TM4SF20 Ancestral Deletion and Susceptibility to a Pediatric Disorder of Early Language Delay

14 Pages·2013·2.15 MB·English

by  Wojciech Wiszniewski

ARTICLE
TM4SF20 Ancestral Deletion and Susceptibility
to a Pediatric Disorder of Early Language Delay
and Cerebral White Matter Hyperintensities
Wojciech Wiszniewski,1,30 Jill V. Hunter,2,30 Neil A. Hanchard,1,3,4 Jason R. Willer,5 Chad Shaw,1
Qi Tian,1 Anna Illner,2 Xueqing Wang,1 Sau W. Cheung,1 Ankita Patel,1 Ian M. Campbell,1
Violet Gelowani,1 Patricia Hixson,1 Audrey R. Ester,1 Mahshid S. Azamian,1 Lorraine Potocki,1
Gladys Zapata,1 Patricia P. Hernandez,1 Melissa B. Ramocki,6 Regie L.P. Santos-Cortez,1 Gao Wang,1
Michele K. York,7 Monica J. Justice,1 Zili D. Chu,2 Patricia I. Bader,8 Lisa Omo-Griffith,8
Nirupama S. Madduri,9 Gunter Scharer,10 Heather P. Crawford,1 Pattamawadee Yanatatsaneejit,11
Anna Eifert,12 Jeffery Kerr,13 Carlos A. Bacino,1 Adiaha I.A. Franklin,14 Robin P. Goin-Kochel,15
Gayle Simpson,16 Ladonna Immken,16 Muhammad E. Haque,6 Marija Stosic,6 Misti D. Williams,17
Thomas M. Morgan,17 Sumit Pruthi,18 Reed Omary,18 Simeon A. Boyadjiev,19 Kay K. Win,20
Aye Thida,21 Matthew Hurles,22 Martin Lloyd Hibberd,23 Chiea Chuen Khor,23
Nguyen Van Vinh Chau,24 Thomas E. Gallagher,25 Apiwat Mutirangura,11 Pawel Stankiewicz,1
Arthur L. Beaudet,1 Mirjana Maletic-Savatic,6 Jill A. Rosenfeld,26 Lisa G. Shaffer,27 Erica E. Davis,4
John W. Belmont,1,4 Sarah Dunstan,28 Cameron P. Simmons,28 Penelope E. Bonnen,1,29
Suzanne M. Leal,1 Nicholas Katsanis,5 James R. Lupski,1,3 and Seema R. Lalani1,4,*
Whitematterhyperintensities(WMHs)ofthebrainareimportantmarkersofagingandsmall-vesseldisease.WMHsarerareinhealthy
childrenand,whenobserved,oftenoccurwithcomorbidneuroinflammatoryorvasculiticprocesses.Here,wedescribeacomplex4kb
deletionin2q36.3thatsegregateswithearlychildhoodcommunicationdisordersandWMHin15unrelatedfamiliespredominantly
fromSoutheastAsia.TheprematurebrainagingphenotypewithpunctateandmultifocalWMHswasobservedin~70%ofyoungcarrier
parentswhounderwentbrainMRI.Thecomplexdeletionremovesthepenultimateexon3ofTM4SF20,ageneencodingatransmem-
braneproteinofunknownfunction.Minigeneanalysisshowedthattheresultantnetlossofanexonintroducesaprematurestopcodon,
which,inturn,leadstothegenerationofastableproteinthatfailstotargettotheplasmamembraneandaccumulatesinthecytoplasm.
Finally, we report this deletion to be enriched in individuals of Vietnamese Kinh descent, with an allele frequency of about 1%,
embeddedinanancestralhaplotype.OurdatapointtoaconstellationofearlylanguagedelayandWMHphenotypes,drivenbyalikely
toxicmechanismofTM4SF20truncation,andhighlighttheimportanceofunderstandingandmanagingpopulation-specificlow-fre-
quencypathogenicalleles.
Introduction With a prevalence that ranges from 11% to 21% in
individuals ~64 years of age and increasing to more than
Age-related WMHs, observed as hyperintensities on T2 90%byage82,1theselesionsareknownto beassociated
weighted and fluid-attenuated inversion recovery (FLAIR) with an increased risk of cerebrovascular events and
brain MRI sequences, are recognized to be neuroimaging cognitive decline.2 Multiple large studies indicate that
expressionofbrainagingandcerebralsmall-vesseldisease. white matter signal abnormalities are infrequent in
1DepartmentofMolecularandHumanGenetics,BaylorCollegeofMedicine,Houston,TX77030,USA;2DepartmentofPediatricRadiology,TexasChil-
dren’sHospital,Houston,TX77030,USA;3DepartmentofPediatrics,TexasChildren’sHospital,Houston,TX77030,USA;4Children’sNutritionResearch
Center,DepartmentofPediatrics,BaylorCollegeofMedicine,Houston,TX77030,USA;5CenterforHumanDiseaseModeling,DukeUniversityMedical
Center,Durham,NC27710,USA;6JanandDanDuncanNeurologicalResearchInstituteatTexasChildren’sHospital,DepartmentsofPediatricsand
Neuroscience,BaylorCollegeofMedicine,TX77030,USA;7DepartmentofNeurology,BaylorCollegeofMedicine,Houston,TX,77030,USA;8Northeast
IndianaGeneticCounselingCenter,FortWayne,IN46845,USA;9DivisionofDevelopmentalMedicine,VanderbiltUniversity,Nashville,TN37232,USA;
10DepartmentofPediatrics,TheChildren’sHospital,Aurora,CO80045,USA;11CenterofExcellenceinMolecularGeneticsofCancerandHumanDiseases,
Chulalongkorn University, Bangkok 10330, Thailand; 12Department of Speech and Language, Texas Children’s Hospital, Houston, TX 77030, USA;
13DepartmentofNeurology,Dell’sChildren’sMedicalCenter,Austin,TX78723,USA;14DepartmentofDevelopmentalPediatrics,TexasChildren’sHos-
pital,Houston,TX77030,USA;15DepartmentofPediatrics,PsychologySection,BaylorCollegeofMedicineandTexasChildren’sHospital,Houston,
TX77030,USA;16SpeciallyforChildren,Dell’sChildren’sMedicalCenter,Austin,TX78723,USA;17DivisionofMedicalGeneticsandGenomicMedicine,
VanderbiltUniversity,Nashville,TN37232,USA;18PediatricRadiology&Neuroradiology,VanderbiltUniversity,Nashville,TN37232,USA;19Sectionof
Genetics,DepartmentofPediatrics,UniversityofCalifornia,Davis,Sacramento,CA95817,USA;20DepartmentofMedicine,HarlemHospitalCenter,New
York,NY10037,USA;21AllcarePediatrics,MissouriCity,TX77459,USA;22WellcomeTrustSangerInstitute,WellcomeTrustGenomeCampus,Hinxton,
Cambridge,CB101SA,UK;23GenomeInstituteofSingapore,Singapore138672,Singapore;24TheHospitalforTropicalDiseases,HoChiMinhCity,Viet-
nam;25TriplerArmyMedicalCenter,Honolulu,HI96859,USA;26SignatureGenomicLaboratories,PerkinElmer,Inc.,Spokane,WA99207,USA;27PawPrint
Genetics,GeneticVeterinarySciences,Inc.,Spokane,WA99202,USA;28OxfordUniversityClinicalResearchUnit,HoChiMinhCity,Vietnam;29Human
GenomeSequencingCenter,BaylorCollegeofMedicine,Houston,TX77030,USA
30Theseauthorscontributedequallytothiswork
*Correspondence:[email protected]
http://dx.doi.org/10.1016/j.ajhg.2013.05.027.(cid:1)2013byTheAmericanSocietyofHumanGenetics.Allrightsreserved.
TheAmericanJournalofHumanGenetics93,197–210,August8,2013 197
healthy individualsintheir20s and30s3,4 and areinfini- the TM4SF20 deletion were evaluated by geneticists, develop-
tesimal(~1%)inaneurologicallyhealthypediatricpopula- mentalpediatricians,and/orneurologistsforlanguagedelayand/
tion.5Conversely,inpathologicalconditionssuchaspedi- orbrainimagingabnormalities.Writteninformedconsentswere
atric systemic lupus, causing occlusive vascular disease, obtainedfromtheparentsofsubjectswiththeTM4SF20deletion,
identifiedontheclinicalarrayCGH.ManySoutheastAsianfam-
WMHs are reported in almost 50% of children, primarily
ilies in the study were either refugees (Burmese) or young Viet-
in the subcortical frontal regions.6 Children with unex-
nameseimmigrantstotheUnitedStates.VietnameseandBurmese
plained cognitive and/or developmental delay also have
languageinterpreterswereemployedforrecruitmentofthenon-
white matter signal abnormalities in an estimated 17%–
English-speakingfamilies.Thefirstandseconddegreerelativesof
26% of cases.7–9 Although the imaging appearance of
the probands were enrolled whenever available and written
whitematter pathologyinsuchcasesmaybenonspecific informedconsentswereobtained.
and suggestive of injury resulting from prematurity, hyp- DNA was extracted from whole blood by the Puregene DNA
oxia, ischemia, or fetal-maternal infection,10 WMHs are BloodKit(Gentra)accordingtothemanufacturer’sinstructions.
never considered innocuous findings when observed in The procedures for DNA digestion, labeling, and hybridization
thepediatricagegroup.11–13 for the oligo arrays were performed according to the manu-
In a genomic study of 15,493 children referred to the facturers’instructions.ArrayCGHwasperformedwithV8OLIGO
custom-designedgenome-widearraywithapproximately180,000
Medical Genetics Laboratories (MGL) at Baylor College of
interrogatingoligonucleotidesmanufacturedbyAgilentTechnol-
Medicine(BCM)(Houston,TX),byusing180,000oligonu-
ogies as previously described.14,15 The clinical array is designed
cleotide-based whole-genome exon-focused array compara-
by the MGL at BCM with exon-by-exon coverage for about
tive genomic hybridization (CGH),14 we identified a 4 kb
1,700genesand700microRNAstodetectintragenicsingleexon
deletionat2q36.3thatremovedthepenultimateexon3of
deletionsandduplications.
TM4SF20 in 12 unrelated individuals with language delay The clinical array includes tiling coverage of mitochondrial
andWMHs(penetranceof~70%).Withtwootherindepen- genome and provides whole-genome coverage at the resolution
dent study groups (group 2, Vietnamese ethnicity, Texas of30kb.Log ratioswereanalyzedforcopy-numberchangevia
2
Children’sHospital,n¼76;group3,Vietnameseethnicity, circular binary segmentation16 to minimize the possibility for
SignatureGenomics,n¼171),weconfirmedthesegregation false-negative results; we performed postprocessing of the oligo
of the deletion allele with familial WMHs and disorders of level data to ensure all copy-number variation (CNV) events
compriseatleastthreeoligoswithconsistentmedianlog-ratios.
communication. We show that this highly penetrant 4 kb
deletioncosegregateswithWMHsandearlychildhoodlan-
Long-RangePolymeraseChainReactionand
guage delay in multiple families extending from Burma to
Sequencing
Micronesia.Wepresentevidencetoillustratethatthispopu-
Long-rangePCR(LR-PCR)reactionswereperformedtoamplifythe
lation-specificdeletionwithallelefrequencyof~1%causes
predicted junction fragments in the breakpoint regions in
truncation of the cognate protein and is associated with
TM4SF20deletioncarriersaccordingtothemanufacturer’sspecifi-
communication disorders, with near complete penetrance
cations(TakaraBio).The1kbjunctionfragmentofthedeletion
in the Vietnamese, Thai, Burmese, Filipino, Indonesian, was amplified by LR-PCR with primers forward 50-ACAAGCA
andMicronesianpediatricsubpopulations.Wealsodemon- TAAGCCATTTGAGATCAACTAGTCC-30 and reverse 50-CAACA
strate that the frequency of WMHs related to TM4SF20 is GAACTGGAGTAAGTATGAAGCAGTCG-30. The PCR products
significantlyhigherthanthatreportedinotherlargepediat- were purified with the PCR Purification Kit (QIAGEN) and
ric studies of WMHs with unexplained cognitive and/or sequenced(LoneStar).
developmentaldelay,suggestingalinkbetweentheobserved
brainimagingabnormalitiesandthedeletionallele.7–9 BrainMagneticResonanceImaging
BrainMRIofall32subjectswiththedeletion(14unrelatedchil-
dren,13carrierparents,and5relatedindividuals)wasperformed
Subjects and Methods
at1.5Torhigherwith5mmslicethickness.Sequencesincluded
sagittalT1-weightedimaging,axialT2-wimaging,andT2-wFLAIR
Subjects imagingacquiredinallsubjects,inadditiontodiffusion-weighted
Thestudywasperformedinaccordancewithprotocolsapproved imagingandgradientechoimaging.Eachcasewasevaluatedbyat
by the appropriate human subjects ethics committees at Baylor least two neuroradiologists, blinded to the other’s radiological
College of Medicine and each participating institution. Written interpretationandthegeneticdata.
informed consents were obtained from the parents of subjects
withtheTM4SF20deletion.
LinkageAnalysis
Families TM200, TM800, TM900, TM1000, TM1100, and 017-1
StudyGroup1:ArrayCGHatMGL wereevaluatedforlinkage.Two-pointparametriclinkageanalysis
BetweenMay2009andJuly2012,15,493childrenofallethnicities was performed with MLINK of the FASTLINK package.17 An
were studied with the 180,000 oligonucleotide-based whole- autosomal-dominantmodeofinheritanceandadiseaseallelefre-
genome exon-focused array CGH for intellectual disability, epi- quencyof1%wereusedintheanalysis.Forthelanguagedelay
lepsy, facial dysmorphisms, congenital heart defects, multiple phenotype, a fully penetrant model with a phenocopy rate of
congenitalanomalies,and/orspeechandlanguagedelay.Theclin- 0.05 was used. The phenocopy rate of 5% was selected because
icalarraywasperformedattheMGLatBCM.Twelvesubjectswith this was approximately the prevalence of language delay in the
198 TheAmericanJournalofHumanGenetics93,197–210,August8,2013
general population. For the WMH phenotype, a reduced pene- constructwasgeneratedbyamplificationofthepartialTM4SF20
trancemodelwasusedwithapenetranceof0.7.Becauseoftherar- ORF (exons 1 and 2) from whole human brain cDNA with the
ityofthisphenotypeinhealthychildren,nophenocopieswere introductionofastopcodoninthereverseprimer.BothWTand
incorporatedinthemodel. DEX3pCR8/GWconstructswererecombineeredintopcDNA6.2-
N-EmGFP(Invitrogen)withLRclonaseII(Invitrogen).
DeterminationofTM4SF20DeletionHaplotype
TosetphaseofthehaplotypeonwhichtheTM4SF20deletionis CellCultureandImmunostaining
segregating,trios(mother,father,child)fromfivefamiliessegre-
mRNASplicingAssay
gating the TM4SF20 deletion were genotyped with either the
HEK293-FTcellswereseededin6-wellplates,andat70%conflu-
Omni 2.5-8 or Human Omni Express arrays from Illumina per ency, 1 mg pcDNA6.2-N-EmGFP-gDNA-DEX3 was transfected
manufacturer’sinstruction.OnefamilywasofVietnameseorigin,
withX-tremeGENEtransfectionreagent(Roche).Cellswerehar-
oneFilipino,andthreefamilieswereofBurmeseancestry.Geno-
vestedinTrizol(Invitrogen)at48hraftertransfectionsubsequent
types for 139 SNPs typed across all individuals and spanning toeither8hroftreatmentwithEmetine(100mg/ml)ornotreat-
500 kb across TM4SF20 were used to determine haplotypes by
ment. We generated oligo-dT-primed cDNA with Quantitect
familyinheritancepatterns. reversetranscriptase(QIAGEN),PCRamplifiedTM4SF20accord-
ingtostandardprocedures,andSangersequencedPCRproducts.
AssessmentofDeletionFrequencyinWorldwide ProteinLocalization
Populations Neuro-2acellswereseededonglasscoverslipsin6-wellplatesand
Population-baseddatawereobtainedfromgenotyping2,089Viet- were transfected with 1 mg pcDNA6.2-N-EmGFP-WT or 1 mg
nameseKinhcordbloodsamples,initiallyrecruitedandutilizedas pcDNA6.2-N-EmGFP-DEX3 with Dharmafect1 transfection re-
controlsinastudyofgeneticsusceptibilitytodenguefever.18The agent (Dharmacon). Cells were fixed with cold methanol at
2,089 cord blood controls were genotyped with the Illumina 24hraftertransfectionandimmunolabeledwitha-tubulinanti-
Human660WQuadBeadChipsaccordingtothemanufacturer’s body (Sigma; 1:500), mouse secondary antibody conjugated to
instructions.Afterexclusionsresultingfromqualitycontrol(relat- Alexa Fluor 568 (Invitrogen; 1:1,000), and nuclei were stained
ednessandgenome-widegenotypingsuccessrate),SNPgenotype with 10 mg/ml Hoeschst 3342 (1:1,000, Invitrogen). Confocal
datafrom2,018VietnameseKinh(1,022males,996females)were imageswerevisualizedwithaNikonEclipse90imicroscopeand
availableforanalysis.Genotypes,logRratio(LRR),andBallelefre- C1confocalscannercontrolledbyEZ-C1v.3.10software(Nikon).
quency(BAF)foreachprobesetweredeterminedwiththeIllumina Confocalimageswereacquiredat603,1.4ODobjective,and5mm
BeadStudiosoftware. z-stack.
TheIlluminaHuman660WQuadBeadChiparraycontainsone
SNPin thedeletionofinterest,rs10933181.GenotypeplusLRR
andBAFforthisSNPwereusedtoinferthepresenceorabsence Results
ofthedeletionineachindividual,agenotypeandaBAF(>0.97
or<0.01)thatwasconsistentwithahomozygouscallandalogR StudyGroup1:ArrayCGHatMGL
ratio that was less than two SDs from the mean LRR for By using 180,000 oligonucleotide-based whole-genome
rs10933181 in this cohort. Subsequently, each individual sus- exon-focused array CGH14 in 15,493 children referred to
pected of having the deletion based on these criteria (n ¼ 53)
the MGL at BCM, we identified an ~4 kb deletion at
plus an additional 72 samples were genotyped by PCR assay, as
2q36.3thatremovedthepenultimateexon3ofTM4SF20
describedabove,inordertoconfirmtheTM4SF20deletionstatus
(RefSeqaccessionnumberNM_024795.3) in12unrelated
foreachindividual.Atotalof46outof53individualstypedpos-
individuals(Figures1A,1B,and2A).Group1haddiverse
itive for the deletion by PCR and one of these 53 subjects was
ethnic distribution and wide-ranging referral indications,
equivocal. The 72 samples that were negative for deletion by
SNPdatawerealsonegativebyPCRassay.Insummary,theperfor- including facial dysmorphisms, congenital cardiovascular
manceoftheSNParray-basedassayforassessmentoftheTM4SF20 malformations, multiple congenital anomalies, intellec-
deletionshowedsensitivity¼1,specificity¼0.92,positivepredic- tualimpairment,and/orspeechandlanguagedelay(Table
tivevalue¼0.87,andnegativepredictivevalue¼1inthiscohort. S1availableonline).Theindexcase,individualTM101(II-
The frequency of this deletion in worldwide populations was 1inFigure1A)wasa3-year-oldBurmeseboy,referredfor
basedondatafromthe1000GenomesProjectvariantcallsfrom evaluation of language delay and left-sided hemiparesis.
thecallsetreleased2012-02-14.
His brain MRI showed bilateral periventricular confluent
WMHs with a thin corpus callosum. The same deletion
GenerationofDNAConstructsforInVitroStudies wasalsofoundinhis35-year-old,apparentlyhealthy,Bur-
Aminigeneconstruct(gDNA-DEX3)toassaymRNAsplicingwas mesemother,TM102(I-2inFigure1A)However,herbrain
generated by PCR amplification from genomic DNA harboring
MRI subsequently showed similar multifocal T2 hyperin-
the TM4SF20 deletion with the forward primer beginning at
tensitiesintheperiventricularandsubcorticaldeepwhite
the 50 end of exon 2 and the reverse primer at the stop codon
matterofbothcerebralhemispheres,suggestiveofcerebral
of exon 4. This fragment was cloned into pCR8/GW-TOPO
small-vessel disease with mild diffuse cerebral and cere-
(Invitrogen), sequence confirmed, and recombineered into
bellar volume loss. This identical deletion was observed
pcDNA6.2-N-EmGFP(Invitrogen)withLRclonaseII(Invitrogen).
TheWTproteinexpressionconstructwasgeneratedbyamplifi- in 11 other unrelated pediatric subjects, all of whom
cationofthefull-lengthTM4SF20openreadingframe(ORF)from shared a diagnosis of communication disorder, ranging
whole human brain cDNA (QUICK-clone, Clontech); the DEX3 from early language delay to autism spectrum disorder.
TheAmericanJournalofHumanGenetics93,197–210,August8,2013 199
Figure1. IdentificationofTM4SF20Exon3DeletioninMultipleFamiliesPredominantlyofSoutheastAsianDescent
(A)BrainMRIstudyinfamilyTM100ishighlighted,revealinghyperintensitiesonaxialT2-weightedspinechoandconfirmedonaxialT2
FLAIR,consistentwithfociofglioticchanges,andobservedintheperiventricularanddeepsubcorticalwhitematter(markedwithlong
whitearrow)inthecarrierchild(II-1)andparent(I-2).ThematernalbrainMRIimages(TM102)areshownatthetop,andtheimagesofthe
probandareillustratedbelow.Comparisonismadewiththeage-matchednormalcorrespondingMRIimage.Notethethincorpuscal-
losum(cc)inTM101.Generousbiparietalextra-axialCSFspaceswithmilddiffusevolumelossisobservedinthecarrierparent(marked
withshortwhitearrow).NotetheprominentVirchow-Robin(VR)spaceshighlightedbytheyellowarrow.
(B)Fourteenadditionalpedigreesareshown.Positivesignindicatespresenceofthedeletionandnegativesignhighlightsabsenceofthe
deletion.
200 TheAmericanJournalofHumanGenetics93,197–210,August8,2013
Figure2. TM4SF20HeterozygousDeletionandBreakpointAnalysis
(AandB)High-resolutionarrayCGHanalysisrevealeda4kbdeletionwithin2q36.3involvingexon3ofTM4SF20(UCSCGenome
BrowserbuildGRCh37/hg19:228,230,759–228,234,864).
(CandD)Long-rangePCRanalysisidentifiedacommon1kbjunctionfragmentinalltheaffectedsubjects.Sequenceanalysisrevealeda
complexrearrangementwitha2.3kbdeletionwithmicrohomologyofGTatthefirstjunction,followedbyaneutralcopynumberre-
gionofapproximately100bp(markedasgraybar-interruptedgreen),thena1.7kbdeletionwithinsertionofaTatthesecondjunction.
(E)ThehaplotypeonwhichtheTM4SF20deletionsegregateswasdeterminedforoneVietnamese,oneFilipino,andthreeBurmesefam-
ilies.Ahaplotypeof30kb(bracketlabeledA,red)wasfoundtobesharedacrossallfamilies.Alongerhaplotypeof90kb(bracketlabeled
B,green)wassharedbetweentheBurmeseandFilipinofamilies.Burmesefamiliessharedalonger467kbhaplotype(bracketlabeledC,
blue).AllSNPstypedintheseindividualsandutilizedinhaplotypeanalysesareshownasblacktickmarksandtheSNPsdelineating
haplotypesarelabeled.RefSeqgenesforthisregionareshown.
TheAmericanJournalofHumanGenetics93,197–210,August8,2013 201
Noneofthemhadanysignificantfacialdysmorphismsor GenomeBrowserGRCh37/hg19)(Figure2C).Theidentical
other congenital anomalies. Driven by the discovery of complex rearrangement in these unrelated pediatric sub-
thisgeneticlesion,weperformedsystematicbrainMRIin jects suggested that the deletion occurred on a single
otherchildrenwiththedeletionandinall,foundpunctate ancestral haplotype and was not due to recurrent de
WMHsinthedeepwhitematterwithenlargedperivascular novoeventssecondarytosusceptibilitytogenomicinsta-
(VR) spaces to multifocal T2 hyperintensities in the peri- bilityrenderedbytheinnategenomicarchitecture.
ventricular white matter in 8 out of 11 pediatric subjects
(72%) imaged (TM101, TM301, TM501, TM601, TM801, StudyGroup2:BrainMRIStudyof76Childrenof
TM1101, TM1301, and TM1401) (Figure 3; Table 1). The VietnameseOriginfromTexasChildren’sHospital
parentsdeclinedbrainimagingstudiesforTM1201.Inter- Toinvestigatefurtherthepotentialroleofthisgeneinlan-
estingly, most children with the 4 kb TM4SF20 deletion guage delay and WMHs, we reviewed the brain-imaging
originated from Southeast Asia or the Far East, including studies in an ethnicity-specific independent group of 76
Burma, Vietnam, Philippines, Thailand, Indonesia, and children of Vietnamese descent (group 2), evaluated at
Micronesia (Table 1). TM301 was reported to be Hispanic Texas Children’s Hospital in Houston, for speech and
and TM1301 was of unspecified Asian origin. We noted languagedelay,unexplaineddevelopmentaldelay,and/or
that the frequency of WMHs in this group was signifi- autism spectrum disorder (Figure S1). The two indepen-
cantly higher than previously reported for healthy chil- dent neuroradiologists blinded to the TM4SF20 deletion
dren between the ages of 1 month and 18 years (p ¼ status observed diverse brain imaging abnormalities in
2.589 3 10(cid:2)11),5 and 3- to 4-fold higher than reported multiplecases,includingdistinctwhitematterabnormal-
for children with unexplained intellectual disability with ities in 19/76 (25%) children. The TM4SF20 deletion was
IQ<70(p¼1.966310(cid:2)4)7orthosewithidiopathicdevel- confirmed by PCR in 4/76 (~5%) cases, three of whom
opmental delay (p ¼ 2.922 3 10(cid:2)3)9 from large studies had distinct white matter signal abnormalities (TM201,
(TableS2).Inaddition,thepresenceofpunctateandmulti- 017-1, 18-01) (Table S3). One child (TM701) without the
focal T2 hyperintensities in the periventricular and deep WMHswasalsofoundtohavethedeletion.Thefrequency
white matter in 8/10 unrelated carrier parents similar to ofWMHsinthisstudywasnotedtobedifferentindeletion
thoseobservedintheaffectedchildrenstronglysuggested versus nondeletion cases (p ¼ 0.0461, Fisher’s exact test),
a link between the observed brain imaging abnormalities indicating that the deletion carriers were more likely to
andthedeletionallele(Table2).Childrenexposedtoearly have WMHs than the nondeletion carriers. TM201,
hypoxic or infectious insult sometimes demonstrate 017-1, and TM701 were then followed further for family
nonspecific T2hyperintensitiesonbrainimaging,butthe studies. Parents declined further evaluation of 18-01,
extentofthefindingsandtheconsistencyacrossunrelated whoharboredthedeletionwithWMHs.
families from a distinct geographical region strongly sug- The individual TM201 (III-1 in Figure 1B), one of the
gestedthatthesewererelatedtotheshareddeletionallele. severely affected individuals with the deletion, was born
Infurthersupportofthis,ofallthe15,493cases(allethnic- prematurely at 33 weeks gestation with extensive loss of
ities) studied for the deletion allele, the variant was periventricularwhitematterandevidenceofstrokeinthe
observed only in those individuals referred for evaluation posterior cerebral artery (PCA) distribution. Pedigree anal-
of developmental delay, speech/language impairment, ysis identified periventricular subcortical T2 hyperinten-
and/or brain imaging abnormalities (n ¼ 6,390) and not sitiesinherotherwisehealthycarrierfather,TM202(II-2),
in those with craniofacial dysmorphisms, congenital car- and her paternal aunt, TM206 (II-4), both of whom were
diovascular malformations, multiple anomalies, musculo- <40 years of age with no cerebrovascular risk factors
skeletal concerns, urogenital abnormalities, and other (FigureS2).Subject017-1(II-1inFigure1B)presentedat7
concerns(n¼ 9,103)(TableS1).Thedifferenceinthefre- years of age with significant language delay. We subse-
quencyofdeletionalleleinthesetwoclassesofphenotypes quently found his 5-year-old sibling (II-2) to also harbor
is highly significant(p ¼ 1.7 3 10(cid:2)6),suggesting that the the deletion andexhibit severe language impairment, but
deletionisdistincttoindividualswithcommunicationdis- his brain imaging study was within normal limits. Their
ordersandbrainimagingabnormalitiesinthisgroup. 37-year-old asymptomatic mother (I-1), who also carried
thedeletion,showedmultifocalperiventricularandsubcor-
DetectionofaComplexGenomicRearrangement ticalT2hyperintensities(FigureS3).SubjectTM701(II-1in
LR-PCRinchildrenandtheircarrierparents’genomicDNA Figure 1B) with normal brain imaging had early language
yieldedan~1kbdeletion-specificjunctionfragment(Fig- delay and was diagnosed with autism spectrum disorder.
ures 2B–2D). Subsequent sequencing revealed a complex Her mother (I-1) was also scanned and had normal brain
rearrangement consisting of a 2.3 kb deletion with a GT imaging,confirmedbytwoneuroradiologists.
microhomologyatthefirstjunctionanda1.7kbdeletion Combininggroups1and2,atotalof15unrelatedpedi-
withinsertionofaTatthesecondjunction;bothdeletions atric subjects with the TM4SF20 deletion were studied
areseparatedbyaneutralcopy-numbergenomicsegment (Table 1). Of the 14 individuals imaged, 10 had WMHs
of approximately 100 bp that is oriented directly with (~71%). Further, with the exception of two individuals
respect to the reference human genome sequence (UCSC (TM201 and TM301) with moderate to severe disease,
202 TheAmericanJournalofHumanGenetics93,197–210,August8,2013
Figure3. SpectrumofWMHsintheChildrenwithTM4SF20Deletion
T2weightedandfluid-attenuatedinversionrecovery(FLAIR)sequencemagneticresonanceimaging(MRI)scansofnineunrelatedsubjects
withTM4SF20deletionarepresented,showingvaryingdegreesofwhitematterdiseaseobservedaspunctateT2hyperintensitiesinthe
subcorticalwhitematterandmultifocalT2hyperintensitiesintheperiventricularanddeepwhitematterofbothcerebralhemispheres.Sub-
jectTM201,bornprematurely,hasgrossenlargementofthethirdandlateralventricleswithnear-completelossofperiventricularwhitemat-
ter.SubjectTM301hasbilateralopenlipschizencephalyinthefrontallobes.Prominentperivascular(VR)spacesareseeninTM501.
TheAmericanJournalofHumanGenetics93,197–210,August8,2013 203
Table1. ClinicalCharacteristicsofChildrenwithTM4SF20HeterozygousDeletion
Study Current Languageand/or
No. PatientID Group Ancestry Inheritance Age Gender Gestation SpeechDisordera WMH MRIStudyResults
1 TM101 1 Burmese maternal 3years male term languagedelay þ periventricularwhite
matterlosswithT2
hyperintensities,not
typicalofperiventricular
leukomalacia
2 TM301 1 Hispanic/ unknown 3years male term moderatetosevere þ bilateralopen-lipped
unknown (adopted) globaldelay, schizencephalyand
nonverbal,epilepsy deformityofthecorpus
callosum;right-sided
peridentatenuclearT2
hyperintensity
3 TM401 1 Burmese paternal 7years male 32weeks languagedelay (cid:2) slightprominencetothe
T2hyperintensity
returnedfromthe
terminalzonesinthe
bilateralperitrigonal
regions;oneortwo
prominentVRspaces
4 TM501 1 Burmese paternal 2years male term languagedelay, þ prominentVRspaceswith
autismspectrum prominencetothe
disorder terminalzoneswith
increasedT2
hyperintensityreturned
fromtheperiventricular
whitematteroutlining
theoccipitalhornswith
delaysinmyelinationto
thesubcorticalU-fibersin
thehighparietalregions
5 TM601 1 Thai/Hispanic maternal 4years male term nonverbal,autism þ twosmallfocalT2
spectrumdisorder hyperintensitiesreturned
fromthebilateraldeep
whitematter
6 TM801 1 Pakistani/ maternal 3years male term autismspectrum þ multifocalpunctateT2
Filipino disorder hyperintensitiesreturned
fromthebilateralfronto-
parietalregions
7 TM901 1 Filipino maternal 2years female term languagedelay (cid:2) normalstudy
8 TM1001 1 Indonesian/ paternal 1year male term languagedelay, (cid:2) withinnormallimits
European globaldelay appearancestothebrain
whichisincompletely
myelinated,butage
appropriate,with
prominentterminalzones
9 TM1101 1 Burmese paternal 1year female term languagedelay þ multifocalsubcorticaland
deepT2hyperintensities
returnedfromalllobesof
bothcerebral
hemisphereswithout
masseffect
10 TM1201 1 Vietnamese/ maternal 4years male term speechand NA MRInotdone
European languagedisorder
11 TM1301 1 unspecified unknown 1year female term languagedelay þ symmetricpatchyT2
Asianancestry (adopted) hyperintensesignalsin
theoccipitalsubcortical
whitematter
12 TM1401 1 Micronesian/ maternal 13years male term languagedelay, þ fewscatterednonspecific
European developmental highT2FLAIRfociseen
delay throughoutthe
subcorticalwhitematter,
mostprominentlyinthe
leftfrontallobe
(Continuedonnextpage)
204 TheAmericanJournalofHumanGenetics93,197–210,August8,2013
Table1. Continued
Study Current Languageand/or
No. PatientID Group Ancestry Inheritance Age Gender Gestation SpeechDisordera WMH MRIStudyResults
13 TM201 2 Vietnamese paternal 1year female 33weeks severeglobaldelay, þ nearcompleteagenesisof
epilepsy thecorpuscallosumwith
evidenceforgliotic
changereturnedinaleft
posteriorcerebralartery
distribution;ex-vacuo
dilationofthe
supratentorialventricular
system
14 017-1 2 Vietnamese maternal 7years male term severelanguage þ atleastonefocalT2
delay hyperintensityreturned
fromtherightposterior
frontalsubcorticalwhite
matter
15 TM701 2 Vietnamese maternal 4years female term languagedelay, (cid:2) normalstudy
autismspectrum
disorder
Abbreviationisasfollows:VR,Virchow-Robinspaces.
aDetailsofdeficitsareasfollows.ForTM101:receptivelanguageabilities,3-year-oldlevel;expressivelanguage,2-year-oldlevel(PreschoolLanguageScale,
FourthEdition[PLS-4]).ForTM301:generaldevelopmentalscore,40;communication,50;cognitive,50;adaptivebehavior,50(DP-3).ForTM401:deficiencies
inLNF(letter-namingfluency),inPSF(phoneme-segmentationfluency),andinNWF(nonsense-wordfluency)(mCLASS:DynamicIndicatorsofBasicEarlyLiter-
acySkills).ForTM501:visualproblem-solvingskills,DQ68;language,DQ43(CognitiveAdaptiveTest/ClinicalLinguisticAuditoryMilestoneScale).ForTM601:
responsesexceedcutoffsonautismspectrumdisorderquestionnaires(SocialCommunicationQuestionnaire-Lifetime[SCQ;2003]formandaSocialResponsive-
nessScale[SRS;2005]parentform).ForTM701:adaptivebehaviorcomposite,68;communication,54;dailylivingskills,75;socialization,63;motorskills,94
(VinelandAdaptiveBehaviorAssessmentIISurveyInterview).ForTM801:expressivecommunication,standardscore56,ageequivalentof6monthsat19months
ofchronologicalage;auditorycomprehension,standardscoreof61,ageequivalentof9months;totallanguagescore54,percentilerank1,ageequivalentof
7months(PLS-4).ForTM1101:visualproblem-solvingskills,DQ87;language,DQ67;overalldevelopmentalquotientis77.ForTM1201:atage3.1,informal
observationofexpressivelanguageskillsshoweduseofuptofivesinglewordspersentencetodescribe,request,andinteractcommunicatively,andtheuseof
simplequestionformulation.Prepositionswereapparentbutfrequentlyomitted.Atage4.4,theexpressiveSpeechProfileresultsshowedanimprovementfrom
themoderatetothemildtomoderatelevelofarticulationproficiency.ForTM1301:expressivelanguageat6monthsandreceptivelanguageat10months,when
evaluatedat12months.ForTM1401:40pointdifferencebetweenverbalandperformanceabilities,withverbalscoressignificantlylower(PeabodyPictureVo-
cabularyTestFourthEdition[PPVT-4]);TestofNonverbalIntelligenceSecondEdition[TONI-2]).
the phenotype of most children with the deletion was Thus, combining all Vietnamese cases from groups 1
consistentwithlanguagedelaywithoutsignificantcranio- (n¼168),2(n¼76),and3(n¼171),therewere415in-
facial or other congenital anomalies and with preserved dividuals with a diverse range of phenotypes studied for
motorskills.Fewhyperpigmentedmaculeswereobserved theTM4SF20deletion.Inaggregate,wefoundthedeletion
variably within this group. Formal speech/language and in 10/189 ¼ 0.053 (95% CI 0.026–0.095) cases evaluated
development assessment in the deletion carriers showed forearlylanguagedelay,developmentaldelay,autismspec-
significant discrepancies between verbal and nonverbal trum disorder, and brain MRI abnormalities, whereas no
skills(Table1). deletionsweredetectedinchildrenwithcongenitalheart
defects, multiple congenital anomalies, facial dysmor-
StudyGroup3:VietnameseGroupfromSignature phisms,urogenitalorskeletalabnormalities,orothercon-
Genomics cerns (0/207 ¼ 0.0 [95% CI 0.0–0.018]) (Table S4). The
The study of the 15 ascertained families indicated near- difference in the frequency of the deletion in these two
completepenetranceforearlylanguagedelayinthepedi- groupsisstatisticallysignificant(p¼5.4310(cid:2)4),demon-
atric population, with high penetrance of WMHs in stratingthatthedeletionisuniquetoVietnameseindivid-
carriers (~70%). To exclude the possibility that this low- uals with communication disorders and brain imaging
frequency,population-specificdeletionallelewasabenign abnormalities.
polymorphismintheVietnamesepopulation,weobtained
unidentified DNA samples from a third independent set,
comprised of 171 pediatric cases of Vietnamese origin PedigreeandLinkageAnalysis
referredtoSignatureGenomicLaboratories,LLCforarray Thesedatasuggestedacausallinkbetweenthedeletionin
CGH analysis. Of these, 79 Vietnamese children were TM4SF20 and WMHs with associated language delay of
referredforevaluationofcommunicationdisorders,devel- variable expressivity. We determined that the early lan-
opmental delay, or brain imaging abnormalities. Via LR- guage and/or speech delay phenotype was self-reported
PCR, we determined the TM4SF20 deletion allele in 4 by parents in pedigrees TM200, TM900, TM1000,
children, all from within this subgroup of 79 (4/79 ¼ TM1200,andTM1400andinsiblingsofTM1100and17-
0.050), and none in those referred for other concerns 01evenbeforethedeletionwasidentifiedintheprobands.
including multiple congenital anomalies, facial dysmor- We performed further genetic and phenotypic family
phisms,congenitalheartdefects,orgrowthfailure(0/92). studies by using standardized criteria for early language
TheAmericanJournalofHumanGenetics93,197–210,August8,2013 205
Table2. ClinicalCharacteristicsofParentswithTM4SF20Deletion
Study Carrier
No. ParentID Group Parent Ancestry Age Comorbidity WMH MRIStudyResults
1 TM102 1 mother Burmese 35years none þ T2multifocalpunctatesubcentimeterhyperintensities
returnedfromthebifronto-parietalsubcorticalwhite
matterwithdilatedVRspaces
2 TM402 1 father Burmese 52years end-stageliverdisease þ cerebellargreaterthancerebralatrophywithmild
(alcoholiccirrhosis, ex-vacuodilatationofthesupraventricularsystemin
hepatitisC) associationwithoneortwosubcorticalwhitematter
focalT2hyperintensities
3 TM502 1 father Burmese 39years none þ punctatefocal2–3mmsubcorticalwhitematterT2
hyperintensitiesreturnedfromthehighbilateral
posteriorfronto-parietalsubcorticalwhitematter
4 TM602 1 mother Thai 35years none þ tinyfociofT2hyperintensitiesreturnedfrom
predominatelytheleftposteriorparietalsubcortical
whitematter
5 TM802 1 mother Filipino 36years none þ bilateralfronto-parietal,lessthan5mmsubcorticalT2
brightwhitematterhyperintensities
6 TM902 1 mother Filipino 32years none (cid:2) normalstudy
7 TM1002 1 father Indonesian 30years none þ multifocalpunctateT2hyperintensitiesreturnedfrom
thebifronto-parietalsubcorticalwhitematter,slightly
moremarkedontherightwhencomparedtotheleft
8 TM1102 1 father Burmese 42years none (cid:2) normalstudy
9 TM1203 1 mother Vietnamese 36years none þ thereareatleast1–2,3mmfocusofabnormalT2
hyperintensityreturnedfromthesubcorticalwhite
matterintherightinsularregion
10 TM1402 1 mother Micronesia 50years hypertension þ multiplescatteredfociofincreasedT2signalthroughout
theperiventricularwhitematter
11 TM202 2 father Vietnamese 37years none þ multifocalpunctateT2hyperintensitiesreturnedfrom
thebilateralfronto-parietalsubcorticalwhitematter
12 017-3 2 mother Vietnamese 37years none þ multifocalpunctate,lessthan5mm,T2hyperintensities
returnedfromthebifronto-parietalsubcorticalwhite
matter
13 TM702 2 mother Vietnamese 29years none (cid:2) normalstudy
Abbreviationisasfollows:VR,Virchow-Robinspaces.
delay,definedasfewerthan50wordsornowordcombina- onlyfourwereinformativeforWMHs.Whenthedeletion
tions between 20 and 34 months of age,19 as well as by wastestedforlinkageattheta¼0,thetotalLODscorefor
brain imaging studies. In six families (TM200, TM900, these six families for the language delay phenotype was
TM1000,TM1100,TM1200,and017;Figure1B),wefound 3.1, whereas the combined LOD score at theta ¼ 0 for
the deletion allele to fully segregatewith earlychildhood the four families for WMHs (TM200, TM800, TM1000,
language delay. In family TM900, the proband’s mother and017)was0.75.Althoughthepenetranceforlanguage
(III-2 in Figure 1B), maternal uncle (III-3), and maternal delayappearstobehigh(inpediatriccases)withanauto-
grandfather(II-1)reportedlateexpressivelanguagedevel- somal-dominant mode of inheritance, there is reduced
opment,between3and4yearsofagepriortothedeletion penetrancefortheWMHphenotype.
testing in the proband; all three were found to have the Overall,weperformedbrainMRIstudyinthefollowing
deletion (Figure S4). These individuals demonstrated deletioncarriers:14unrelatedchildren,13parents,and5
higheducationalachievement,holdingdoctoratedegrees. related individuals (TM205, TM206, 017-02, TM1103,
Thedeletionwasnotpresentintheproband’sbrother(IV- and TM1104) from 15 families (Table S5). Abnormalities
2),father(III-1),maternalgrandmother(II-2),ormaternal consistent with WMHs were observed in 22/32 (~69%)
great-grandmother(I-1).Thepaternalgreat-grandfather(I- deletion carriers, indicating a high (but not complete)
2) (aged 92 years) declined genetic testing. Brain MRI penetranceprobablyduetothedeletionallele(FiguresS5
studies were carried out in the proband and her 32-year- and3).Eachcasewasevaluatedbyatleasttwoneuroradi-
oldmother,whichwereessentiallynormal. ologists who were blinded to the radiological interpreta-
There were six families (TM200, TM800, TM900, tion and the genetic data. An individual was designated
TM1000, TM1100, and 017) who were sufficiently large to have WMHs only if the diagnosis was concurred by
for linkage analysis for language delay. Of these families, two neuroradiologists. Across all individuals studied, the
206 TheAmericanJournalofHumanGenetics93,197–210,August8,2013