Posted on Authorea 8 Oct 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.160218236.66387607/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. srcts(seta o ev ciiy fcru alsm(ags ht atrsrcue.Ti limited This to speech in in tried structure). only lies matter study noticeable abnormality white bit, model that tiny (largest mouse indicated a by callosum and recent communication corpus stuttering inter-hemispheric A down of of slows mystery said activity) speech. population the are nerve astrocytes for to deficits for responsible central these (essential However are neurons astrocytes that unknown. specific neurons is on the stuttering impact of illuminate the but phenomena unique dysfunction the have lysosomal to to implicating genes disorders, these neurological linking in mechanism concept emerging ge- an the studies deficits, until trafficking elucidate possible not to was linkage genes disorder, trait, of , Identification complex inherited families a studies. consanguineous any is worldwide across on stuttering loci to focused since studies multiple Nevertheless applicable mapping multiple in stuttering. as resulted to in analysis relevant studies, etiology indicated linkage non-genetic and were with netic began factors and genes genetic anxiety causative stress, for of etc evidence conditions genes temperament, The genetic, with under child’s interact includes dynamics, dysfluency can that family the multifactorial cause, on aggravate but is a depends may children stuttering not also and by of though outgrown modifications) factors Emergence generally epigenetic Environmental is problem. (cause (PWS) condition factors. stutter enduring The neurological who an it. people and as saying wherein environmental some trouble speech have in of but flow persists say normal it to the want disrupts they that what fluency know of disorder a is Stuttering Keywords: stuttering. INTRODUCTION of genesis under- the the in understand involved to be prospects to pathways research a mechanisms neural the in is combined sequencing, drive stuttering variant and can exome that heterozygous study multiple fact Stuttering, our the a hypothesize variants) in We support and identified results (with mechanisms. gene Our targets genes lying gene NLRP11 confirmation. 14 putative further in The of needs variant that list disorder. homozygous family putative polygenic enriched STU-65 a for an in Additionally, identified in search were resulted gene functions. to clin- NAGPA analysis neural implemented two gen- data in was from five ES involved approach individuals studied, across SPTBN5) bioinformatics families six stuttering sequential multiplex understanding in showing and two a TMOD2,HTR2B,RSC1A1,TRPV2,WNK1,ARSD STU-66) performed for (COL4A2,COL6A3,COL6A6,ITGAX,LAMA5,ADAMTS9,CSGALNACT1, the prioritization, and was In scope variant (STU-65 ES and targets. Analyzing to families study gene genes fami- ES Indian this identified. From putative afflicted south In multiple be of affected, erations. pathways. multiple with identification could implicated characterized, persuasive, etiology in well speech is genetic help ically for trait conclusive may burden this no sequencing(ES) mutational yet of exome the heritability generations, using and across families disorder segregation multiplex speech phenotypic complex showing a lies is Stuttering Abstract Abstract 2020 8, October 1 CR SRIKUMARI sequencing exome highly using in families stuttering Indian to multiplex linked genes putative novel Identifying nvriyo Madras of University GNPTG , NAGPA and 1 ADIIG NANDHINI , AP4E1 htepan 0 fsutrn cases stuttering of 20% explains that 3 h astv ee ikdt tteigwr dnie obe to identified were stuttering to linked genes causative The . 1 n hnr J Chandru and , 1 1 . 1 4 l hs ee on ointracellular to point genes these All . 2 olwd erhfor search Worldwide . GNPTAB 5 . Posted on Authorea 8 Oct 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.160218236.66387607/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. pt 9konvrat n 2nvlvrat,sra cos3 ee tbeA1). (table genes sums 30 thus across interest spread of methodology) variants, genes in family: novel The (detailed 12 variants). STU-65 (dbSNP criteria and heterozygous exclusion the variants known (all of step known genes previously 12 four 18 19 were and a to to variants list Applying 218 known up the 218 variants, region. shortened the variants, coding common ensued of known the 85 them 260 to in Only in these common variants fall unreported). variants common were Of novel (hitherto the variants that 42 novel for genes 15 genes. were further 276 42 filtering produced 248 and revealed On that in database) combination A1). diagram harbored such (figure Venn variants One proband using the 260 analyzed 3). from and were (ranging (figure father variants individuals affected data 2245 to four the of the of mean of combinations a in data possible resulted variant methodology), The in proband. 2188-2296). (detailed in variants criteria filter 73,198 variant and Applying sibling unaffected in 73,141 mother, unaffected family: Further STU-66 (V-33;V-35). STU-66 section. siblings methodology affected two the stuttering: brother filtering includes in severe unaffected Variant mentioned 2) (IV-3), with are (figure mother families families family unaffected the STU-65 multiplex (IV-2), of (V-5); heritability. father highly details proband missing affected two the the of from and unravel comprising (V-4) and individuals 1) architecture (figure six genetic family complex in nuclear the performed reveal was to technology ES in powerful two a present is in not ES employed but was families ES multiplex the series. in RESULTS this inherited variants in pathogenic second individuals. the databases. the in affected public analyzing as help in by would paper common stuttering, database families this are existing for multiplex from that of families variants analyzing concept affected or families, the multiple variants in forms rare segregates this penetrant that Subsequently highly genes series. trait these of this complex to identification in ascribed a either paper be first is could the stuttering that as Since (2/64) reported 3.1% is of which the resolution elusive, preliminary reported minimal remains Our Nadu, a Tamil but of showed unexplored. state genes nearly the stuttering in and be may children reported limited school to stuttering 74,544 are stuttering of that India of cohort in large hint prevalence a stuttering findings in brain. of study these in epidemiology the genetics genetic involved All of the parts genes on different deficits. and studies with communication Research circuits communication neural to in in delay point slight deficits all from dopamine, - result overactive disposal - who waste research children cellular stuttering in in hemisphere findings brain. The left the of the the parts in different among connecting attenuated those rather is and regions stuttering callosum auditory developmental corpus and involving Basal motor connectivity motor speech stutter - to matter) cortical (BGTC feedback (white and auditory movements of Structural speech putamen integration Stutter in of Who and process. timing Putamen) Children control self-initiated and in SMA support differences loop, that Thalamo-cortical connectivity Ganglia networks functional neural and pathophysiology involving structural of (CWS) understanding revealed stuttering, our studies to system, enhance imaging contribute speech could Brain may of children processes anatomy in maturational in studies developments these imaging articulatory the in brain phonatory, parallels Interferences hence respiratory, stuttering skills. of innervate articulatory onset that and of nerves language Age (cranial systems). system resonance sub-cortical nervous and and (cortical peripheral system nervous and central regions) between communication efficient requires production Speech 7,8 vratvt ftedpmn ertasitrhsbe mlctda oeta atrin factor potential a as implicated been has neurotransmitter dopamine the of activity Over . Sdt ftetoaetdsb V3,poadadV3,aetdbohr eutdin resulted brother) affected V-35, and proband (V-33, sibs affected two the of data ES Sdt ftefu ebr,rsle n7,2 ainsi ffce ahr 369in 73,689 father, affected in variants 73,426 in resulted members, four the of data ES 9 hsi seietta h rbe sntcnndt igepr fbrain of part single a to confined not is problem the that evident is it Thus . 0.46% 10 . netgtoa uainsreigfrtercrec fpreviously of recurrence the for screening mutation Investigational 10 h i ftesuyi oietf e addt genes candidate new identify to is study the of aim The . 2 6 . Posted on Authorea 8 Oct 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.160218236.66387607/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. h 2 ee lo nw nlssmlfnto,btddntietf n infiatvraini hmin them in variation significant any pathways identify to putative not analyses other the did for extended but Search we pathway, function, lysosomal lysosomal single in families. a known two to our also, limited Consid- are genes stuttering discussed. 123 in the are genes stuttering implicated severe far so with (c.322G ( families variant genes multiplex missense two implicated heterozygous of previously sequencing the exome ering in findings The multiple harbored members affected the genes. DISCUSSION All seven these A4). Seven (figure of identified each STU-65). variants in in to the variants 294 exclusive of and was irrespective family that STU-66 families, variations, in both with genes (276 genes compared of ( was list family genes each expansive in the individuals stuttering, affected 14 causing the families of genes stuttering the comprised multiplex determine list two To gene the 2). enriched in and the 1 observed and Thus (tables commonly p-value approaches their Genes A3). two on (table the based this interactors in sorted In recurred as were that 2), indicated over-represented. genes (table were browser are genes genes pathway 14 Similarly, that remaining encompassing pathways. pathways the pathways common relevant determine nine most (https://reactome.org) into 25 fit database manner they Reactome but families, in two tool the encompassing for pathways different putative are nine yielded genes genes 64 the approach genes of 41 relevance biology phenotypic systems the for using manually pathways Checking common in sibs. (c.322G genes in gene of observed NAGPA Identification also in was variant stuttering heterozygous in reported implicated recently database) a addition In first. variants homozygous scrutinized we generations, cerebro- sibs. three in for involved consanguinity one are had Only and family lesions membrane STU-65 matter basement the white of Since and component porencephaly structural include language major that of a disease vascular fluency forms in to that variant collagen related missense IV disorder one a only in family, reported than STU-66 more of the count genes. In databases allele 35 available an the to with trait, list variants in common the variations, less of narrowing a missense Comparison excluded, be and were to indels database (4 stuttering gnomAD the variants in Considering in variants, than 34 families. novel 20 other both 3 to of detected, and list data was known step exome gene variation 31 four to intronic/UTR the known same up significant down were the sum No narrowed interest 158 Applying of variants, these, A2). regions. genes known (table Of the coding 158 genes. Thus the genes. 34 to in heterozygous). 153 30 found STU-66) both in and all to in homozygous harbored were common (as variants variants that Those variants criteria common A3). novel 161 exclusion (figure were in chart variants 3 flow the resulting diagram and the of Venn out variants in analysis downstream using depicted sorted The is above were genes A2). described sibs common (figure sib-pair 294 as the the criteria to in common similar observed found Applying were genes respectively. 294 approach, variants 51,062 and 58,346 eto uim hr pehi loaffected also is speech where Autism, of NF- ment represses to said pathways is it but inflammasome NLRP11 L3A,Cof6,SN,T4F,MC,SPTBN5 MUC6, TM4SF1, SYNC, C1orf167, SLC36A1, . 14 hscnb ietyo nietylne osutrn tbe1.Itiunl,tepsil candidate possible the Intriguingly, 1). (table stuttering to linked indirectly or directly be can This NLRP11 \ ot er-mueadnuoiflmainhsbe salse skyfcosi h develop- the in factors key as established been has neuro-inflammation and Neuro-immune sout. samme fNDlk eetrpoenwt yi oanrsosbefrfrainof formation for responsible domain pyrin with protein receptor like NOD of member a is ain (c.1052G variant > ;pAg5Gn a rsn nhmzgu odto nbt the both in condition homozygous in present was p.Arg351Gln) A; > GNPTAB ;pGu0Ls in p.Glu108Lys) A; 15 κ n yeIitreo epne,ivle ninflammation in involved responses, interferon I type and B . NLRP11 , GNPTG 3 11 COL4A2 a bevdi u study. our in observed was ol eapsil addt eei hsfamily. this in gene candidate possible a be could NAGPA , NAGPA ee(c.461G gene and 12,13 MSRB1) a eetdi u td.Snethe Since study. our in detected was . and AP4E1 > eefudt ecmo to common be to found were > ;pGy5Gu previously p.Gly154Glu) A; ;pGu0Ls gnomAD p.Glu108Lys; A; COL4A2 nyoepathogenic one only ) noe type encodes Posted on Authorea 8 Oct 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.160218236.66387607/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. aegns(ncrmsm 8 iedsoli/emclnfml n ernlcdei eeta helps that gene 2 cadherin neuronal and family desmoglein/desmocolin like Shugart 18) stuttering. chromosome for (on neuroimaging cause genes of major indi- date the findings functions as with signaling deficits coincide in communication tracts) well involved cell very matter molecules, pathways white matrix These in cellular deficits. (deficits extra communication form cell plasticity identified overall synaptic genes cating and the development of system most nervous Thus in role important an plays that neuro- pseudogenes speech. release disrupt may neurons that muscles motor ARSD NMJ of and (NMJ), at contraction cells junctions to occur muscle leading muscular might and receptors, system neuro neuron post-synaptic signaling At to between bind communication speech. that in of tranmitters involved production are them coordinate of may All channels. and receptors, stimuli-sensing neuroplasticity serotonin in are in signaling study present role in the important in observed variants has gene and which Other signaling membrane basement in cells disrupt role nerve synapses. may plays maintaining and laminins membrane, muscle and basement like collagen of ECM cells, networks. form of perineuronal the neuroplasticity and functioning in for matrix stability interstitial critical lamina, structural are basal provides involves that dynamic, form parts also is basic that three proteoglycans has as protein ECM core to ECM associated of GAGs component the the of one biosynthesis, sulfate chondroitin common ADAMTS9 a was have integrin genes to candidate for possible tend 14 significantly stuttering. like of to interact Genes dissection related functional that pathways pathway, genes the same identify the the to in Since involved attempted be may 4). and (figure function ( 0.125-0.0001 genes from target resulted browser mechanisms, ranging 14 pathway biological to of common subjected TMOD2 list for set when CSGALNACT1, a gene look genes in 64 to enriched pathways), These resulted an biological members STU-65. Reactome in affected common in with among genes interact a only 34/294 (that overlap and in tool that STU-66 involved genes in coordination be of genes in 30/276 analysis act to of downstream that seem the second family per they events each neromuscular but might In junctions 40,000 other neuromuscular involves at speech each deficits as These with stuttering communication. using to interaction cell-cell contribute or genes any cell seven within show interactions mechanism: these not biological protein-protein of did of products products Analysis protein gene the (https://www.genecards.org). of repolymerization (https://string-db.org/) actin in involved lmnsadas id oknsnadatnproteins. actin and kinesin to binds also and filaments SPTBN5 rtndpnetaioai rnpre,hgl xrse nban uain nti eeinvolves in gene Mutations disease. this 45. neuromuscular in ataxia Mutations of spinocerebellar marker and brain. a iminoglycinuria in as with gene. expressed each associated Seven highly in speech C1orf167 variants transporter, members. of multiple affected unaffected acid of coordination the the amino sets poor in with different dependent absent had overlapping proton but they variants a STU-65) though gene in stuttering those 294 with for families and account family STU-66 to genes in used genes was (276 strategy members based segregation A i. L3A,Cof6,SN,T4F,MC,SPTBN5 MUC6, TM4SF1, SYNC, C1orf167, SLC36A1, viz., eog ocutro ufts ee hthdoyeGG htaefudt ersn truncated represent to found are that GAGs hydrolyse that genes sulfatase of cluster to belongs sascae ihnua uedefects. tube neural with associated is eeecdspoenta ik ebaelpd,poen n yooi atr ocytoskeletal to factors cytosolic and proteins lipids, membrane links that protein encodes gene O42 O63 COL6A6 COL6A3, COL4A2, 18 eecdsfrmtlortiaeivle nEMdegradation ECM in involved metalloproteinase for codes gene 27 and . SPTBN5 LAMA5 21 sivle nvscetasotadNA inln nua elahso molecule) adhesion cell (neural signaling NCAM and transport vesicle in involved is . , o laminin for HTR2B 22,23 8,29 RSC1A1 C rvdsa prpit niomn o h eeomn and development the for environment appropriate an provides ECM . n eei nmlsuis(ect ncru callosum) corpus in (deficits studies animal genetic and , 19 RSC1A1 httgte omteetaclua arx(C)components. (ECM) matrix cellular extra the form together that r novdi omto n erdto fcollagens of degradation and formation in involved are TM4SF1 , O42 O63 O66 TA,LM5 ADAMTS9, LAMA5, ITGAX, COL6A6, COL6A3, COL4A2, TRPV2 SYNC 24,25 , RV,WNK1 TRPV2, and ntepeetsuy aitosi ee encoding genes in variations study, present the In . 4 and eei novdi ucecnrcinadi regarded is and contraction muscle in involved is gene TMOD2 MSRB1 MUC6 WNK1 eeivle nmsl contraction, muscle in involved gene eeat nrsos osrs n salso is and stress to response in acts gene ee omteetaellrcomponents. extracellular the form genes ee ntasoto ml oeue in molecules small of transport in genes and , tal., et ARSD MSRB1 20 30 . nslc tool silico in CSGALNACT1 mlctd neetn candi- interesting implicated, and eecmo obt the both to common were 26 SPTBN5) 28 ec itracsin disturbances Hence . . SLC36A1 5 fiueA) the A5), (figure hc conclude which ihP-values with sivle in involved is 17 , HTR2B encodes ITGAX 16 . Posted on Authorea 8 Oct 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.160218236.66387607/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. i one rte V6 a oeaebtporsiesutrn,wt g tosto . er;hselder first his two years; and 2.5 IV-7) of stuttering; onset (mild and uncle at parents, (V-5) age III-1), proband non-consanguineous (recovered; with the to grandmother stuttering, Both His born progressive unaffected. unaffected. but was was was moderate (IV-3) (V-5) (V-4) had mother brother proband (V-6) but The stuttering brother mild younger 1). had his (IV-2) (figure from father pedigree siblings the the where affected in two loops and inbreeding family STU-66 family: sequencing. from exome STU-66 individuals comprehensive uL four for 20 Bengaluru individuals; and Ltd., quantified six family. was Labs in STU-65 sample Medgenome performed to per sent was DNA their was of ES and sample method 500ng probands each extraction About from of PCI USA). collected volume was using Wilmington, blood isolated Scientific, of was Fisher milliliters DNA (Thermo- Eight Genomic for noted. members. characterized were details clinically family Instrument-3 other and Severity and database Stuttering history from using family recruited pathologist were speech STU-66) by and stuttering these (STU-65 of families multiplex role Two the ensure in would However validation functional stuttering. METHODOLOGY further to and linked members putatively stuttering. additional be in in may genes in ES transport variant identified. and homozygous also signaling a family, in 65 role diagnosis ( having STU genes of prevention, genes target 14 etiology to 14 these the implications of of with list TMOD2 enriched understanding targets CSGALNACT1, and an better new ADAMTS9, generated a LAMA5, we of ES, to ITGAX, identification Using contribute the treatment. only facilitate and not also will but architecture stuttering of genetic number the the in Dissecting dynamics. only regulatory not huge lies the complexity to regu- the due in Thus CONCLUSION involved be regions. may RNAs coding also functional to protein but make believed than involved was 20% bigger genes that which times DNA of our 20 of RNA, are in 80% makes regions disorder (1.5%); regulatory that The genome active the within (http://jonlieffmd.com/blog/mind-and-molecular-genetics-in-the-neuron-3-evolution). biologically combined of lation genes now and expressivity of is regions variable multiple junk coding The be hypothesize protein stuttering we only heterogeneity. stuttering. that addresses and genetic of ES fact this background genesis by the genetic explained the support be complex in results could ES involved a our families disorder. be Yet, with variants the to identified disorder of mechanism. mechanisms the development polygenic biological of the studies a the in Functional understanding role with is variants. viewed direct the in have down be help narrow should not further identified would may would variants genes members candidate gene additional potential the in the of validation. and some functional type because through descriptive caution also a and of is family investigation the Our of members affected other the signaling in in in involved variant genes homozygous putative (haploinsufficiency) a interesting family, in protein STU-65 to in point However functional families stuttering of transport. multiplex and level two in reduced approach ES with Our copy different mutant connect absence one that total circuits only be than the have rather could in to language found and reported fact in speech were are of in stuttering bases origin involved with neural that associated neurons today thinking brain brain, in traditional our of important to of regions are part opposed communications one As to Such localized speech. adhesion. of cell production and the communication cell-cell in NAGPA SPTBN5) eewr loietfid h oeo hs ainsms eepoe ycnrigissegregation its confirming by explored be must variants these of role The identified. also were gene novdi erlrltdptwy,i h w utpe aiissuid epooethat propose We studied. families multiplex two the in pathways, related neural in involved 31 namr lbrt WSsuy 0cniaegnswr dnie ob strongly be to identified were genes candidate 10 study, GWAS elaborate more a In . euusekn aiysoe ee ffce costregnrtoswt three with generations three across affected seven showed family speaking Telugu A 33 32 . lblytegnsipiae nsutrn ildt,i l ffce individuals affected all in date, till stuttering in implicated genes the Globally . NLRP11 eeadahtrzgu ain in variant heterozygous a and gene 5 , HTR2B NLRP11 , RSC1A1 34 sn tutrdquestionnaire structured a Using . 35 eeadahtrzgu variant heterozygous a and gene O42 O63 COL6A6, COL6A3, COL4A2, n unie yNn drop Nano by quantified and , RV,WNK1 TRPV2, NAGPA eewere gene , ARSD Posted on Authorea 8 Oct 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.160218236.66387607/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. ae et o aho h aperne rm8.2t 99.27X. average to The 80.42 from genes. ranged was the 23690 sample analysis and covers The the alignment which overall respectively. of The % (74,557,381bp) each 97.28 for panel 99.9%). and depth is SS-V5-UTR 99.99 of accuracy panel using around probability call was alignment the base samples after indicates the the Q30 means performed all which and in 1000 probabilities percentage in alignment error measures 1 passed score calling (Q of base Q30 call above estimating base scores by quality incorrect variant accuracy showed calling data the base of the 93% than More sequenced. individuals family: etc.). of STU-66 function, impact of of predicted data loss Sequencing and (missense, type protein variant the variants prediction, on (https://www.coriell.org/1/NHGRI/ information pathogenicity variant contain computational variants the and frequency, annotated genomes VariMAT population variants. the prediction the 1000 on annotating for class tools multiple nih.gov/snp/), prediction pathogenicity variant integrates UniProt It Collections/1000-Genomes-Collections/1000-Genomes-Project)], https://gnomad.broadinstitute.org), (https://www.omim.org), (https://www.ncbi.nlm. broadinstitute.org; an- Toolkit). OMIM dbSNP variant (http://exac. Annotation in-house (https://www.ncbi.nlm.nih.gov/clinvar), ExAC MedGenome Mutation (https://www.uniprot.org/), (https://www.genome.gov/genetics-glossary/Genome-Wide-Association- ClinVar using and [GWAS annotated Variation databases Studies), and - grade software (VariMAT GATK clinical analyzed using were pipeline genes called the notation were of Coverage variants (small file. to The INDELs format) converted and call Variation) is (Variant Human Nucleotide format VCF (Single to standard SAM Bedtools. SNVs aligned a in using obtain were in output to deletions) tool) The processed and line and insertions tool. command Samtools) BWA-MEM 100x fastq-mcf (using using at (using file hg19) generate reads reads BAM to to sequence and (alignment processed bp trimmed genome quality and 2X150 Reference was qualified generate data as to amplification. raw considered X10) Cluster The analysis. was Seq variant The to values downstream (Hi subjected hybridization. Q30 further sequencer by and for with the (exome) files 10ul data FASTQ to interest in Sequenced of on 2nm region depth. loaded of the sequencing was (V5+UTR) concentration enrich cell final probes to flow to capture used The diluted were oligonucleotide was exons Biotinylated obtained coding using library prepared the (Illumina). was all library Kit ES for Reagent facility. designed Bangalore XT Ltd., extensively Labs Select been MedGenome aunts have at Agilent-Sure out members maternal carried family commercially (mild), extended was had ES grandmother The also Their stuttering. He severe brother etc. A4). with His education. (table fidgeting, affected fair. phenotyped hand school also was grimace, were speech beyond facial in cousins continued intelligibility blinking, and and jerking eye but slow jaw with was neck, increase speech along the of situational prolongations in rate tension and behaviours His flaring, repetitions Secondary nod. nose had head 2-3. posture, side syllable, of articulatory left initial iterations fixed frequent in sounds, and with and contacts school clicking repetitions hard years from blinks, part-word include dropout eye 2.5 and observed a included at syllable was Dysfluencies gradually pauses, proband strangers. The silent stuttering with prolongation, complications. developed increase, birth also (V-35) situational no Father had brother with and younger respectively clenching. severe his jaw and and and moderate were (V-33) rate her proband speech days, the fast early Both of the reminiscence in clenching. stuttering jaw a who severe and with of (III-21,IV-8) repetitions but parents complained had now mother consanguineous his to reduced Though born have were 2). stuttering. dysfluencies (V-35) (figure with brother affected I-2 affected also founder trace his were female to and common helped (V-33) a generation proband third to The from phenotype starting the informants tracing senior inbreeding, ES. of multigenerational Availability for intense selected generations. were six (V-5) across PWS proband father the affected and of family: comprising (V-4) STU-65 family brother nuclear unaffected The (IV-3), stuttering. mother with unaffected affected (IV-2), also were V-2,V-3) (severe; cousins ai paigfml rmamjredgmu uaircsehdmr hn20 than more had caste Mudaliar endogamous major a from family speaking Tamil A h ardedE,gnrtdadt f1-4G o aho h four the of each for Gb 10-14 of data a generated ES, paired-end The 6 Posted on Authorea 8 Oct 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.160218236.66387607/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. .Fiei-oige ,Dan .Gntccnrbtost tteig h urn vdne o Genet Mol et evidence. Phoniatrica http://doi.wiley.com/10.1002/mgg3.276 current Folia the from: 64, Available stuttering: Vol. Mar;5(2):95–102. to 2011. 2017 contributions art, [Internet]. Genetic the Med D. Genomic of Drayna state C, Frigerio-Domingues The 3. stuttering: of who 34–47. bases children p. Genetic young 2011. E. of Yairi Logopaedica. characteristics SJ, Temperamental Kraft 2003; KE. 2. Res. EG Hear Conture Lang Speech, MW, J Pellowski stutter. JD, Anderson 1. Bibliography other any or stuttering to attributed disorders genes, language reported and previously speech with matched were variants shortlisted These databases available with variants of Comparison exclusion following the list variant resulting this unaffected To and diagram). affected criteria Venn Exclusion the InteractiVenn (creates in called sets present tool employed. data variants were source six gene criteria open to uncommon An up and family. handle common a the in profiling members on rely often prioritization: Variant profile to criteria, filtering variant to members. of: subjected family comprising sample were additional investigation, from each files under data variant samples for the individual on depth the based variants of panel of Each average prioritization by The followed in individuals, score hg19) affected respectively. quality to showed % (alignment filtering: data 94.89 Variant percentage the X. and alignment 100 of 99.99 to passed 90% 80 around than and from was More alignment ranges sib overall Gb. and The 8-11 proband of Q30. data above total distribution generating family: sequenced were STU-65 V-35, of data Sequencing Tegnss a itdfrsec n agaedsresby disorders language and speech for listed far card/lysosome). so (https://pathcards.genecards.org/ genes pathway The lysosomal 3. the in listed genes 123 The 2. Kraft by reported SNPs Significant 1. role. omnipresent very have they as excluded were genes encoding finger zinc at were in placed Variants phenotypes variants OMIM 4. site unmatched splice completely Proximal with gene 3. in variants phenotype, known the sorting In 2. Met- and (SVM)) machine vector support (meta-analytic MetaSVM using tolerant as predicted those 1. variants. impact splicing proximal or donor/acceptor splice and of indels, comprise and variants type, loss loss site stop start missense, splice termination, being Intronic frameshift, variants includes 3. impact that moderate type The variant 2. impact high The 1. ic h he ee ofrkont eipiae nsutrn eeietfidi h lysosomal the in identified were pathway. stuttering this in to related implicated genes be in variants to potential known any observed far carefully so study genes this pathway, three the Since ancestry. European northern of stuttering’ tool prediction one than more by excluded. damaging were be disease, to with predicted associated variants genes those considered. consensus words, was detect other to In data omics excluded. multiple accommodate which aLR MAF and 3X being depth databases. variation) for (MedGenome cutoff Medvar a proprietary with and filtered are variants These h ontemvrataayi rtclivle hr itn ftevrat nthe in variants the of listing short involves protocol analysis variant downstream The h ain roiiainadcniaegn dnicto nfml ae cohort based family in identification gene candidate and prioritization variant The 32 nte’eoewd soito td fpritn developmental persistent of study association ’Genome-wide the in nti aiytepoadadhsaetdbohr -3and V-33 brother, affected his and proband the family this In > uloie wyfo xnwr loexcluded. also were exon from away nucleotides 8 7 35 37 a elydfrti ups htcan that purpose this for deployed was . < %i xC 00genomes, 1000 ExAC, in 1% aevariants rare nall in Posted on Authorea 8 Oct 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.160218236.66387607/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. 0 ewc,R,Dsni,I,Welr .N,&EwrsD.TeAAT ADsnernadMetallo- and Disintegrin (A ADAMTS The & DR. 2015;16(1):113. . Edwards . Biol. . & Genome A., N., family. motifs) G. D. Thrombospondin Wheeler, Nickerson, with I., J., proteinase Desanlis, M. R., 2018;1413(1):119–25. Bamshad, Kelwick, Sci. variant X., 20. sequence Acad homozygous J. Y a N Chong, to Ann attributed V´azquez, J., syndrome LAMA5. J., myasthenic in 2007;8(5):215. congenital Arredondo, presynaptic Biol. A A., Genome CM. R. McDonald Biotech. integrins. 3 The Maselli, S. diversity. Simon gene 19. & collagen X., of Ye, analysis Y., Takada, genomic Comparative 18. M. Qasim & Disord. N., Fluency Ahmed, 2019;9(3):83. J F., Emotion. Haq, and 17. Behaviour, Stuttering-Neurology, of dysregula- Dimensions neuro-immune Three and The 2000;4(25):387–388. Inflammation R. N. Logan 2018;11(2):56. Antonucci NLR Pharmaceuticals. 16. & The L., TA. disorders. A. Kufer spectrum Biol Brigida, & autism J S., in Schultz, . tions . signaling. . D., inflammatory Siniscalco, C., of Y. 15. regulation Gloria, the Y., to Postma, contributes A., 2018;293(8):2701–10. protein Sowa, Chem. I., 11 novo domain–containing Kienes, De pyrin E., N. Becker, family Matsumoto K., collagen & Ellwanger, IV . 14. type . . the H., Doi, encoding 2012;90(1):86–90. Y., COL4A2, Genet. Tsurusaki, Halley in Hum S., & Yamaoka, mutations Genet. H., . Hum inherited . Arai, . J K., and S., Haginoya, Eur D. Y., Yoneda, Kuo, disease. J., small-vessel 13. D. and Licht, P., porencephaly P. familial L. Govaert, with Chang W., 2012;20(8):844. & associated F. Verheijen, mutation . E., . COL4A2 . M. DJ. S., Meuwissen, C. E., Bloss, Verbeek, Genes, G., 12. impairment. D. Disord. language Amaral, and Fluency N., disability J 2013;12(8):792–801. Akshoomoff, reading of L., Behav. components L. Brain India. shared Miller, of R., Nadu, study N. Tamil association Powers, Genome-wide of D., J. state Eicher, the 11. Genetic in CR. Srisailapathy children Srikumari school NP, Karthikeyen among 2018;58. S, stuttering Valarmathi A, of Thalamuthu epidemiology G, Devi D Nandhini genes—DRD2, 10. dopaminergic three in the inheritance 31;67(3):264–88. disorder: differences Polygenic of defiant 1996;May MD. Brain. neuroanatomical effect G oppositional Genet. Dietz subtractive and matter JP, MacMurray conduct, and White C, hyperactivity, additive Ahn deficit stutter. M. R, The attention Gade Angstadt stuttering, RH, who Ring syndrome, & C, Tourette Chiu children L., of S, Wu A. DE, in Comings Choo, 2015;138(3):694–711. 9. differences C., Brain. D. connectivity stutter. Zhu, who network E., children young S. Neural Chang, DC. Drayna 8. & Zhu J SE, abilities. . . language . Chang expressive 2013;136(12):3709–26. S., of status W. Initial Lee, 7. III: 1999;Oct,42(5):1125–35. J., stuttering Res. childhood Hoffmann, astrocyte Hear Early du and Lang AN. 2019;116(35):17515–24. deficits B., Speech, E Sci. Yairi vocalization E. RV, Acad Watkins cause Natl Huchinson, 6. Proc mice D., callosum. into L. corpus engineered Reyes, the mutations in J., Genet. III stuttering pathology and Hum Root, GNPTAB II J U., Human types Eur T. D. Mucolipidosis genes. Han, al. same et the 5. R, in Rahn variants E, different Paris with E, associated Sainz 2016; are R, stuttering Webster non-syndromic CEF, and Domingues MH, Raza 4. 8 α hi as oecpay mJ Am porencephaly. cause chain 2 β ,adDT.A Med J Am DAT1. and H, Posted on Authorea 8 Oct 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.160218236.66387607/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. SPTBN5, 1 studied S.No families can two that the pathways, in putative 9 stuttering, in to identified linked 2019;3(9). genes indirectly Genom. candidate* or Genet of directly Transl list be disorders. curated language tool Manually and web-based 1: speech a Table of InteractiVenn: 2015;16:169. R. Genomics Bioinformatics. Minghim CR. BMC P.; J diagrams. G. Guerra Telles, Venn 37. R.; through F. sets Silva, of da analysis V.; the G. editor. for CSHL, Meirelles, Press H.; V. Heberle, ed., 36. 3rd Manual. Laboratory A Cloning: Molecular D. 2001. Russell Vol. and TX. J.F. stuttering. 1994;Austin, Sambrook, developmental (SSI-3). 35. Instrument-3 persistent Severity in Stuttering deficits GD. 276–80. metabolic Riley p. inherited 34. 2012. for Metabolism. role and A Genetics Molecular D. Illinois; 107, Drayna of University C, stuttering. Kang developmental 33. persistent of genome- study a association Tracts Vocal 2010. Genome-wide of speech-Comparing 2007;49(2):15–20. SJ. Results Pennsylvania. and Kraft Univ language al. 32. Mag of evolution Exped et from: the Capabilities. Available Tracking J, Speech 15;124A(2):133–5. R. Wanyee Identify McCarthy Jan to B, & 2004 P., [Internet]. Doan Lieberman, Genet 31. K, Med Doheny J J, Am stuttering. Kilshaw http://doi.wiley.com/10.1002/ajmg.a.20347 for pa- J, scan matter linkage Mundorff White 2018;55:68–83. wide M. Disord. YY, Fluency Ben-Shachar Shugart J & tractography. R., 30. from NCAM NCAM- Ezrati-Vinacour, Lessons of M. stuttering: O., developmental import Schachner Amir, persistent nuclear & O., in on thways Civier, . . and V., . NCAM Kronfeld-Duenias, C., to Schulze, 29. 2010;30(32):10784–98. calmodulin U., Neurosci. of Bormann, J binding I., fragments. on fak Kalus, and depends G., pseudoautosomal outgrowth Joshi, ancestral neurite M., an induced Mzoughi, in R., duplications gene Characteri- Kleene, junction suggests A. 28. 3 Ballabio neuromuscular 1996;5(4):423–31. & Xp22. in M., Genet. on pathways Rocchi, genes Mol G., sulfatase Hum signaling Andolfi, of region. S., synapse: cluster Messali, a a N., of Archidiacono, build zation B., To Franco, G., L. Meroni, Mei 27. & C., W. 2010;137(7):1017–33. Development. Xiong, assembly. and development H., 2016;3-24. muscle Wu, skeletal Bodyq. 26. Hum in Matrix matrix Extracell extracellular Funct of Compos importance function. The K. Grzelkowska-Kowalczyk M 25. & 2011;3(1):a005108. J., Biol. S. Biol. Perspect Franco, Open Harb Soc S., Spring R C. development. neural Barros, shapes 24. matrix extracellular the 2009;326(5957)):1216–9. How ). WB. (80- Huttner 2019;9(1):180216. Science & R., fibrils. K. pretty Long, just 23. Not and matrix: cloning extracellular Molecular The K. Hynes. 2003;278(5):3072–8. Sugahara 22. Chem. & Biol elongation T., and J Ogawa, initiation sulfate. the I., chondroitin/dermatan in involved J. of N-acetylgalactosaminyltransferase Tamura, chondroitin J., second Tanaka, a of H., expression Kitagawa, T., Uyama, 21. T-6fml Pathways RAPIGAP family STU-66 from list Gene le .Etaellrmti:fntosi h evu ytm Cold system. nervous the in functions matrix: Extracellular U. uller ¨ PLXNA1 , 9 pathways developmental neural DSCAML1 COL6A6 family STU-65 from list Gene NELL2, , Posted on Authorea 8 Oct 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.160218236.66387607/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. 12 11 10 8 7 6 5 enzyme Lysosome 4 3 2 1 be can that S.No studied pathways, families enrichment two in the identified in stuttering, genes - - to candidate* linked of indirectly or list directly Tentative 2: state; Table homozygous the in segregating pathways NLRP11 for except expression status, gene Note: 9 SPTBN5, 8 CD3EAP 7 ADAMTS9, 6 5 4 3 2 S.No Tecniaegnspooe r xlsv oaetdidvdas ergtn nheterozygous in segregating individuals, affected to exclusive are proposed genes candidate *The ATXN2 KIF13B, CCNJL, COL4A2 O42 COL6A3, COL4A2, TMOD2 SPTBN5 COL6A3, COL4A2, COL6A3, COL4A2, COL4A2 COL6A3, COL4A2, COL6A3 COL4A2, COL6A3, COL4A2, COL6A3, COL4A2, ADAMTS9, Pathways COL6A3, COL4A2, COL6A3 COL4A2, family STU-66 from list Gene ABCA13 GPR97 ALAS1 SPTBN5, ADAMTS9, CSGALNACT1 RSC1A1 Pathways TMOD2 family STU-66 from list Gene SPTBN5, , , , , SYNC NIPA2 ZNF763, UNKL COL6A3, , 10 eil transport vesicle transduction/ signal targeting metabolism contraction muscle muesystem immune molecules small of transport organisation/degradation matrix cellular extra olgnformation Collagen Contraction Muscle Striated out-growth neurite for signaling NCAM enzymes modifying and biosynthesis Collagen interactions Laminin structures multimeric other and fibrils collagen of Assembly degradation Collagen arylsulfatases of activation The matrix extracellular the of Degradation trimerization chain Collagen proteoglycans ECM bold niae ee novdi multiple in involved genes indicates RP KDM5A MRIP, LAMA5 MYH1 STARD13, HTR2B, ATXN2L, ITGAX LAMA5 COL6A6, COL6A6 COL6A6 LAMA5 COL6A6, COL6A6 COL6A6 LAMA5 COL6A6, COL6A6 LAMA5 ITGAX, COL6A6, family STU-65 from list Gene NAGPA HSIT3H2A WDR27, ARSD, PAN3, MYHI family STU-65 from list Gene NLRP11 CRTAM, WNK1 TRPV2, NEK4, , COL6A6, , ITGAX , Posted on Authorea 8 Oct 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.160218236.66387607/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. iue4 lwcatdpcigeoeaayi ftomlilxsutrn families stuttering multiplex (15 two family of analysis STU-66 exome the depicting in chart individuals Flow four 4: the Figure among shared variants the STU-65 showing - combinations) diagram family Venn stuttering 3: a of Figure pedigree STU-66 generational - Six family 2: stuttering Figure of pedigree generation Five 1: Figure individuals 24 among legends: findings Figure associated most other family 25 and STU-65 showing phenotype the families stuttering in the ES examined of two Characterization in database A4: Reactome identified Table using variants stuttering pathogenicity language of to for speech results related predictions to pathways analysis bioinformatic correlating significant the Over-representation family with STU-65 along A3: of observed Table pair variants sib novel to in 4 common correlating includes variants that family the disorders predictions STU-66 of bioinformatic Compilation of the A2: pair with Table along offspring observed parent variants novel in 13 pathogenicity common includes for variants that the disorders language of speech Compilation A1: Table tables: Supplementary Note: 25 24 23 22 21 20 19 18 17 16 15 14 13 S.No Tecniaegnspooe r xlsv oaetdsgeaigi eeoyosstatus heterozygous in segregating affected to exclusive are proposed genes candidate *The O42 COL6A3 COL4A2, RSC1A1 CSGALNACT1 COL4A2 ARSD RSC1A1 COL6A3 COL4A2, COL4A2 Pathways family STU-66 from list Gene 11 metabolism Glycosphingolipid absorption hexose Intestinal interactions surface cell Integrin formation fibril Anchoring receptors Serotonin activation arylsulfatase and formation hypusine carboxylation, Gamma signaling Interleukin-13 and Interleukin-4 R channels TRP organization matrix Extracellular absorption Intestinal channels Stimuli-sensing biosynthesis sulfate Chondroitin fibrils collagen of Crosslinking TRPV2 LAMA5 ITGAX, COL6A6, WNK1 TRPV2, ITGAX COL6A6, HTR2B ARSD LAMA5 ITGAX, family STU-65 from list Gene Posted on Authorea 8 Oct 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.160218236.66387607/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. rtisietfidi omnfrtetomlilxsutrn families stuttering seven multiplex for two connection any the show for not common did families analysis) in multiplex (STRING identified stuttering network proteins two association between protein Functional shared A5: genes Figure the stuttering showing with family diagram STU-65 Venn a of A4: data Figure sequence exome to applied the pipeline in Bioinformatic-analysis pair A3: Figure sib affected the between shared family variants STU-65 the stuttering showing with family diagram STU-66 Venn a of A2: data Figure sequence exome to applied pipeline Bioinformatic-analysis A1: Figure figures: Supplementary 12 Posted on Authorea 8 Oct 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.160218236.66387607/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. 13 Posted on Authorea 8 Oct 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.160218236.66387607/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. 14