American Journal of Medical Genetics 124A:133135 (2004)

Results of a Genome-Wide LinkageScan for Stuttering

Yin Yao Shugart,1 Jennifer Mundorff,2 James Kilshaw,3,4 Kimberly Doheny,1 Betty Doan,1

Jacqueline Wanyee,1 Eric D. Green,5 and Dennis Drayna3*1Center for Inherited Disease Research, Johns Hopkins University Bayview Research Campus, Baltimore, Maryland2Hollins Communications Research Institute, Roanoke, Virginia3National Institute on Deafness andOther Communication Disorders, National Institutes of Health, Rockville, Maryland4Stuttering Foundation of America, Memphis, Tennessee5National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland

We performed a linkage study of stutteringusing 392 markers distributed across thegenome in a series of 68 families identifiedin the general outbred population of NorthAmerica and Europe. Standardized diagno-sis was performed using recorded samplesof both conversation and reading, in whichstuttering dysfluencies were scored as per-centage of dysfluent words and syllables.Analysis was first performed using non-parametric methods implemented in GENE-HUNTER, where we obtained maximumstatistical support for markers of chromo-some 18, with a maximum NPL (Sall) of 1.51 atD18S976. The single largest pedigree withinour sample (pedigree 0006) alone gave anNPL of 4.72 at D18S976. For fine mapping,we analyzed 18 markers on chromosome 18across all families using ALLEGRO. OverallNPL (Srobdom) scores >5 were obtained withmarkers on 18p, and Zlr scores 2.5 on 18pand proximal 18q. Furthermore, pedigree0006 alone gave an NPL (Srobdom) of 5.35.Overall our results suggest chromosome 18may harbor a predisposing locus for thisdisorder, and additional genes may exist.Published 2003 Wiley-Liss, Inc.{

KEY WORDS: stuttering; linkage; speechdisorder; non-parametricanalysis

INTRODUCTION

Stuttering is a disorder of the rhythm of speechcharacterized by involuntary repetition or prolongationof syllables, and by interruptions in the smooth flow ofspeech, known as blocks. Although no clear cause of thisdisorder has been identified, genetic factors in stutter-ing have long been suggested [Bloodstein, 1995; Yairiet al., 1996]. A number of factors, however, have hinder-ed genetic studies of stuttering, including a highlydistorted sex ratio, an inability to ascribe a mode ofinheritance, the high frequency of the trait in normalyoung children, and greatly variable expression withinfamilies. Our previous studies have suggested that atleast one of these complications, the distorted sex ratio,may be less significant in familial stuttering than in thegeneral stuttering population [Drayna et al., 1999]. Toaddress the other complicating factors, we employed asimplified approach for a linkage study, using primarilyaffected family members who displayed persistentstuttering beyond young childhood. Our strategy wasto perform a genome-wide linkage survey and use non-parametric analysis methods to identify genomic re-gions of interest, and then gather additional data andperform additional analysis.

MATERIALS AND METHODS

Families were recruited from the general outbredpopulation of North America and Great Britain viatargeted appeals to stuttering interest groups and to thegeneral public. Subjects were individuals over the age of8 (mean age 31 years) who currently stuttered, and haddone so for at least 6 months. Families were ascertainedand enrolled under National Institutes of Health IRB-approved protocol no. 97-DC-0057, originally approv-ed by NCI IRB on 1/8/97, and annually reviewed andapproved by the NINDS IRB thereafter. Informed

Grant sponsor: NIDCD; Grant number: Z01 00046-03; Grantsponsor: National Institutes of Health (The Johns HopkinsUniversity); Grant number: N01-HG-65403.

Yin Yao Shugarts present address is Department of Epide-miology, Johns Hopkins School of Public Health, 615 N. Wolfe St.,Baltimore, MD 21205.

*Correspondence to: Dr. Dennis Drayna, NIDCD/NIH, 5Research Court, Room 2B-46, Rockville, MD 20850.E-mail: drayna@nidcd.nih.gov

Received 5 December 2002; Accepted 22 April 2003

DOI 10.1002/ajmg.a.20347

Published 2003 Wiley-Liss, Inc.{This article was prepared by a group consisting of both UnitedStates Government employees and non-United States Governmentemployees, and as such is subject to 117 U.S.C. Sec. 105.

consent was obtained from all subjects or their legalguardians, and an additional assent was obtained fromminor subjects. A complete diagram of the pedigrees ofall families included is available from the correspondingauthor. We excluded families with documented or re-ported stuttering in both the maternal and paternallineages, as such bilineal families can obscure non-parametric statistical analysis. The sample consisted of68 families; 23 of these families consisted of one gene-ration, 33 contained two generations, and 12 containedthree generations. Overall, the families contained anaverage of 2.72 affected individuals per family. Thesample contained 48 sibships with 2 affected, 9 sibshipswith 3 affected, 2 shipships 4 affected, and one pair ofaffected half-sibs. Of the 48 sibships with 2 affected, 22had zero parents, 13 had 1 parent, and 13 had 2 parentsgenotyped and included in the analysis.For diagnostic consistency, stuttering was evaluated

by a single clinician (J.M.), using a standardized readingtext of 500 words containing balanced numbers of eachclass of speech sounds [Webster, 1978] plus 5min of freeconversation speech. Text of the standard reading pas-sage can be obtained from the corresponding author at:drayna@nidcd.nih.gov. Speech samples were recordedand stuttering dysfluencies (repetitions, prolongations,and blocks) were counted and evaluated as both percentof words spoken and percent of syllables spoken. Indi-viduals were classified as affected if they demonstrated4% word dysfluencies in either reading or free speech,a cut-off typically regarded asmild-to-moderate stutter-ing in clinical settings. All subjects were over the ageof 8 and had documentation of stuttering for 6 monthsor more. DNA samples were obtained as previouslydescribed [Muellenbeldt et al., 1995].A total of 226 individuals in 68 familieswere included,

188 of whom were affected.Genotyping was performed by standard fluorescent

methods on an ABI 3700. The markers used were amodification of the CHLC version 9 marker set (392markers, average spacing 9 cM, average heterozygosity0.76). See www.cidr.jhmi.edu for details on genotypingmethods. The error rate for the genome scan, based on2,993 paired genotypes from blind duplicate samples,was 0.15%. The overall missing data rate was 14.4%,largely due to failed genotyping reactions with buccalswab DNA in the high throughput system employed.After accounting for failed genotyping reactions, a totalof 81,928 genotypes were generated and analyzed in theinitial genome-wide scan. Marker allele frequencieswere computed using data from one individual ran-domly chosen from each pedigree. Initial genome-widelinkage analysis was performed coding unaffectedindividuals as such. Because of diagnostic uncertaintiesin subsequently sampled individuals in pedigree 0006,high resolution analysis on chromosome 18 was per-formed in this family coding individuals as eitheraffected or unknown.

RESULTS

Initial analysis across the entire genome usingGENEHUNTER [Kruglyak et al., 1996] indicated

evidence for linkage to a number of markers distributedbroadly across chromosome 18p and proximal 18q. Thesummed NPL Sall scores for all families was 1.51 atD18S976 (P0.03). Analysis of our largest family alone(pedigree 0006, containing six affected individuals)gave an NPL of 4.72 at D18S976. This genome scan alsogave NPL scores greater than 1 but less than 1.5 at locion chromosome 1, 2, 10, and 13. Interestingly, pedigree0006 only gave highly positive scores on chromosome18q. Therefore, we then focused attention on chromo-some 18 chromosomal region for additional analysis.For fine mapping, we typed 12 additional markers

spanning approximately 60 cMon chromosome 18 for allfamilies, resulting in a final combinedmarker spacing of3.3 cM in this region. We then performed both para-metric and non-parametric statistical analyses usingALLEGRO [Gudbjartsson et al., 2000]. For the para-metric analysis, an incomplete dominant mode of in-heritance model was assumed, based on the overallpattern of stuttering in our family sample. Specifically,the penetrance was set to be 0.9 and the diseasefrequency was set to be 0.001.While the overall LOD score across all families were

negative (LOD

desmocolin family on 18q12.1, and the neuronal cad-herin 2 gene on 18q11.2, both of which are known to beinvolved in cell adhesion and intercellular communica-tion. Such communications may be important in theneurons involved in speech production in the brain.There has been one other report of the results from a

genome-wide linkage survey, which was performed inthe Hutterites, a highly genetically isolated population[Cox and Yairi, 2000]. This survey also gave support forlinkage on chromosomes 1, 13, and 16, where we obtain-ed NPL scores of 1.1, 1.38, and 0.518, respectively.Although it is not clear whether the markers that gavepositive NPL scores from our genome scan overlap withthe markers investigated by Cox and Yairi, it appearsthat chromosomes 1 and 13 may warrant further study.The lack of linkage on chromosome 18 in the Hutteritessuggests stuttering may display locus heterogeneity indifferent study populations. Because stutteringdisplayscomplex inheritance, it will be important to confirm ourresults in additional independent samples. We proposeto collect additional families, and to make our genotypicdata available to facilitate these necessary confirmatorystudies.

ACKNOWLEDGMENTS

We thank the assistance from the Stuttering Founda-tion of America and theBritish StammeringAssociation

in our family ascertainment and enrollment. Geno-typing services at C.I.D.R. were funded through theNational Institutes of Health via The Johns HopkinsUniversity, Contract Number N01-HG-65403.

REFERENCES

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Kruglyak L, Daly MJ, Reeve-Daly MP, Lander ES. 1996. Parametric andnonparametric linkage analysis: A unified multipoint approach. AmJ Hum Genet 58(6):13471363.

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