ARTICLE OPEN ACCESS

Integrated sequencing and array comparativegenomic hybridization in familial ParkinsondiseaseLaurie A Robak MD PhD Renqian Du PhD DDS Bo Yuan PhD Shen Gu PhD

Isabel Alfradique-Dunham MD Vismaya Kondapalli BS Evelyn Hinojosa BS Amanda Stillwell BS

Emily Young BA Chaofan Zhang BS Xiaofei Song PhD Haowei Du MS Tomasz Gambin PhD

Shalini N Jhangiani PhD Zeynep Coban Akdemir PhD Donna M Muzny MS Anusha Tejomurtula MS

Owen A Ross PhD Chad Shaw PhD Joseph Jankovic MD Weimin Bi PhD Jennifer E Posey MD PhD

James R Lupski MD PhDdagger and Joshua M Shulman MD PhDdagger

Neurol Genet 20206e498 doi101212NXG0000000000000498

Correspondence

Dr Shulman

JoshuaShulmanbcmedu

or Dr Lupski

jlupskibcmedu

AbstractObjectiveTo determine how single nucleotide variants (SNVs) and copy number variants (CNVs)contribute to molecular diagnosis in familial Parkinson disease (PD) we integrated exomesequencing (ES) and genome-wide array-based comparative genomic hybridization (aCGH)and further probed CNV structure to reveal mutational mechanisms

MethodsWe performed ES on 110 subjects with PD and a positive family history 99 subjects were alsoevaluated using genome-wide aCGHWe interrogated ES and aCGH data for pathogenic SNVsand CNVs at Mendelian PD gene loci We confirmed SNVs via Sanger sequencing and furthercharacterized CNVs with custom-designed high-density aCGH droplet digital PCR andbreakpoint sequencing

ResultsUsing ES we discovered individuals with known pathogenic SNVs in GBA (pGlu365LyspThr408Met pAsn409Ser and pLeu483Pro) and LRRK2 (pArg1441Gly andpGly2019Ser) Two subjects were each double heterozygotes for variants in GBA and LRRK2Based on aCGH we additionally discovered cases with an SNCA duplication and heterozygousintragenic GBA deletion Five additional subjects harbored both SNVs (pAsn52Metfs29pThr240Met pPro437Leu and pTrp453) and likely disrupting CNVs at the PRKN locusconsistent with compound heterozygosity In nearly all cases breakpoint sequencing revealedmicrohomology a mutational signature consistent with CNV formation due to DNA repli-cation errors

ConclusionsIntegrated ES and aCGH yielded a genetic diagnosis in 193 of our familial PD cohort Ouranalyses highlight potential mechanisms for SNCA and PRKN CNV formation uncovermultilocus pathogenic variation and identify novel SNVs and CNVs for further investigation aspotential PD risk alleles

These authors contributed equally to this manuscript as condashfirst authors

daggerThese authors contributed equally to this manuscript as condashlast authors

From the Department of Molecular and Human Genetics (LAR RD BY SG VK EH AS EY CZ XS HD TG ZCA AT CS WB JEP JRL JMS) Department of Neurology(IA-D JJ JMS) and Human Genome Sequencing Center (SNJ DMM JRL) Baylor College of Medicine Houston TX Baylor Genetics (WB) Houston TX Department of Neurology(OAR) Department of Neuroscience (OAR) and Department of Clinical Genomics (OAR) Mayo Clinic Jacksonville FL ParkinsonrsquosDiseaseCenter andMovementDisordersClinic (JJ) andDepartment of Pediatrics (JRL JMS) Baylor College of Medicine Houston TX Department of Pediatrics (JRL) Texas Childrenrsquos Hospital Houston Department of Neuroscience (JMS)Baylor College of Medicine Houston TX and Jan and Dan Duncan Neurological Research Institute (JMS) Texas Childrenrsquos Hospital Houston

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

The Article Processing Charge was funded by the Burroughs Wellcome Fund

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

Up to 20 of patients with Parkinson disease (PD) report apositive family history1 and genetic risk factors are more com-mon in these families2 Identification of specific genetic risk fac-tors can reveal prognostic information such as risk of cognitiveimpairment andor rate of progression and may soon highlighteligibility for personalized therapies3 In addition discovery of riskvariants may inform genetic counseling of unaffected familymembers Indeed surveys of patients with PD and caregiversreveal a high level of interest in genetic testing for PD4

More than 40 different loci that increase PD susceptibilityhave been identified in familial and sporadic PD56 Exomesequencing (ES) is ideally suited to identify single nucleotidevariants (SNVs) in genetically heterogeneous diseases In astudy of adult patients referred for diverse clinical indicationsES had a diagnostic yield of 10 in individuals older than 30years7 In a recent study of 80 early-onset sporadic PD casesES yielded an overall diagnostic rate of 11 with GBA allelesaccounting for 58 Nevertheless clinical genetic testing isnot routinely performed for PD and ES remains poorlystudied as a potential genetic diagnostic tool

Although most identified PD risk alleles are SNVs chromo-somal structural rearrangements or copy number variants(CNVs) also play an important role9 Despite notable recentadvances10 ES remains insensitive for detection of small CNVs(lt50 kb)11 Several complementary approaches includingmultiplex ligation-dependent probe amplification bacterial ar-tificial chromosome arrays and single nucleotide poly-morphism arrays1213 have shown mixed success foridentification of CNVs in PD cohorts In contrast genome-wide array-based comparative genomic hybridization (aCGH)is a highly-validated sensitive clinical screening tool for CNVdetection offering exon-by-exon coverage for a multitude ofdisease-associated genes1415 Although not yet adopted inmostdiagnostic laboratories droplet digital PCR (ddPCR) is alsoemerging as a rapid and cost-efficient targeted approach for theassessment of small CNVs at specific loci Compared withstandard quantitative PCR digital PCR offers enhanced copynumber and gene dosage sensitivity precision and reliabilitydue to sample partitioning16 In addition mechanisms of CNVformation in PD remain understudied

To our knowledge integrated ES and aCGH for analysis ofSNVs and CNVs respectively have not previously beensystematically used in PD We hypothesized that ES andaCGH in combination will yield an increased genetic mo-lecular diagnostic rate We also evaluated ddPCR as a novel

strategy for confirmation of pathogenic CNVs in PD andusing breakpoint sequencing we investigated potentialmechanisms for CNV formation

MethodsSee supplementary information (linkslwwcomNXGA305)for complete methods including further details andreferences

SubjectsWe studied 110 PD cases evaluated in the Baylor College ofMedicine (BCM) PDCenter andMovement Disorders Clinicin Houston TX with a family history of PD As a positivecontrol for aCGH we included a sample from a known sub-ject with an SNCA triplication17ndash19 We also interrogated aBaylor Genetics diagnostic laboratory sample including12922 clinical referral samples for aCGH from peripheralblood using either v9 or v10 Baylor arrays Subject numbersthroughout the text are consistent with clinical and de-mographic details provided in table e-1 (linkslwwcomNXGA306)

Standard protocol approvals registrationsand subject consentsAll subjects provided informed consent The BCM In-stitutional Review Board approved this study along with theanalysis of aggregate clinical genomic data

Gene set definition and variant criteriaWe focused our analyses on genes and variants established tocause familial PD including the autosomal dominant lociSNCA (PARK1 MIM168601) GBA (MIM168600)LRRK2 (MIM607060) GCH1 (MIM600225) DNAJC13(MIM616361) and VPS35 (MIM614203) as well as theautosomal recessive loci PRKN (PARK2 MIM600116)PINK1 (MIM605909) and PARK7 (DJ1 MIM606324)based on the available literature in April 2015 when this studywas initiated56 In our CNV analyses we also considereddeletions at 22q112 Gene names in this study conform tocurrent guidelines from the HUGO Gene NomenclatureCommittee (genenamesorg) All pathogenic alleles includedin this study are well-established nonsynonymous codingvariants with moderate to high penetrance (odds ratio [OR]gt2) meeting stringent evidence for replication across studiesor within the same study We considered all other variantsdiscovered in these genes but not previously reported in PDto be variants of unknown significance (VUSs)

GlossaryaCGH = array-based comparative genomic hybridization BCM = Baylor College of Medicine CNV = copy number variantddPCR = droplet digital PCR DUP-TRPINV-DUP = duplication-inverted triplication-duplication ES = exome sequencingFoSTeS = fork stalling and template switching MMBIR = microhomology-mediated break-induced replication OR = oddsratio PD = Parkinson disease SNV = single nucleotide variant VUS = variant of unknown significance

2 Neurology Genetics | Volume 6 Number 5 | October 2020 NeurologyorgNG

Detection and confirmation of SNVs and CNVsWe extracted genomic DNA from peripheral blood samplesobtained from each participant and performed ES using the Illu-mina HiSeq 2000 at the BCM Human Genome SequencingCenter Samples achieved an average of 95 of targeted exomebases covered to a depth of 20X or greater All pathogenic SNVsdetected were confirmed via Sanger sequencing Genome-widearray CGH was performed on 99 of 110 subjects for which suf-ficient DNA remained using Baylor Genetics v10 2x400K clinical-grade oligonucleotide microarrays We defined potential CNVs asthose regions with 3 or more consecutive probes with consistentdirection of effect For confirmation we used a custom 8x60Khigh-density array through Agilent (Santa Clara CA) To confirmCNVs atPRKN andGBA we additionally performed ddPCR (seee-Methods for detailed protocol linkslwwcomNXGA305)

Data availabilityFor all subjects in the BCM cohort who consented to allow forpublic data sharing ES and aCGH are in process for release inrelevant genomic databases The complete ES and aCGH datasets are also available on request by contacting the corre-sponding author Dr Shulman (joshuashulmanbcmedu)

ResultsWe pursued genetic diagnostic evaluation of 110 total subjects(including 109 unrelated probands) with familial PD Themean age at onset was 50 years (SD = 15) 51 were maleThe ethnic composition of the cohort was 72 Caucasian17 Hispanic 6 East Asian South Asian or Middle Easternand 6 undefined (not reported)

Single nucleotide variantsWe first examined subject ES data for pathogenic SNVsin established PD genes (see Methods) Among the domi-nant PD loci 15 individuals had variants in LRRK2(c6055GgtApGly2019Ser and c4321CgtGpArg1441-Gly) and GBA (c1093GgtApGlu365Lys c1223CgtTpThr408Met c1448TgtCpLeu483Pro and c1226AgtGpAsn409Ser) (table 1 and table e-1 linkslwwcomNXGA306) Two subjects each harbored heterozygous SNVsin both GBA and LRRK2 ie were double heterozygotes Onesuch subject had a combination ofLRRK2pGly2019Ser andGBApGlu365Lys (subject 2) whereas the other had LRRK2pGly2019Ser and GBA pLeu483Pro (subject 13) Both subjectshad onset of PD symptoms in their 40s On initial examinationsubject 2 had tremor at rest rigidity bradykinesia and dystonicposturing in both hands She reported a history of PD in her fatherand paternal grandfather (figure e-1A linkslwwcomNXGA305 and table e-1 linkslwwcomNXGA306) There was nohistory of cognitive impairment or dementia Subject 13 presentedwith resting tremor rigidity and bradykinesia She reported afamily history of PD in her paternal uncle (figure e-1B linkslwwcomNXGA305 and table e-1 linkslwwcomNXGA306)Ten years after PD diagnosis she developed visual hallucinationsand delusions The subjects were of European and Hispanic an-cestry respectively neither reported Ashkenazi Jewish heritage

ES also revealed 7 individuals with pathogenic variants in lociusually associated with autosomal recessive PD including PRKN(6 individuals) and PARK7 (1 individual) However all subjectswere heterozygous SNV carriers and therefore isolated ES wasnondiagnostic (table 1) Therefore based on ES alone weidentified a pathogenic variant accounting for PD in 138 (n =15 of 109 probands) of our familial PD cohort We confirmed allimplicated variants via Sanger sequencing Besides the pathogenicvariants noted above ES also identified heterozygous VUSs inmany PD risk genes (table e-2 linkslwwcomNXGA306)

Copy number variantsWe next interrogated aCGH data for pathogenic CNVsamong PD genes Our analyses included 99 of 110 totalsubjects evaluated by ES We did not detect any CNVs inVPS35 LRRK2DNAJC13GCH1 PARK7 or PINK1 nor didwe identify any candidate deletions at the 22q112 locusHowever we discovered CNVs in SNCA (n = 1) GBA (n =1) and PRKN (n = 5) We confirmed all reported CNVsthrough custom high-density arrays and breakpoint se-quencing CNVs in SNCA andGBA affect dominant PD genesand were therefore diagnostic based on aCGH alone Weconsider all heterozygous CNVs in the recessive PD genePRKN in combination with ES results (see next section)Overall isolated aCGH identified a diagnostic genetic riskfactor for PD in 20 of our cohort (n = 2 of 99 probands)Based on aCGH we also detected numerous large CNVs (gt1Mb) within our cohort that affect other genomic loci thesevariants remain of uncertain clinical significance (table e-3linkslwwcomNXGA306)

In subject 3 we detected a 248-kb duplication encompassingSNCA as well as the adjacent geneMMRN1 We confirmedthis CNV by high-density aCGH and breakpoint analysis(figure 1A) On initial examination this subject exhibitedrigidity tremor and gait impairment along with hyper-reflexia and clonus The subject was of Hispanic and NativeAmerican ancestry the subjectrsquos father had PD with de-mentia Besides providing independent confirmationbreakpoint sequencing can provide clues to mechanisms ofCNV formation In the case of subject 3 we identified a 1-bpmicrohomology domain which is a short sequence that isidentical to another region in the genome reduced from 2copies to 1 during the template switch accompanying rep-licative repair20 Microhomology is characteristic of certainDNA replication errors that can generate CNVs (seeDiscussion)2122 As a positive control for our aCGH anal-ysis we also included a known SNCA triplication samplefrom the index family in which SNCA locus multiplicationwas first discovered as a cause for PD17ndash19 Breakpoint se-quencing revealed that this copy number alteration is a 17-Mb complex genomic rearrangement (figure 1B) consistingof a duplication-inverted triplication-duplication (DUP-TRPINV-DUP) This finding confirms and extends priorinvestigation of this particular structural variant23 and is alsoconsistent with a likely replication-based mechanism forCNV formation24

NeurologyorgNG Neurology Genetics | Volume 6 Number 5 | October 2020 3

We also discovered a heterozygous intragenic deletion inGBAin 1 subject (figure 2) who presented at age 28 years withtremor bradykinesia and rigidity She had an excellent re-sponse to levodopa in her early 30s and subsequently de-veloped dyskinesia The subject was of European descent andboth of her maternal grandparents were also diagnosed withPD (figure e-1C linkslwwcomNXGA305) Because vari-ant confirmation at the GBA locus can be complicated by anadjacent pseudogene with significant homology we con-firmed the 47-kb deletion of exons 2ndash8 using long-rangePCR Using breakpoint sequencing we also confirmed het-erozygosity and further revealed a 5-bp microhomology do-main consistent with CNV formation due to nonhomologousrecombination or replication errors (figure 2B)

Integrated analysis of SNVs and CNVsAs highlighted above isolated ES and aCGH each identified anumber of subjects with heterozygous SNVs or CNVs af-fecting recessive genes To determine whether these changesmight be diagnostic we next examined the results together forpotential biallelic variation due to both an SNV allele and aCNV allele Indeed 3 subjects in our cohort were newlyidentified as potential compound heterozygous carriers ofboth a pathogenic CNV and SNV in PRKN (figure 3) Subject6 had a 364-kb duplication of exons 4ndash6 and a frameshiftdeletion c155delApAsn52Metfs29 Subject 20 had a

222-kb deletion of exons 8 and 9 and a stopgain c1358GgtApTrp453 Subject 11 harbored a pathogenic PRKN SNV(c1310CgtTpPro437Leu) and a complex locus rearrange-ment including a copy number neutral region flanked by404 kb and 199 kb duplications affecting exons 2 and 3 andexons 5 and 6 respectively (figure 3B) Our cohort also in-cluded 2 brothers with known PRKN-PD25 however neitherES nor aCGH was previously performed on these subjectsOur analysis confirmed compound heterozygosity for theknown pathogenic SNV in exon 6 (c719CgtTpThr240Metapparently homozygous on ES) and a CNV (178-kb deletionof exons 5 and 6) (figure 3C) Of interest ES also discoveredan additional VUS (c2TgtCpMet1Thr) Based on availableclinical information (table e-1 linkslwwcomNXGA306)all subjects with PRKN variants had young-onset PD (agerange 15ndash36 years)

We again confirmed all CNVs using custom high-densityarrays as well as breakpoint sequencing In the case of subject6 we were unable to successfully amplify breakpoint junctionsdespite multiple attempts suggesting a more complex geno-mic rearrangement or raising the possibility that this dupli-cation is located elsewhere in the genome For all other PRKNCNVs figure 3 shows the junction structures highlightinglikely mechanisms of CNV formation Overall integrated ESand CNV identified a genetic cause for PD in 4 additional

Table 1 SNVs associated with increased PD risk detected via exome sequencing

Gene Variant Diagnostic Subjects (n)

Dominant

LRRK2 c6055GgtApGly2019Ser Y 3ab

LRRK2 c4321CgtGpArg1441Gly Y 1

GBA c1093GgtApGlu365Lys Y 5a

GBA c1223CgtTpThr408Met Y 5

GBA c1448TgtCpLeu483Pro Y 1b

GBA c1226AgtGpAsn409Ser Y 2

Recessive

PRKN c155delApAsn52Metfs29 Y 1

PRKN c1310CgtTpPro437Leuc Y 1

PRKN c1358GgtApTrp453 Y 1

PRKN c719CgtTpThr240Met Y 2

PRKN c823CgtTpArg275Trp N 1

PARK7 c310GgtApAla104Thr N 1

Abbreviations CNV = copy number variant PD = Parkinson disease SNV = single nucleotide variantAll indicated SNVs were heterozygous except PRKN c719CgtTpThr240Met which was hemizygous as the variant is in trans to a deletion allele Pathogenicvariants were considered diagnostic (Y) if discovered in an autosomal dominant gene or in the case of autosomal recessive genes if in combination with aCNV (asterisk see also figure 3) Nondiagnostic (N) heterozygous SNVs were also discovered in PRKN (pArg275Trp) and PARK7 (pAla104Thr) In 2 subjectsSNVs in both LRRK2 and GBA were identified (double heterozygotes)a LRRK2 pGly2019Ser and GBA pGlu365Lysb LRRK2 pGly2019Ser and GBA pLeu483Proc Interpretations of c1310CgtTpPro437Leu are conflicting (see e-References linkslwwcomNXGA306)

4 Neurology Genetics | Volume 6 Number 5 | October 2020 NeurologyorgNG

probands increasing the overall genetic diagnostic yield to193 (n = 21)

Digital droplet PCRGiven the observed high frequency of PRKN CNVs in thisfamilial PD cohort (5) and the high cost of clinical-gradeaCGH we examined the feasibility of an exon-by-exon ddPCRassay to detect PRKN CNVs as a proof of principle ddPCR isan emerging cost-effective method for sensitive and reliableassessment of specific CNVs Indeed ddPCR revealed allPRKNCNVs detected using aCGH (subjects 6 11 20 21 and22) (figure 4A) We also interrogated an additional 92 casesfrom our cohort with available DNA for intragenic PRKNCNVs using ddPCR without discovery of additional CNVs(figure 4A and figure e-2 linkslwwcomNXGA305)We alsoapplied ddPCR to screen for potential CNVs at theGBA locusThe 12-exon GBA shares high homology with the nearby 13-exon pseudogene GBAP1 (figure 4B) As shown in figure 4Cusing ddPCR we successfully amplified all 12 exons ofGBA Sixexons (1 2 4 7 9 and 10) identified unique amplicons with a

positive droplet ratio of 1 whereas the other 6 exons (3 5 6 811 and 12) demonstrated shared amplicons with the pseudo-gene resulting in a droplet ratio of 2 Importantly ddPCR alsoconfirmed the deletion of GBA exons 2ndash8 in subject 1 (figure4C) and did not reveal evidence for additional CNVs in 85other samples tested (figure 4C and figure e-3 linkslwwcomNXGA305) Of note ddPCR initially suggested a single exondeletion (exon 6) in subject 48 however further investigationusing Sanger sequencing revealed an intronic SNV likelydegrading ddPCR amplification (figure e-4 linkslwwcomNXGA305) We redesigned the affected primer and demon-strated full amplification of exon 6 (e-Methods and figure e-4linkslwwcomNXGA305) Overall our results suggest thatddPCRmay be a sensitive and specific diagnostic tool for CNVdetection in PD including at loci such as GBA complicated bygenomic regions with high sequence homology

CNV burden in clinical cohortsCompared with SNVs limited reference data are availableon the population frequency of CNVs especially in

Figure 1 aCGH plots and breakpoint junction sequences of 2 CNVs involving SNCA

(A) In subject 3 a 248-kb duplicationwas identified In this case thewhole SNCA genewas duplicated The junction sequence (bottom) is alignedwith upstreamand downstream reference sequences with the blue and pink colors indicating their different origins and the red indicating inserted nucleotides andmicrohomology (B) A 17-MbDUP-TRPINV-DUP rearrangementwas identified in the index subject with a known SNCAmultiplication17ndash19 The x-axis indicatesthe chromosomal regions surrounding SNCA The y-axis indicates the subject vs control log2 ratio of the aCGH results with duplications at 058 triplications at1 and heterozygous deletions at minus1 based on theoretical calculations Red dots in the graph represent probes with log2 ratio gt025 black dots with log2 ratiofrom 025 to minus025 and green dots with log2 ratio ltminus025 The normal-duplication-triplication transition regions are magnified in boxes above the plot Theentire SNCA gene is triplicated In addition an SNP (rs12651181 underlined) was detected close to JCT2 aCGH = array-based comparative genomic hy-bridization CNV = copy number variant DUP-TRPINV-DUP duplication-triplication inverted-duplication SNP = single nucleotide polymorphism

NeurologyorgNG Neurology Genetics | Volume 6 Number 5 | October 2020 5

neurologically healthy adult samples hampering the in-terpretation of CNV frequencies detected in our cohort Wetherefore leveraged data from the Baylor Genetics diagnosticlaboratory including 12922 aCGH clinical referral samplesThis large cohort is skewed for pediatric cases (mean age = 74years SD = 97 years range 0ndash79 years) reflecting the morecommon use of aCGH in this population Although the co-hort includes a substantial proportion of individuals withdevelopmental delay autism and dysmorphic features therewere no recorded submissions for PD Based on stringentcriteria (see e-Methods linkslwwcomNXGA305) at mostPD loci CNVs were either absent (DNAJC13 LRRK2PINK1 and SNCA) or very rare at VPS35 (n = 2)GCH1 (n =1) and PARK7 (n = 6 all subjects had 1p36 deletion syn-drome) By contrast CNVs were more common at 22q112(n = 90 all losses affecting the critical region) and PRKN (n =95) Notably the frequency of PRKNCNVs in our PD cohort(51) represents a significant increase when compared withthat of the Baylor Genetics clinical reference sample (fre-quency = 074 OR = 72 95 confidence interval 29ndash181p = 28 times 10minus5) Because of the pseudogene GBAP1 andsuboptimal probe coverage the array does not reliably captureGBA CNVs

DiscussionEstablishing a specific genetic diagnosis can provide in-formation about PD risk and progression relevant to patientsand their families and may soon influence treatment deci-sions26 In our familial PD sample ES and aCGH in-dependently identified a genetic cause for PD in 138 and20 respectively The diagnostic yield for ES was slightlyhigher than that recently reported for an early-onset PD co-hort (1125)8 and was also greater than the 107 diagnosticrate in an unselected adult series referred for clinical

diagnostic ES7 Given incipient treatment trials for GBA-PDand the potential importance of identifying eligible subjects inthe future26 our analyses considered lower-risk pathogenicalleles (OR 24)27 pGlu365Lys and pThr408Met alongwith higher-penetrance variants (eg pLeu483Pro ORgt5)28 Importantly integrated ES and aCGH identified 5additional subjects (4 unrelated probands)mdashincluding asubject with a GBA deletionmdashyielding an overall combineddiagnostic rate of 193 We also uncovered numerous VUSincluding SNVs within Mendelian PD genes (table e-2 linkslwwcomNXGA306) as well as large CNVs affecting otherloci (table e-4 linkslwwcomNXGA306) Although addi-tional evidence will be required to confirm or refute patho-genicity our genetic diagnostic rate would nearly double ifthese VUS in PD genes are bona fide risk factors Overall ourfindings suggest that integrated ES and aCGH analysis is es-sential for routine high-confidence genetic diagnosis in fa-milial PD

Most genetic diagnostic studies in PD cohorts to date haveignored the potential contribution of CNVs Similarly exceptin several notable targeted CNV studies1229 research-basedPD gene discovery has almost exclusively focused on SNVsusing ES or genotyping arrays Importantly we would havemissed multiple pathogenic CNV alleles at both autosomaldominant (SNCA and GBA) and recessive (PRKN) lociwithout performing aCGH In 5 subjects pathogenic allelesdiscovered at PRKNwould have been nondiagnostic based onisolated ES leading to misclassification as heterozygous car-riers whereas integrated SNV-CNV analyses successfullyestablished the molecular diagnosis of PRKN-PD Our find-ings suggest caution for interpretation of studies attributingPD risk to either PRKN CNV or SNV heterozygous carrierstates in isolation consistent with prior studies30 Althoughwe did not detect any CNVs at PINK1 or PARK7 in ourcohort the importance of integrated SNV-CNV analysis may

Figure 2 aCGH plot and breakpoint junction sequence of GBA deletion

(A) aCGH plot and (B) junction sequence of a 47-kb deletion identified involvingGBA in subject 1 The deletion (shadowed) encompasses 7 exons of GBA (fromexon 2 to exon 8) (C) By agarose gel electrophoresis the amplification of the deleted region (Del) in subject 1 showed a5 kb discrepancy compared with acontrol (Ctl) consistent with aCGH findings PCR showed preferential amplification of the shorter fragment in the Del lane aCGH = array-based comparativegenomic hybridization

6 Neurology Genetics | Volume 6 Number 5 | October 2020 NeurologyorgNG

extend to other autosomal recessive PD loci besides PRKNBecause our CNV and SNV data are unphased and parentalgenotypes are not available we cannot definitively exclude thepossibility that certain CNVs and SNVs at PRKN were in cis-rather than trans-configuration Nevertheless our data sug-gest that structural variants may co-occur with SNVs morecommonly than previously recognized making considerationof both allele types important for comprehensive genetic di-agnosis in PD

ES has significantly accelerated the scope of gene discovery inPD and other neurologic disorders but remains insensitive toallele classes such as trinucleotide repeat expansions and

CNVs Although bioinformatic tools may help identify CNVsfrom ES data available algorithms have high false-positiverates when compared with aCGH31 and this method maymiss up to 30 of clinically relevant CNVs11 To ourknowledge genome-wide aCGH with exon-by-exon coveragehas not been previously applied in PD Limitations of aCGHinclude significant cost and the possibility of missing smalldeletionsduplications Alternative methods such as ddPCRmay offer a cost-effective alternative for screening specificgenes32 including for small CNVs In our study ddPCRshowed high sensitivity and specificity for detection of CNVsat both PRKN and GBA Moreover ddPCR successfully dif-ferentiated copy number changes affecting exons unique to

Figure 3 aCGH plots and breakpoint junction sequences of PRKN CNVs

aCGH plots (left panel) and breakpoint junction sequences (right panel) of CNVs identified involving the PRKN gene in the cohort At the top a schematic genestructure demonstrates the 12 exons of PRKN (A) In subject 20 a 222-kb deletion covering exons 8 and 9 was accompanied by a known pathogenic nonsensemutation c1358GgtApTrp453 (gnomAD frequency = 0) in exon 12 (B) In subject 11 in addition to a missense variant c1310CgtTpPro437Leu (exon 12) aduplication-normal-duplication (DUP-NML-DUP) was identified (C) Siblings 21 and 22 share a pathogenic missense variant c719CgtTpThr240Met (in exon 6)and a 178-kb deletion (disrupting exons 5 and 6) (D) In subject 6 a 364-kb duplication encompassed exons 4 to 6 A known pathogenic frameshift variantc155delApAsn52Metfs29was identified in exon 2 (gnomAD frequency = 25 times 10minus4) Breakpoint sequencingwasnot successful in this sample aCGH=array-based comparative genomic hybridization CNV = copy number variant gnomAD = Genome Aggregation Database

NeurologyorgNG Neurology Genetics | Volume 6 Number 5 | October 2020 7

GBA avoiding potential confounding by the adjacent pseu-dogene GBAP1

Despite evidence of an important role in disease risk themechanism(s) for generating CNVs relevant to PD remainlargely unknown Broadly CNVs may form through mecha-nisms associated with DNA recombination DNA replicationandor DNA repair20 Nonallelic homologous recombinationcan result in recurrent rearrangements In contrast non-homologous end joining20 fork stalling and templateswitching (FoSTeS) and microhomology-mediated break-induced replication (MMBIR)21 lead to nonrecurrent CNVs

In our study all junction breakpoint sequencing results wereconsistent with the FoSTeSMMBIR mechanism includingfor CNV alleles discovered at SNCA PRKN and GBA Theseresults are consistent with a recent analysis of SNCA dupli-cations from 6 independent cases33 These findings haveimportant implications for screening assays because the de-tection of nonrecurrent CNVs requires methods sensitive forheterogeneous exon-by-exon changes The FoSTeSMMBIRmechanism can also trigger multiple iterative templateswitches in a single event leading to the generation of morecomplex genomic rearrangements Breakpoint sequencing ofan SNCA CNV first observed in the Spellman-MuenterIowa

Figure 4 PRKN and GBA ddPCR results of representative subjects

(A) Positive droplet concentrations in 8 subjects Primer pairs for the 12 exons of PRKN and 2 control genes RPPH1 and TERT were used to obtain positivedroplet concentrations from PCR in each individual (e-Methods and figure e-4A linkslwwcomNXGA305) The y-axis shows exon-by-exon results in 13columnswith different colors showing comparable results to the average value of RPPH1 and TERT A y-axis value of 05 indicates a deletion 1 copy neutral (nodeletion no duplication) and 15 a duplication In subject 6 a duplication involving exons 4 to 6 was identified as shown by aCGH in subject 11 exons 2 4 5and 6 demonstrated copy number gains in subject 20 there is a copy number loss involving exons 8 and 9 similarly in subjects 21 and 22 a copy number lossof exons 5 and 6 is detected In subjects 1 23 and HapMap NA10851 no amplicons showed altered copy number See also figure e-2 (linkslwwcomNXGA305) Copy number variants are denoted with asterisks () (B) GBA and its nearby pseudogene GBAP1 share a high degree of sequence homology withddPCR primer pairs for 6 of the 12 exons of GBA producing amplicons concurrently from GBA and GBAP1 GBA exons 3 5 6 8 11 and 12 are color coded todemonstrate their homologous regions withinGBAP1 which result in a doubling of the apparent copy number identified by ddPCR 4 instead of 2 copies (ratio= 2) indicate copy number neutrality for these exons GBA exon 5 is homologous with an intragenic region between exons 4 and 5 of GBAP1 (C) ddPCRdetected potential exonic CNVs in GBA Here we demonstrate a deletion identified in subject 1 compared with HapMap subject NA10581 and other 2subjects ratios of exons 2 to 8were each reduced by 05-fold consistent with a deletion involving these exons Deleted exons are denotedwith an asterisk ()deleted exons with a droplet ratio of 15 due to GBAP1 amplification are denoted with an arrowhead See also figure e-3 (linkslwwcomNXGA305) aCGH =array-based comparative genomic hybridization CNV = copy number variant ddPCR = droplet digital PCR

8 Neurology Genetics | Volume 6 Number 5 | October 2020 NeurologyorgNG

kindred1819 confirmed the DUP-TRP-DUP structure23 andfurther revealed an internal inversion (DUP-TRPINV-DUP) and microhomology This rearrangement must havearisen during mitosis via FoSTeSMMBIR20 and thereforelikely represents a de novo triplication in contrast to themeiotic PMP22 triplications observed in Charcot-Marie-Tooth (MIM118220) which derives from a duplication inthe previous generation34 Our results therefore demonstratethe essential role of breakpoint junction sequencing in de-finitively resolving CNV structure and responsiblemechanisms

Deletions in GBA rarely contribute to autosomal recessiveGaucher disease (MIM230800)35 Our discovery of a GBAdeletion allele in a subject with PD expected to cause glu-cocerobrosidase haploinsufficiency adds to other emergingevidence supporting a loss-of-function mechanism in GBA-PD36 It will be informative to screen for additional GBACNVs in additional casecontrol cohortsmdashperhaps usingddPCRmdashto determine how commonly these alleles are as-sociated with PD risk and estimate their effect size and pen-etrance We also identified 2 subjects doubly heterozygous forSNVs in both GBA and LRRK2 consistent with prior re-ports37 We expect that additional PD cases compatible witholigogenic inheritance models will emerge following wide-spread adoption of comprehensive genome-wide diagnosticapproaches including ES and aCGH Future studies mustaddress how such alleles may interact to modify PD risk andor clinical manifestations Finally although our study focusedon pathogenic alleles in established Mendelian loci futureassessment of a more complete spectrum of genetic variationthrough integrated SNV-CNV analysis is also likely to en-hance power for novel PD gene discovery

AcknowledgmentThe authors thank all the families who participated in thisstudy They also thank Dr Dennis W Dickson andDr Zbigniew Wszolek (Mayo Clinic Jacksonville FL) forproviding the SNCA-positive control sample They alsoacknowledge Anita Kaw (Baylor College of MedicineHouston TX) for help with sample preparation and MitchellA Rao and Ido Machol (Baylor Genetics Houston TX) fordata retrieval from the clinical database

Study fundingThis work was supported in part by The American Academyof Neurology (LAR) Baylor College of Medicine MedicalGenetics Research Fellowship T32 GM07526 (LAR)NINDS K08 NS112467 (LAR) the Baylor-Hopkins Centerfor Mendelian Genomics BHCMG (UM1 HG006542 toJRL) which is jointly funded by the National Human Ge-nome Research Institute (NHGRI) National Heart Lung andBlood Institute (NHLBI) and NINDS R35 NS105078(JRL) JEP was supported by NHGRI K08 HG008986JMS was supported by grants from the Huffington Foun-dation Jan and Dan Duncan Neurological Research Instituteat Texas Childrenrsquos Hospital and a Career Award for Medical

Scientists from the Burroughs Wellcome Fund The MayoClinic Brain Bank is supported by the Mangurian FoundationLewy Body Dementia Program at Mayo Clinic

DisclosureLA Robak R Du B Yuan S Gu I Alfradique-DunhamV Kondapalli E Hinojosa A Stillwell E Young C ZhangX Song H Du T Gambin SN Jhangiani Z Coban Akde-mir DM Muzny A Tejomurtula OA Ross C Shaw JJankovic W Bi and JE Posey report no disclosures relevantto the manuscript JR Lupski has stock ownership in23andMe and Lasergen and is a paid consultant for Regen-eron and a coinventor on multiple US and European patentsrelated to molecular diagnostics for inherited neuropathieseye diseases and bacterial genomic fingerprinting JMShulman consults for the Adrienne Helis Malvin amp DianaHelis Henry Medical Research Foundations Go to Neurol-ogyorgNG for full disclosures

Publication historyReceived by Neurology Genetics December 27 2019 Accepted in finalform June 15 2020

Appendix Authors

Name Location Contribution

Laurie ARobak MDPhD

Baylor College ofMedicineHouston TX

Conceptualization investigationdata generation data analysisdata curation writingmdashoriginaldraft and writingmdashreview andediting

Renqian DuPhD DDS

Baylor College ofMedicineHouston TX

Conceptualization investigationdata generation data analysisdata curation writingmdashoriginaldraft and writingmdashreview andediting

Bo Yuan PhD Baylor College ofMedicineHouston TX

Conceptualization investigationdata generation data analysisdata curation andwritingmdashreview and editing

Shen Gu PhD Baylor College ofMedicineHouston TX

Conceptualization investigationdata generation data analysisdata curation andwritingmdashreview and editing

IsabelAlfradique-Dunham MD

Baylor College ofMedicineHouston TX

Data curation andwritingmdashreview and editing

VismayaKondapalli BS

Baylor College ofMedicineHouston TX

Data generationwritingmdashoriginal draft andwritingmdashreview and editing

EvelynHinojosa BS

Baylor College ofMedicineHouston TX

Data curation andwritingmdashreview and editing

AmandaStillwell BS

Baylor College ofMedicineHouston TX

Data curation andwritingmdashreview and editing

Emily YoungBA

Baylor College ofMedicineHouston TX

Data curation andwritingmdashreview and editing

Continued

NeurologyorgNG Neurology Genetics | Volume 6 Number 5 | October 2020 9

References1 Elbaz A Grigoletto F Baldereschi M et al Familial aggregation of Parkinsonrsquos dis-

ease a population-based case-control study in Europe EUROPARKINSON StudyGroup Neurology 1999521876ndash1882

2 Karimi-Moghadam A Charsouei S Bell B Jabalameli MR Parkinson disease fromMendelian forms to genetic susceptibility new molecular insights into the neuro-degeneration process Cell Mol Neurobiol 2018381153ndash1178

3 Espay AJ Brundin P Lang AE Precision medicine for disease modification in Par-kinson disease Nat Rev Neurol 201713119ndash126

4 Falcone DC Wood EM Xie SX Siderowf A Van Deerlin VM Genetic testing andParkinson disease assessment of patient knowledge attitudes and interest J GenetCouns 201120384ndash395

5 Bras J Guerreiro R Hardy J SnapShot genetics of Parkinsonrsquos disease Cell 2015160570ndash570e1

6 Hernandez DG Reed X Singleton AB Genetics in Parkinson disease Mendelianversus non-Mendelian inheritance J Neurochem 2016139(suppl 1)59ndash74

7 Posey JE Rosenfeld JA James RA et al Molecular diagnostic experience of whole-exome sequencing in adult patients Genet Med 201618678ndash685

8 Schormair B Kemlink D Mollenhauer B et al Diagnostic exome sequencing in early-onset Parkinsonrsquos disease confirms VPS13C as a rare cause of autosomal-recessiveParkinsonrsquos disease Clin Genet 201893603ndash612

9 La Cognata V Morello G DrsquoAgata V Cavallaro S Copy number variability in Par-kinsonrsquos disease assembling the puzzle through a systems biology approach HumGenet 201713613ndash37

10 Gambin T Akdemir ZC Yuan B et al Homozygous and hemizygous CNV detectionfrom exome sequencing data in a Mendelian disease cohort Nucleic Acids Res 2017451633ndash1648

11 Dharmadhikari AV Ghosh R Yuan B et al Copy number variant and runs of ho-mozygosity detection by microarrays enabled more precise molecular diagnoses in11020 clinical exome cases Genome Med 20191130

12 Pankratz N Dumitriu A Hetrick KN et al Copy number variation in familial Par-kinson disease PLoS One 20116e20988

13 Simon-Sanchez J Scholz S del Mar Matarin M et al Genomewide SNP assay revealsmutations underlying Parkinson disease Hum Mutat 200829315ndash322

14 Wiszniewska J Bi W Shaw C et al Combined array CGH plus SNP genome analysesin a single assay for optimized clinical testing Eur J Hum Genet 20142279ndash87

15 Cheung SW Shaw CA YuW et al Development and validation of a CGHmicroarrayfor clinical cytogenetic diagnosis Genet Med 20057422ndash432

16 Harmala SK Butcher R Roberts CH Copy number variation analysis by dropletdigital PCR Methods Mol Biol 20171654135ndash149

17 Singleton AB Farrer M Johnson J et al alpha-Synuclein locus triplication causesParkinsonrsquos disease Science 2003302841

18 Spellman GG Report of familial cases of parkinsonism Evidence of a dominant traitin a patientrsquos family JAMA 1962179372ndash374

19 Muenter MD Forno LS Hornykiewicz O et al Hereditary form of parkinsonismdementia Ann Neurol 199843768ndash781

20 Carvalho CMB Lupski JR Mechanisms underlying structural variant formation ingenomic disorders Nat Rev Genet 201617224ndash238

21 Zhang F Khajavi M Connolly AM Towne CF Batish SD Lupski JR The DNAreplication FoSTeSMMBIR mechanism can generate genomic genic and exoniccomplex rearrangements in humans Nat Genet 200941849ndash853

22 Beck CR Carvalho CMB Akdemir ZC et al Megabase length hypermutation ac-companies human structural variation at 17p112 Cell 20191761310ndash1324e10

23 Zafar F Valappil RA Kim S et al Genetic fine-mapping of the Iowan SNCA genetriplication in a patient with Parkinsonrsquos disease NPJ Parkinsons Dis 2018418

24 Carvalho CMB Ramocki MB Pehlivan D et al Inverted genomic segments andcomplex triplication rearrangements are mediated by inverted repeats in the humangenome Nat Genet 2011431074ndash1081

25 Deng H Le W-D Hunter CB et al Heterogeneous phenotype in a family withcompound heterozygous parkin gene mutations Arch Neurol 200663273ndash277

26 Espay AJ Brundin P Lang AE Precision medicine for disease modification in Par-kinson disease Nat Rev Neurol 201713119ndash126

27 Benitez BA Davis AA Jin SC et al Resequencing analysis of fiveMendelian genes andthe top genes from genome-wide association studies in Parkinsonrsquos disease MolNeurodegener 20161129

28 Sidransky E Nalls MA Aasly JO et al Multicenter analysis of glucocerebrosidasemutations in Parkinsonrsquos disease N Engl J Med 20093611651ndash1661

29 Kim JS Yoo JY Lee KS et al Comparative genome hybridization array analysis forsporadic Parkinsonrsquos disease Int J Neurosci 20081181331ndash1345

30 Kay DM Stevens CF Hamza TH et al A comprehensive analysis of deletions multi-plications and copy number variations in PARK2 Neurology 2010751189ndash1194

31 de Ligt J Boone PM Pfundt R et al Detection of clinically relevant copy numbervariants with whole-exome sequencing Hum Mutat 2013341439ndash1448

32 Gu S Posey JE Yuan B et al Mechanisms for the generation of two quadruplicationsassociated with split-hand malformation Hum Mutat 201637160ndash164

33 Seo SH Bacolla A Yoo D et al Replication-based rearrangements are a commonmechanism for SNCA duplication in Parkinsonrsquos diseaseMovDisord 202035868ndash876

34 Liu P Gelowani V Zhang F et al Mechanism prevalence and more severe neu-ropathy phenotype of the Charcot-Marie-Tooth type 1A triplication Am J HumGenet 201494462ndash469

35 Beutler E Gelbart T West C Identification of six new Gaucher disease mutationsGenomics 199315203ndash205

36 Ysselstein D Shulman JM Krainc D Emerging links between pediatric lysosomalstorage diseases and adult parkinsonism Mov Disord 201934614ndash624

37 Yahalom G Greenbaum L Israeli-Korn S et al Carriers of both GBA and LRRK2mutations compared to carriers of either in Parkinsonrsquos disease risk estimates andgenotype-phenotype correlations Parkinsonism Relat Disord 201962179ndash184

Appendix (continued)

Name Location Contribution

ChaofanZhang BS

Baylor College ofMedicineHouston TX

Data generation andwritingmdashreview and editing

Xiaofei SongPhD

Baylor College ofMedicineHouston TX

Data generation andwritingmdashreview and editing

Haowei DuMS

Baylor College ofMedicineHouston TX

Data generation andwritingmdashreview and editing

TomaszGambin PhD

Baylor College ofMedicineHouston TX

Data analysis andwritingmdashreview and editing

Shalini NJhangianiPhD

Baylor GeneticsHouston TX

Data generation andwritingmdashreview and editing

Zeynep CobanAkdemir PhD

Baylor College ofMedicineHouston TX

Data analysis methodology andwritingmdashreview and editing

Donna MMuzny MS

Baylor GeneticsHouston TX

Data generation andwritingmdashreview and editing

AnushaTejomurtulaMS

Baylor College ofMedicineHouston TX

Data generation andwritingmdashreview and editing

Owen A RossPhD

Mayo ClinicJacksonville FL

Writingmdashreview and editing andresources

Chad ShawPhD

Baylor College ofMedicineHouston TX

Data curation andwritingmdashreview and editing

JosephJankovic MD

Baylor College ofMedicineHouston TX

Data curation writingmdashreviewand editing resources andsupervision

Weimin BiPhD

Baylor College ofMedicineHouston TX

Data curation writingmdashreviewand editing and supervision

Jennifer EPosey MDPhD

Baylor College ofMedicineHouston TX

Data analysis data curationwritingmdashoriginal draftwritingmdashreview and editing andsupervision

James RLupski MDPhD

Baylor College ofMedicineHouston TX

Conceptualizationwritingmdashreview and editingresources and supervision

Joshua MShulman MDPhD

Baylor College ofMedicineHouston TX

Conceptualizationwritingmdashoriginal draftwritingmdashreview and editingresources and supervision

10 Neurology Genetics | Volume 6 Number 5 | October 2020 NeurologyorgNG

DOI 101212NXG000000000000049820206 Neurol Genet

Laurie A Robak Renqian Du Bo Yuan et al Parkinson disease

Integrated sequencing and array comparative genomic hybridization in familial

This information is current as of July 28 2020

ServicesUpdated Information amp

httpngneurologyorgcontent65e498fullhtmlincluding high resolution figures can be found at

References httpngneurologyorgcontent65e498fullhtmlref-list-1

This article cites 37 articles 1 of which you can access for free at

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

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 2020 The Author(s)

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