Conference Report

Eighth International Workshop on the Fragile XSyndrome and X-Linked Mental Retardation,August 16–22, 1997

Jeanette J.A. Holden,1* Maire Percy,2 Diane Allingham-Hawkins,3 W. Ted Brown,4Pietro Chiurazzi,5 Gene Fisch,6 Louise Gane,7 Chris Gunter,8 Randi Hagerman,7Edmund C. Jenkins,4 R. Frank Kooy,9 Herbert A. Lubs,10,11 Anna Murray,12 Giovanni Neri,5Charles Schwartz,13 Lisbeth Tranebjaerg,11 Laurent Villard,14 and Patrick J. Willems9

1Department of Psychiatry, Queen’s University and Cytogenetics & DNA Research Laboratory, Ongwanada, Kingston,Ontario

2Department of Neurogenetics, Surrey Place Center, Toronto, Ontario3Molecular Genetics Laboratory, North York General Hospital, North York, Ontario4New York State Institute for Basic Research, Staten Island, New York5Institute of Medical Genetics, Catholic University, Rome, Italy6General Clinical Research Center, Yale University, New Haven, Connecticut7Child Development Unit, The Children’s Hospital, Denver, Colorado8Department of Biochemistry, Emory University, Atlanta, Georgia9Department of Medical Genetics, University of Antwerp, Antwerp, Belgium10Department of Pediatrics, Genetic Division, School of Medicine, University of Miami, Miami, Florida11Department of Medical Genetics, University Hospital of Tromso, Norway12Wessex Regional Genetics Laboratory, Salisbury District Hospital, Wiltshire, England, United Kingdom13JC Self Research Institute, Greenwood Genetic Center, Greenwood, South Carolina14INSERM U491, Faculte de Medecine, Marseille, France

INTRODUCTION

The Eighth International workshop on the fragile Xsyndrome and X-linked mental retardation was held atthe Isaiah Tubbs Convention Centre near Picton, On-tario from August 16–22, 1997. This was the first timethe workshop has been held in Canada. It was hostedby Jeanette Holden, Maire Percy, and Diane Alling-ham-Hawkins, who gratefully acknowledge the sup-

port provided by the chairs of the various sessions notonly for their assistance during the workshop but alsoduring its organization and the review of manuscriptsfor the proceedings. Approximately 90 participantsfrom 20 countries attended the workshop (Fig. 1).

Attendees were met at the airport in Toronto byKathy Yang and Chris Trevors (Kingston, Ontario),and guided to the Days Inn for the Welcome Receptionon Saturday evening. In keeping with some of the pre-vious workshops, luggage did not arrive with one of theattendees. Most participants arrived on Saturday totake advantage of a preworkshop tour. Followingbreakfast on Sunday morning, the workshop partici-pants began the preworkshop tour, travelling by char-tered buses south to view the spectacular Niagara Falls(from the Canadian side). After a walk around the gar-dens, we toured the area, arriving at Chateau desCharmes for a guided tour of the winery and tastingsession. A lunch of salmon, salads, ‘‘the most tenderand juicy corn in the world,’’ and desserts galore ca-tered by Pillar and Post (Niagara on the Lake) wasserved outside the Chateau des Charmes with a view ofthe vineyards. Following a walk through the vineyardsand the purchase of the Chateau’s famous ice wine,participants continued the tour north through Toronto.We arrived at the workshop site late on Sunday eve-ning where we were greeted with a dinner buffet and a

Contract grant sponsor: the National Institute of Health; Con-tract grant number: R13 HD/NS35269-01; Contract grant spon-sors: the Ontario Mental Health Foundation; Ongwanada Re-source Centre; the Department of Psychiatry and the Faculty ofMedicine at Queen’s University; Surrey Place Centre; Universityof Toronto; the National Research Council of Canada; PerceptiveSystems Inc.; Vysis Inc.; Fisher Scientific; BioRad Laboratories;Perkin Elmer Applied Biosystems; Bio Synth International Inc.;Nature Genetics; Genomyx; Beta Electronics Inc.; MJ Research;Mary Ann Liebert; Ilford Corporation; Sanford Corporation;Clonex; Mills Office Supplies; Northwood Press Inc.; CanadianImperial Bank of Commerce; Rock Springs Beverage Corpora-tion; Warner Music; BMG Music Canada Inc.; National GrocersLimited; Molson Breweries.

*Correspondence to: Jeanette J.A. Holden, Department of Psy-chiatry, Queen’s University and Cytogenetics and DNA ResearchLaboratory, Ongwanada, Kingston, Ontario, Canada K7M BA6.

Received 22 August 1997; Accepted 2 October 1998

American Journal of Medical Genetics 83:221–236 (1999)

© 1999 Wiley-Liss, Inc.

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222 Holden et al.

speaker from Sandbanks Provincial Park who de-scribed the vegetation and wildlife on the local fresh-water sand dunes.

After a restful sleep, participants awoke to the splen-dor of the morning sun on the lake. The sessions beganwith an exciting presentation by Ivan Jean Weiler (Ur-bana, IL) who described her recent work with BillGreenough and colleagues on the role of the FMR1 pro-tein (FMRP) in synaptic pruning. They have examinedspine density in brains from the ‘‘FMR1 knockout’’mice, and found increased density compared with wildtype, suggesting a deficit in the process of pruning syn-apses during development. As FMRP binds tightly topolyribosomes and is produced at synapses, Ivan Jeanand Bill and their colleagues have proposed that FMRPsynthesis at the synapses is essential for specific as-pects of synapse maturation and elimination andtherefore is key to normal brain development.

Following the keynote address, there were severalsessions devoted to different aspects of fragile X syn-drome—clinical features in males and females, thegene, FMRP, population studies and founder effects,etc. Parents (two mothers and one father) of childrenwith fragile X syndrome joined us during the evening toexpress their thoughts about the importance of re-search to them and what they would like to see interms of new research efforts in the future. After asecond day discussing the population genetics of fragileX syndrome, FMRP, and FMR1 knockout mice, a ses-sion was devoted to the fragile X E syndrome andFMR2 as well as other triplet repeat expansion disor-ders. Meetings were held in the mornings and eve-nings, allowing free time in the afternoons to swim,bicycle around the area with Jeanette, rent canoes, orrelax. We had an afternoon and evening break onWednesday, August 20; participants again gathered inbuses and toured part of Prince Edward County, driv-ing east to Kingston where we took the ‘‘Tour Train’’ tolearn about historic Kingston. From Kingston, wedrove along the scenic highway adjacent to the St.Lawrence River to Gananoque where we boarded aboat for a 4-hour tour of the Thousand Islands, whichincluded dinner and dancing on board. Wednesdaymorning and all day Thursday were devoted to issuesrelated to X-linked mental retardation (XLMR), includ-ing both syndromic and nonsyndromic forms. DavidNelson (Houston, TX) presented the ‘‘state of the X-chromosome map,’’ with a discussion led by David andHerb Lubs (Miami, FL) on how important the X-chromosome mapping effort is to the localization ofgenes for XLMR and, conversely, the essential role thatthose persons involved in XLMR mapping efforts havein the construction of the functional map of the X-chromosome. The workshop sessions concluded with anupdate of XLMR genes (Herb Lubs, Miami), a discus-sion of the XLMR database (David Cabezas, Miami,FL), and a thoughtful hypothesis about the importanceof genes for intelligence and evolution of the X-chromosome presented by Herb Lubs. The workshopitself concluded with a discussion about the site of thenext workshop, which will be hosted by Jean-PierreFryns and held at Le Bischenberg near Strasbourg,France—a ‘‘farewell BBQ,’’ door prizes provided by

many sponsors, and a slide show of previous workshopsand workshop attendees (presented by JeanetteHolden). Most participants enjoyed the exceptionalweather (which held all week) and relaxed late into thenight, reflecting on the past few days, on old and newacquaintances, and on the peace of the surroundings.

On to the reports by the Session Chairs.

FRAXA SYNDROME: CLINICAL ISSUES MALESRandi Hagerman

Previous work regarding clinical issues in males hasfocused on their cognitive performance, particularlythe spectrum of involvement related to molecular vari-ables [Merenstein et al., 1996; Steyaert et al., 1996].Many papers have been written on longitudinal IQ de-cline in males, although long-term longitudinal followup from infancy has not been carried out. Previous be-havioral studies in males with fragile X syndrome fo-cused on the autistic features, an approach-avoidancebehavior in social interactions, hyperactivity, aggres-sion, anxiety, and hyperarousal. Several controlledstudies with various comparison groups, including IQmatched patients and children with autism, suggestthat this combination of findings represents a uniquebehavioral phenotype in patients with fragile X syn-drome [Cohen et al., 1989; Sudhatter et al., 1990;Lachiewicz et al., 1994; Baumgardner et al., 1995; Maz-zocco et al., 1997]. The underpinnings of the behavioraland cognitive phenotype have been studied by neuro-imaging work by Reiss et al. [1991, 1994, 1995]. Sha-piro et al. [1995] and others have found an overall en-larged brain with particular enlargement of the hippo-campus, caudate, thalamus, and ventricles, as well as adecreased volume of the cerebellar vermis, which maybe related to the sensory integration problems exhib-ited by males with fragile X syndrome. The larger brainvolume fits with neuroanatomical studies of the FMR1knockout mouse by Comery et al. [1997], which foundan increase in dendritic branching compared with nor-mal littermates, suggesting a lack of normal pruning inthe absence of the FMR1 protein.

The session began with a study by Don Bailey et al.(Chapel Hill, NC) on their longitudinal study of 39males with the full mutation who were enrolled be-tween age 24 to 66 months. The Battelle developmentalinventory was carried out at 6-month intervals. Theslope of the growth curve for total development corre-sponded to a developmental rate approximately halfthat of normal. Although 30% of those assessed at 24–30 months had a developmental quotient >65, all had aDQ of <65 by 66 months. Motor skills were the highest,followed by adaptive skills, but communication andcognitive skills were the lowest. Significant variationwas seen among individuals, although more detailedstudies of the methylation status, FMRP levels, andenvironmental variability are needed to better under-stand this variation.

Gene Fisch (New Haven, CT) presented two studies.The first focused on maladaptive and adaptive behav-iors in 27 males with the full mutation using the Vine-land adaptive behavior scale. Communication skills,particularly expressive language, were the most se-

Eighth International Workshop on the Fragile X 223

verely affected and socialization skills were the leastimpacted; play and leisure activities accelerated withage. There was a decline in maladaptive behavior withage, although this was unrelated to cognitive ability.Maladaptive and adaptive behaviors were not corre-lated with each other. In a second study, 13 males and4 females between the ages of 5 and 17 years wereassessed using the clinical evaluation of language fun-damentals—preschool (CELF-P). Language skills werelower in the males, reaching a plateau after age 6 yearswith a language age of 3–4 years.

Randi Hagerman (Denver, CO) presented a study ofelectrodermal reactivity as measured by the skin con-ductance response to repetitive sensory stimuli (includ-ing visual, auditory, olfactory, tactile, and vestibularstimuli) given 10 times to assess habituation. The sub-jects were 25 individuals with the FMR1 mutation, 25age-matched normal controls, and 5 individuals withmental retardation without fragile X syndrome. Maleswith fragile X syndrome had remarkably higher ampli-tude of response to all stimuli, which did not habituatenormally with repetitive stimuli compared with con-trols. Significant differences were seen between maleswith fragile X syndrome and controls in maximum am-plitude, number of peaks and valleys, and degree ofhabituation with repetitive stimuli. No significant dif-ferences were seen between females and controls. Thedegree of abnormality in the skin conductance responsecorrelated inversely with the level of FMRP. Therefore,the electrodermal abnormalities appear to be intrinsicto the degree of involvement in fragile X syndrome. Theelectrodermal response represents the activity of thesympathetic nervous system and these findings reflectenhanced responsiveness or hyperarousal, which isconsistent with the clinical findings of hyperarousalwith stimuli leading to tantrums, aggression, or anxi-ety.

Helle Hjalgrim (Glostrup, Denmark) presented theresults of SPECT scans of the cerebrum in six patientswith fragile X syndrome. Relative hypoperfusion of theright side compared with the left was seen in the cortexand in the basal ganglia. These results are consistentwith recent behavioral and neuropsychological studiesthat demonstrate right-sided problems, including vi-sual-spatial perceptual deficits in patients with fragileX syndrome [Thompson et al., 1994; Bennetto and Pen-nington, 1996; Borghgraef et al., 1996].

Further brain imaging studies were presented byMarkku Ryynanen (Kuopio, Finland) who comparedmagnetic resonance imaging (MRI) studies in adultsincluding 10 males and 10 females with the full muta-tion as well as 10 males and 10 females with the pre-mutation. Although raw hippocampal volumes werelarger in those with the full mutation, when the hippo-campal volumes were normalized for intracranial orbrain area, there was no difference between those withthe full mutation and those with a premutation. This isin contrast to previous studies by Reiss et al. [1994,1995] Ryynanen and colleagues did find that more thanhalf of those with the full mutation had nonspecificchanges on MRI, including focal hyperintensities in thetemporal pole white matter, atypical appearance of

hippocampal morphology, enlargement of perivascularspaces, and enlargement of the ventricles.

Deborah Hatton (Chapel Hill, NC) reported on 51males with fragile X syndrome in the longitudinalstudy, who were rated using the Carey temperamentscales. Compared with normative data on these scales,a larger than expected percentage of boys with fragile Xsyndrome exhibited a difficult temperament. Fathersand mothers showed a high degree of agreement andthere was a moderately high degree of stability overtime using these scales. The degree of difficulty on thetemperament scales did not correlate with IQ.

Don Bailey (Chapel Hill, NC) reported on autisticbehavior in 57 males (mean age of 66.7 months) fromthe same longitudinal study. Twenty-five percent of theboys with fragile X syndrome received a score in theautism range on the childhood autism rating scale(CARS). Their profiles were similar to a comparisongroup of 391 children with autism but without fragile Xsyndrome. Within the group with fragile X syndrome,those with autism demonstrated more problems withverbal communication, object use, and imitation com-pared with those without autism. Of the 14 boys withfragile X syndrome who were in the autistic range onthe CARS, five were previously diagnosed as autistic byoutside centers, and four were nonverbal. There was asignificant correlation between degree of developmen-tal delay and the severity of the rating on the CARSscale. Although there is a continuum of autistic-likebehaviors in fragile X syndrome, low IQ, nonverbalmales with fragile X syndrome are at high risk for adiagnosis of autism.

Lastly, Deborah Hatton (Chapel Hill, NC) presenteda prospective study of 44 boys with fragile X syndromefrom their longitudinal project who had undergone anophthalmic examination. Clinically significant abnor-malities were seen in 25% of the subjects with strabis-mus in 9%, refractive errors in 18% (primary hyperopiaand astigmatism), and nystagmus in 2%. Althoughthese findings are lower than in previous studies,which likely had a selection bias, they do not changethe recommendation that all children with fragile Xsyndrome should be seen by an ophthalmologist or op-tometrist within the first 4 to 5 years of age [Hager-man, 1996].

FRAXA SYNDROME: CLINICAL ISSUESFEMALES

Louise Gane

During the 1990’s, numerous centers worldwide fo-cused on the neurocognitive, psychological, and emo-tional presentation of females who carry the FMR1 pre-and full mutations. This has led to a clearer under-standing of the impact of the gene mutation on femalecarriers as well as affected females. However, our cur-rent understanding would not be possible without ear-lier work published in previous decades that docu-mented mental retardation and the physical findingsassociated with fragile X syndrome in females [Esca-lante, 1971; Vianna-Morgante et al., 1982; Fryns et al.,1986; Loesch and Hay, 1988; Reiss et al., 1989]. Asearly as 1980, Turner et al. recommended an evalua-

224 Holden et al.

tion for fragile X syndrome among all females present-ing with mental retardation. And, with time, the term‘‘affected’’ was used to refer to a female with fragile Xsyndrome who had cognitive deficits. It is now recog-nized that 50–70% of females with the full mutationhave an IQ in the borderline (IQ 70–84) or retardedrange (IQ <70) [Hagerman et al., 1992; Rousseau et al.,1994; de Vries et al., 1996]. The literature of the mid1990’s also established that females with the full mu-tation and a normal IQ often exhibit learning dis-abilities related to executive function deficits (includ-ing attention problems, topic maintenance difficulties,impulsivity) and have been identified by neuropsycho-logical testing [Mazzocco et al., 1993; Sobesky et al.,1994b]. A higher incidence of schizotypal features (in-cluding inappropriate affect, odd communication, so-cial isolation, social anxiety) in females with the fullmutation has been reported [Sobesky et al., 1994a]. Astudy by Reiss et al. [1993] showed that many femaleswith the premutation are cognitively unimpaired. Inaddition, with the discovery of the FMR1 gene muta-tion came further investigation into those females whocarry the premutation. These females have been re-ported to be at high risk for premature ovarian failure(POF), and problems with math, shyness, and moodlability [Sobesky et al., 1995]. Recommendations re-garding identification, testing, as well as the treatmentand intervention needs of female gene carriers weremade by Ave Lachiewicz in 1995 [Lachiewicz, 1995].Franke et al. [1996] found an increased incidence (ap-proximately 40%) for affective disorders and a three-fold increased incidence of anxiety disorders with socialphobia being particularly striking. In the remainingyears of this decade, it is hoped that the clinical pictureassociated with the gene mutation will continue to beelucidated. The studies presented during this work-shop session have added considerably to the growingbody of knowledge regarding clinical phenotypes inwomen with the fragile X gene mutation.

This session began with a presentation by DeborahHatton (Chapel Hill, NC), who described a longitudinalstudy of six young girls with fragile X syndrome whoare being assessed at 3- to 6-month intervals from age3 months to 6 years. Development, behavior, tempera-ment, social and adaptive characteristics, biological,home, and school factors, services, and family contextare being analyzed. Home visits are an integral part ofthis study. The goal is to provide a ‘‘holistic description’’of young girls with fragile X syndrome. Similarities be-tween young girls and young boys with fragile X syn-drome in the developmental area have been noted. Theyoung girls being studied were found to be reticentwith delays apparent prior to age 4 and 5 years.

The second presentation was by Gene Fisch (NewHaven, CT), who described a multicenter prospectivestudy that followed two groups of females with the fullmutation from May 1991. One group of eight females(age 4–14 years) and a second group of six females (age6–14 years) are being studied. The Stanford Binet 4thEdition, the Vineland adaptive scales, and the WISC-Rwere used to assess cognitive abilities and adaptivebehavior. The subjects were divided into two age co-horts: 1) those tested initially prior to age 7 years and

2) those tested initially after age 7 years. In the firstcohort, 5/6 (93%) females showed an IQ decline. In thesecond cohort, 4/5 (80%) females showed an IQ decline.Adaptive behavior decline was seen in 5/8 (62%) fe-males. All of the females studied had higher adaptivebehavior than cognitive scores.

Selective mutism in a 12-year-old girl with the fullmutation was the subject of a case report presented byRandi Hagerman (Denver, CO). Diagnosed at age 8years, this patient had mild speech development delayprior to age 3 years. Although she would speak well toher mother and sisters, she would only respond to herfather when he asked her questions. She also had adiagnosis of generalized anxiety disorder. Individualand family counseling sessions, fluoxetine (Prozac),continued speech and language therapy, and specialeducation support services were the recommendedtreatment to which she responded well. Her sister withthe full mutation was also selectively mute in fifth andsixth grade but was talkative after puberty. It is pos-sible that females with fragile X syndrome may be atan increased risk for selective mutism at a young agebecause of significant problems with shyness and socialanxiety associated with fragile X syndrome. Additionalresearch in this area was encouraged.

Randi Hagerman also presented a case report of acompound heterozygous female with fragile X syn-drome, who has a full mutation (∼363 repeats) on one Xchromosome and a premutation (103 repeats) on theother X chromosome. Southern blot analysis showed anactivation ratio of 0.78 for the premutation. Immuno-histochemistry revealed FMRP expression in 63.5% ofher lymphocytes. Her mother and father both have pre-mutations: 146 and 98 repeats, respectively. Thewoman has a history of extreme shyness, attentionproblems, and learning disabilities with problems inmath and reading. She developed mood swings aftermenarche and has a history of anxiety. Physical exami-nation demonstrated clinical findings associated withthe full mutation. Cognitive testing at 16 years of agedocumented a VIQ of 75, a PIQ of 63, and a full scale IQ(FSIQ) of 67. Her freedom from distractibility scorewas low at 58 as were scores on subtests requiringattention and concentration. She has responded totreatment with methylphenidate (Ritalin), which hashelped her attention and concentration problems.Fluoxetine (Prozac) has resulted in a decrease in anxi-ety at home and school.

A case report on a female with an isodicentric X andfragile X syndrome, first reported by Debra Freeden-berg, was presented by Louise Staley-Gane (Denver,CO). Physical examination at age 19 years demon-strated poor eye contact and perseverative speech. Alow hairline was noted in addition to physical findingsassociated with fragile X syndrome. Cognitive testingat age 16 years had demonstrated a VIQ of 59, a PIQ of51, and FSIQ of 51, and Vineland adaptive behaviorscores were low. Fluoxetine (Prozac) was prescribed fortreatment of anxiety and shyness. She has respondedwell to this treatment with increased sociability, de-creased anxiety, and improved self-sufficiency.

The importance of clinical issues in females whocarry the gene mutation is becoming more widely rec-

Eighth International Workshop on the Fragile X 225

ognized and hopefully will provide further impetus tofocus future research upon the physical, neurocogni-tive, psychological, and emotional issues seen in fe-males who carry the gene mutation, particularly inthose who carry the premutation. Such studies maylead to further understanding of the molecular basis ofmild gene dysfunction and aid in providing informationthat will eventually lead to more effective treatmentsfor the difficulties experienced by many female genecarriers.

FRAXA SYNDROME: PREMATURE OVARIANFAILURE

Diane J. Allingham-Hawkins

For several years, women who are carriers of thefragile X syndrome have been considered to be at anincreased risk of premature menopause (prior to theage of 40) based on anecdotal evidence. Three recentstudies support this view. In 1991, Cronister et al. re-ported that 13% of carriers in her study experiencedpremature menopause. In 1994, Schwartz et al. re-ported a larger, multicenter study in which 24% of pre-mutation carriers were found to experience menopausebefore age 40, whereas only 6 and 8% of full mutationcarriers and noncarriers, respectively, experiencedearly menopause. Finally, Partington et al. [1996] re-ported that fragile X carriers experience menopause 6to 8 years earlier, on average, than women in the gen-eral population of the United Kingdom. Because of therelatively small numbers in these studies and ques-tions about appropriate control populations, an inter-national collaboration was formed at the 7th Interna-tional workshop on the fragile X and X-linked MentalRetardation held in Tromso, Norway in 1995. It wasdecided that each center would collect menstrual andreproductive histories on women in fragile X familieswhose mutation status was known. The control popu-lation would be first degree relatives of carriers whowere known by molecular testing to be noncarriers. Allwomen age 25 years and older would be eligible for thestudy. The three main study questions were: do fragileX full/premutation females experience premature ovar-ian failure at an increased frequency over noncarrierfemales? Do fragile X full/premutation females have anexcess of twinning? Do fragile X full/premutation fe-males have an excess or deficiency of aneuploidy and/orspontaneous abortions? A questionnaire was developedby Dr. Patricia Jacobs (Salisbury, England) in the en-suing months and distributed to all interested centers.This session of the 8th International workshop on frag-ile X and X-linked mental retardation was devoted topresentation of interim findings from the internationalcollaboration as well as to other related studies.

Anna Murray (Salisbury, UK) presented the resultsof a study examining fragile X carrier status in womenwith idiopathic POF. Of 147 women studied, 6.1% car-ried an FMR1 premutation suggesting a significantcontribution of fragile X syndrome to idiopathic POF.Dr. Murray also presented the Wessex data from theInternational POF study: 0/19 carriers of an FMR1 fullmutation, 8/54 (15%) premutation carriers, and 3/45(7%) controls exhibited POF. Concern was expressed

over the high level of POF among controls, although itwas suggested that this could be caused by the smallsample size. Angela Vianna-Morgante (Sao Paulo, Bra-zil) presented the Brazilian POF data: 0/26 full muta-tion carriers, 10/80 (12.5%) premutation carriers, and0/38 controls exhibited POF. The frequency among pre-mutation carriers was significantly higher than amongcontrols. Sally Nolin (Staten Island, NY) presented pre-liminary data from her center: 0/6 full mutation carri-ers, 4/68 (5.9%) premutation carriers, and 0/2 controlsexhibited POF. Pat Howard-Peebles (Fairfax, VA) pre-sented the Virginia POF data: 0/3 full mutation carri-ers, 0/12 premutation carriers, and 0/1 controls exhib-ited POF. Maria Syrrou (Ioannina, Greece) presenteddata on families in Greece and Cyprus: 1/12 (8.3%) pre-mutation carriers and 0/3 controls exhibited POF. Inaddition, 2/5 (40%) women from two fragile X familiesfor whom FMR1 allele size was not known exhibitedPOF. Dr. Syrrou also presented data from three groupsof women with ovarian dysfunction: 1/6 (16.7%) womenwith familial POF carried an FMR1 premutation,whereas 0/9 women with poor response to ovarianstimulation and 0/10 women with sporadic POF carriedFMR1 premutations. These data suggested a highprevalence of fragile X syndrome among familial POFcases. Jeanette Holden (Kingston, Ontario) presentedthe Kingston POF data: 3/11 (27.2%) premutation car-riers and 7/17 (41.2%) of women from fragile X familieswith unknown FMR1 allele sizes exhibited POF. Dr.Holden also noted that 2/3 (67%) women from a singlefamily who are compound heterozygotes for two FMR1premutations had experienced POF. Diane Allingham-Hawkins (Toronto, Ontario) presented the combineddata from all contributing centers to date: 0/49 full mu-tation carriers, 64/215 (30%) premutation carriers, and3/95 (3%) controls exhibited POF. In addition, 10/22(45%) of women with unknown mutation status and 2/3of premutation compound heterozygotes exhibitedPOF. There is clear evidence of an increased risk ofPOF among premutation, but not full mutation, carri-ers. During a working group meeting, the contributorsagreed to expand the study population to include allwomen 18 years and older and to attempt to collectadditional data from full mutation and control women.

Aileen Kenneson (Atlanta, GA) presented data from216 women with early menopause compared with acontrol group of 107 women with menopause after age47. None of the women with early menopause and one(1%) of the women in the control group was shown tocarry an FMR1 premutation. Mathematical modelsbased on these data were presented that indicated thatthe fragile X premutation is more likely to be a riskfactor for familial POF than for sporadic POF. The au-thors concluded that the FMR1 premutation is not amajor causative factor for idiopathic early menopause.

Arie Smits (Nijmegen, The Netherlands) presenteddata that suggested a high incidence of elevated FSHlevels and short follicular lengths among fragile X car-riers. These findings are consistent with the higher in-cidence of POF in carriers. The authors suggested in-vestigation of postmenopausal symptoms, such as car-diovascular disease and osteoporosis, among fragile Xcarriers.

226 Holden et al.

Lisbeth Tranebjaerg (Tromso, Norway) presented aninteresting family ascertained because of gonadal dys-genesis due to Perrault syndrome in two young women.In the course of evaluating the family, their motherwas found to carry an FMR1 premutation. The affecteddaughters did not carry the premutation. The motherhad experienced fertility problems prior to her firstpregnancy, which had resulted in interventions involv-ing partial resection of her ovaries and hormonalstimulation. She went on to have three successful preg-nancies. The authors stressed the importance of inves-tigating FMR1 allele sizing in females with reducedfertility.

METHODS, INCLUDING PRENATALDIAGNOSIS

Edmund C. Jenkins

Prenatal diagnostic studies in the early 1980’s reliedon cytogenetic fragile site detection, whereas those ofthe mid-to-late 1980’s were a combination of cytogenet-ics and DNA linkage studies. Where possible, the ac-tual fragile X chromosome could be followed from onegeneration to the next by using restriction fragmentlength polymorphisms, thereby detecting the chromo-some, not the mutation, in the fetus. Much of the time,families were not studied sufficiently in advance ofpregnancy in order for the linkage studies to be helpful.After the FMR1 gene was cloned in 1991, Southernanalyses provided the critical data as to the size andmethylation status of the full FMR1 mutation, untilpolymerase chain reaction (PCR) technology could bemodified so that the CGG-repeat region could be sized.Some laboratories use Southern analysis alone to de-termine the fragile X status of the fetus, whereas oth-ers, like our own, use a combination, because PCR canprovide results within a few days. In our experience, ifthe results are unequivocal, the pregnant woman isinformed of these results with the knowledge that wecomplement every study with Southern blot analysis.

Looking back, the last study of fragile X chromosomeprenatal detection appeared in 1996 [for review, seeJenkins and Brown, 1998], and it was likely used in theprocess of validating molecular protocols within thatlaboratory. There have been, overall, at least 149 am-niotic fluid, chorionic villus, and percutaneous bloodsamples found to be fragile-X positive by 21 laborato-ries or groups of investigators. Although the chromo-some test was good in detecting the full mutation, atleast 23 false negatives and one false positive havebeen studied. Excluding maternal cell contamination,there have been no false positives or false negativesreported for full mutation detection using moleculartechnology in over 110 cases of full mutations reportedfrom 16 laboratories or groups of investigators between1991 and 1996. Because molecular technology detectsnot only full mutations but also premutations and hasdefined normal as a function of CGG repeat size andmethylation status, it is really the only technology touse today for both pre- and postnatal fragile X detec-tion. Although not yet possible for females, it is possiblethat in the near future, methylation and CGG-repeat

length may be determined solely by PCR as indicatedin a recent study by Das et al. [1997/1998].

For this session, Ted Brown (Staten Island, NY) pro-vided an update in carrier screening and fragile X pre-natal diagnosis. He reported that the PCR protocol inuse at our institute accurately identifies normals, pre-mutations, and approximately 90% of the full muta-tions. He also indicated that CVS appears to be thetissue of choice and that we have found consistentlyreliable results using CVS specimens. He concludedthat screening at-risk pregnant women for their fragileX status, followed by prenatal diagnosis, is now pos-sible. Ted then presented Carl Dobkin et al.’s innova-tive PCR protocol for the identification of fragile X fullmutations, which significantly reduced sample size andconsequent turnaround time to provide results compa-rable with Southern analysis. This protocol allows pre-natal diagnosis of full mutations 2–4 weeks earlierthan standard Southern analysis.

Ed Jenkins (Staten Island, NY) then presented evi-dence that full mutations can be detected as early as 10weeks of gestation using monoclonal antibodies with afeasible turnaround time of 1 day after specimen re-ceipt.

Ed then shared with everyone some of the first trans-mission electron micrographs of longitudinal sectionsof the fragile X chromosome and even entire metaphasespreads and showed some evidence that suggested den-sity variation (not seen at the light microscope level)precedes the appearance of the fragile site.

Sally Nolin (Staten Island, NY) summarized her re-sults on single cell analysis in sperm and stated thatoverall the FMR1 CGG repeat was significantly moreunstable in sperm than in lymphocytes.

Maire Percy (Toronto, Ontario) reported her analy-ses of technical factors affecting electrophoretic pat-terns of FMR1 repeats and urged that electrophoresisconditions, including the choice of molecular size mark-ers, should be better quality assured and standardizedto optimize intra- and inter-laboratory reliability.

In line with Maire’s presentation, Pietro Chiurazzi(Rome, Italy) announced that he has collaborated withlaboratories in Italy, the UK, and Canada to establisha panel of DNA samples that will allow standardizedgenotyping between laboratories. He said that thepanel is available to all research groups interested insharing their standardization studies.

Pietro’s was the last presentation of this session,which elicited lively discussion including a question byDavid Nelson as to what our nuclear-specific monoclo-nal antibody looked like on Western blots. This infor-mation is provided in the Proceedings.

POPULATION STUDIES AND SCREENING;FOUNDER CHROMOSOMES

Anna Murray and Jeanette Holden

Prior to 1991, the fragile X syndrome was diagnosedby cytogenetic analysis, and screening surveys re-ported an incidence in males of between 1 in 1,000 and1 in 2,600. The two surveys most often cited [Turner etal., 1986; Webb et al., 1986], screened individuals witheither mild or moderate/severe mental retardation;

Eighth International Workshop on the Fragile X 227

however, both populations have been retested usingmolecular techniques and a significant number of cy-togenetically positive cases did not have an expansionmutation in the FMR1 gene [Turner et al., 1996; Mor-ton et al., 1997]. There are several possible explana-tions for the false positive rate in some original studies.First, fragile site expression in 3–5% of cells was oftenconsidered ‘‘positive’’ and current criteria might ex-clude such cases. Second, the cytogenetic studies aretechnically difficult and require considerable expertiseto distinguish the FRAXA site from other fragile sitesin the region, including the common fragile site atXq26, FRAXE, and FRAXF; in some cases, it is impos-sible to distinguish these using conventional methods,especially since the quality of the chromosomes can bepoor. Recalculation of the incidence of FRAXA syn-drome gives a population prevalence of approximately1 in 4,000 in both studies [Turner et al., 1996; Mortonet al., 1997].

The incidence of the FRAXA full mutation in re-tarded populations has been studied by many groupsand varies considerably among studies, ranging from0.1 to 4.2% [Meadows et al., 1996; van den Oweland etal., 1997]. While it is possible that these variations rep-resent true population differences in the frequency ofthe full mutation, intuitively it is more likely that theyare due to qualitative differences between the groupstested.

The incidence of the FRAXA premutation has beendetermined in a large population-based survey of10,624 random female blood samples. In this popula-tion, 41 women carried premutations with between 55and 101 repeats, a frequency of 1 in 259 women [Rous-seau et al., 1995]. Spence et al. [1996] found a similarlyhigh frequency of premutations in women in Virginia,USA (1/248). However, other surveys show a muchlower frequency, e.g., 1 in 1,538 chromosomes [Reiss etal., 1994] and 1 in 1,000 chromosomes [Holden et al.,1995]. The differences among studies can be explainedin part by variation in the definition of a premutationand also by the techniques used to measure triplet re-peat number. However, these factors do not seem toaccount for all of the variation.

The incidence of FRAXE full mutations and premu-tations is not known, but they are much rarer thanFRAXA mutations. From six reported studies, only twofull mutations were detected in a total population of3,919 mentally impaired individuals [Murray et al.,1996; Meadows et al., 1996; Allingham-Hawkins andRay, 1995; Knight et al., 1996; Holinski-Feder et al.,1996; Wang et al., 1995]. The frequency of the FRAXEfull mutation in the population has been estimated tobe approximately 1 in 50,000 males [Brown, 1996].

There were 11 presentations in the populationscreening session. Sultana Faradz (Indonesia) pre-sented the results of screening 237 mildly retarded in-dividuals, finding 2.1% carried FRAXA full mutations.Screening a Hellenic population of 866 identified eightfull mutations (0.92%) and two premutations (0.23%)(Philippos Patsalis, Cyprus). Stephen Claes (Leuven,Belgium) reported the highest frequency with 4.3% ofdevelopmentally disabled males carrying full muta-

tions. However, when this population was divided ac-cording to degree of retardation, 8.5% of the moder-ately retarded group carried full mutations. Populationincidence figures for the full mutation were provided bySheila Youings (Salisbury, England) (1 in 6,075) andPhilippos Patsalis (Cyprus and Greece) (1 in 4,200).These two groups also tested FRAXE in a total of 2,075individuals, but neither found any full mutations. Cal-vin Pang (Hong Kong) tested 649 normal and 324 re-tarded Chinese children and, although the frequency offull mutations in the retarded group was similar toother studies (0.6%), the distribution of normal allelesdiffered from Caucasian series. The modal repeat inthe Chinese population was 29 compared with 30 inCaucasians.

Four groups reported the frequency of intermediateor grey-zone alleles in developmentally disabled popu-lations compared with controls, following studies of apossible adverse effect of such alleles on intellectualfunction [Murray et al., 1996)]. Sheila Youings andAnna Murray (Salisbury, England) confirmed theirpublished work on a larger population and presentedevidence against other possible explanations for the ob-servation, e.g., type 1 errors, a gene in linkage disequi-librium with the intermediate alleles and meiotic drive.Angela Vianna-Morgante (Sao Paulo, Brazil) gave ashort presentation on her poster in which she showed asignificant excess of FRAXA intermediate alleles inmentally retarded males (6.3%) compared with controlmales (2.4%). However, Calvin Pang (Hong Kong) andJeanette Holden (Kingston, Ontario) did not find a sig-nificant excess of grey zone alleles in their studies.

Three groups had investigated haplotype associa-tions. Angela Vianna-Morgante (Sao Paulo, Brazil)compared fragile X and control chromosomes using themarkers DXS548 and FRAXAC1 and showed signifi-cant differences in haplotype frequencies in agreementwith previous studies. Priscilla Poon (Hong Kong)looked at the same markers in the Chinese populationand interestingly found a difference in the modalFRAXAC1 allele compared with Caucasians. She alsoreported a significant difference in DXS548 alleles innon-fragile X individuals with mental retardation com-pared with unaffected controls and suggested theremay be a second mental retardation gene linked toDXS548. The third study on haplotypes, by ChrisGunter (Atlanta, GA), described a new biallelic singlenucleotide polymorphism (SNP) within the FMR1 genethat showed a significant difference between fragile Xchromosomes and controls. Chris suggested that the Gallele for this SNP may be a good indicator of FRAXAalleles at high risk of expansion.

Ergul Tuncbilek (Ankara, Turkey) confirmed thevariability of the fragile X phenotype in a group ofTurkish patients and suggested that behavioral char-acteristics were a better diagnostic indicator in chil-dren than physical phenotype. Finally, MarkkuRyynanen (Kuopio, Finland) looked at the impact offragile X screening for pregnant women and found thatwith an 85% uptake, the test proved very acceptable.

228 Holden et al.

FMR1 GENE SESSIONPietro Chiurazzi and Chris Gunter

Although more than 90% of fragile X cases resultfrom expansion of the FMR1 CGG repeat, several pa-tients have been reported with deletions, rearrange-ments, and more rarely, point mutations. The firstthree presentations of this session focused on muta-tions in the FMR1 gene other than the 58CGG repeatexpansions.

Karen Gronskov (Glostrup, Denmark) performedsingle strand conformational polymorphism (SSCP) tolook for point mutations in the coding regions of FMR1.One hundred, thirty males with mental retardationwith no CGG repeat expansions were tested and nocausative mutations were found. However, potentiallyuseful polymorphisms in intron 1 and exons 1 and 5were described in the screening process, including theFMRb polymorphism (exon 5) previously characterizedby Kunst and Warren [1994].

Helle Hjalgrim (Glostrup, Denmark) described aphenotypically normal female with an Xq24→qter de-letion on one chromosome and a microdeletion in exon1 of the FMR1 gene, presumably derived from the ma-ternal full mutation. Although the microdeletion elimi-nated the CGG repeats and some flanking sequences,expression of FMRP was detected in lymphoblastoidcells, indicating that these were not required for tran-scription. Helle also reported on a 47,XY,fra(X)(q27.3)male born to a mother with a premutation.

Pietro Chiurazzi (Rome, Italy) reported on a moder-ately retarded male with a large deletion (200–500 kb)encompassing the FMR1 gene inherited from his hemi-zygous mother and on two unrelated mildly retardedmales who are mosaic for a normal FMR1 allele inher-ited from their mothers and a gross rearrangement ofthe promoter region in more than 50% of their lympho-cytes. These are the first cases in which a postzygoticevent involving the FMR1 promoter region has notarisen in the context of an expanded CGG repeat.

Presentations regarding the stability of the CGG re-peat were then discussed. Philippos Patsalis (Nicosia,Cyprus) described the transmission of the FMR1 CGGrepeat in 100 three-generation families of Hellenic ori-gin, totaling 751 individuals. One unstable transmis-sion was observed (1/651 meioses) and alleles rangedfrom 11 to 55 repeats.

Marie-Lou Morel (Quebec, PQ) reported a largescreening of 2,515 Canadian females and their new-borns for CGG repeat size, using a new high through-put mini-Southern blot method. They were able to re-fine their frequency estimates of grey-zone (40–53 re-peats) and premutation alleles in this population to1/27 and 1/258 females, respectively. Only 4 out of 45transmissions of grey-zone alleles resulted in a changeof repeat size and these were all contractions (−1 to −5triplets). One premutation out of two resulted in anexpanded allele with two more triplets and, interest-ingly, a 10 triplet expansion (22 to 32 repeats) was alsoobserved.

Anna Murray (Salisbury, England) presented evi-dence that a general instability mechanism, possibly

mediated by genes such as the mismatch repairgenes, may determine the initial destablilization of theFMR1 triplet repeat. In particular, she reported onefamily with hereditary nonpolyposis colorectal cancer(HNPCC) with at least three unstable transmissions ofan FMR1 allele within the intermediate size range.

Peter Steinbach (Ulm, Germany) presented data onthe mitotic instability of expanded CGG repeats in cul-tured fibroblasts derived from a male carrier with anunmethylated full mutation. In contrast, their grouphad previously published data on the mitotic stabilityof hypermethylated full mutations and speculated onthe involvement of a methylation-mediated DNA mis-match repair system in ensuring this stability.

Flora Tassone (Denver, CO) examined blood and sev-eral tissues from a male premutation carrier. Such re-sults on postmortem specimens are of great relevancein view of the mosaicism observed in fragile X patients,which limits any phenotype-genotype correlation. Inthis case the same 94-repeat allele was found in alltissues studied, including various brain regions andtestes. This repeat did, however, expand upon trans-mission to his two daughters (to 95 and 111 repeats).

Marina Grasso (Genova, Italy) described eight casesof mosaicism in males harboring a full mutation and aband of normal (4 cases) or smaller size (4 cases). Thenormal band presumably originated by reduction of thefull mutation within the CGG repeat itself, and proteinproduction could be detected in blood samples fromthese individuals. This result also warns against theexclusive use of PCR-based diagnostic methods that donot allow the detection of full mutations and wouldhave given false-negative results in these instances.The other four cases are also likely derived from aninternal deletion comprising the CGG repeat and someflanking sequences and can be extremely useful formeasuring FMRP production and correlating proteinlevels with gene changes.

Finally, Peter Steinbach (Ulm, Germany) illustratedan extension of their footprinting analysis of the FMR1promoter, demonstrating the presence on premutatedalleles of footprints that are typical of normal tran-scribed genes. When ‘‘high-functioning’’ males withonly a partially methylated full mutation were exam-ined, footprints were absent in a proportion of cells.

Two posters were also presented. The first one byDrouin et al. (Quebec, PQ) described four footprints inthe promoter region of transcriptionally active FMR1alleles. The second one by Beatrice Schmucker et al.(Erlangen, Germany) reported two cases of mosaicswith a full mutation and alleles in the normal range (47and 37, respectively).

FMR1 PROTEINW. Ted Brown and Maire Percy

A summary of the current understanding of theFMR1 protein (FMRP) was presented by Ted Brown(Staten Island, NY). The fragile X gene includes 17exons spanning a genomic region of 38 kb. There are atleast 12 alternately spliced isoforms of FMRP. How-ever, there appears to be no clear tissue isoform speci-

Eighth International Workshop on the Fragile X 229

ficity, and a 4.4-kb mRNA transcript is the most abun-dant one in all tissues. The full-length protein is 631amino acids (AA), whereas the smallest isoform is 568AA. The AA sequence is highly conserved: the humansequence shows 97% identity with mouse, 92% withchicken, and 90% with frog. In the chick and frog, exons11 and 12 are missing, which suggests that these exonswere a relatively recent evolutionary addition. The 38untranslated region (38UTR) is long with predictedstem-loop structures and is also highly conserved.FMRP is localized mainly in the cytoplasm with high-est tissue concentrations in the brain and gonads. Anuclear export signal (NES) consisting of 17 AAs islocated within exon 14. A nuclear localization signal(NLS) is located in the amino terminus region ofFMRP. The protein contains two copies of a KH motif,an RNA binding motif whose name comes from homol-ogy to hnRNP K. It also contains a second RNA bindingmotif, an RGG box region, located in the distal part ofthe protein. The KH motif sequences show 100% con-servation between humans and frogs. One fragile Xsyndrome patient has been identified with a point mu-tation (Ile304Asp) that destroys RNA binding. Three-dimensional analysis of KH domains indicates theyhave a beta-sheet on one side and alpha helices on theother side. FMRP binds strongly to poly(G) and poly(U)tracts. It forms dimeric and multimeric complexes withitself and with the FXR1 and FXR2 proteins, in vitro.FMRP reportedly binds to approximately 4% of brainmRNAs including its own mRNA. It binds loosely to the60-S ribosomal subunit.

Sequences in exons 13 and 14, which are distal to theKH motifs, appear to mediate 60-S ribosomal binding.FMRP is found in highest concentrations associatedwith active polysomes. It is found in neuronal cyto-plasm in the rough endoplasmic reticulum, at dendriticbranch points, in dendritic spine necks and heads.FMR1-related mRNA sequences have also been local-ized to dendrites. In synaptoneurosomes, followingstimulation of metabotropic glutamate receptors,FMR1 mRNA is translated into FMRP, which associ-ates with polysomes.

Ted’s overview was followed by eight presentations/posters. The session began with a presentation dealingwith the recently isolated FXR1 and FXR2 proteinsthat are homologous to FMRP. An important questionis why they do not complement for lack of FMRP inpersons with the fragile X syndrome. To address thisissue, Andre Hoogeveen (Rotterdam, The Netherlands)and colleagues studied the interaction of FXR1, FXR2,and FMR1 proteins with poly(A)+ RNA. They also stud-ied the interaction between the three proteins using invitro binding assays and characterized the expressionof the three proteins in fetal and adult brain and testisusing immunohistochemistry. In conjunction withother biochemical studies of lymphoblasts, they con-cluded that although the three proteins can bind topoly(A)+ RNA and interact with one another in vitro,they are not always expressed in the same cells. Fur-thermore, when they are expressed in the same cells,most of the complexes formed appear to be of homo-meric origin, suggesting that there is no functional het-eromeric complex formation of the three proteins.

The function of FMRP, absent in individuals affect-ed with fragile X syndrome, has remained unknownalthough its affinity for RNA and sedimentation withribosomes have been described. F. Corbin (Quebec, PQ)and colleagues reported that 95% of endogenousFMRP in normal cells is associated with activelytranslating ribosomes. They showed that it is not as-sociated with the 60-S ribosomal subunit, but rathersediments as a 60-S messenger-ribonucleoparticle(mRNP). Also, FMRP associates almost exclusivelywith poly(A)+ RNA and binds very close to the poly(A)+

tail of mRNAs, suggesting that FMRP is part ofmRNP complexes and remains bound to mRNAs dur-ing the translation process.

Nan Zhong (Staten Island, NY) and colleagues hy-pothesized that FMR1 is a ‘‘trigger’’ gene whose ab-sence initiates a pathogenic pathway with effects ontranscription and translation. Using reverse differen-tial display analysis (RDDA), they found the transcrip-tion pattern to be different in lymphoblasts from per-sons with fragile X syndrome and normal individuals.They identified Ras-GTPase-activating protein SH3-binding protein (G3BP) as having reduced transcrip-tion in fragile X lymphoblasts. They also observed aribosomal RNA transcript to be absent or greatly re-duced in FMR1 knockout mouse brains and oocytes.Their findings suggest that these differentially reducedtranscripts result from the absence of the FMRP func-tions of RNA-binding or transport.

Julia Currie and Ted Brown (Staten Island, NY) de-veloped a KH motif search strategy and conducted acomputer-assisted analysis of the completed yeast se-quence for homologues to FMRP. Nine yeast proteinswere identified that contain KH domains: five withknown functions and four with unknown function. Noclose match to FMRP or to the FXR proteins was found.The closest match was to one of the eight KH domainswithin the yeast homologue of Vigilin, a vertebrategene containing 14 KH domains. They speculated thatthe absence of a yeast FMR1 homologue might indicatethat it evolved at a later stage of evolution.

Giovanni Neri (Rome, Italy) and colleagues reportedbriefly on an ongoing project aimed at reactivating thefully methylated FMR1 gene in vitro. Preliminary re-sults indicate that treatment of cell lines from fragile Xsyndrome patients with the demethylating agent5-azadeoxycytidine results in restoration of FMR1transcription and translation.

Flora Tassone (Denver, CO) reported on a majorstudy with Annette Taylor and Randi Hagerman to in-vestigate the relationship between FMRP expressionand phenotypic measures in 80 individuals with fragileX syndrome. Strong correlations were found betweenFMRP expression determined by immunocytochemis-try of blood smears and 1) IQ in mosaic males andmales with a partially methylated full mutation and 2)physical index in females with a full mutation. Resultssuggest that FMRP expression in >50% of lymphocytesis an indicator of nonretarded IQ in males. Nonethe-less, the presence of fragile X behavioral and physicalcharacteristics in three males with FMRP expressionranging from 60–70% of lymphocytes suggests that

230 Holden et al.

even a small FMRP deficit results in some manifesta-tions of the disorder.

The objectives of the retrospective study described byArie Smits (Nijmegen, Netherlands) and colleagueswere twofold: 1) to establish the social and mental sta-tus of females with a full mutation and 2) to determineif the degree of FMRP expression in blood smears pre-dicts the cognitive profile. One hundred, fifty femaleswith a full mutation were included (60 were selected forIQ testing and FMRP expression). Females with a fullmutation were 1.7 times more frequently unmarriedthan women in the general population. Sixty percent ofthose older than 40 years were self-supporting. Fromthis latter group, 75% had children. Mentally impairedfemales had children at a younger age than those whowere cognitively normal. The FMRP expression ap-peared not to be a useful predictor of intellectual statusin this group and accounted for only 20% of the vari-ance in IQ.

STUDIES ON TRANSGENIC MOUSE MODELSFOR FRAGILE X SYNDROME

Frank Kooy and Patrick Willems

A knockout mouse for the fragile X syndrome wasgenerated in 1994 [Bakker et al., 1994] and distributedto various laboratories around the world. In his generaloverview, Patrick Willems (Antwerp, Belgium) summa-rized the results obtained by different investigators. Inthe knockout mouse, the murine Fmr1 gene is replacedby a nonfunctional copy. Although the nature of themutation in the mouse model (gene interruption) is dif-ferent from that in most human patients (CGG repeatamplification), like fragile X patients, the knockoutmouse has no functional mRNA or protein. The fragileX knockout mouse reflects the phenotypic characteris-tics of the fragile X syndrome in having macroorchi-dism and cognitive deficits, as observed in the Morriswater maze task [Bakker et al., 1994; Kooy et al., 1996;D’Hooge et al., 1997].

Carola Bontekoe from Ben Oostra’s (Rotterdam,Netherlands) lab presented two new transgenic fragileX mouse lines. In one line, a construct with a longinterspersed CGG repeat (CGG)11AGG(CGG)60CAG(CGG)8 of premutation size was introduced at randompositions in the genome by microinjection into mousefertilized oocytes. In three independent mouse lines, atotal of 263 transgenic animals was studied. All ani-mals examined inherited the original allele withoutany evidence for size alterations. It was concluded thatfactors other than repeat length per se might play arole in repeat stability [Bontekoe et al., 1997].

Carola also presented results on a second mouse line,created by introducing an FMR1 transgene under aCMV promoter into the existing knockout mouse.Western blotting showed similar levels of FMRP ex-pression in the ‘‘rescue’’ mouse as in wild-type mice, butimmunochemistry of brain sections showed differencesin expression. In wild type mice, protein expression isvisible in neurons but not in glia. However, in the res-cued mouse, FMRP was detected in all types of braincells. Therefore, the level of FMRP expression in neu-rons might be too low to rescue the knockout mice from

developing the fragile X phenotype. The testicularweight of the rescued mice was in the range of that ofthe knockout mice and was therefore not corrected bythe additional transgene. Cognitive and behavioralfunction tests have not yet been performed extensively.

Ted Brown (Staten Island, NY) presented experi-mental results, suggesting that the learning deficits ofknockout mice in cognitive tests such as the Morriswater-maze or the plus-maze were much more pro-nounced when the mice were trained in ‘‘massed’’ tri-als, e.g., training sessions with very brief intervals be-tween trials. In the discussion following the presenta-tion, Frank Kooy and Patrick Willems pointed out thatthese results were not confirmed under similar testconditions in Antwerp.

Gene Fisch (New Haven, CT) used operant condition-ing to examine learning and memory of fragile X mice.He illustrated that knockout mice could acquire a bar-press response to discriminate visual and auditorystimuli. Surprisingly, the three knockout mice ac-quired the discrimination tasks better than the twocontrols.

MRI was used by Frank Kooy (Antwerp, Belgium)from Patrick Willem’s lab to study the brain anatomyof fragile X mice. Abnormalities, such as a reduced sizeof the cerebellar vermis, which have been described inhuman patients [Reiss et al., 1991; Reiss et al., 1995],could not be detected. Additionally, Frank Kooy pre-sented a knockout mouse model for X-linked hydro-cephalus. MRI images showed an increase in the size ofthe ventricular system in the knockout mouse.

Testes from fragile X mice were studied by AndreHoogeveen and Ben Oostra (Rotterdam, Netherlands).Although significantly larger, the testes from theknockout mouse appeared identical in structure to thecontrol testes. The macroorchidism appeared to be theresult of increased Sertoli cell proliferation betweenembryonic day 12 (E12) to 15 days postnatally. FSHlevels appeared to be normal, and the cause of the pro-liferation could not be found.

In the final presentation, Maire Percy (Toronto, On-tario) showed results from a search with her colleaguesSharon Shtang and Marc Perry, for an FMR1 homo-logue in the nematode Caenorhabditis elegans. She hy-bridized a probe containing the two FMR1 KH domainsto a C. elegans cDNA library and identified a cDNAwith homology to some regions of the FMR1 gene. How-ever, it remains to be determined whether this gene isa C. elegans FMR1 homologue.

In conclusion, this session presented the first set ofresults of experiments on the transgenic mouse modelsof the fragile X syndrome. As the knockout mice arenow available from the Jackson laboratory, more ex-perimental work is expected to be presented at the nextfragile X workshop.

FRAXE AND OTHER TRIPLET REPEATDISORDERSGene Fisch

The fragile site at FRAXE (gene name, FMR2) wasfirst identified as one easily confused with FRAXA[Sutherland and Baker, 1992]. Indeed, individuals

Eighth International Workshop on the Fragile X 231

with FRAXE syndrome were originally detected fromcytogenetically positive but DNA negative cases ofFRAXA. FRAXE syndrome is notable because it sharesmany genetic properties with FRAXA syndrome. It isproduced by an abnormal expansion of GCC repeats inthe Xq27-28 region, approximately 600 kb distal to theFRAXA site. Hypermethylation is apparent when thenumber of repeats exceeds 200 [Knight et al., 1993]. Inthe general population, FMR2 alleles range from 4 to35 repeats [Knight et al., 1993; Murray et al., 1996;Zhong et al., 1996]. The prevalence of FMR2 repeatexpansion in the general population is estimated to belower than FMR1 repeat expansions, possibly becausethe phenotype has fewer pronounced clinical manifes-tations and milder cognitive deficits.

Presentations in the session on FMR2 and other trip-let repeats explored several avenues of research: 1)elaboration of the genetic characteristics of FMR2; 2)the prevalence of FRAXE syndrome in various popula-tions; and 3) further characterization of the cognitive/behavioral phenotype. In a related issue, the mecha-nism of CTG expansion in the DMPK gene comparedwith other triplet repeat disorders was also discussed.

Monserrat Mila (Barcelona, Spain) presented a caseof a mentally retarded male with fewer than 200 GCCrepeats who exhibited methylated mosaicism forFMR2. This suggests that the threshold for methyl-ation in the FRAXE syndrome may be lower than forFRAXA syndrome. These researchers suggest thatmethylation status should also be evaluated when in-dividuals with small mutations are identified.

Ted Brown (Staten Island, NY) reported on theirstudy of founder effects in FMR2 by looking for corre-lations in haplotypes of five nearby microsatellites.While no significant correlations were found betweenproximal markers, a significant negative correlationbetween two distal markers was observed. The fre-quency of this haplotype in larger alleles suggests aFRAXE founder effect.

Charles Schwartz (Greenwood, SC) described theircenter’s experience regarding referrals for DNA test-ing. Having examined 600 samples, these researchersfound two unrelated cases of FRAXE syndrome. Theirresults suggest that FMR2 repeat expansion occursless frequently than FMR1 repeat expansion even in apopulation at risk for genetic disorders.

Jeanette Holden (Kingston, Ontario) presented twostudies on molecular testing for FRAXE syndrome inselected populations. In her first study, 444 develop-mentally disabled Canadian males were tested, andone FMR2 repeat expansion was identified. In her sec-ond study, families with single affected males diag-nosed as autistic or pervasively developmentally dis-abled were tested for repeat expansion and repeatnumbers in FMR1 and FMR2 and at a third folate-sensitive fragile site, FRAXF. After comparing frequen-cies of allele sizes at these loci with those from thegeneral population, these researchers found no signifi-cant differences. Their results suggest that repeat ex-pansions and mutations in FMR1 and FMR2 have nogenetic role in autism, but differences observed at theFRAXF locus suggest that it may be an etiological fac-

tor for autism and/or pervasive developmental disor-ders and that additional testing at that locus is war-ranted.

Gene Fisch (New Haven, CT) reported on longitudi-nal changes in cognitive-behavioral levels in young in-dividuals with FMR2 repeat expansions. Results indi-cate that subjects tested do not follow the typical pat-tern of declining IQ and DQ scores observed in childrenwith the fragile X syndrome of comparable ages. It fur-ther suggests that the pattern of neurological develop-ment in persons with FRAXE syndrome differs fromthat which occurs in FRAXA syndrome.

Peter Steinbach (Ulm, Germany) discussed methyl-ation patterns 58 of the CTG repeat in the DMPK gene.He and his colleagues found constitutive methylationat two upstream restriction sites, but other sites in theregion were unmethylated. However, in young, se-verely affected DM patients, they found completemethylation of the other sites on the mutated chromo-some. This finding may prove valuable in identifyingthe mechanism that leads to methylation at the CpGisland in the FMR1 gene.

XLMR: KNOWN AND NEW SYNDROMESLisbeth Tranebjaerg, Herb Lubs, and

Giovanni Neri

This session began with an update on informationregarding known XLMR syndromes, including sevenpresentations. The session continued with a number ofstudies on new XLMR-syndromes. This number is con-stantly increasing (see Update 1998 by Herb Lubs etal., this issue). All the new syndromes that were re-ported during this session are included in the updatechapter.

Patrick Willems (Antwerp, Belgium) began the ses-sion with a report on a family with three boys withdevelopmental disability who had eight CAG repeats inthe androgen receptor (AR) gene (normal range is 10-35 repeats, based on both reported alleles and a studyin Belgium of 200 controls). Examination of the ARprotein from one affected boy showed absence of recep-tor phosphorylation, and Patrick suggested that men-tal retardation in the family might be caused by short-ening of the CAG repeat region in the AR gene.

Lisbeth Tranebjaerg (Tromso, Norway) then pre-sented a review of findings on the DDP gene in theMohr-Tranebjaerg syndrome and results on two fami-lies. The first was a family with Jensen syndrome,which was found to have a mutation in the DDP gene.The second family had X-linked deafness and dementiaand was found to have an insertion of a triplet in thenoncoding region. In the Jensen family, neuropatholog-ical findings included optic atrophy, diffuse demyelin-ation, gliosis, and calcium deposits. The results indi-cate that the Mohr-Tranebjaerg and Jensen syndromesare allelic and that other mutations in the DDP genemay cause inherited or isolated deafness/dystonia, de-mentia or blindness, or even psychiatric symptomsseen in the patients with either of these syndromes.

Stephan Claes (Leuven, Belgium) described mappingthe locus for X-linked infantile spasms (MIM 308350)

232 Holden et al.

to Xpter-Xp11.4 using two families with this syndrome.A maximum lod score of 2.36 was obtained for markersDXS989 and DMD49 at a recombination fraction of 0.

Charles Schwartz (Greenwood, SC) reviewed find-ings on the original Renpenning family and reportedlinkage studies of this family, characterized phenotypi-cally by a tendency toward microcephaly, short stature,and small testes. Linkage was established to DXS1003(Xp11.2-p11.4) with a lod score of 2.36 at zero recom-bination. He proposed that the designation of Renpen-ning syndrome be reserved for XLMR entities mappingto this general region, which share clinical findingswith the original family.

Vincent des Portes (Paris, France) et al. reported afamily with dominant X-linked subcortical laminarheterotopia and mild mental retardation in heterozy-gous females and lissencephaly and severe mental re-tardation in hemizygous males. They examined linkagein three unrelated families and excluded linkage formost of the X chromosome, localizing the gene toXq22.3-q23. They have constructed a YAC contig forthe Xq22.3 region and are searching for expressed se-quences that may represent the gene responsible forthis interesting disorder of cortical development.

Laurent Villard (Marseilles, France) presented ananalysis of the XNP/ATR-X gene in normal and patho-logical conditions. This gene is involved in severalXLMR disorders, including ATR-X syndrome, Juberg-Marsidi syndrome, and severe mental retardation(MR) without a-thalassemia. A description of the genewas presented. Villard and his colleagues have carriedout yeast two-hybrid assays and identified two proteinsinteracting with the XNP protein: the first of these(PIX1) interacts with the carboxy terminus of the XNPprotein. They are also carrying out in situ hybridiza-tion studies to rat embryos to determine the cells inwhich XNP is expressed. Using these various ap-proaches, the investigators hope to determine the func-tion of the XNP protein. They are prepared to examinethe XNP gene in families mapping to the XNP/ATR-Xgene region and also in patients with clinical signsoverlapping those for the syndromes described above.Please contact Dr. Villard directly if you have suitablesubjects for study.

Stephan Claes (Leuven, Belgium) described linkagein three families with XLMR and spastic paraplegia.Two regions had previously been assigned for XLMRand complicated spastic paraplegia: SPG1 results frommutations in the L1CAM gene and SPG2 is caused bymutations in the proteolipid protein gene (PLP) atXq22. Their first SPG family was found to have a mu-tation in L1CAM. The second family showed linkage toXq28, but did not have a mutation in L1CAM or inthree other candidate genes in this region. The thirdfamily showed linkage to Xp21.1-q21.3, and both PLPand L1CAM were eliminated as candidate genes in thisfamily. The results suggest that there may be othergenes on the X chromosome that result in XLMR andspastic paraplegia.

Ben Hamel (Nijmegen, Netherlands) reported twonew XLMR conditions, one with cleft lip/palate andvariable occurrence of preaxial polydactyly, mapping to

Xp11.3-Xq21.3 with lod score of 2.78 (recombinationfraction of 0) with the marker DXS441 and flankingmarkers DXS337and DXS990. In another XLMR con-dition, he reported a severe phenotype with mental re-tardation, blindness, convulsions, spasticity, dysmy-elination, and early death. This disorder was mappedto the pericentromeric region, with lod score of 3.15with marker DXS1204 and flanking markers DXS37and PGK1p1, defining the genetic region to a distanceof approximately 7 cM.

Nancy Carpenter (Tulsa, OK) described two newfamilies with distinct syndromes. The first was a four-generation family with XLMR and a highly variablephenotype in males (high arched palate, recessed nasalbridge, aggression, microcephaly in some members).Linkage analysis showed linkage to DXS207 in Xp22.2and DXS989 in Xp22.1 with lod score of 2.41, a theta of0. The second family had coarse facies, wide distal pha-langes, short stature, and mapped to the proximal re-gion of Xq with tight linkage to DXS1003, ALAS2, AR,DXS986, DXS990, and DXS454 in Xp11.3 to Xq21.3(Zmax 4 2.53 at theta 4 0).

From the Miami-Greenwood study, CharlesSchwartz (Greenwood, SC) presented four XLMR enti-ties that mapped to Xq12-q21, four entities mapping toXq28, and another in Xq22.1-Xq23.2. Two of these havebeen published [Pai et al., 1997; Stevenson et al., 1997].The first family had macrocephaly and severe MR, withlinkage to DXS1111, DXS566, and DXS986 with lodscore of 2.96 at theta 4 0. The second family had dis-tinctive facies, hypotonia, areflexia, tapered fingerswith excessive fingerprint arches, and mild-moderateMR; tight linkage was found with DXS1166 andDXS995 in Xq13and Xq21, respectively, with Zmax 42.53. The third family had clinical findings consistentwith Allan-Herndon-Dudley syndrome and gave lodscore of 2.88 at theta 4 0 with DXS995 and DXS458.The fourth family had variable, mild symptoms, includ-ing a tendency to short stature, small head circumfer-ence, small testes, etc., and lod score of 4.41 with zerorecombination found for DXS1166 in Xq13.2. A familywith dysmorphic facial features, seizures, mild im-mune deficiency, and progressive gait disturbanceshowed linkage to DXS1120, PLP, and DXS101 withlod score of 2.3 at theta 4 0, loci in Xq22.1-q23.2. Thenext four families all showed linkage with markers inXq28. The first Xq28 family had severe MR, seizures,midface hypoplasia, ears with unfolded helices, stubthumbs, and had lod score of 2.4 with markers distal toDXS8091 (theta 4 0). The second Xq28 family had se-vere MR but had relative macrocephaly, adult-onsetglaucoma, cleft palate, seizures, short stature, andsmall hands and feet. A lod score of 2.11 was obtainedfor p39, DXS8061, and DXS8103 at theta 4 0. Thethird Xq28 family also had severe MR but had hyper-telorism, hypotonia, mitochondrial myopathy, andchildhood death. It showed a maximum lod score of3.42 with DXS8103, DXS1177, DXS1108, and p39. Thefinal Xq28 family had severe MR, recurrent respiratoryinfections, seizures, and variable mild facial dysmor-phism with death in childhood. A lod score of 3.65 wasobtained with DXS1684 at theta 4 0.

Eighth International Workshop on the Fragile X 233

NONSPECIFIC XLMRHerb Lubs and Giovanni Neri

In the morning session on nonspecific XLMR, Vin-cent des Portes (Paris, France) first reported on theplans for a European consortium to combine clinicaland genetic data from 25 families in the Netherlands,Belgium, France, and Germany.

Elke Holinski (Munchen, Germany) described fournew families with significant linkage data. In one, het-erozygotes were mildly affected and, in another, af-fected males had a downhill rather than stable course.

Ben Hamel (Nijmegen, Netherlands) presented fiveadditional families without specific findings (MRX 43,44, 45, 46, and 52), all with lod scores greater than 2.0.

Stephan Claes (Leuven, Belgium) reported three ad-ditional families (MRX49, MRX50, and MRX51) withsignificant lod scores, two of which were found to havemacrocephaly. A fourth family with a borderline lodscore was also presented.

The details in MRX58, a Newfoundland family alsowith macrocephaly, were given by David Macgregor(St. John’s, Newfoundland). The need to use appropri-ate norms and tables that also relate head circumfer-ence to adult height rather than the usual pediatrictables (which extend only to age 18), was stressed dur-ing the discussion [see Bushby et al., 1992].

The variation in IQ scores within some XLMR fami-lies was stressed in a study by T. van Roosmalen.

The session concluded with a study by Chris Schutz(Kingston, Ontario) discussing a possible relationshipbetween the MAOA gene and autism or autistic-likebehavior in pervasive developmental disorders.

A summary of the status of the MRX disorders wasgiven later in the day by Herb Lubs (Miami, FL andTromso, Norway).

MAPPING STRATEGIES/GENEIDENTIFICATION FOR XLMR SYNDROMES

Charles Schwartz and Laurent Villard

This session was devoted to mapping strategies forXLMR loci and included taking advantage of X-chromo-somal breakpoints in translocations as a method forrapidly localizing specific genes and examining YACand cosmid contigs for expressed sequence tags thatmay be good candidate genes. An update of XLMRgenes was presented as well as information regardingthe XLMR database. The session concluded with anexciting presentation by Herb Lubs in which he pre-sented a hypothesis regarding the evolution of the Xchromosome and the localization of so many genes con-tributing to intelligence on this chromosome.

Vincent des Portes (Paris, France) described cloningthe breakpoints involved in an apparently balancedtranslocation [t(X;12)(q11;q15)] from a female patientwith mild mental retardation. This region is of interestbecause at least one XLMR locus has been mappedto Xq11. They identified a YAC spanning the X-chromosome breakpoint and are constructing a cosmidcontig. This will be screened for expressed sequencetags, which will then be tested for their roles in XLMRentities mapping to this region.

In a second study, des Portes and colleagues mappedan XLMR locus to Xp21.3-p22.1 using deletion analysisand constructed a cosmid contig spanning 550 kb of thecritical region. They narrowed the critical region to 20overlapping cosmid clones using DXS1218 (which is de-leted in a patient with MR) and have sequenced ap-proximately 100 kb of the critical region. When com-pleted, the map will provide a detailed map of the re-gion and should allow for the identification of at leastone XLMR gene mapping to this part of the chromo-some.

Laurent Villard (Marseille, France) and colleaguesare developing a physical map of the Xcen-Xq21 regionand identifying ESTs throughout this region. Villardpresented their progress, which included the localiza-tion of 42 ESTs, some of which were shown to identifyknown human or mouse genes (using a Blast search).They are currently being tested as candidate genes fordisorders mapping to the Xcen-Xq21 region.

Herb Lubs (Miami, FL and Tromso, Norway) pre-sented the 1998 update for XLMR genes, pointing outthat two new genes had been identified since the pre-vious workshop: the gene for Coffin-Lowry syndromeand the gene for Mohr-Tranebjaerg syndrome. In addi-tion, 18 new syndromes and 19 nonspecific XLMR en-tities were reported. The total of known disorders andMRX families was 182 as of July 31, 1997—this in-cluded information provided through the abstracts ofthe present workshop. Fifty-two of the 123 XLMR dis-orders have been mapped, and 20 disorders and 1 non-specific XLMR (FRAXE) have been cloned or the genesidentified. Particularly impressive is the fact that mu-tations in 59 families (including FRAXE) have beenmapped (lod scores >2.0). The next workshop shouldsee more localizations as investigators share familiesand resources to identify culprit genes.

David Cabezas (Miami, FL) demonstrated the cur-rent XLMR database, originally developed by Fern-ando Arena and Herb Lubs in 1988. Various aspectshave been revised, allowing a more comprehensivesearch using 717 keywords. For each disorder, infor-mation is provided on clinical findings, references, key-words, chromosomal location, access to photographs,and OMIM numbers. Maps for each disease locus arealso being developed. The database is a wealth of in-formation and is critical for determining suitable can-didate disorders for new families, narrowing the searchfor candidate genes during mapping studies, providinginformation on specific XLMR entities to families, etc.

The final presentation of the workshop was given byHerb Lubs (Miami, FL) and was fitting considering hispivotal role in the identification of the fragile X chro-mosome. Herb pointed out that not only are there moremales than females who exhibit lower IQs, but thereare also more males than females with higher IQs. Hesuggested that there are alleles of X-linked genes thatresult in high intelligence and that such alleles wouldlikely have a significant survival (and possibly repro-ductive advantage) in males, possibly contributing tothe rapid evolution of intelligence in man. It is alsopossible that there was preferential retention of suchgenes (affecting either the brain generally or intelli-gence) on the X-chromosome during mammalian evo-

234 Holden et al.

lution. Herb suggested testing this by examining link-age of high IQ scores in large families with males hav-ing high scores on subtests that are usually variable inmales. He further suggested that a comparison of thenumbers of X-linked ESTs in brain tissue relative to allX-linked ESTs or brain-specific autosomal ESTs in sev-eral species might shed light on whether genes affect-ing brain development might have been retained on theX-chromosome during evolution. Closing the loop of hisinvolvement with the fragile X syndrome, Herb sug-gested that the FMR1 gene is a good candidate for thetype of gene that might be involved in high intelligence,based on the quantitative effects of variations onFMRP levels and its likely role in brain maturation.We look forward to groups ‘‘testing’’ this hypothesis inthe next few years!

The closing discussion centered around questionsthat still needed to be addressed during the next fewyears. Among the issues that need to be considered are:1) the precise incidence of fragile X syndrome (includ-ing both full mutations and premutations) in the popu-lation (we need to examine large numbers of individu-als from several populations to determine this); 2) theclinical/psychological phenotype of girls with FMR1premutations and girls with FMR1 full mutations; 3)the clinical/psychological phenotype of premutationcarrier males (are there specific strengths); 4) the bio-logical basis for premature ovarian failure in premuta-tion carrier women; 5) the mechanism of CGG-repeatincrease from grey-zone alleles to premutation alleles;6) the nature of unstable grey zone alleles (i.e., arethere interspersions other that AGG-triplets). The nextworkshop will be hosted by Jean-Pierre Fryns and welook forward to continued discussions and perhapsmore answers to the above.

Following the workshop, some brave souls spent 3days canoeing and camping in Algonquin Park. Al-though no moose were seen or wolves heard during ourstay, the still waters of the lakes with mirrored imagesof the tall pines and spruce, the epicurean delights(simple but delicious), and the precious company (withconversations in a mixture of English, Italian, Greek,and Indonesian) made this a trip to remember. Whenwe returned to reality back home, we learned that thelost luggage of one attendee was waiting at the homeairport.

ACKNOWLEDGMENTS

We thank the Chairs of the various sessions for theirinput into the program and for their work during andafter the workshop. We thank Ted Brown and Ed Jen-kins for their assistance in securing funds from theNational Institutes of Health to support part of theworkshop activities. We thank the many reviewers ofthe manuscripts found on the following pages; theirsincere input into making this issue a success is verymuch appreciated. The workshop would not have beenpossible without the enthusiasm and excellent work ofall the staff in the Cytogenetics and DNA ResearchLaboratory at Ongwanada: Kathy Yang, Chris Trevors,and John Hucker worked all summer to ensure a suc-cessful workshop, and Gerri Pitcher, who assisted with

manuscript distributions.. We are grateful for the fi-nancial support and door prizes provided by the follow-ing: National Institute of Health (grant # R13 HD/NS35269-01), a grant from the Ontario Mental HealthFoundation, Ongwanada Resource Centre, the Depart-ment of Psychiatry and the Faculty of Medicine atQueen’s University, Surrey Place Centre, University ofToronto, the National Research Council of Canada,Perceptive Systems Inc., Vysis Inc., Fisher Scientific,BioRad Laboratories, Perkin Elmer Applied Biosys-tems, Bio Synth International Inc., Nature Genetics,Genomyx, Beta Electronics Inc., MJ Research, MaryAnn Liebert, Ilford Corporation, Sanford Corporation,Clonex, Mills Office Supplies, Northwood Press Inc.,Canadian Imperial Bank of Commerce, Rock SpringsBeverage Corporation,Warner Music, BMG MusicCanada Inc., National Grocers Limited, and MolsonBreweries.

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