WGS and RNA Studies Diagnose Noncoding DMD Variants in ...

ARTICLE OPEN ACCESS

WGS and RNA Studies Diagnose NoncodingDMD Variants in Males With High CreatineKinaseLeigh B Waddell PhD Samantha J Bryen BSc (Hons) Beryl B Cummings PhD AdamBournazos BSc (Hons)

Frances J Evesson PhD Himanshu Joshi B Software Engineering B Business (Finance) Jamie LMarshall PhD

Taru Tukiainen PhD Elise Valkanas BA (Biology) Ben Weisburd BS (Computer Sc) Simon Sadedin PhD

Mark R Davis PhD Fathimath Faiz PhD Rebecca Gooding PhD Sarah A Sandaradura MBChB FRACP PhD

Gina L OrsquoGrady MBChB FRACP PhD Michel C Tchan MBBS FRACP PhD David R Mowat MBBS FRACP

Emily C Oates MBBS FRACP PhD Michelle A Farrar MBBS FRACP PhD

Hugo Sampaio MBBCh FRACP MPhil Alan Ma MBBS FRACP Katherine Neas MBChB FRACP

Min-Xia Wang PhD Amanda Charlton MBChB FRCPA Charles Chan MBBS (Hons) FRCPA PhD

Diane N Kenwright MBBS FRCPA Nicole Graf MBBS FRCPA Susan Arbuckle MBBS FRCPA

Nigel F Clarke MBChB FRACP PhD Daniel G MacArthur PhD Kristi J Jones MBBS FRACP PhD

Monkol Lek PhD and Sandra T Cooper PhD

Neurol Genet 20217e554 doi101212NXG0000000000000554

Correspondence

Dr Cooper

Sandracoopersydneyeduau

AbstractObjectiveTo describe the diagnostic utility of whole-genome sequencing and RNA studies in boys withsuspected dystrophinopathy for whom multiplex ligation-dependent probe amplification andexomic parallel sequencing failed to yield a genetic diagnosis and to use remnant normalDMDsplicing in 3 families to define critical levels of wild-type dystrophin bridging clinical spectrumsof Duchenne to myalgia

MethodsExome genome andor muscle RNA sequencing was performed for 7 males with elevatedcreatine kinase PCR of muscle-derived complementary DNA (cDNA) studied consequencesfor DMD premessenger RNA (pre-mRNA) splicing Quantitative Western blot was used todetermine levels of dystrophin relative to control muscle

ResultsSplice-altering intronic single nucleotide variants or structural rearrangements in DMD wereidentified in all 7 families Four individuals with abnormal splicing causing a premature stopcodon and nonsense-mediated decay expressed remnant levels of normally spliced DMDmRNA Quantitative Western blot enabled correlation of wild-type dystrophin and clinicalseverity with 0ndash5 dystrophin conferring a Duchenne phenotype 10 plusmn 2 a Beckerphenotype and 15 plusmn 2 dystrophin associated with myalgia without manifesting weakness

RELATED ARTICLE

EditorialMolecular Diagnosisin 100 ofDystrophinopathies AreWe There Yet

Page e529

Deceased

From the Kids Neuroscience Centre (LBW SJB AB FJE HJ SAS GLO ECO NFC KJJ STC) Kids Research Institute The Childrenrsquos Hospital at Westmead New South WalesAustralia Disciplineof Child andAdolescent Health (LBW SJB AB FJE SAS GLO ECO NFC KJJ STC) Faculty ofMedicine andHealth TheUniversity of SydneyWestmeadNewSouthWales Australia Analytic and Translational Genetics Unit (BBC JLM TT EV DGM ML)Massachusetts General Hospital BostonMedical andPopulationGenetics (BBC JLMTT EV BW SSDGMML) andCenter forMendelianGenomics (BBC JLM EV BW SS DGMML) Broad InstituteofMITampHarvardCambridgeMA FunctionalNeuromics (FJESTC) Childrenrsquos Medical Research Institute Westmead New South Wales Australia Murdoch Childrenrsquos Research Institute (SS) Parkville Victoria Australia Department of DiagnosticGenomics (MRD FF RG) PathWest Laboratory MedicineWA Nedlands Australia Department of Clinical Genetics (SAS AM KJJ) Childrenrsquos Hospital at Westmead New SouthWalesAustralia Department of Genetic Medicine (MCT) Westmead Hospital New SouthWales Australia Discipline of GenomicMedicine (MCT AM) SydneyMedical School The University ofSydneyNewSouthWales Australia Centre for Clinical Genetics (DRM) Sydney Childrenrsquos Hospital Randwick NewSouthWales Australia School ofWomenrsquos and ChildrenrsquosHealth (DRMMAF) UNSWMedicine UNSWSydney Australia Department of Neurology (MAF HS) Sydney Childrenrsquos Hospital Randwick NewSouthWales Australia Department of Clinical Genetics(AM) NepeanHospital Sydney Australia Genetic Health ServiceNZ (KN)WellingtonNewZealand Neurology Laboratory (M-XW) Royal Prince AlfredHospital CamperdownNew SouthWales Australia Central Clinical School (M-XW) Faculty of Medicine and Health The University of Sydney Camperdown New South Wales Australia Anatomic Pathology (AC CC NGSA) TheChildrenrsquosHospital atWestmeadNewSouthWales Australia Anatomic Pathologist (DNK)Department ofPathology andMolecularMedicineUniversity ofOtagoWellingtonNewZealand and Harvard Medical School (DGM) Boston MA

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

The Article Processing Charge was funded by the authors

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 2021 The Author(s) Published by Wolters Kluwer Health Inc on behalf of the American Academy of Neurology 1

ConclusionsWhole-genome sequencing relied heavily on RNA studies to identifyDMD splice-altering variants Short-read RNA sequencingwas regularly confounded by the effectiveness of nonsense-mediated mRNA decay and low read depth of the giant DMDmRNA PCR of muscle cDNA provided a simple yet informative approach Highly relevant to genetic therapies for dystro-phinopathies our data align strongly with previous studies of mutant dystrophin in Becker muscular dystrophy with thecollective conclusion that a fractional increase in levels of normal dystrophin between 5 and 20 is clinically significant

Duchenne muscular dystrophy (DMD) is a severe X-linkeddisorder primarily affecting approximately 1 in 5000 malebirths1ndash3 DMD shows a relentlessly progressive courseresulting in loss of ambulation in teens and early mortalitydue to cardiac or respiratory involvement45 Dystrophino-pathies range clinically from the severe DMD to asymp-tomatic hyperCKemia5ndash12 DMD is associated with theabsence of dystrophin in muscle due to loss-of-functionvariants in the DMD gene encoding dystrophin56 whereasBecker muscular dystrophy (BMD) is associated with vari-ants in DMD that result in reduced levels of (mutated)dystrophin56

TheDMD gene is the largest gene in the human genome withnumerous enormous introns1314 One-third of pathogenicDMD variants are de novo1516 with most affected individualsbearing insertions or deletions (indels) of coding exons1517

Pathogenic DMD missense variants are rare61518 and non-coding variants are emerging as an important rare cause ofdystrophinopathy15171920 Approximately 5 of patientsclinically diagnosed with DMD do not have a genetic di-agnosis after mutational analysis5

Herein we show the diagnostic application of whole-genomesequencing transcriptomics and dystrophin protein

Glossarybp = base pair CK = creatine kinase DMD = Duchenne muscular dystrophy gnomAD = Genome Aggregation DatabaseGTEx = Genotype-Tissue Expression IGV = Integrative Genomic Browser MLPA = multiplex ligation-dependent probeamplificationmRNA =messenger RNA nt = nucleotide RNA-seq = RNA sequencing RT-PCR = reverse transcription PCRSNV = single nucleotide variant WB = Western blot WGA = wheat germ agglutinin WT = wild type

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

biochemistry to secure a genetic diagnosis for 13 affectedmales from 7 families with elevated creatine kinase (CK) whoremained undiagnosed following multiplex ligation-dependent probe amplification (MLPA) and exomic se-quencing Importantly we identify 3 families with DMDsplicing variants who produce varying levels of mis-splicedtranscripts that encode a premature stop codon and are tar-geted by nonsense-mediated decay though express varyinglevels of remnant normally spliced DMD mRNA Thereforequantitative Western blot (WB) of muscle biopsy specimensfrom these 3 dystrophin hypomorphs has uniquely enabledspecific correlation of levels of wild-type (WT) dystrophinwith clinical severity

MethodsStandard Protocol Approvals Registrationsand Patient ConsentsThis study was approved by the Childrenrsquos Hospital atWestmead Human Research Ethics Committee (Biospeci-men Bank_10CHW45) with informed written consentfrom all participants

We describe a retrospective cohort of boys diagnosed withDMD variants from genomic and RNA studies who had el-evated CK and dystrophic muscle biopsies and were un-diagnosed after MLPA and exomic parallel sequencing

Immunohistochemistry and Western BlottingImmunohistochemistry21 and Western blotting22 were per-formed as previously described WB used NuPAGE 3ndash8Tris-Acetate precast gels (Invitrogen by Thermo FisherScientific NSW Australia) Antibodies for immunohisto-chemistry muscle fiber membranes were stained with anti-dystrophin DYS1 DYS2 DYS3 and anti-spectrin SPEC1(Leica Biosystems VIC Australia) with anti-mouse AlexaFluor 555 secondary antibody membranes were counter-stained with wheat germ agglutinin-AF488 (WGA) and nu-clei were stained with DAPI (Invitrogen Thermo FisherScientific) WBs were probed with DYS1 (Leica Biosystems)rabbit polyclonal dystrophin antibody (Rb-DMD ab15277Abcam) α-actinin-2 (4A3 gift from A Beggs ChildrenrsquosHospital Boston Boston MA) sarcomeric actin (clone 5C5A2172 Sigma-Aldrich) and the anti-mouse or anti-rabbit IgGlight chain HRP-conjugated secondary antibodies (GEHealthcare NSW Australia) The rabbit polyclonal dystro-phin antibody (Rb-DMD ab15277 Abcam) detects a 10-foldserial dilution whereas DYS2 is less sensitive (detects a 4-foldserial dilution) Therefore ab15277 was selected due toprovision of a more informative standard curve for semi-quantification of dystrophin levels in the probands ImageJ23

was used to measure the densities of the patient and seriallydiluted controls bands to create a standard curve as pre-viously described19 Semiquantitation of dystrophin levels wasperformed by comparing densities of the dystrophin band inpatient sample relative to the standard curves of dystrophin in

2 age- and sex-matched controls across 3 experimentalreplicates

Massively Parallel SequencingWhole-exome sequencing (probands and AI1 AI2 and AII2) PCR-free whole-genome sequencing (probands fromfamilies A and B DndashG) and RNA sequencing (RNA-seqprobands from families A B D E and G) were performed atthe Broad Institute of Harvard and MIT as previously de-scribed20 RNA-seq was performed for CII2 at PathWestLaboratory Medicine WA as previously described for the fetalsamples in reference 24

Sanger Sequencing and RT-PCRRNA was extracted and reverse transcription PCR (RT-PCR)was performed as previously described25 Primers used forAII1 have been previously described20 The remaining primerdetails are as follows Ex42F 59-CAATGCTCCTGACCTCTGTG-39 Ex4344R 59-CTGTCAAATCGCCCTTGTCG-39LINC00251Ex3R 59-CTGAAATGGGTGGGATGAAG-39LINC00251Ex2F 59-GATGCCCCTTAACCAAGGAC-39Ex26F 59-GATGCACGAATGGATGACAC-39 Ex27R59-TGTGCTACAGGTGGAGCTTG-39 Ex2627F59-GCAGTTGAAGAGATGAAGAGAGC-39 Ex29R59-TGGGTTATCCTCTGAATGTCG-39 In26PF 59-AAA-CTTAGTTCGGCCCCATG-39 Ex48F 59-GTTAAAT-CATCTGCTGCTGTGG-39 Ex54R 59-ACTGGCG-GAGGTCTTTGG-39 Ex4952F 59-ACTCAGCCAGTGA-AGGCAAC-39 Ex53R 59-TCCTAAGACCTGCTCAGCT-TC-39 Ex51F 59-CGACTGGCTTTCTCTGCTTG-39Ex5052F 59-CAAATCCTGCATTGTTGCAGG-39GAPDHEx3F 59-TCACCAGGGCTGCTTTTAAC-39 andGAPDHEx6R 59-GGCAGAGATGATGACCCTTT-39 Con-firmation and segregation analysis of DMD variants was per-formed by Sanger sequencing21 except for family F in whichDNAwas not available Primers used for families A D E andGhave been previously described20 The remaining primer detailsare as follows family BmdashIn43F 59-TTTAGTTTCCAGC-CACTCCTGTC-39 with chr8R 59-TAGCAGGGGCAAGG-GTTG-39 and chr8F 59-TGCCTCTCCAGAATGAGGAC-39with In43R 59-CGGGGAACATCACACACC-39 to confirminsertion breakpoints family CmdashIn26F 59-CGAAGGAAAC-TGGTATGTAG-39 with In26R 59-AAAGCCGTATGACA-GATTCG-39 to determine causative variant PCR conditionswere 5 minutes 95degC 35 cyclesmdash30 seconds 95degC 30 seconds58degC and 1 minute 72degC 8 minutes 72degC or as described inreference 20

Whole-Genome Sequencing AnalysisPCR-Free whole-genome sequencing was performed on anIllumina HiSeq X Ten using 2 times 150 paired end reads at30times mean coverage The sequencing reads were aligned tothe GRCh37 genome reference and single nucleotidevariants (SNVs) small insertions and deletions (indels)were detected using methods previously described in ref-erence 20 A reanalysis of rare (Genome AggregationDatabase [gnomAD] AF lt 0005) SNVs and indels

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

revealed no pathogenic DMD variants The Manta toolfrom Illumina (PMID 26647377) was used to identifystructural variants or split read abnormalities within theDMD gene Putative structural variants were manuallyinspected within Integrative Genomic Browser (IGV)to validate and resolve exact breakpoints of structuralrearrangements

RNA-seq AnalysisRNA-seq analysis was performed as described in reference20 Briefly all samples were jointly processed and alignedwith the Genotype-Tissue Expression Consortium (GTEx)26 to identify spliced reads only seen in patients or groups of

patients and missing in controls In addition given the na-ture of the previously suspected diagnosis of a dystrophin-opathy in cases in which this approach did not lead to adiagnosis exonic read depth was mapped in each patient andcompared with controls and sashimi plots of patients weremanually inspected using the IGV for the DMD gene Incases in which RNA-seq identified a mis-splicing eventpatient exome and genomes were manually evaluateddepending on availability

Data AvailabilityData not published within this article are available by requestfrom any qualified investigator

Table Clinical Presentation DMD Variants and Dystrophin Western Blot

AII1 BIV1 CII2 DII1 EII1 FII6 GII1

Clinicalsymptoms

Muscle painfatigue andmyoglobinuriawith exercise

Proximal weaknessand bilateral calfhypertrophy

Progressivelimb-girdleweakness andfalling regularly

Proximalweaknesscalfhypertrophyand positiveGowers sign

Muscleweaknessand calfhypertrophy

Proximal muscleweaknesspositive Gowerssign and calfhypertrophy

Calfhypertrophyand positiveGowers signproximalweaknesselbowcontracturesand learningdifficulties

Onset 15 y 5 y 9 y 35 y 6 y 35 y 5 y

Familyhistory

2 affectedbrothersreporting myalgiaand serum CKlevels of300ndash14700 UL

Four-generationfamily segregatingwith an X-linkedmuscular dystrophywithcardiomyopathy

Nil Nil Has asimilarlyaffectedbrother

The mother (FI6)also has musclepain and elevatedserum CK levels of500 UL

Nil

Serum CKUL

1400ndash7500 9964 420 14500 18889 24000 gt12000

Ambulance Remainsambulant

Remains ambulant Intermittentuse of awheelchairfrom 13 y

Wheelchairdependent at13 y

Wheelchairdependentat 9 y

Remainsambulant and toewalking at 9 y

Wheelchairdependent at 7y

Cardiac andrespiratoryinvolvement

Nil Normalechocardiogramcardiomyopathy inBIII2 and BIII7

Nocturnalbilateralpositive airwaypressure at 28y normalcardiacfunction

Cardiac-reducedcontractility(ejectionfraction30ndash35)with normalleft ventriclesize

Nil Nil Borderlineincrease inheart size at 9 ydied at age 10 yfrom cardiaccomplications

DMD variant Pseudoexoninclusion in DMDintron 43 NM_0040062c6290+30954CgtT

116 kb chr8insertion in DMDintron 43 NM_0040062c6290+28627_6290+28628ins[TGTGGGCAAAGGCNR_0389011-100749_430-3036NM_00400626290+28628_6290+28751]

Pseudoexoninclusion inDMD intron 26NM_0040062c3603+820GgtT

Inversion ofDMD exon 51NM_0040062c7310-2629_7542+1338inv

Inversion ofDMD exons1ndash18 NM_0040062c-1950935_2293-1933inv

Inversion of DMDexons 1ndash44 NM_0040062c[NM_00130454816818-10658_NM_00400626438+112319inv4233+10599_5325+387dup6117+6701_6438+112319dup]

Inversion ofDMD exons1ndash60 NM_0040062c[-117965533_9085-12259inv-117965534_-117965551del9085-12258_9085-12196del]

Westernblot

15 plusmn 2 10 plusmn 2 0ndash5 0ndash5 0ndash5 0ndash5 0ndash5

Abbreviations CK = creatine kinase DMD = Duchenne muscular dystrophy DMD = DMD gene or transcript


ResultsClinical PresentationFour families have been described previously in reference 20AII1 as N33 DII1 as C3 EII1 as C4 andGII1 as C2 Clinicalpresentation DMD variants and dystrophin WB results aresummarized in the table Briefly AII1 presented at 15 yearswith muscle pain fatigue and episodes of myoglobinuria withexercise and elevated serumCK (CK 1400ndash7500 UL normalrange lt200 UL) He has 2 affected brothers with myalgia andelevated serumCK (300ndash14700 UL (figure 1A) Family B is a4-generation family with an X-linked muscular dystrophy withcardiomyopathy BIII2 was diagnosed with dilated cardiomy-opathy in his 20s underwent cardiac transplantation at age 29years and died of transplant-related complications at age 31years BIII7 was diagnosed with BMD in his mid-teens He hasno known history of cardiomyopathy and remains ambulant inhis 40s (figure 1B) BIV1 showed elevated serumCK 9964 UL at age 6 months Now age 5 years he has proximal muscleweakness bilateral calf hypertrophy and normal echocardio-gram CII2 presented at age 9 years with progressive limb-girdleweakness requiring intermittent use of a wheelchair from age 13years and nocturnal bilateral positive airway pressure (BiPAP)from age 28 years He has normal cardiac function with serumCK of 420 UL at age 31 years (figure 1C) DII1 presented atage 35 years with proximal weakness calf hypertrophy positiveGowers sign and serumCKof 14500ULHe required use of awheelchair from age 13 years Echocardiogram at age 17 yearsshowed reduced contractility (ejection fraction 30ndash35) withnormal left ventricle size (figure 1D) EII1 presented at age 6years with muscle weakness enlarged calves and serum CK of18889 UL He required use of a wheelchair at age 9 years and

has no known cardiac or respiratory involvement EII1 has asimilarly affected brother (figure 1E) FII6 presented at age 35years with proximal muscle weakness positive Gowers signprominent calves and serum CK of 24000 UL He remainsambulant but is toe walking at age 9 years He has no knowncardiac or respiratory involvement FII6rsquos mother (FI6) re-ports muscle pain and has elevated serum CK of 500 UL(figure 1F) GII1 presented at age 5 years with waddling gaitcalf hypertrophy positive Gowers sign and serum CK levels ofgt12000 UL He required use of a wheelchair from age 7 yearsEchocardiogram at age 9 years showed borderline increase inheart size and he died at age 10 years from cardiac complica-tions (figure 1G)

DMD Diagnostic Genetic TestingDMD MLPA and Sanger sequencing were performed andreported normal for AII1 BIV1 CII2 DII1 EII1 and GII1 DMD MLPA performed for FII6 revealed duplications ofexons 31ndash37 and 43ndash44 which were predicted to be in-frameand therefore considered inconsistent with his severeDuchenne-like phenotype though with high clinical suspicionof causality A genetic basis could not be identified via whole-exome sequencing (AII1 BIV1 DII1 EII1 FII6 and GII1 with duplications of exons 31ndash37 and 43ndash44 confirmed forFII6) or massively parallel sequencing of a targeted neuro-muscular gene panel (CII2)

Immunohistochemistry DemonstratesDystrophin Abnormalities in SkeletalMuscle BiopsiesSkeletal muscle immunohistochemistry for AII1 BIV1 DII1 EII1 FII6 and GII1 confirms abnormalities in dystrophin

Figure 1 Pedigree of Families AndashG

Index patient for each family denoted with black arrow Affected members colored in red and carriers part colored in red


Figure 2 Muscle RNA Studies of DMD in Patients

(A) RNA-seq read coverage of DMD exons inmuscle RNA from AII1 BIV1 DII1 EII1 and GII1 and 2 GTEx controls Red arrows indicate the reduction in readdepth which corresponds with the location of DMD structural variants for BIV1 DII1 EII1 and GII1 (BndashG) RT-PCR studies ofmuscle-derived RNA of patientswith splicing abnormalities and 3male controls (C1 quadriceps 65 years C2 vastus lateralis 17 years C3 unknown 20 years) Primers used are listed at thebottom right of each gel image and are labeled according to their location (exon Ex intron In pseudoexon P) and orientation (forward F reverse R)Bridging primers span a splice junction and are denoted by XY where X and Y are exons the primer spans All results were confirmed by Sanger sequencing(B) RT-PCR showing reduced levels of correctly splicedDMD transcript (exons 43 and 44) in AII1 and BIV1 comparedwith controls AII1 shows the inclusion ofa 128-bp pseudoexon (C) Primers specific to the 128 bp pseudoexon revealed that the inclusion is specific to AII1 (Sanger sequencing showed that the faintbands in C1were non-DMD sequences) Sequencing reveals that faint bands in AII1 correspond tomultiple pseudoexons inDMD incorporated into aminorityof DMD transcripts (D) Various chr8 pseudoexons and LINC00251 exons are included in DMD transcripts as a result of the chr8 insertion in BIV1 The lowestband detected in all samples in the top gel corresponds to non-DMD sequences (E) RT-PCR confirms the inclusion of a 84-bp pseudoexon in CII2 in themajority of DMD transcripts Normal splicing can only be detected in very low levels in CII2 by bridging primers The 92 bp pseudoexon is absent in controlsamples (F) RT-PCR of DII1 confirms that exon 51 is absent fromallDMD transcripts A bridging primer indicates that skipping of both exons 50 and 51 is a lowfrequency event observed in both controls and DII1 (G)GAPDH loading controls to indicate that similar concentrations of complementary DNAwere used forboth control and patient samples DMD = Duchenne muscular dystrophy DMD = DMD gene or transcript RT-PCR = reverse transcription PCR


Figure 3 Schematics of Variants Identified in Families AndashG

(A) Family A intronic c6290+30954CgtT (black arrow) creates a cryptic donor splice site leading to inclusion of a 128-bp pseudoexon (red within DMD intron43) into theDMDmRNA causing a frameshift and stop codon (red arrow) encoded by exon 44 (ex44) Gene direction is demonstrated by gray arrows Readingframe between exons is shown by shape complementarity (B) Family B insertion of 116284 bp of chr8 (red sequence) into DMD intron 43 The insertionincludes LINC00251 exons 1ndash3 (black outlined exons) A 124-bp sequence of intron 43 of DMD (chrX32276895-32277018) is duplicated as part of thestructural rearrangement and now flanks the chr8 insertion In addition there is an insertion of 13 bp (insGCCTTTGCCCACA shown in green) adjacent to 1copy of the 124-bp duplication mRNA studies show evidence for numerous different abnormal splicing events from DMD exon 43 to various pseudoexons(red exons) and LINC00251 exons (red exons with black outlines) within the chr8 insertion Low levels of normal DMD splicing (from exons 43 and 44 blueexons) are also observed Frameof splicing in pseudoexons and LINC00251 exons not shown (C) Family C intronic c3603+820GgtT (black arrow) increases thestrength of the polypyrimidine tract leading to use of a cryptic acceptor splice site (35 algorithms within Alamut Visual biosoftware predictions MaxEntScanNNSPLICE andGeneSplicer) leading to inclusion of a 84-bp pseudoexon (red withinDMD intron 26) into theDMDmRNA encoding a stop codon (red arrow) 39nucleotides into the pseudoexon Gene direction is demonstrated by gray arrows Reading frame between exons is shown by shape complementarity (D)Family D inversion ofDMD exon 51 and flanking adjacent intronic sequence Flanking the structural rearrangement are 2 intronic deletions (orange 35 kb andpurple 44 bp) and an insertion of CCAATA (green) mRNA studies show exon 51 skipping causing a frameshift and a premature stop codon (TAG encoded byexon 52 red arrow) (E) Family E A 26-Mb inversion on the X chromosomebetween 2 breakpoints A in intron 45 of CFAP47 19Mbupstreamof exon 1 ofDMD(GRCh37chrX35180364) and B in intron 18 of DMD (GRCh37chrX32521892 NM_0040062) This reverses the orientation of exons 1ndash18 of DMD which arenow joined to CFAP47 sequences upstream of exon The DMD gene is in blue exons dark blue and introns light blue Intergenic sequence (non-DMD ChrX ingreen) (F) Family F A 41-Mb inversion on the X chromosomebetween 2 breakpoints A is 38Mbupstreamof exon 1 ofDMD (GRCh37chrX36236087) andB inintron 44 of DMD (GRCh37chrX32122714) This reverses the orientation of exons 1ndash44 of DMD which are now joined to intergenic sequence upstream ofexon 1 This is accompanied by duplication of exons 31ndash37 (orange) and exons 43 and 44 (purple) around the breakpoint (G) Family G A 1198-Mb inversionon the X chromosomebetween 2 breakpoints A in an intergenic region on the q armof the X chromosome 118Mbupstreamof exon 1 ofDMD (GRCh37chrX151194962) and B in intron 60 ofDMD (GRCh37chrX31379010 NM_0040062) This reverses the orientation of exons 1ndash60 ofDMD which are now joined tointergenic sequence upstream of exon 1 In addition 2 deletions were identified at these breakpoints an intronic 63 bp deletion (orange GRCh37chrX31378947-31379009) and an intergenic 18 bp deletion (purple GRCh37chrX151194963-151194980) X chromosome displayed in unusual orientationwith q arm to the left so the DMD gene is presented with exons in order DMD = DMD gene or transcript mRNA = messenger RNA


(figure e-1 linkslwwcomNXGA367) Using 3 anti-dystrophin antibodies AII1 and BIV1 showed reduceddystrophin staining whereas DII1 EII1 FII6 and GII1showed absent staining (figure e-1 absent dystrophin stainingshown only for GII1) WGA outlines the myofibers and la-bels the endomysium in patient and control skeletal musclesamples

Correlation of Splicing Analyses WithWhole-Genome Sequencing IdentifiesPathogenic Intronic and Structural VariantsInducing Abnormal DMD SplicingSix individuals (AII1 BIV1 DII1 EII1 FII6 and GII1)were subject to whole-genome sequencing 6 individuals weresubject to RNA-seq (AII1 BIV1 CII2 DII1 EII1 andGII1) and 4 individuals (AII1 BIV1 CII2 and DII1)were analyzed by RT-PCR of muscle-derived mRNA Scru-tiny of DMD transcripts (NM_0040062 11058 nucleotides[nt] in length) shows typical 39 bias in read depth (vastlymore reads at the 39 end compared with the 59 end of DMDtranscripts) Acknowledging 39 bias an abnormal profile ofDMD transcript read depth was apparent for BIV1 DII1EII1 and GII1 (figure 2A) relative to multiple musclecontrols from the GTEx consortium26

Standard variant filtering approaches of genomic sequencingfailed to identify most causal variants RNA-seq identified

abnormal pseudoexon inclusion into DMD transcripts forfamilies A and C The remaining pathogenic variants wereidentified only through the combination of whole-genomesequencing bioinformatics and RNA analyses

A genetic diagnosis in AII1 was identified in a previousstudy20 with a deep intronic pathogenic variant GRCh37ChrX32274692GgtA c6290+30954CgtT inducing partialmis-splicing of DMD The DMD c6290+30954CgtT variantcreates a cryptic donor 59 splice site resulting in inclusion of avariant-activated pseudoexon of 128 nt inserted between exon43 and exon 44 which encodes 59 missense amino acids andeffects a frameshift resulting in a premature termination co-don encoded by exon 44 (figure 3A) RT-PCR confirmedabnormal inclusion of the variant-activated pseudoexon andresidual normal splicing ofDMD exons 42-43-44-45 (figure 2B and C)

RNA-seq for BIV1 showed low levels of DMD transcriptswith a distinct drop in reads from exon 44 onward (figure 2Aarrow) Bespoke realignment and analyses of WGS dataidentified insertion of 118000 nt of chromosome 8 (chr8)sequences within DMD intron 43 encompassing theLINC00251 gene locus RT-PCR showed that the chr8 in-sertion induced abnormal splicing of the DMD gene (figure3B) Multiple adverse events were detected that involvedsplicing from exon 43 of DMD to various pseudoexons and

Figure 4 Western Blot Panel for All Patients

(Aa) Western blot was performed on skeletalmuscle from index patients from families A and B(AII1 and BIV1) against DYS1 (rod domain epi-tope) and Rb-DMD (C-terminal epitope) with serialdilutions (12 34 56 and 910) human controlskeletal muscle Muscle lysate derived from anindividual with Duchenne muscular dystrophyand undetectable levels of dystrophin byWesternblot (DMD control deltoid 14-year-old boyGRCh37chrX32364116GgtA NM_0040062c5530CgtT pArg1844) were added to dilutedcontrols to normalize total protein loading in eachlane of the gel Loading controls a-actinin-2 andmyosin (coomassie) (Ab) Image J23 was used tomeasure the densities of the patient and seriallydiluted controls bands to create a standard curveQuantification of relative dystrophin levels wasperformed by comparing patient sample densi-ties to the control standard curves across the 3gels shown AII1 demonstrates 155 plusmn 19levels of dystrophin protein relative to controlsBIV1 demonstrates 96 plusmn 17 levels of dystro-phin protein relative to control (B) Western blotanalysis on skeletal muscle from patient CII2against DYS1 shows undetectable levels of dys-trophin compared with controls Loading con-trols myosin (coomassie) (C) Western blotanalysis on skeletalmuscle frompatients DII1 EII1 FII6 and GII1 against DYS1 compared withhuman control skeletal muscle DII1 shows verylow levels of dystrophin EII1 FII6 and GII1 showundetectable levels of dystrophin Loading con-trols a-actinin-2 and sarcomeric actin Male con-trols used C1 tibialis anterior 16 years C2unknown 55 years C3 unknown 14 years C4quadriceps 45 years DMD =Duchennemusculardystrophy


LINC00251 exons within the chr8 insertion Sanger sequenc-ing with bespoke PCR over the breakpoints on gDNA con-firmed the chr8 inclusion in intron 43 and provided a diagnosticassay that confirmed segregation of the insertion within thefamily pedigree Normal splicing of DMD exons 42-43-44-45was observed as a low-frequency event (figure 2 B and D)

For CII2 manual analysis of RNA-seq data identified abnormalinclusion of 84 nt from intron 26 into a majority of DMDtranscripts (figure 3C) Sanger sequencing of the genomic regionin gDNA fromCII2 identified a deep intronic variant GRCh37ChrX32471959CgtA c3603+820GgtT that was absent in gno-mAD The DMD c3603+820GgtT variant in intron 26 disruptsan AG creating an AG-exclusion zone between an availableconsensus lariat branch point and 39 splice site27 Spliceosomaluse of a naturally occurring consensus 59 splice site sequence andthis strengthened 39 splice site result in the inclusion of a variant-activated pseudoexon into a majority of DMD transcriptsencoding 19 missense amino acids followed by a stop codon(figure 3C) RT-PCR confirmed abnormal inclusion of thevariant-activated pseudoexon intoDMD transcripts and residuallow levels of DMD transcripts with normal splicing of exons 25-26-27 (figure 2E) Sanger sequencing confirmed that thec3603+820GgtT variant was de novo in CII2

For DII1 RNA-seq in a previous study20 showed low levels ofDMD transcripts with exon 51 skipping inducing a frameshiftand premature stop codon encoded by exon 52 (r7310_7542del pSer2437Cysfs33 figure 3D) Interrogation ofWGS determined presence of a DMD structural rearrange-ment rendering DMD exon 51 in the reverse orientation andunable to be spliced into the DMD mRNA confirmed bySanger sequencing RT-PCR confirms exon 51 skipping as thepredominant mis-splicing event in DII1 with skipping ofexons 50 and 51 a low-frequency in-frame event observed inbothDII1 and controls (figure 2F) Low levels of exon 50 and51 skipping are consistent with low levels of dystrophindetected by WB analysis (figure 4C)

RNA-seq showed an abrupt loss of transcripts after exon 18 inEII1 as previously described in reference 20 (figure 2) WGSshowed evidence for an inversion within the DMD gene re-versing the orientation of exons 1ndash18 ofDMD which are nowjoined to intergenic sequences upstream of exon 1 explainingthe presence of abruptly terminating exon 1ndash18 transcriptstranscribed from theDMD promoter (figure 3E) The 19 Mbintergenic region included in the inversion contains FAM47AFAM47B and TMEM47 genes Sanger sequencing of geno-mic DNA over the breakpoints confirmed the inversion

FII6 with in-frame duplications of exons 31ndash37 and 43ndash44identified on DMD MLPA was shown by WGS to have alarger more complex structural rearrangement (figure 3F)which reverses the orientation of exons 1ndash44 of DMD whichare now joined to intron 45 of CFAP47 upstream of exon 1Expression of CFAP47 is likely to be disrupted However theclinical significance of loss of CFAP47 expression is unknown

In GII1 RNA-seq in a previous study20 showed low readcount forDMD transcripts with evidence for even fewer readsfrom exon 60 Closer scrutiny of whole-genome sequencingdata identified a structural rearrangement reversing the ori-entation of exons 1ndash60 of DMD which are now joined tointergenic sequences upstream of exon 1 (figure 3G) Sangersequencing of genomic DNA over the breakpoints confirmedthe inversion

WB Analyses Define the Threshold of WTDystrophin Conferring Clinical Phenotypes ofDuchenne to MyalgiaOur splicing studies reveal that AII1 BIV1 andCII2 each haveresidual levels of normally spliced DMD transcripts with ab-normal splicing events apparently targeted for degradation bynonsense-mediated decay (figure 2 BndashE) Therefore these in-dividuals uniquely provide an opportunity to quantify levels ofWT dystrophin and correlate with clinical phenotype Quanti-tative WB (figure 4) using skeletal muscle biospecimens reveals(1) 15 plusmn 2 normal dystrophin levels in AII1 correlatingwith a myalgia phenotype without apparent weakness (2)10 plusmn 2 levels of dystrophin in BIV1 (figure 4A) withBecker muscular dystrophy mild weakness and cardiac phe-notype and (3) 0ndash5 levels of dystrophin in affected indi-viduals who present with a severe Becker (CII2) or Duchennephenotype (DII1 EII1 FII6 and GII1) (figure 4 B and C)

DiscussionOur study further substantiates DMD splicing variants as animportant causal basis for males presenting with symptomsconsistent with a dystrophinopathy for whom exomic se-quencing approaches or MLPA return negative findings Acausal splicing variant in DMD was identified in all 7 familieswithin our dystrophinopathy cohort and includes 13 affectedmales presenting with hyperCKemia with pain andor muscleweakness andor cardiac involvement

Importantly identification of the causative variant in DMDwithin this hard-to-diagnose cohort required deployment ofWGS RNA-seq andor bespoke RT-PCR studies of mRNAisolated from skeletal muscle For example for CII2 RNA-seq was crucial to identify the inclusion of an 84 base pair (bp)pseudoexon encoding a frameshift which prompted Sangersequencing of this region which lead to the identification ofthe casual intron 26 c3603+820GgtT variant which was un-detectable by gene panel testing Sanger sequencing of theindividual exons or MLPA Although multiple genetic inves-tigations are costly and not available currently to many di-agnostic laboratories costs incurred through muscle biopsyWGS or RNA studies are insignificant relative to the costburden to health services for dystrophinopathy cases for ex-ample the heart transplantation for family B A precise geneticdiagnosis for an X-linked disorder has important and wide-reaching implications for genetic prenatal and prognosticcounseling across the wider family unit and can inform


reproductive decision making In addition a genetic diagnosiscould enable future customizable treatments such as splice-modulating antisense oligonucleotide drugs28 which wouldtheoretically be applicable to families A C and D

Although WGS and RNA-seq bring powerful adjunct teststo clinical genomics shortcomings of short read massivelyparallel sequencing were clearly observed in this study Ashuman exons are typically 100ndash150 bp in length short-readRNA-seq is limited in that a single read does not effectivelybridge multiple exons Most significantly RNA-seq forBIV1 DII1 EII1 and GII1 was confounded by the ef-fectiveness of nonsense-mediated decay an innate sur-veillance mechanism that degrades mRNA bearing apremature stop codon1517 We suspect that the reason wedo not see a profound reduction in read depth for AII1(figure 2A) is due in part to higher read depth across thetranscriptome including DMD and in part to the residualnormal splicing of a significant proportion of DMD tran-scripts (27) Nonsense-mediated decay amplifies in-herent challenges associated with RNA-seq of very largemRNAs where mRNA capture and sequencing libraryconstruction result in a characteristic bias in read depthwith vastly more reads at the 39 end than the 59 end of avery long mRNA Notably common disease genes inneuromuscular disorders are among the largest codingmRNAs in humans with DMD mRNA 14000 nt NEBmRNA 50000 nt and TTN mRNA 100000 ntTherefore ribosomal RNA depletion andor long readRNA-seq approaches which display reduced 39 bias maybe more effective for diagnosing neuromuscular disorders

Regular data filtering approaches of genomic sequencing failedto identify most of the causal variants (excluding families A andC found on RNA-seq) This is likely due to the nature of thevariants themselves (noncanonical splice affecting variants orstructural variants) small read lengths and mapping restrictionsagainst the reference sequence The structural rearrangementwithin DMD intron 43 of family B took extensive bioinformaticanalysis to delineate even when our RT-PCR (data not shown)and RNA-seq studies had indicated intron 43 as the likely lo-cation of the problem Although (in retrospect) the copynumber variation of the duplicated region of chr8 is evidentinformatics approaches to map split reads to precisely define thebreakpoints were challenging and ultimately required both in-formatics and Sanger sequencing of PCR amplicons to fullyresolve Of note the bespoke PCR uniquely identifying theDMD intron 43 structural rearrangement was clinically preferredas the diagnostic test for segregation and carrier testing due to itsgreater specificity relative to the microarray to detect the chr8copy number variation The availability of a validated bespokePCR also means that carrier females in this family could haveprenatal diagnosis of male pregnancies

Although families B D and G have cardiac involvement that iscommon in dystrophinopathy families A C E and F do nothave reported cardiac symptoms and are being monitored for

possible development of cardiac symptoms The profoundcardiac involvement in family B raises suspicion of potentialdifferences in DMD pre-mRNA mis-splicing between cardiacand skeletal muscle activated by the insertion of 118 kb of Chr8sequences containing the LINC00251 gene It is plausible thatthe severe cardiac involvement in family B is due to more fullypenetrant DMD mis-splicing in cardiac tissue compared withskeletal muscle Unfortunately no stored cardiac specimenswere available for mRNA studies from other affected familymembers who had undergone transplant surgery It is alsopossible that levels of inclusion of the frameshifting pseudoexonin family C may differ between skeletal muscle (and potentiallybetween different skeletal muscles) and cardiac muscle

In conclusion we highlight DMD splicing variants as animportant causal basis in individuals with a suspecteddystrophinopathy who remain undiagnosed after exomicsequencing or MLPA approaches Causative DMD variantsidentified in AII1 BIV1 and CII2 that induce partial mis-splicing of DMD mRNA provided us with a unique op-portunity each affected individual produced varying levelsof remnant normally spliced DMDmRNA with all mis-splicedtranscripts encoding a premature stop codon and targeted bynonsense-mediated decay Therefore we were able to usequantitative WB to correlate levels of WT dystrophin withclinical severity We establish a steep therapeutic range of WTdystrophin protein levels (figure 4A) with 15 WT dystro-phin associated withmyalgia without apparent weakness10levels of WT dystrophin associated with Becker muscular dys-trophy mild weakness and cardiac phenotype and lt5 WTdystrophin associated with a severe Becker or Duchenne-likephenotype Our findings broadly concur with previous studiescorrelating levels of mutated dystrophin in BMD with clinicalseverity729ndash31 supporting the notion of a functional redundancywithin the spectrin-like repeats of the dystrophin rod domainOf great relevance to international efforts to develop genetictherapies in DMD our data provide compelling evidence thatwith early intervention only fractional increases in levels ofdystrophin are likely to result in clinical improvement

AcknowledgmentThe authors thank the families for their invaluablecontributions to this research and the clinicians and healthcare workers involved in their assessment and manage-ment For cytogenomic analysis on family B the authorsthank the laboratory of Artur P Darmanian SydneyGenome Diagnostics within the Childrenrsquos Hospital atWestmead NSW Australia For bespoke PCR segregationanalysis on extended family members from family B theauthors thank Dr Bruce Bennetts Dr Gladys Ho andSydney Genome Diagnostics Western Sydney GeneticsProgram The Childrenrsquos Hospital at Westmead NSWAustralia For histopathology on family C the authorsthank the Department of Anatomical Pathology SouthEastern Area Laboratory Services Sydney ChildrenrsquosHospital Randwick NSW Australia The Genotype-TissueExpression (GTEx) Project was supported by the Common


Fund of the Office of the Director of the NIH(commonfundnihgovGTEx) and by the NCI NHGRINHLBI NIDA NIMH and NINDS The datasets used forthe analyses described in this manuscript were obtainedfrom dbGaP at httpwwwncbinlmnihgovgap throughdbGaP accession number phs000424v7p2

Study FundingThis study was supported by the National Health and Medi-cal Research Council of Australia (APP1048816 andAPP1136197 STC APP1080587 STC DGM) SJ Bryen issupported by a Muscular Dystrophy New South Wales PhDscholarship EC Oates is supported by NHMRC ECRGNT1090428 WES WGS and RNA-seq was provided by theBroad Institute of MIT and Harvard Center for Mendelian Ge-nomics (Broad CMG) and was funded by the National HumanGenome Research Institute the National Eye Institute and theNational Heart Lung and Blood Institutersquos NIH grant UM1HG008900 to DGM and Heidi Rehm

DisclosureSTCooper is director of FrontierGenomics Pty Ltd (Australia)Frontier Genomics has not traded (as of September 25 2020)Frontier Genomics Pty Ltd (Australia) has no existing financialrelationships that will benefit from publication of these data NFClarke is deceased disclosures are not included for this authorThe remaining coauthors do not have any relationships financialor otherwise thatmay result in a perceived conflict of interest Goto NeurologyorgNG for full disclosures

Publication HistoryReceived by Neurology Genetics May 1 2020 Accepted in final formNovember 19 2020

Appendix Authors

Name Location Contribution

Leigh B WaddellPhD

The ChildrenrsquosHospital atWestmead NewSouth WalesAustralia

Concept and design ofthe study acquisitionand analysis of dataand drafting themanuscriptfigures forintellectual content

Samantha J BryenBSc (Hons)



Beryl B CummingsPhD

Broad Institute of MITamp HarvardCambridge MA

Acquisition and analysisof data and drafting themanuscriptfigures forintellectual content

Adam BournazosBSc (Hons)



Appendix (continued)


Frances J EvessonPhD



Himanshu Joshi BSoftwareEngineering BBusiness (Finance)



Jamie L MarshallPhD


Acquisition and analysisof data

Taru Tukiainen PhD Broad Institute of MITamp HarvardCambridge MA


Elise Valkanas BA(Biology)



Ben Weisburd BS(Computer Sc)



Simon Sadedin PhD Broad Institute of MITamp HarvardCambridge MA


Mark R Davis PhD PathWest LaboratoryMedicine WANedlands Australia


Fathimath Faiz PhD PathWest LaboratoryMedicine WANedlands Australia


Rebecca GoodingPhD

PathWest LaboratoryMedicine WANedlandsAustralia


Sarah ASandaraduraMBChB FRACP PhD



Gina L OrsquoGradyMBChB FRACP PhD



Michel C TchanMBBS FRACP PhD

Westmead HospitalNew South WalesAustralia


David R MowatMBBS FRACP

Sydney ChildrenrsquosHospital RandwickNew South WalesAustralia


Emily C OatesMBBS FRACP PhD

The ChildrenrsquosHospital atWestmeadNew South WalesAustralia


Michelle A FarrarMBBS FRACP PhD



Continued


References1 Moat SJ Bradley DM Salmon R Clarke A Hartley L Newborn bloodspot screening

for Duchenne muscular dystrophy 21 years experience in Wales (UK) Eur J HumGenet 2013211049ndash1053

2 Helderman-VanDen Enden ATJMMadan K BreuningMH et al An urgent need fora change in policy revealed by a study on prenatal testing for Duchenne musculardystrophy Eur J Hum Genet 20132121ndash26

3 Mendell JR Shilling C Leslie ND et al Evidence-based path to newborn screeningfor Duchenne muscular dystrophy Ann Neurol 201271304ndash313

4 Emery AEH The muscular dystrophies Lancet 2002359687ndash6955 Flanigan KM Duchenne and Becker muscular dystrophies Neurol Clin 201432

671ndash6886 Flanigan KM Dunn DM Von Niederhausern A et al Mutational spectrum of DMD

mutations in dystrophinopathy patients application of modern diagnostic techniquesto a large cohort Hum Mutat 2009301657ndash1666

7 Bushby KMD Gardner-Medwin D The clinical genetic and dystrophin character-istics of Becker muscular dystrophy I Natural history J Neurol 199324098ndash104

8 Yazaki M Yoshida K Nakamura A et al Clinical characteristics of aged Becker musculardystrophy patients with onset after 30 years Eur Neurol 199942145ndash149

9 Heald A Anderson LVB Bushby KMD Shaw PJ Becker muscular dystrophy withonset after 60 years Neurology 1994442388ndash2390

10 Bushby KMD Cleghorn NJ Curtis A et al Identification of a mutation in thepromoter region of the dystrophin gene in a patient with atypical Becker musculardystrophy Hum Genet 199188195ndash199

11 Minetti C Tanji K Chang HW et al Dystrophinopathy in two young boys withexercise-induced cramps and myoglobinuria Eur J Pediatr 1993152848ndash851

12 Melis MA Cau M Muntoni F et al Elevation of serum creatine kinase as the onlymanifestation of an intragenic deletion of the dystrophin gene in three unrelatedfamilies Eur J Paediatr Neurol 19982255ndash261

13 Muntoni F Torelli S Ferlini A Dystrophin and mutations one gene several proteinsmultiple phenotypes Lancet Neurol 20032731ndash740

14 Ferlini A Neri M Gualandi F The medical genetics of dystrophinopathies moleculargenetic diagnosis and its impact on clinical practice Neuromuscul Disord 2013234ndash14

15 Bladen CL Salgado D Monges S et al The TREAT-NMD DMD global databaseanalysis of more than 7000 Duchenne muscular dystrophy mutations Hum Mutat201536395ndash402

16 Laing NG Molecular genetics and genetic counselling for DuchenneBecker mus-cular dystrophy Mol Cel Biol Hum Dis Ser 1993337ndash84

17 Juan-Mateu J Gonzalez-Quereda L Rodriguez MJ et al DMD mutations in 576dystrophinopathy families a step forward in genotype-phenotype correlations PLoSOne 201510e0135189

18 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

19 Jones HF Bryen SJ Waddell LB et al Importance of muscle biopsy to establishpathogenicity of DMD missense and splice variants Neuromuscul Disord 201929913ndash919

20 Cummings BB Marshall JL Tukiainen T et al Improving genetic diagnosis inMendelian disease with transcriptome sequencing Sci Transl Med 20179eaal5209

21 Waddell LB Tran J Zheng XF et al A study of FHL1 BAG3 MATR3 PTRF andTCAP in Australian muscular dystrophy patients Neuromuscul Disord 201121776ndash781

22 Cooper ST Lo HP North KN Single section Western blot improving the moleculardiagnosis of the muscular dystrophies Neurology 20036193ndash97

23 Schneider CA Rasband WS Eliceiri KW NIH Image to ImageJ 25 years of imageanalysis Nat Methods 20129671ndash675

24 Bryen SJ Ewans L Pinner J et al Recurrent TTN metatranscript‐only c39974‐11TgtG splice variant associated with autosomal recessive arthrogryposis multiplexcongenita and myopathy Hum Mutat 202041403ndash411

25 Bryen SJ Joshi H Evesson FJ et al Pathogenic abnormal splicing due to intronicdeletions that induce biophysical space constraint for spliceosome assembly Am JHum Genet 2019105573ndash587



Hugo SampaioMBBCh FRACPMPhil



Alan Ma MBBSFRACP



Katherine NeasMBChB FRACP

Genetic Health ServiceNZ Wellington NewZealand


Min-Xia Wang PhD Royal Prince AlfredHospitalCamperdown NSWAustralia


Amanda CharltonMBChB FRCPA



Charles Chan MBBS(Hons) FRCPA PhD



Diane N KenwrightMBBS FRCPA

University of OtagoWellington NewZealand


Nicole Graf MBBSFRCPA



Susan ArbuckleMBBS FRCPA



Nigel F ClarkeMBChB FRACP PhD


Concept and design ofthe study andacquisition andanalysis of data

Daniel GMacArthur PhD


Concept and designof the study andacquisition andanalysis of data

Kristi J Jones MBBSFRACP PhD

TheChildrenrsquosHospitalat Westmead NewSouth Wales Australia

Concept and design ofthe study acquisitionand analysis of dataand drafting themanuscriptfiguresfor intellectualcontent

Monkol Lek PhD Broad Institute of MITamp HarvardCambridge MA




Sandra T CooperPhD


Concept anddesign of the studyacquisition and analysisof data and drafting themanuscriptfiguresfor intellectualcontent


26 GTEx Consortium The Genotype-Tissue Expression (GTEx) project Nat Genet201345580ndash585

27 Wimmer K Schamschula E Wernstedt A et al AG‐exclusion zones revisited lessonsto learn from 91 intronic NF1 39 splice site mutations outside the canonical AG‐dinucleotides Hum Mutat 2020411145ndash1156

28 Kim J Hu C El Achkar CM et al Patient-customized oligonucleotide therapy for arare genetic disease N Engl J Med 20193811644ndash1652

29 Muntoni F Is a muscle biopsy in Duchenne dystrophy really necessary Neurology200157574ndash575

30 Hoffman EP Kunkel LM Angelini C Clarke A JohnsonMHarris JB Improved diagnosisof Becker muscular dystrophy by dystrophin testing Neurology 1989391011ndash1017

31 Van Den Bergen JC Wokke BH Janson AA et al Dystrophin levels and clinicalseverity in Becker muscular dystrophy patients J Neurol Neurosurg Psychiatry 201485747ndash753


DOI 101212NXG000000000000055420217 Neurol Genet

Leigh B Waddell Samantha J Bryen Beryl B Cummings et al Creatine Kinase

Variants in Males With HighDMDWGS and RNA Studies Diagnose Noncoding

This information is current as of January 29 2021

reserved Online ISSN 2376-7839Published by Wolters Kluwer Health Inc on behalf of the American Academy of Neurology All rightsan open-access online-only continuous publication journal Copyright Copyright copy 2021 The Author(s)

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

ServicesUpdated Information amp

httpngneurologyorgcontent71e554fullhtmlincluding high resolution figures can be found at

References httpngneurologyorgcontent71e554fullhtmlref-list-1

This article cites 31 articles 2 of which you can access for free at

Citations httpngneurologyorgcontent71e554fullhtmlotherarticles

This article has been cited by 2 HighWire-hosted articles

Subspecialty Collections

httpngneurologyorgcgicollectionmuscle_diseaseMuscle disease

httpngneurologyorgcgicollectiondiagnostic_test_assessment_Diagnostic test assessment

httpngneurologyorgcgicollectionall_neuromuscular_diseaseAll Neuromuscular Disease

httpngneurologyorgcgicollectionall_geneticsAll Genetics

httpngneurologyorgcgicollectionall_clinical_neurologyAll Clinical Neurologyfollowing 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



ConclusionsWhole-genome sequencing relied heavily on RNA studies to identifyDMD splice-altering variants Short-read RNA sequencingwas regularly confounded by the effectiveness of nonsense-mediated mRNA decay and low read depth of the giant DMDmRNA PCR of muscle cDNA provided a simple yet informative approach Highly relevant to genetic therapies for dystro-phinopathies our data align strongly with previous studies of mutant dystrophin in Becker muscular dystrophy with thecollective conclusion that a fractional increase in levels of normal dystrophin between 5 and 20 is clinically significant

Duchenne muscular dystrophy (DMD) is a severe X-linkeddisorder primarily affecting approximately 1 in 5000 malebirths1ndash3 DMD shows a relentlessly progressive courseresulting in loss of ambulation in teens and early mortalitydue to cardiac or respiratory involvement45 Dystrophino-pathies range clinically from the severe DMD to asymp-tomatic hyperCKemia5ndash12 DMD is associated with theabsence of dystrophin in muscle due to loss-of-functionvariants in the DMD gene encoding dystrophin56 whereasBecker muscular dystrophy (BMD) is associated with vari-ants in DMD that result in reduced levels of (mutated)dystrophin56

TheDMD gene is the largest gene in the human genome withnumerous enormous introns1314 One-third of pathogenicDMD variants are de novo1516 with most affected individualsbearing insertions or deletions (indels) of coding exons1517

Pathogenic DMD missense variants are rare61518 and non-coding variants are emerging as an important rare cause ofdystrophinopathy15171920 Approximately 5 of patientsclinically diagnosed with DMD do not have a genetic di-agnosis after mutational analysis5

Herein we show the diagnostic application of whole-genomesequencing transcriptomics and dystrophin protein

Glossarybp = base pair CK = creatine kinase DMD = Duchenne muscular dystrophy gnomAD = Genome Aggregation DatabaseGTEx = Genotype-Tissue Expression IGV = Integrative Genomic Browser MLPA = multiplex ligation-dependent probeamplificationmRNA =messenger RNA nt = nucleotide RNA-seq = RNA sequencing RT-PCR = reverse transcription PCRSNV = single nucleotide variant WB = Western blot WGA = wheat germ agglutinin WT = wild type


















Clinicalsymptoms








Onset 15 y 5 y 9 y 35 y 6 y 35 y 5 y

Familyhistory





Nil

Serum CKUL

1400ndash7500 9964 420 14500 18889 24000 gt12000



















Westernblot
















































Appendix Authors


Leigh B WaddellPhD






Beryl B CummingsPhD














Jamie L MarshallPhD

















Rebecca GoodingPhD





















Continued

































Alan Ma MBBSFRACP




































Sandra T CooperPhD































Reprints




















Clinicalsymptoms








Onset 15 y 5 y 9 y 35 y 6 y 35 y 5 y

Familyhistory





Nil

Serum CKUL

1400ndash7500 9964 420 14500 18889 24000 gt12000



















Westernblot
















































Appendix Authors


Leigh B WaddellPhD






Beryl B CummingsPhD














Jamie L MarshallPhD

















Rebecca GoodingPhD





















Continued

































Alan Ma MBBSFRACP




































Sandra T CooperPhD































Reprints
















































Appendix Authors


Leigh B WaddellPhD






Beryl B CummingsPhD














Jamie L MarshallPhD

















Rebecca GoodingPhD





















Continued

































Alan Ma MBBSFRACP




































Sandra T CooperPhD































Reprints



































Alan Ma MBBSFRACP




































Sandra T CooperPhD































Reprints































Reprints




WGS and RNA Studies Diagnose Noncoding DMD Variants in ...

Documents

Transcript of WGS and RNA Studies Diagnose Noncoding DMD Variants in ...