeurology (I Litvinenko) University Pediatric Hospital ldquoProf Ivan Mitevrdquo Sofia Department Pediatrics (II) St George University Hospital Medical University Plovdiv BulgariaPediatric Neurology Department (YV) Pediatric Neurologist County Clinical Emergency Hospital of Constanta G CurteanuMunicipal Clinical Hospital Oradea (OAI) Department ofNeuroscience (MV M Militaru) Iuliu Hatieganu University of Medicine and Pharmacy Cluj Napoca Pediatric Neurology Department (CB NB) ldquoAlexandru Obregiardquo ClinicalPsychiatry Hospital Bucharest ldquoGrigore T Popardquo University of Medicine and Pharmacy (BL CR MP) ldquoSfanta Mariardquo Childrenrsquos Hospital (BL CR MP) Pediatric Clinical HospitalSibiu (GV) ldquoDr Victor Gomoiurdquo Childrenrsquos Hospital (DE D Vasile MS) ldquoCarol Davilardquo University of Medicine and Pharmacy (MS NB) Bucharest Romania Russian ChildrenNeuromuscular Center (D Vlodavets) Veltischev Clinical Pediatric Research Institute of Pirogov Russian National Research Medical University Moscow Russia Department ofNeuroscience Neurology and Pediatric Neurology ldquoIuliu HatieganurdquoUniversity ofMedicine and Pharmacy (MMager) Faculty ofMedicine Pediatric Neurology Department (MMager)Emergency Clinical Hospital for Children Cluj-Napoca Romania Department of Basic and Clinical Sciences (TK) University of Nicosia Cyprus Department of Pediatrics (SD) GeneralHospital Zadar Zadar Croatia Department of Paediatrics (I Lehman JSF) University Hospital Centre Zagreb Faculty of Medicine University of Osijek (JSF) Croatia UniversityHospital of Neurology and Psychiatry Sveti Naum (VB) Clinic of Neurology (VG) University Hospital Sofiamed Sofia University ldquoSt Kliment Ohridskirdquo (VG)Bulgaria Institute ofBiomedical Sciences (BB) Faculty of Medicine Vilnius University Lithuania Ali Ait Idir Hospital (SDB SM-M) Algiers Algeria University of Algiers I Algeria (SDB SM-M) Centerof GenomicMedicine (ACE) University ofMedicine and Pharmacy Victor Babes Timisoara Regional Center ofMedical Genetics Timis (ACE) Clinical Emergency Hospital for ChildrenLouis Turcanu Timisoara Romania Department of Neurology (AL AP AK-P) Medical University ofWarsaw Poland Institute of Neurology (AS) Psychiatry andNarkology NationalAcademy of Medical Science of Ukraine Neurologie (DBK OD) CHU Tidjani Damerdji Tlemcen Algerie and BGI-Shenzhen (MF ZL) Shenzhen China

Go to NeurologyorgNG for full disclosures Funding information is provided at the end of the article

The Article Processing Charge was funded by Consorzio Futuro in Ricerca

This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License 40 (CC BY-NC-ND) which permits downloadingand sharing the work provided it is properly cited The work cannot be changed in any way or used commercially without permission from the journal

Copyright copy 2020 American Academy of Neurology 1

its adoption could allow a detection rate close to 90 of all occurring mutations Gene modifiers haplotypes were identified withsome ethnic-specific configurations

ConclusionsOur data provide unreported mutation landscapes in different countries suggesting that ethnicity may orient genetic diagnosisflowchart which can be adjusted depending on the mutation type frequency with impact in drug eligibility

Dystrophinopathies are a group of X-linked allelic diseasescaused bymutations in the dystrophin gene (DMD gene OnlineMendelian Inheritance in Man [OMIM] 300377) affectingevery 1 in 5000 male births worldwide Mutations in the DMDgene lead to a spectrum of diseases from the severe Duchenne(DMD 310200) and the milder Becker (BMD 300376)muscle dystrophies to the isolated dilated cardiomyopathy(302045) and other mild phenotypes or asymptomaticmales1ndash3 The frame rule assumes that the functional conse-quences ofDMDmutations are related to the maintaining of theopen reading frame allowing a shorter and partially functionaldystrophin to be translated Therefore mutations that maintainthe frame result in the milder BMD phenotype whereas out-of-frame mutations cause multiple premature stop codons down-stream and are associated with the severe DMD phenotypealthough some exceptions occur4 Genetic testing is required toconfirm a dystrophinopathy diagnosis and allows geneticcounseling prevention and patients eligibility for already-approved drugs or novel personalized therapeutic treatments56

We approached 328 patients referred to us by 10 Eastern Eu-ropean countries (Poland Hungary Lithuania Romania SerbiaCroatia Bosnia Bulgaria Ukraine and Russia) and 2 non-European countries (Algeria and Cyprus) with the aim ofidentifying DMD mutations We also validated whole-exomesequencing (WES) as an accurate and highly sensitivemethod ofdetecting all DMD deletions and small mutations although notduplications and also gene modifiers Our study allowed toidentify different mutation landscapes in the studied populationsand primed up considerations for personalized treatments andreadjusted diagnostic flowcharts based on the ethnicities

MethodsPatient EnrollmentPatient enrollment was conducted based on the clinical di-agnosis with a suspicion of a dystrophinopathy We enrolled328 male patients (June 2016 and December 2019) fromAlgeria (68) Cyprus (2) Bosnia (1) Bulgaria (12) Croatia

(5) Hungary (14) Lithuania (6) Poland (66) Romania(62) Russia (1) Serbia (2) and Ukraine (89) A total of 328affected males and 28 females were tested for DMD genemutations Among the 328 males 229 were clinically di-agnosed as DMD 27 as BMD 33 as dystrophinopathy and 39as high CK The 28 at-risk females were from Bosnia (1)Bulgaria (1) Croatia (3) Hungary (4) Poland (1) Romania(13) Ukraine (1) and Algeria (4) The clinical diagnosis wasconducted according to established standard clinical outcomemeasures and scales for DMD mutation7

Ethical IssuesEthical consent forms for genetic testing were collected ateach clinical center which referred the patients to our in-stitution as part of the routine diagnostic procedures forDMD genetic diagnosis approved by each local ethical com-mittee Whole-exome sequencing data were analyzed forDMD mutations and gene modifiers (for scientific purpose)only as per the ethical consent forms Study design and dataanalyses described in this article were performed based onEthical Approval N 552016 and N 662020OssAOUFe

DMD Deletion and Duplication IdentificationGenomic DNA was extracted from EDTA-preserved wholeblood using QIAsymphony Instrument and Kits following themanufacturerrsquos instructions Multiplex ligation-dependent probeamplification (MLPA) assay was performed on all 328 malepatients using the P034P035 DMD Kit (MRC HollandAmsterdam the Netherlands) according to the manufacturerrsquosinstructions The reaction products were analyzed using a DNAanalyzer (ABI 3130 XL Applied Biosystems Foster City CAUSA) and data analysis was performed using Coffalyser (MRCHolland Amsterdam the Netherlands) and GeneMarker(SoftGenetics State College PA USA) software for MLPAWhen the MLPA result suggested a single exon deletion orduplication the result was always confirmed by an alternativemolecular technique which was PCR and sequencing for single-deleted exons (including the 59 and 39 adjacent exons) and exon-specific real-time PCR for single exon duplications according tocurrent DMD diagnosis guidelines89

GlossaryACMG = American College of Medical Genetics and Genomics BMD = Becker muscular dystrophy CK = creatine kinaseDMD =Duchennemuscular dystrophy IGV = Integrative Genomics Viewer LGMD = limb-girdle muscular dystrophyNGS =next-generation sequencing OMIM = Online Mendelian Inheritance in Man VUS = variant of uncertainunknownsignificance WES = whole-exome sequencing WGS = whole-genome sequencing

2 Neurology Genetics | Volume 7 Number 1 | February 2021 NeurologyorgNG

DMD Small Mutation IdentificationThe DMD gene was sequenced in all patients resulted negativeby MLPA testing using next-generation sequencing (NGS) ap-proach DMD MASTRtrade assay (Multiplicom Niel Belgium)was used according to the manufacturerrsquos instructions All 79exons were sequenced with a minimal coverage per allele of 50Xand run on MiSeq (Illumina San Diego CA USA) Dataanalyses were performed using Sophia Genetics pipeline Alldetected variants were validated by Sanger sequencing

WES AnalysisWES pilot validation study was performed on 28 male patientsalready tested using MLPA and NGS sequencing and provenpositive for the presence of aDMDmutation MGIEasy ExomeCapture V4 Probe Set (MGI Shenzhen China) was performedto enrich the coding regions and constructed libraries werethen sequenced by using IlluminaHiSeq2000 platformwith 90-bp paired-end reads Standard analysis of the raw data wasperformed as previously described10 including alignmentvariant (single nucleotide polymorphism [SNP] insertiondeletion polymorphism [InDel] and copy number variation[CNV]) calling and annotation Integrative Genomics Viewer(IGV) software11 was also used to determine the coverage ofevery exon of the DMD gene and the quality of the reads

Data Analysis and Bioinformatics ToolsAll mutationsvariations recorded in this study were annotatedaccording to the standards established by the Human GenomeVariation Society (HGVS hgvsorg) and available databasesestablished by the LeidenMuscular Dystrophy pages (dmdnl)the Leiden Open Variation Database 30 (lovd nl30home)and UMD-DMD (umdbeDMD)12 All variations werechecked for their presence in databases (LOVD ClinVar andgnomAD) and their possible pathogenic meaning was verifiedusing the MutationTaster PolyPhen and Human SplicingFinder in silico prediction tools All known and novel variations(known pathogenic likely pathogenic and variant ofuncertainunknown significance [VUS]) identified in thisstudy were deposited in the LOVD database

Data were analyzed looking for mutation type (deletions dupli-cations and small mutations) frequency and distribution alongtheDMD gene andmutation hotspotsWe consideredmutationsoccurring in DMD and BMD as main clinical categories Patientswere categorized based on their country of origin to identifymutation type frequency differences related to ethnicity

We also categorized mutations amenable for personalizedtherapies (exon 44 45 51 and 53 skipping by antisense oli-goribonucleotides and stop codon reversion)1314 We cate-gorized previously unreported variants based on the ACMGclassification15 as likely pathogenic and variant of uncertainsignificance and reported them separately

Data AvailabilityThe data generated and analyzed in the current study will beavailable and shared by request to the corresponding author (AF)

from any qualified investigator These data include clinical data andraw genome files (NGS MLPA WES and Sanger sequences)

ResultsTable 1 shows the result of DMD testing in all patients withDMDBMD Of the 328 male patients 222 resulted positive forcarrying a hemizygous DMD variation (the detection rate was68) whereas 106 resulted negative Among the 222 patientscarrying aDMDmutationvariation DMDwere 188 and BMDwere 24 In 10 patients with DMD (1) BMD (1) high CK (7)and dystrophinopathy (1) phenotypes we identified VUSDystrophinopathy definition was adopted by clinicians when aphenotypewas not typical or when the boysrsquo age (younger than 7years) did not allow unequivocal clinical diagnosis Among the106 negative cases 34 were defined as DMD 8 as BMD 39 ashigh CK and 25 as dystrophinopathy Among all identifiedvariants 26 (all fromEastern European patients) were previouslyunreported and categorized as likely pathogenic (table e-1 linkslwwcomNXGA338) Another 9 variations (in 10 patients)were categorized as VUSs (table e-2 linkslwwcomNXGA339) Novel likely pathogenic variants were included in thedata analysis whereas VUSs were not included in the statistics

Frame RuleIn the 72 patients with DMD and 16 patients with BMDcarrying DMD deletions the frame rule applies to 985 ofDMDs (being out of frame) and 95 of BMDs (being inframe) For duplications the frame rule applies to 99 ofDMDs (18 of 19 patients) and to the only 1 duplicated BMDwe have identified being in frame

Deletion and Duplication HotspotsWe observed a total of 88 deletions (regardless of the country oforigin) 72 in patients with DMD and 16 in patients with BMDwith a very low frequency of deletions in Eastern Europeancountries Deletion distribution shows the absence of the usualhotspot around exons 2ndash7 with all exons until exon 37 rarelyinvolved in deletion events The well-known hotspot withbreakpoint in intron 44 is present in 21 patients Also evident is arecurrent breakpoint in intron 45 (18 patients) and intron 50 (25patients) with therefore 73 of deletions having 1 breakpointwithin introns 44 45 and 50 (figure e-1a linkslwwcomNXGA342) The statistical analysis of duplications is hampered by thevery low number of duplicated patients (18 DMDs and only 1BMD) (figure e-1b linkslwwcomNXGA342)

Mutation Frequency and Distribution inPatients With DMDWe considered our patients as belonging to 2 distinct groupsEastern Europe (Bosnia Bulgaria Croatia Hungary SerbiaLithuania Poland Romania Ukraine and Russia) and Alge-ria Cyprus was not included in the statistics (only 2 cases)

The ethnicity of Eastern Europe is quite heterogeneous beingcomposed by more than 40 different ethnic groups16 never-theless a few studies demonstrated high genetic background

NeurologyorgNG Neurology Genetics | Volume 7 Number 1 | February 2021 3

Table 1 Centers and Genotypic Data of the Patients

Patientsenrolled

Negativefor DMDmutations

Positivefor DMDmutations DMD BMD

VUS(a)

Deletionsall

DeletionsDMD

DeletionsBMD

Duplicationsall

DuplicationsDMD

DuplicationsBMD

Nonsenseall

NonsenseDMD

NonsenseBMD

Missenseall

MissenseDMD

MissenseBMD

Frameshiftingall

FrameshiftingDMD

FrameshiftingBMD

Canonicalsplicesites all

Canonicalsplicesites DMD

Canonicalsplicesites BMD

Splicingconsensussites all

Splicingconsensussites DMD

Splicingconsensussites BMD

Algeria 68 16 52 39 9 4 36 30 6 2 2 0 4 2 2 3 1 1 2 2 0 2 2 0 3 0 0

Bosnia 1 0 1 1 0 0 0 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0

Bulgaria 12 0 12 11 1 0 1 1 0 1 1 0 6 6 0 0 0 0 0 0 0 4 3 1 0 0 0

Croatia 5 0 5 4 1 0 0 0 0 0 0 0 3 3 0 0 0 0 1 0 1 1 1 0 0 0 0

Hungary 14 5 9 9 0 0 1 1 0 0 0 0 4 4 0 0 0 0 3 3 0 1 1 0 0 0 0

Lithuania 6 2 4 4 0 0 0 0 0 1 1 0 2 2 0 0 0 0 1 1 0 0 0 0 0 0 0

Poland 66 30 36 31 1 4 2 2 0 2 2 0 13 12 1 3 0 0 11 11 0 5 5 0 1 0 0

Romania 62 25 37 29 7 1 23 17 6 3 3 0 4 4 0 0 0 0 4 4 0 2 1 1 1 0 0

Russia 1 0 1 1 0 0 0 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0

Serbia 2 0 2 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 2 0 0 0 0 0 0 0

Ukraine 89 27 62 56 5 1 25 21 4 9 8 1 15 15 0 2 1 0 7 7 0 3 3 0 1 1 0

Cyprus 2 1 1 1 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Total 328 106 222 188 24 10 88 72 16 19 18 1 53 50 3 7 2 1 31 30 1 18 16 2 6 1 0

Abbreviations BMD = Becker muscular dystrophy DMD = Duchenne muscular dystrophy VUS = variant of uncertainunknown significancea See table e-2 (linkslwwcomNXGA339)All the variants identified were submitted to the LOVD database (lovdnl) The table reports on countries and clinical centers which have referred the patients to our Unit and the mutations identified in DMD and BMDphenotypes only Patients with BMD showing a DMD mutation were only 24 Among these 16 patients resulted as carrying a DMD deletion and only 1 patient carried a duplication 3 BMD carried a nonsense 1 a missensemutation 1 a small in frame deletion and 2 patients with BMD showed splicingmutations Algerian patients showed 6 deletions (5 in frame and 1 out of frame) Romanian patients showed 6 in-frame deletions and 1 canonicalsplice site mutation Ukrainian patients showed 4 in-frame deletions and 1 duplication Polish patients showed 1 nonsense patients from Bulgaria and Croatia showed 1 canonical splice site and 1 small in-frame deletionrespectively

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admixture for which we estimated these countries as a singlegroup for our DMD mutation statistical analysis From theEastern European countries a total of 258 patients were gen-otyped and 169 patients were diagnosed with a DMD muta-tion Mutations were predominantly nonsense (31) anddeletions (29) followed by small frameshifting variations(18) duplications (11) and canonical site splicing variants(9) Missense and consensus splicing variations were alsoidentified (1) Eighty-nine patients did not show a typicalDMD mutation (detection rate 65) (figure 1A)

A DMDmutation was identified in 52 of the 68 samples fromAlgeria (detection rate 76) (figure 1B) The vast majority ofAlgerian patients carried deletions (77) followed by allother mutations in very similar percentages In a previouspublication a similar mutation landscape was reported17 Bymerging these data 86 of Algerian patients carry deletionsand 14 all others

Figure 2 shows the overview of the mutation landscape ineach Eastern European country and in Cyprus The numberof patients with BMD (24) is too low to allow statisticsAmong the 28 at-risk females 11 were identified as hetero-zygous carriers

WES AnalysisTwenty-eight patients with proven DMD mutations (12 de-letions 15 small mutations and 1 duplication) were usedto validate WES analysis (table e-3 linkslwwcomNXGA340)

As expected all DMD small variants previously detected usingNGS sequencing were identified byWES analysis In addition allMLPA identified deletions were also picked up byWES Figure 3A and B shows software visualization of 2 deletions (exon 51 andexons 18ndash19) where no reads were visible within the deletedinterval In both cases WES analysis defined the deletion inter-vals within intron borders thus excluding intraexonic deletionsThe duplication was not identified by WES (data not shown)

DMD Gene Modifier HaplotypingWES analysis performed in the 28 DMDs allowed to call SNPsvariations in genes known to be DMD genetic modifiers asSPP1 LTBP4 CD40 THBS1 and ACTN318 We could notobtain genotypes of SPP1 and THBS1 SNP regions(rs28357094 and rs2725797 respectively) because they werenot covered by WES enrichment LTPB4 CD40 and ACTN3were fully covered and we profiled the LFTB4 haplotypes andtheCD40 andACTN3 genotypes Results are shown in table 2

We also identified in 8 patients other exonic SNPs occurringin 3 gene modifiers namely 3 SNPs within the LTPB4 gene 9SNPs in ACTN3 and 2 SNPs in THBS1 (table e-4 linkslwwcomNXGA341) The majority are synonymous (6) andmissense changes (7) whereas 1 was a nonsense variant inexon 21 of the ACTN3 gene

Mutations Amenable of NewTherapeutic ApproachesPatients carrying deletions amenable of exon-skipping thera-peutic approaches were evaluated In total 41 of patients are

Figure 1 Distribution of DMD Mutations in Patients With DMD in Countries

Overview of mutation distribution in patients with DMD from Eastern Europe (A) and Algeria (B) Nonsense mutations were the most frequently occurringmutations in Eastern European patients accounting for 31 of mutation types in the patients with DMD followed by deletions (29) frameshifting (18)duplications (11) and splicing canonical sites (9) missense and consensus splicing are the least frequent (1 each) Deletions were the most frequentmutations inAlgerianpatients (77) whereas nonsense (5) frameshifting (5) splicing canonical sites (5) duplication (5) andmissense (3) variationswerethe least frequent No splicing consensus sequence mutations were identified in patients from Algeria The reported numbers include known and novel likelypathogenicmutationsvariations butnot VUSAmong the28 females tested for carrier detectionwe found11carriers all heterozygous for largedeletions (6) andsmallmutations (5) The remaining femaleswerenot carriers The 2 patients fromCypruswerenot included in the statistical analysis as 1 patient showedanexon2 duplication whereas the other resulted negative DMD = Duchenne muscular dystrophy VUS = variant of uncertainunknown significance

NeurologyorgNG Neurology Genetics | Volume 7 Number 1 | February 2021 5

eligible for exon 51 skipping (orphan drug being eteplirsen)30 might be treated by exon 45 skipping 18 by exon 53skipping and 11 by exon 44 skipping Considering non-sense mutation correction by read-through mechanisms forstop codon reversion 53 patients overall (50 DMDs or 27)were found to be carrying a nonsense mutation for whichataluren is an approved orphan drug (figure 1)

DiscussionDMD mutation identification is an integral part of the di-agnostic flowchart for patients and their family allowing ge-netic diagnosis family planning prenatal testing andeligibility for personalized treatments19 Because of theenormous size of the dystrophin gene the mutational spec-trum is tremendously heterogeneous and the genetic di-agnostic approach must be both accurate and sensitive

Here we provide DMD gene genetic characterization in alarge group of 328 patients with a clinical diagnosis of DMDBMD from Eastern European and non-European patientsdiagnosed between June 2016 and December 2019 Standarddiagnostic approach based on best practice guidelines iden-tified mutations in 222 patients

Our analysis highlighted a relevant diversity in the muta-tion type across countries Indeed the frequency of dele-tions in Algeria (77) vs Eastern Europe (29) greatlyvaries Conversely nonsense mutations (31) and small

mutations (29) represent the most frequent mutationtype in these last countries while rarely occurring in Algeria(18) Ethnic origin and genotype assortments may play arole in this evident variable mutation distribution The highpercentage of still undiagnosed negative cases (34 East-ern Europe and 274 Algeria) is remarkable We mayfirstly consider that some DMD mutations may have es-caped MLPA and sequencing testing as for atypical mu-tations occurring in regulatory regions or due to deepintronic variantsrearrangements20 Because these are es-timated to be less than 1 in various patient series21222324

their impact on the detection rate should be low and only afew cases might have been missed Ethnicity may also play arole in mutation type frequency as already described23only the adoption of a single and fully accurate method ableto detect all mutation types such as whole-genome se-quencing (WGS) will allow us to unravel these events andtheir frequency in the various populations25 Second a fewpatients referred as DMDBMD or generally as possibledystrophinopathies may represent phenocopies of otherclinical entities like limb-girdle muscular dystrophies(LGMDs) or other rare hereditary myopathies2627 In-deed many patients with high CK-related phenotypes andresulted as not carrying a DMD mutation showed DMDVUSs which might be difficult to interpret (see below)LGMDs may indeed deserve a more intensive investigationbut this requires a multigene approach for example genepanel testing WES or in the near future WGS28 Finallyin countries with high consanguinity like Algeria29 a lowerdetection rate due to the occurrence of many recessive

Figure 2 Country Distribution of DMD Mutations in Patients With DMD in Eastern European Countries

Geographical distribution of DMD mutations inEastern European countries and relative numberof patients (in brackets) The percentages for eachtype of DMDmutation are shown for each countryIn countries with more than 30 patients with DMDcarrying a DMDmutation differences can be notedbecause Polish and Ukrainian patients have asimilar mutation landscape with a very high fre-quency of small mutations (average of 55)whereas in Romania 31 of mutation types aresmall mutations with deletions and 69 accountfor duplications DMD = Duchenne musculardystrophy

6 Neurology Genetics | Volume 7 Number 1 | February 2021 NeurologyorgNG

LGMDs might be expected In Algeria however the DMDdetection rate was higher than in the Eastern Europeancountries Although the lack of muscle biopsy availability innegative patients did not allow an RNA study to look forDMD mutations in some negative cases the new tool ofurinary stem cells to profiling the DMD transcript might beconsidered30 Because the DNA testing remains the goldstandard to achieve a genetic diagnosis only a genome-based diagnostic approach will allow us to define the fullDMD mutation scenario

We identified 9 previously unreported VUSs (1 shared by 2brothers) in the DMD gene The pathogenic effect of theseVUSs is often based on an in silico prediction model (tablee-2 linkslwwcomNXGA339) although VUS requiresfunctional validation possibly by RNA profiling or segrega-tion studies in larger families to assess their pathogenicmeaning Therefore their meaning remains hypothetical

We fully validated WES analysis in our pilot study All smallmutations and large deletions were correctly identifiedTherefore WES may cover more than 90 of all DMD genemutations for certain populations and MLPA might only beperformed in negative cases to identify duplications

The adoption of this approach especially if running manypatients in parallel could reduce costs and time to diagnosisbringing to light more than 90 of the causative mutationsof dystrophinopathies Furthermore the use of WES as afirst approach would not only allow the study of the

variations of the DMD gene but also all other genes ofclinical interest

WES provides the great advantage to possibly interrogate thedata for SNPs in gene modifiers Indeed we were able toprofile the genotypes of LTBP4 CD40 and ACTN3 Al-though the small numbers (28 patients) do not allow agenotype-phenotype correlation some differences in geno-type assortment are visible if comparing Algerian and EasternEurope (Ukraine) patients Looking at the allele frequency ofLTBP4 SNPs (which form a 4-SNP haplotype) Ukrainianpatients have higher frequency of the A (1928) G (1928)G (1928) and T (1828) alleles compared with Algerianpatients This implies that the VAAM (AGGT) protectivehaplotype frequency in patients may vary therefore having aless powerful value for predicting milder DMD phenotype (interms of loss of ambulation) Indeed ethnic differences inSNPs allele frequency may hamper a meaningful statisticalassociation in population studies WES also identified pre-viously unreported SNPs in LTBP4 ACTN3 and THBS1The heterozygous ACTN3 exon 21 nonsense variation isunreported and may have a deleterious effect with a possiblerole in modulating the DMD phenotype

The mutation landscape also allows for the evaluation ofpersonalized treatment eligibility31 Eteplirsen (Exondys 51)which inducesDMD exon 51 skipping in amenable patients32

is an approved orphan drug for DMD in the USA Othermolecules that induce exon 45 and 53 skipping are also cur-rently in clinical trials

Figure 3 Integrative Genome Viewer (IGV) Visualization

(A) IGV visualization of sample 253318 Visualization of sample 253318 with deletion of exon 51 (a) there is no coverage or readings of exon 51 (b and c)Visualization of coverage and reads of exons 50 and 52 which precede and follow the deletion of exon 51 respectively (B) IGV visualization of sample 252618 Visualization of sample 252618with deletionof exons 18ndash19 (a) This figure shows that there is no coverageor readings corresponding to exons 18 and 19(b and c) Visualization of coverage and reads of the flanking exon 17 preceding the deletion and of exon 20 following the deletion

NeurologyorgNG Neurology Genetics | Volume 7 Number 1 | February 2021 7

Table 2 Known Gene Modifiers Genotyping of Patients Studied by WES

CountryIDnumber DMD mutation

LTBP4 (chr19) rs2303729GA MAF = 047564 (G)

LTBP4 (chr19) rs1131620AG MAF = 049521 (G)

LTBP4 (chr19) rs1051303AG MAF = 049501 (G)

LTBP4 (chr19) rs10880CT MAF = 041713 (T)

CD40 (chr20) rs1883832CT MAF = 022883 (T)

ACTN3 (chr11) rs1815739CT MAF = 040076 (T)

1 Algeria GM217318

Exon 10 c1012GgtT(pGlu338)

GA AG AG CC CT CT

2 Algeria GM217718

Exon 18 c2253delG(pLys752Argfs8)

GG AA AA CC CC CC

3 Algeria GM251018

Gene deletion exons 45-47 AA GG GG TT TT CC

4 Algeria GM251118

Gene deletion exons 48-50 GG AA AA CC CC CC

5 Algeria GM251518

Gene deletion exons 2-26 AA GG GG TT CT CT

6 Algeria GM251618

Gene duplication exons 52-62

GA AG AG CC CC TT

7 Algeria GM251718

Gene deletion exons 7-12 GA AA AA CC CT CT

8 Algeria GM252418

Gene deletion exons 45-50 GA AG AG TT CC CC

9 Algeria GM252518

Gene deletion exons 18-19 GG AA AA CC CT CC

10 Algeria GM252618

Gene deletion exons 18-19 GG AA AA CC CC CT

11 Algeria GM252818

Gene deletion exons 35-45 GA AG AG CT CC CT

12 Algeria GM252918

Gene deletion exons 52-54 GG AA AA CT CT CT

13 Algeria GM253018

Gene deletion exons 4-7 GA AG AG CT CC CC

14 Ukraine GM257318

Intron 54 c8027+1GgtT GG AA AA CC CC CT

15 Ukraine GM201018

Exon 38 c5444AgtG(pAsp1815Glufs2)

GG AG AG CC CC CT

16 Ukraine GM204318

Exon 4 c206dupC(pArg70Lysfs19)

AA GG GG TT CC CT

17 Ukraine GM204618

Intron 68 c9975-2AgtT GG AA AA CC CC CT

Continued

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Table 2 Known Gene Modifiers Genotyping of Patients Studied by WES (continued)

CountryIDnumber DMD mutation

LTBP4 (chr19) rs2303729GA MAF = 047564 (G)

LTBP4 (chr19) rs1131620AG MAF = 049521 (G)

LTBP4 (chr19) rs1051303AG MAF = 049501 (G)

LTBP4 (chr19) rs10880CT MAF = 041713 (T)

CD40 (chr20) rs1883832CT MAF = 022883 (T)

ACTN3 (chr11) rs1815739CT MAF = 040076 (T)

18 Ukraine GM205018

Intron 26 c3603+1GgtT AA GG GG CT CC CC

19 Ukraine GM209718

Exon 20 c2512CgtT(pGln838)

GA AG AG CT CC CT

20 Ukraine GM213118

Exon 44 c6292CgtT(pArg2098)

AA GG GG TT CT CT

21 Ukraine GM213218

Exon 38 c5341AgtT(pLys1781)

AA AG AG CT CC CT

22 Ukraine GM253318

Gene deletion exon 51 GA AG AG TT CC CC

23 Ukraine GM253418

Exon 20 c2521CgtT(pGln841)

AA GG GG TT CT CC

24 Ukraine GM253618

Exon 16 c1961TgtG(pLeu654)

GA AG AG CT CC CC

25 Ukraine GM253818

Exon 8 c794delAinsCT(pHis265Profs22)

AA GG GG TT CT TT

26 Ukraine GM253918

Intron 5 c358-1GgtT GA AG AG CT CT CT

27 Ukraine GM254018

Gene deletion exons 48-50 GA AG AG CT CC CC

28 Ukraine GM254118

Exon 55 c8034_8037delTGAG(pGlu2681Leufs44)

AA GG GG TT CT CT

Abbreviations DMD = Duchenne muscular dystrophy SNP = single nucleotide polymorphism WES = whole-exome sequencingThemodifier SNPs of genes LTBP4 CD40 and ACTN3 already known to be associated with the DMD phenotypic variability are reported Fourmissense SNPs (rs2303729 rs1131620 rs1051303 and rs10880) along the codingsequence of LTBP4 SNP rs1883832 in the 59-UTR of CD40 and SNP rs1815739 of ACTN3 (R577X) were studied and the genotypes are reported For each sample ID number DMDmutation and SNPs genotypes are reportedSNP rs28357094 of the SPP1 gene and SNPs rs2725797 and rs2624259 of the THBS1 gene could not be studied due to their location in the promoter (5 bases upstream of the transcription start site) and in a telomeric regionsituated about 750 kb flanking a strong enhancer site respectively

Neurolo

gyorgN

GNeurologyG

enetics|

Volume7N

umber

1|

February

20219

Indeed 51 is the most skippable exon (orphan drug ete-plirsen) 30 might be treated by exon 45 skipping 18 byexon 53 skipping and 11 by exon 44 skipping Consideringnonsense mutation correction by read-through mechanismsfor stop codon reversion 53 patients overall (239) werefound to be carrying a nonsensemutation for which ataluren isan approved orphan drug There were however remarkabledifferences when considering country distribution withEastern European countries showing 31 of patients treatableusing a nonsense mutation correction approach and Algeriajust 533

Genetic characterization in different and large patient cohortsprompts many reflections on DMDBMD prevalence mu-tation type gene modifiers allele frequency and personalizedtherapy as we have explored in Eastern Europe and in AlgeriaWe suggest that diagnostic flowcharts should be adjusteddepending on ethnic characteristics In some populationswhere small mutations are more frequent than deletions andor duplications like in Eastern European countries WESmight be indeed the preferred first-step diagnostic tool Thesestudies will have an impact on diagnostic approaches pipelineswith repercussions on care and design of new therapies

AcknowledgmentPTC Therapeutics (United States) is gratefully acknowl-edged for funding the DMD diagnostic project ldquoInterna-tional DMDrdquo (to A Ferlini I-DMD ospfeitreparti-e-servizireparti-dalla-a-alla-m-1genetica-medicagenetica-medica-englishmedical-genetics-unitgenetica-medica-ser-vicesdmd-international-project) The authors thank MsCourtney King (Medical Genetics Unit Department ofMedical Sciences University of Ferrara Ferrara Italy) forthe English revision of this article Thanks are also due to theEU SOLVE-RD project (to A Ferlini) The authors aregrateful to Drs Orza Diana MD Cristea Alexandru MDPuia Virgil-Petru MD (Spitalul Clinic de Urgenta PentruCopii Louis Turcanu Timisoara Romania) RadenkaKuzmanic-Samija MD (University Clinical Hospital SplitCroatia) and Alexandra Grozavu MD (Bacau CountyEmergency Hospital Romania) because they referred tothem patients for DMD diagnosis A Ferlini and otherauthors of this article are full members of the EuropeanReference Network (ERN) Euro-NMD

Study FundingPTC Therapeutics service grant (to A Ferlini) All cliniciansand authors listed in authorship disclose that the patientgenetic diagnosis was supported by this PTC service grant

DisclosureThe authors report no disclosures Go to NeurologyorgNGfor full disclosures

Publication HistoryReceived by Neurology Genetics May 24 2020 Accepted in final formOctober 21 2020

Appendix Authors

Name Location Contribution

Rita SelvaticiPhD

Medical Genetics UnitDepartment of MedicalSciences University ofFerrara Italy

Designed and conceptualizedthe study wrote the paperperformed and interpretedthe DMDmolecular testinginterpreted the WES dataand performed the variantcalling

Rachele RossiPhD

Medical Genetics UnitDepartment of MedicalSciences University ofFerrara Italy

Performed and interpretedthe DMDmolecular testinginterpreted the WES dataperformed the variantcalling and edited thepaper

FernandaFortunato MD

Medical Genetics UnitDepartment of MedicalSciences University ofFerrara Italy

Performed and interpretedthe DMD molecular testing

CeciliaTrabanelli MSc

Medical Genetics UnitDepartment of MedicalSciences University ofFerrara Italy

Performed and interpretedthe DMD molecular testing

Yamina Sifi PhD Neurologie CHU deBenbadis ConstantineAlgerie

Collected and referred tous patients for DMDdiagnosis

Alice MarguttiMSc

Medical Genetics UnitDepartment of MedicalSciences University ofFerrara Italy

Performed and interpretedthe DMD molecular testing

Marcella NeriMD PhD

Medical Genetics UnitDepartment of MedicalSciences University ofFerrara Italy

Performed and interpretedthe DMD molecular testing

FrancescaGualandi MDPhD

Medical Genetics UnitDepartment of MedicalSciences University ofFerrara Italy

Performed and interpretedthe DMD molecular testing

Lena Szabo MD 2nd Department ofPaediatrics SemmelweisUniversity Budapest

Collected and referred tous patients for DMDdiagnosis

Balint FeketeMD

Institute of GenomicMedicine and RareDisorders SemmelweisUniversity BudapestHungary

Collected and referred tous patients for DMDdiagnosis

LyudmillaAngelova MD

Department of MedicalGenetics MedicalUniversity Varna Bulgaria

Collected and referred tous patients for DMDdiagnosis

Ivan LitvinenkoMD

Medical Univesity SofiaDepartment of PediatricsUniversity PediatricHospital ldquoProf Ivan MitevrdquoSofia Department of ChildNeurology Bulgaria

Collected and referred tous patients for DMDdiagnosis

Ivan Ivanov MD Department of PediatricsSt George UniversityHospital Faculty ofMedicine MedicalUniversity Plovdiv Bulgaria

Collected and referred tous patients for DMDdiagnosis

Yurtsever VildanMD

Pediatric NeurologistCounty Clinical EmergencyHospital of ConstantaPediatric NeurologyDepartment Romania

Collected and referred tous patients for DMDdiagnosis

10 Neurology Genetics | Volume 7 Number 1 | February 2021 NeurologyorgNG

Appendix (continued)

Name Location Contribution

Oana AlexandraIuhas MD

G Curteanu MunicipalClinical Hospital OradeaRomania

Collected and referred tous patients for DMDdiagnosis

Mihaela VintanMD

Iuliu Hatieganu Universityof Medicine and PharmacyCluj-Napoca RomaniaDepartment ofNeuroscience

Collected and referred tous patients for DMDdiagnosis

Carmen BurloiuMD

ldquoAlexandru ObregiardquoClinical Psichiatry HospitalPediatric NeurologyDepartment BucharestRomania

Collected and referred tous patients for DMDdiagnosis

ButnariuLacramioaraMD

University of Medicine andPharmacy Grigore T PopaIasi Romania Hospital SfMaria Iasi

Collected and referred tous patients for DMDdiagnosis

Gabriela VisaMD

Pediatric Clinical HospitalSibiu Romania

Collected and referred tous patients for DMDdiagnosis

Diana Epure MD ldquoDr Victor GomoiurdquoChildrenrsquos HospitalBucharest Romania

Collected and referred tous patients for DMDdiagnosis

Cristina RusuMD

1 ldquoGrigore T PopardquoUniversity of Medicine andPharmacy Iasi Romania 2ldquoSfanta MariardquoChildrenrsquosHospital Iasi Romania

Collected and referred tous patients for DMDdiagnosis

Daniela VasileMD

Victor Gomoiu ChildrenrsquosHospital BucharestRomania

Collected and referred tous patients for DMDdiagnosis

MagdalenaSandu MD

Victor Gomoiu ChildrenrsquosHospital Carol DavilaUniversity of Medicine andPharmacy BucharestRomania

Collected and referred tous patients for DMDdiagnosis

DmitryVlodavets MD

Russian ChildrenNeuromuscular CenterVeltischev Clinical PediatricResearch Institute ofPirogov Russian NationalResearch MedicalUniversity Moscow Russia

Collected and referred tous patients for DMDdiagnosis

Monica MagerMD PhD

Iuliu Hatieganursquo Universityof Medicine and PharmacyFaculty of MedicineDepartment ofNeuroscience Neurologyand Pediatric NeurologyEmergency ClinicalHospital for ChildrenPediatric NeurologyDepartment Cluj-NapocaRomania

Collected and referred tous patients for DMDdiagnosis

TheodoreKyriakides MDPhD

Department of Basic andClinical Sciences Universityof Nicosia Cyprus

Collected and referred tous patients for DMDdiagnosis

Sanja Delin MD General Hospital ZadarDepartment of PediatricsZadar Croatia

Collected and referred tous patients for DMDdiagnosis

Ivan LehmanMD

Department of PaediatricsUniversity Hospital CentreZagreb Zagreb Croatia

Collected and referred tous patients for DMDdiagnosis

Appendix (continued)

Name Location Contribution

Jadranka SekeljFures MD

Childrenrsquos Hospital ZagrebFaculty of MedicineUniversity of Osijek Croatia

Collected and referred tous patients for DMDdiagnosis

Veneta BojinovaMD

University Hospital ofNeurology and PsychiatrySveti Naum Sofia Bulgaria

Collected and referred tous patients for DMDdiagnosis

Mariela MilitaruMD

University of Medicine andPharmacy ldquoIuliuHatieganursquorsquoCluj-NapocaRomania andGenetic Center Romania

Collected and referred tous patients for DMDdiagnosis

VelinaGuergueltchevaMD

Clinic of NeurologyUniversity HospitalSofiamed and SofiaUniversity ldquoSt KlimentOhridskirdquo Sofia Bulgaria

Collected and referred tous patients for DMDdiagnosis

Birute BurnyteMD

Institute of BiomedicalSciences Faculty ofMedicine VilniusUniversity Lithuania

Collected and referred tous patients for DMDdiagnosis

Maria JudithMolnar MD PhD

Institute of GenomicMedicine andRareDisordersSemmelweis UniversityBudapest Hungary

Collected and referred tous patients for DMDdiagnosis

NiculinaButoianu MD

Carol Davila Faculty ofMedicine and PharmacyBucharest PediatricNeurology Clinic ldquoAlObregia Hospitalrdquo Romania

Collected and referred tous patients for DMDdiagnosis

Selma DouniaBensemmaneMD

Ali Ait Idir Hospital AlgiersAlgeria University ofAlgiers I

Collected and referred tous patients for DMDdiagnosis

Samira Makri-Mokrane MD

Ali Ait Idir Hospital AlgiersAlgeria University ofAlgiers I

Collected and referred tous patients for DMDdiagnosis

AgnesHerczegfalvi MD

2nd Pediatric Clinic ofSemmelweis UniversityBudapest Hungary

Collected and referred tous patients for DMDdiagnosis

Monica PanzaruMD

ldquoGrigore T PopardquoUniversity of Medicine andPharmacy Iasi RomanialdquoSfanta Mariardquo ChildrenrsquosHospital Iasi Romania

Collected and referred tous patients for DMDdiagnosis

Adela ChiritaEmandi MD

1 Center of GenomicMedicine University ofMedicine and PharmacyVictor Babes TimisoaraRomania 2 RegionalCenter of Medical GeneticsTimis Clinical EmergencyHospital for Children LouisTurcanu TimisoaraRomania

Collected and referred tous patients for DMDdiagnosis

AnnaLusakowska MD

Department of NeurologyMedical University ofWarsaw Poland

Collected and referred tous patients for DMDdiagnosis

Anna Potulska-Chromik MD

Department of NeurologyMedical University ofWarsaw Poland

Collected and referred tous patients for DMDdiagnosis

Anna Kostera-Pruszczyk MDPhD

Department of NeurologyMedical University ofWarsaw Poland

Collected and referred tous patients for DMDdiagnosis

Continued

NeurologyorgNG Neurology Genetics | Volume 7 Number 1 | February 2021 11

References1 Muntoni F Torelli S Ferlini A Dystrophin and mutations one gene several proteins

multiple phenotypes Lancet Neurol 20032731ndash7402 Saengpattrachai M Ray PN Hawkins CE Berzen A Banwell BL Grandpa and I have dys-

trophinopathy approach to asymptomatic hyperCKemia Pediatr Neurol 200635145ndash1493 Zimowski JG Pilch J Pawelec M et al A rare subclinical or mild type of Becker

muscular dystrophy caused by a single exon 48 deletion of the dystrophin gene J ApplGenet 201758343

4 FalzaranoMS Scotton C Passarelli C Ferlini A Duchenne muscular dystrophy fromdiagnosis to therapy Molecules 20152018168ndash18184

5 Straub V Balabanov P Bushby K et al Stakeholder cooperation to overcome chal-lenges in orphan medicine development the example of Duchenne muscular dys-trophy Lancet Neurol 201615882ndash890

6 Sardone V Zhou H Muntoni F Ferlini A Falzarano MS Antisense Oligonucleotide-based therapy for neuromuscular disease Molecules 201722563

7 Birnkrant DJ Bushby K Bann CM et al Diagnosis and management of Duchennemuscular dystrophy part 1 diagnosis and neuromuscular rehabilitation endocrineand gastrointestinal and nutritional management Lancet Neurol 201817251ndash267

8 Fratter C Dalgleish R Allen SK et al Best practice guidelines for genetic testing indystrophinopathies Eur J Hum Genet 2020281141ndash1159

9 Abbs S Tuffery-Giraud S Bakker E Ferlini A Sejersen T Mueller CR Best practiceguidelines on molecular diagnostics in DuchenneBecker muscular dystrophiesNeuromuscul Disord 201020422ndash427

10 Fang M Abolhassani H Lim CK Zhang J Hammarstrom L Next generation se-quencing data analysis in primary immunodeficiency disordersmdashfuture directionsJ Clin Immunol 201636(suppl 1)68ndash75

11 Robinson JT Thorvaldsdottir H Wenger AM Zehir A Mesirov JP Variant reviewwith the Integrative Genomics Viewer (IGV) Cancer Research 20177731ndash34

12 den Dunnen JT Sequence variant descriptions HGVS nomenclature and MutalyzerCurr Protoc Hum Genet 2016907131ndash71319

13 McDonald CM Campbell C Torricelli RE et al Ataluren in patients with non-sense mutation Duchenne muscular dystrophy (ACT DMD) a multicentrerandomised double-blind placebo-controlled phase 3 trial Lancet 20173901489ndash1498

14 Echevarrıa L Aupy P Goyenvalle A Exon-skipping advances for Duchenne musculardystrophy Hum Mol Genet 201827163ndash172

15 Richards S Aziz N Bale S Bick D Das S Gastier-Foster J Standards and guidelinesfor the interpretation of sequence variants a joint consensus recommendation of theAmerican College of Medical Genetics and Genomics and the Association for Mo-lecular Pathology Genet Med 201517405ndash424

16 Bhopal R White DL European Western Caucasian or what Inappropriate la-beling in research on race ethnicity and health Am J Public Health 1998881303ndash1307

17 Dalichaouche I Sifi Y Roudaut C et al γ-sarcoglycan and dystrophin mutationspectrum in an Algerian cohort Muscle Nerve 201756129ndash135

18 Bello L Pegoraro E The ldquousual suspectsrdquo genes for inflammation fibrosis re-generation and muscle strength modify Duchenne muscular dystrophy J Clin Med20198649

19 Shimizu-Motohashi Y Komaki H Motohashi N Takeda S Yokota T Aoki Y Re-storing dystrophin expression in Duchenne muscular dystrophy current status oftherapeutic approaches J Pers Med 201991

20 Aartsma-Rus A Hegde M Ben-Omran T et al Consensus and systematic review onreducing the time to diagnosis of Duchenne muscular dystrophy J Pediatr 2019204305ndash313

21 Bladen CL Salgado D Monges S et al The TREAT-NMD DMD Global Databaseanalysis of more than 7 000 Duchenne muscular dystrophy mutations Hum Mutat201536395ndash402

22 Tuffery-Giraud S Beroud C Leturcq F et al Genotype-phenotype analysis in 2405patients with a dystrophinopathy using the UMD-DMD database a model of na-tionwide knowledgebase Hum Mutat 200930934ndash945

23 Neri M Rossi R Trabanelli C et al The genetic landscape of dystrophin mutations inItaly a nationwide study Front Genet 202011131

24 Flanigan KM Dunn DM von Niederhausern A et al Nonsense mutation-associatedBecker muscular dystrophy interplay between exon definition and splicing regulatoryelements within the DMD gene Hum Mutat 201132299ndash308

25 Barseghyan H Tang W Wang RT et al Next-generation mapping a novel approachfor detection of pathogenic structural variants with a potential utility in clinical di-agnosis Genome Med 2017990

26 Barohn RJ Dimachkie MM Jackson CE A pattern recognition approach to patientswith a suspected myopathy Neurol Clin 201432569ndash593

27 Neri M Selvatici R Scotton C et al A patient with limb girdle muscular dys-trophy carries a TRIM32 deletion detected by a novel CGH array in compoundheterozygosis with a nonsense mutation Neuromuscul Disord 201323478ndash482

28 Donkervoort S Dowling JJ Laporte J MacArthur D Bonnemann CG 214th ENMCworkshop participants 214th ENMC International Workshop establishing an In-ternational Consortium for Gene Discovery and Clinical Research forMuscle DiseaseHeemskerk the Netherlands 6-18 October 2015 Neuromuscul Disord 201929644ndash650

29 Chentouf A Talhi R Dahdouh A et al Consanguinity and epilepsy in Oran Algeria acase-control study Epilepsy Res 201511110ndash17

30 Falzarano MS Ferlini A Urinary stem cells as tools to study genetic disease overviewof the literature AJ Clin Med 20198627

31 Aartsma-Rus A Straub V Hemmings R et al Development of exon skipping therapiesfor Duchenne muscular dystrophy a critical review and a perspective on the out-standing issues Nucleic Acid Ther 2017251ndash259

32 Syed YY Eteplirsen first Global approval Drugs 2016761699ndash170433 Nakamura A Moving towards successful exon-skipping therapy for Duchenne mus-

cular dystrophy J Hum Genet 201762871ndash876

Appendix (continued)

Name Location Contribution

Andriy ShatilloMD

Institute of NeurologyPsychiatry and NarkologyNational Academy ofMedical Science of Ukraine

Collected and referred tous patients for DMDdiagnosis

DjawedBouchenakKhelladi MD

Neurologie CHU TidjaniDamerji Tlemcen Algerie

Collected and referred tous patients for DMDdiagnosis

OussamaDendane MD

Neurologie CHU TidjaniDamerji Tlemcen Algerie

Collected and referred tous patients for DMDdiagnosis

Mingyan FangPhD

BGI-Shenzhen ShenzhenChina

Performed the WES andbioinformatics analysis

Zhiyuan Lu PhD BGI-Shenzhen ShenzhenChina

Performed the WES andbioinformatics analysis

AlessandraFerlini MD PhD

Medical Genetics UnitDepartment of MedicalSciences University ofFerrara Italy

Designed andconceptualized the studyand wrote the paper

12 Neurology Genetics | Volume 7 Number 1 | February 2021 NeurologyorgNG

DOI 101212NXG000000000000053620217 Neurol Genet

Rita Selvatici Rachele Rossi Fernanda Fortunato et al Countries

Ethnicity-related DMD Genotype Landscapes in European and Non-European

This information is current as of December 24 2020

Neurology All rights reserved Online ISSN 2376-7839an open-access online-only continuous publication journal Copyright copy 2020 American Academy of

is an official journal of the American Academy of Neurology Published since April 2015 it isNeurol Genet

ServicesUpdated Information amp

httpngneurologyorgcontent71e536fullhtmlincluding high resolution figures can be found at

References httpngneurologyorgcontent71e536fullhtmlref-list-1

This article cites 32 articles 0 of which you can access for free at

Subspecialty Collections

httpngneurologyorgcgicollectionmuscle_diseaseMuscle disease

onhttpngneurologyorgcgicollectionall_equity_diversity_and_inclusi(IDEAS)Inclusion Diversity Equity Anti-racism and Social Justice

httpngneurologyorgcgicollectiondiagnostic_test_assessment_Diagnostic test assessment

httpngneurologyorgcgicollectioncohort_studiesCohort studies

httpngneurologyorgcgicollectionall_geneticsAll Geneticsfollowing collection(s) This article along with others on similar topics appears in the

Permissions amp Licensing

httpngneurologyorgmiscaboutxhtmlpermissionsits entirety can be found online atInformation about reproducing this article in parts (figurestables) or in

Reprints

httpngneurologyorgmiscaddirxhtmlreprintsusInformation about ordering reprints can be found online

Neurology All rights reserved Online ISSN 2376-7839an open-access online-only continuous publication journal Copyright copy 2020 American Academy of

is an official journal of the American Academy of Neurology Published since April 2015 it isNeurol Genet

ServicesUpdated Information amp

httpngneurologyorgcontent71e536fullhtmlincluding high resolution figures can be found at

References httpngneurologyorgcontent71e536fullhtmlref-list-1

This article cites 32 articles 0 of which you can access for free at

Subspecialty Collections

httpngneurologyorgcgicollectionmuscle_diseaseMuscle disease

onhttpngneurologyorgcgicollectionall_equity_diversity_and_inclusi(IDEAS)Inclusion Diversity Equity Anti-racism and Social Justice

httpngneurologyorgcgicollectiondiagnostic_test_assessment_Diagnostic test assessment

httpngneurologyorgcgicollectioncohort_studiesCohort studies

httpngneurologyorgcgicollectionall_geneticsAll Geneticsfollowing collection(s) This article along with others on similar topics appears in the

Permissions amp Licensing

httpngneurologyorgmiscaboutxhtmlpermissionsits entirety can be found online atInformation about reproducing this article in parts (figurestables) or in

Reprints

httpngneurologyorgmiscaddirxhtmlreprintsusInformation about ordering reprints can be found online

Neurology All rights reserved Online ISSN 2376-7839an open-access online-only continuous publication journal Copyright copy 2020 American Academy of

is an official journal of the American Academy of Neurology Published since April 2015 it isNeurol Genet