moleculargenetic loss of · neuroma and meningioma in bilateral acoustic neurofi-bromatosis and the...

Proc. Nati. Acad. Sci. USAVol. 84, pp. 5419-5423, August 1987Medical Sciences

Molecular genetic approach to human meningioma: Loss of geneson chromosome 22

(brain tumors/neurofibromatosis/recessive oncogenes/DNA markers/inherited disease genes)

BERND R. SEIZINGER*t, SUZANNE DE LA MONTEt, LEONARD ATKINS§, JAMES F. GUSELLA*t,AND ROBERT L. MARTUZA¶*Neurogenetics Laboratory, tNeuropathology Laboratory, §Department of Pathology, and fDepartment of Surgery (Neurosurgery), Massachusetts GeneralHospital, and tDepartment of Genetics, Harvard Medical School, Boston, MA 02114

Communicated by Richard L. Sidman, March 30, 1987

ABSTRACT A molecular genetic approach employingpolymorphic DNA markers has been used to investigate the roleof chromosomal aberrations in meningioma, one of the mostcommon tumors of the human nervous system. Comparison ofthe alleles detected by DNA markers in tumor DNA versusDNA from normal tissue revealed chromosomal alterationspresent in primary surgical specimens. In agreement withcytogenetic studies of cultured meningiomas, the most frequentalteration detected was loss of heterozygosity on chromosome22. Forty of 51 patients were constitutionally heterozygous forat least one chromosome 22 DNA marker. Seventeen of the 40constitutionally heterozygotic patients (43 %) displayedhemizygosity for the corresponding marker in their meningi-oma tumor tissues. Loss of heterozygosity was also detected ata significantly lower frequency for markers on several otherautosomes. In view of the striking association between acousticneuroma and meningioma in bilateral acoustic neurofi-bromatosis and the discovery that acoustic neuromas displayspecific loss of genes on chromosome 22, we propose that acommon mechanism involving chromosome 22 is operative inthe development of both tumor types. Fine-structure mappingto reveal partial deletions in meningiomas may provide themeans to clone and characterize a gene (or genes) ofimportancefor tumorigenesis in this and possibly other clinically associatedtumors of the human nervous system.

Meningiomas, which arise from arachnoidal cells surround-ing the brain, are one of the most common tumors of thehuman nervous system and are a frequent cause of seizuresand other neurological dysfunction (1). Although generallyconsidered to be benign neoplasms, meningiomas may de-velop aggressive or malignant behavior with cortical invasionand multiple recurrences leading to progressive neurologicaldisability and death (2, 3). The pathogenic mechanismsunderlying tumor initiation, progression, malignancy, andrecurrence are not yet understood.

Cytogenetic studies have suggested (4-6) that the majorityof meningiomas are associated with various chromosomalabnormalities, most frequently the loss of one copy ofchromosome 22. However, virtually all of these investiga-tions have been performed on cultured tumor cells, since theoriginal tumor usually contains too few metaphases for directkaryotyping. To avoid the potential difficulty of chromo-somal alterations occurring under the selective pressure of invitro propagation, we have employed a molecular geneticapproach with polymorphic DNA markers for several chro-mosomes to investigate the genetic constitution of primarysurgical tumor specimens. Loss of genes on chromosome 22was detected in 43% of the meningiomas from which our

study collected data. In view of our observation that acousticneuromas lose genes on chromosome 22 (7) and the strikingclinical association ofmeningiomas and acoustic neuromas inpatients with bilateral acoustic neurofibromatosis (BANF)(8-11), this finding suggests a similar pathogenic mechanismin both tumor types.

MATERIALS AND METHODSDNA of high relative molecular weight was isolated fromprimary intracranial meningiomas and corresponding normaltissue (peripheral leukocytes) as described (7). With theexception of tumor M10, which was irradiated, all specimenswere obtained prior to any radiotherapy or chemotherapy.Approximately 5 ,ug of normal and tumor DNA was digestedto completion with appropriate restriction enzyme, fraction-ated by agarose gel electrophoresis, transferred to nylonmembrane, and hybridized to 32P-labeled probe DNA (7). Thefollowing probes known to reveal restriction fragment lengthpolymorphisms (RFLPs) in human genomic DNA for loci onseveral different chromosomes were used: Psisl (Oncor)(SlS) (12, 13); pMS3-18 (D22S1) (14); p22/34 (D22S9) (15);HuXC-2 (IGLC) (15); N8C6 (NGFB) (16, 17); G8 (D4S10) (18,19); Dry5-1 (DiOS1) (15); 1-101 (D1OSPDX1) (20); pEJ6.6(HRASI) (21); pJ19.4 (D11S17) (15); pHINS 321 (INS) (22);p640 (KRAS2) (20); p7F12 (D13S1) (23); pIE8 (D13S4) (23);pHUB8 (D13S5) (24); pHU26 (D13S7) (24); pAW101 (D14S1)(25); CH800 (GHI) (26); B74 (D18S3) (15); pC3 (C3) (27);pGSH8 (D21S17) (28).Autoradiograms were analyzed by scanning densitometry

with a LKB Ultrascan XL. The peak areas corresponding toeach hybridization signal were calculated by electronic inte-gration. To determine whether loss of one allele for chromo-some 22 in the tumor tissue was associated with duplicationof the remaining allele, the hybridization signals for chromo-some 22 probes were normalized to those obtained when thesame Southern blots were rehybridized with probes for locion other chromosomes. The sequence D4S10 on chromo-some 4p was used as a control locus for the tumors M8, M10,M17, M19, and M25; sequence D21S17 on chromosome 21qwas used for the tumors M5, M29, M30, M33, and M40;NGFB on chromosome lp was used for the tumors M15,M20, M31, and M39; and sequence D13S5 on chromosome13q was used for the tumors M2, M11, and M38. Het-erozygosity for RFLPs at these control loci frequentlyprovided clear information that they were not deleted in thetumor DNA. For each tissue sample the hybridization signalsspecific to chromosome 22 were normalized to those forcontrol chromosome probes. Then, a ratio of the normalizedvalues for each tumor/normal tissue pair was obtained (fordetails see ref. 7). To validate our approach of normalizing

Abbreviations: BANF, bilateral acoustic neurofibromatosis; RFLP,restriction fragment length polymorphism.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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the hybridization signals for chromosome 22 probes, wecompared (7) hybridization signals for pairs of markers forother chromosomes. These invariably gave constant ratiosfor tumor and normal DNA.For cytogenetic studies, meningiomas were dispersed with

collagenase II (Cooper Biomedical, Malvern, PA) cultured inF-10 medium (GIBCO) with 15% (vol/vol) fetal calf serum(GIBCO) plus antibiotics at 370C in an atmosphere of 95%air/5% CO2. After 3 days to 2 weeks in culture, Colcemid(CIBA-Geigy) (10 mg/ml) was added for 6 hr. The cells weredispersed with trypsin/EDTA (GIBCO), washed withHanks' balanced salt solution without calcium or magnesium(GIBCO) and centrifuged. The supernatant was decanted,and the pellet was incubated in 4 ml of hypotonic solution(KCl at 3 g/liter; EDTA at 0.2 g/liter; Hepes at 4.8 g/liter, pH7.4) with 1500 units of streptokinase (Hoechst Pharmaceuti-cals, Hounslow, Middlesex, U.K.) for 12 min, at 370C, andfixed with methanol/glacial acetic acid, 3:1 (vol/vol). After24 hr at 40C, air-dried slides were prepared using a modifi-cation ofa standard technique (29). The slides were incubatedat 650C overnight and G-banded with trypsin the followingday. Slides were microscopically evaluated, 20 metaphaseswere counted in most instances, and at least 5 metaphases ofeach specimen were karyotyped in detail.

RESULTSTo detect somatic loss of chromosome 22 sequences in thetumors, DNA was typed with four different polymorphicDNA loci: SIS, the platelet-derived growth factor p-chainlocus homologous to the sis oncogene mapping to 22q12.3-13.1; D22S1, an anonymous DNA segment at 22q12-13;D22S9, an anonymous DNA segment at 22q11; and IGLC,the X-chain immunoglobin constant-region locus at 22q11. Allfour markers, which were the only polymorphic DNA mark-ers available, map to the long arm of chromosome 22.Cytogenetic studies on meningiomas show (4-6) either loss ofthe entire chromosome 22 or partial deletions on the long armbut not on the short arm. Forty of the 51 meningioma patientsinvestigated were heterozygous in their normal tissue for atleast one of the four polymorphic DNA markers and conse-quently could be used to determine whether loss of consti-tutional heterozygosity had occurred in their respectivetumor tissue. The results of this analysis are presented inTable 1. TumorDNA from 17 of the 40 heterozygous patients(43%) displayed a loss or marker reduction in one of the twochromosome 22 alleles (Fig. 1). The remaining hybridizationsignals corresponding to the deleted alleles in some tumorsamples might be due to contaminating nontumor cells(vascular or connective tissue) that might be present inprimary biological specimens (7, 30). Alternatively, thesemeningiomas might consist of a mosaic of cells with normalkaryotype and cells with monosomy for chromosome 22. Thissituation has been observed in cytogenetic studies of culturedmeningiomas (4).

Several types of abnormal mitotic events could potentiallyaccount for somatic loss of constitutional heterozygosity intumors (31-36). To distinguish among the possible mecha-nisms, we compared the intensity of the hybridization signalsin Fig. 1 with those obtained by rehybridizing the same filterswith several probes for loci on other chromosomes. The ratiobetween tumor and normal tissue of the copy number of locion chromosome 22 was -1:2 in all 17 patients (Table 2). Thedensitometric analyses were, therefore, consistent in all 17cases with a loss of one copy of chromosome 22, rather thanwith mitotic recombination or with chromosomal loss andsubsequent reduplication of the remaining homolog. We arepresently unable to distinguish with certainty between loss ofthe entire chromosome 22 and partial deletions of onlyregions on chromosome 22 containing the four marker loci.

Table 1. Loss of heterozygosity at loci on chromosome 22in meningiomas

Marker/enzyme

Patient h

MlM2M3M4M5M6M7M8M9M1oMulM12M13M14M15M16M17M18M19M20M21M22M23M24M25M26M27M28M29M30M31M32M33M34M35M36M37

M38M39M40

SIS/ D22S1/findIll Bgl II

12 -

12 12

-0

12

- 12

12 12

- 1212 -

12 1212 12©2 -12 12

12 12

12 1212 12

12 -

12 -

2 -

D22S9/Taq I

12

1212012

1212

1212

1212

12

12

12

IGLC/EcoRI

12

12

1212

12

1200121212

12

12

121212

00

-0@0D 0

Karyotype

46, XY45, XX, -22

46, XY43, X, -Y, -14, -22

46, XX45, XX, -2246, XY/46, XY, lp+

45, XX, -22

46, XX45, XX, -2246, XX

46, XX/45, XX, -1,-4, -11, -13,+M1+M2+M3,Inv(2)

46, XX

45, XX, -4, -5, -?8(?9), +15, -22, -22,+4 TO 7 markers

DNA from primary surgical tumor specimens and from correspond-ing normal peripheral leukocytes was isolated, digested with appropri-ate restriction enzymes known to reveal RFLPs, fractionated byagarose gel electrophoresis, transferred to nylon membranes, andhybridized to 32P-labeled probe DNA for polymorphic loci on chromo-some 22.The phenotype observed in the tumor tissue is shown for every case,

where the blood DNA displayed heterozygosity. 12, heterozygosity(even though different pairs of alleles may be present for certainmultiallele markers); 1, continued presence of the larger allele restric-tion fragment and loss of the smaller allelic fragment relative to normaltissue DNA; and 2, continued presence of the smaller allelic restrictionfragment and loss of the larger fragment. Where the normal DNA wastested but was uninformative because it did not display heterozygosity,a minus sign (-) has been entered to simplify consideration of the data.The absence ofany entry indicates that a marker was not tested becauseof limited availability of DNA or did not give a readable result for theparticular tumor. Cases of hemizygosity in the meningioma tumortissues are circled. Karyotypes were available for 14 meningiomas.

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M8N T

I -2- -

MICN T

'- ' -fF2- -- s-

M17N T

2- 4

Sis - oncogene2- _2. 2-2 -_

M5N T

M8 M!oN T N T

M19NT

- .. ..

2-

2- -

M25 M39 M40N T N T N T

I- _*0 - is 1I-T

2 2- 2

2- ** 2- i

2 - -

M15 M29N T N T N T

M33NT

I- _ I- i- _ I- - I- w4

2-- 2- 2-- 2- - 2- .wI- _ I - 2I-2- -0 -

2- 40 2- _0

M17 t2 v MNtrN T N T N T

. - -_, I- *l_ -l

2-b ,

M2N T

M!ON T

2- 21- @@

2- 40 - 2- 41 -

M1I M20N T N T

2- 402 2- **It

M30 M31 M38 139N T N T N T N T

1-_* -$§2 i~-*,

2-- 2-9w 2-*9

.-.

FIG. 1. Loss of constitutional heterozygosity at loci on chromosome 22 in meningioma tumor tissue. Patient designations are shown abovethe autoradiograms. Numbers on the left indicate the observed alleles, with 1 and 2 referring to the larger and smaller allelic restriction fragments,respectively. DNA was isolated from tumor specimens and corresponding normal tissue (peripheral leukocytes), digested with appropriaterestriction enzymes, fractionated by agarose gel electrophoresis, and transferred to nylon membrane. Southern blots were hybridized to32P-labeled DNA probes for the following loci on chromosome 22: Psisl (Oncor) (SIS); pMS3-18 (D22S1); p22/34 (D22S9); HuXC2 (IGLC). Psislreveals a RFLP with fragments of 21 kilobases (kb) and 14.5 and 6.5 kb in HindIII-digested human genomic DNA (12, 13). The pMS3-18 probedetects a Bgi II RFLP with fragments of 9.5 kb and 6.5 kb (14). The probe p22/34 reveals allelic fragments of 5.8 kb and 3.2 kb in Taq I-digestedDNA (15). HuXC2 detects a multiallele RFLP with EcoRI fragments of 8 kb, 13 kb, 18 kb, and 23 kb. N, DNA from normal tissue (peripheralleukocytes); T, DNA from meningioma tumor tissue.

Note, however, that in all seven meningiomas where thepatient was constitutionally heterozygous for more than onemarker, the corresponding tumor lost heterozygosity for allchromosome 22 loci tested.Karyotype data, which were available for 14 of the 40

meningiomas in Table 1, provided support for the view thatthe loss usually involved the entire chromosome. In 6 of 7meningiomas with abnormal karyotypes, one complete copyof chromosome 22 was found to be lost. Tumor M40 hadmultiple aberrations with apparent loss of both copies ofchromosome 22 and several marker chromosomes (Table 1).(A marker chromosome is a chromosome or chromosomefragment whose origin cannot be cytogenetically defined).The molecular genetic analysis of this meningioma, however,suggested that only one copy of chromosome 22 was lostindicating that the other copy might be included in one of themarker chromosomes seen in the corresponding karyotype.With the exception of tumors M19 and M38 that displayednormal karyotypes but showed loss ofgenes on chromosome22 in the molecular genetic analysis, the karyotype data wereconsistent with those obtained with polymorphic DNA mark-ers for this chromosome (Table 1).To explore the specificity of the changes involving chro-

mosome 22, we typed DNA from the 40 meningiomas andcorresponding normal tissue with a panel of 17 randomlychosen additional molecular probes for 11 other chromo-somes. Loss of heterozygosity for one or more of these loci

was observed in 8 meningiomas and involved several differ-ent autosomes: chromosome 4 (M40; 1 of 22 informativepatients), chromosome 10 (Ml, M2, and M10; 3 of 21informative patients), chromosome 11 (M4 and M10; 2 of 31informative patients), chromosome 13 (M19; 1 of 31 infor-mative patients), chromosome 14 (M8, M10, and M29; 3 of 28informative patients), and chromosome 19 (M10; 1 of 4informative patients). Investigations of markers for chromo-some 1 (17 informative patients), chromosome 12 (6 infor-mative patients), chromosome 17 (20 informative patients),chromosome 18 (12 informative patients), and chromosome21 (15 informative patients) did not reveal any alterations.Statistical analysis (exact Wilcoxon test for ordering, ref. 37)of the marker losses indicated a significantly higher frequen-cy of reduction to hemizygosity for loci on chromosome 22 (P< 0.01) as compared to any other chromosome. It should benoted that the list of meningiomas with loss of chromosome22 (Table 1) overlaps only partially with that showing losseson other chromosomes. While several meningiomas exhibit-ed alterations only with chromosome 22 markers, two tumors(Ml and M4) without apparent loss of chromosome 22 werefound to have lost heterozygosity for loci on other chromo-somes. Certain chromosomal aberrations seen in thekaryotypes, such as the loss ofchromosome 14 in tumor M20and the loss ofchromosomes 4, 11, and 13 in tumor M37, werenot detected with informative DNA probes for these chro-mosomes. Potential explanations for this apparent discrep-

D22S1

N T

M8N T

1- qw .#

D22S9f-

2 - AW

M39! T

N I.

IGLC

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Table 2. Quantitative densitometry of probe hybridization formeningiomas with loss of heterozygosity on chromosome 22

Normalized hybridization signals

Chromosome 22/ Tumor/Patient Patient control chromosome normal

M2 Meningioma 1.76 0.45Leukocytes 3.95




M1l Meningioma 0.95 0.45Leukocytes 2.13













Southern blots, which had been hybridized to probes for chromo-some 22 (see Fig. 1), were freed of these probes in distilled water for2 hr at 650C and rehybridized with probes for polymorphic loci onother chromosomes (control chromosomes). Heterozygosity forRFLPs at these control loci usually provided clear evidence thatthese were not deleted in the tumor DNA (data not shown). Thehybridization signals on the autoradiograms were analyzed byquantitative densitometry. Hybridization signals specific to chromo-some 22 were normalized to hybridization signals for control chro-mosome probes in the same sample. Then, a ratio of the normalizedvalues for each tumor/normal tissue pair was calculated. Thereliability of this approach has been demonstrated (7).

ancy are that these changes have been present in a smallsubset of the tumor cells that display a growth advantage inculture or that they may have occurred in vitro. There was noevidence for gene amplifications in the genetic analysis ofanyof the 40 meningiomas, nor did any of the karyotypes showdouble minutes or homogeneously staining regions.Of the 40 meningioma patients, 13 (33%) were male, and 27

(67%) were female. Ages ranged from 19 to 77 years (mean ±SEM, 52 + 3.2 years). No correlation was found between theage and sex of the patient and any chromosomal alterationsincluding those on chromosome 22. Furthermore, there wasno correlation between any chromosomal changes and theclassical histological subtypes of meningiomas (38). With theexception of M31, all meningiomas represented sporadiccases without apparent family history of similar tumors.Patient M31, whose meningioma tumor tissue showed loss ofchromosome 22, was diagnosed with bilateral acoustic

neurofibromatosis according to National Institutes of Healthcriteria (39). Unfortunately, acoustic neuroma tissue was notavailable for this patient. Among the nonfamilial cases, lossof alleles on chromosome 22 was seen in both benign and"malignant" meningiomas. Five of the 40 tumors that wereinformative for chromosome 22 markers represented recur-rent cases of atypical or malignant meningiomas showingaggressive clinical behavior. In the following cases, tumorrecurrence necessitated multiple operations subsequent tothe initial surgery: M2, two operations in 3 years; M8, twooperations in 3 years; M10, three operations in 3 years; M11,four operations in 3 years; and M19, two operations in 1 year.For this investigation, only tissue from the most recentoperation was available from each of these cases. All fivemalignant meningiomas displayed loss of genes on chromo-some 22. Among the other 35 meningiomas, 12 tumorsshowed loss of chromosome 22 alleles, but these patientshave not been followed for a sufficient period (all <2 years)to exclude the possibility of tumor recurrence. The appar-ently higher frequency of chromosome 22 loss in malignantmeningiomas raises the possibility of a role for this geneticaberration in the development of aggressive features orrecurrence in this tumor type. However, investigation of amuch larger number of tumors from patients followed over alonger period will be needed to clarify this important issue.

DISCUSSIONWe have used a molecular genetic approach to show thatmeningiomas frequently display loss of genes on chromo-some 22, although this is not the only genetic abnormalitydetected. Our data indicate, in general agreement withkaryotype studies on cultured tumor cells (4-6), that loss ofgenes on chromosome 22 is significantly more frequent thanalterations on other chromosomes in primary tumor speci-mens. Thus, the reduction to hemizygosity on chromosome22 may be one important step in tumorigenesis of menin-gioma. Since it is observed in both benign and malignantmeningiomas, this mechanism might operate at the primarylevel of tumor initiation. Whether the additional chromo-somal changes might represent alternative causes of tumorformation or contribute to progression or malignancy remainsto be determined. Similarly, investigation of meningiomaswith probes for other chromosomes not included in thepresent investigation might reveal additional chromosomeloss relevant to tumor development.The majority of the tumors in the present study were

sporadic meningiomas from individuals with no evidentfamily history of nervous system tumors. However, one ofthe meningiomas that lost heterozygosity on chromosome 22was obtained from a patient with BANF, an inheritedneurological disorder characterized by neoplasia of cells ofneural crest origin. Its hallmark is the bilateral formation ofacoustic neuromas and a high susceptibility to a variety ofother nervous system tumors, particularly to meningioma(8-11). We have shown (7, 40) that both familial and sporadiccases of acoustic neuroma display specific loss of genes onchromosome 22, indicating the possibility of a commonmechanism of tumorigenesis for meningioma and acousticneuroma. Furthermore, this finding suggests that the defec-tive gene causing BANF may reside on chromosome 22. Wehave identified (40) acoustic neuromas in which only aportion of chromosome 22 was deleted, narrowing the pos-sible location of the gene for BANF to the region distal toband 22q11 of the D22S9 locus. The identification of pro-gressively smaller deletions on chromosome 22 in tumorsassociated with BANF, including acoustic neuromas andmeningiomas, may well provide a means to clone andcharacterize the defect.

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It is noteworthy that two meningiomas with loss of geneson chromosome 22 in the molecular genetic analysis (M19and M38) displayed a normal karyotype. This might beexplained by a selective growth of cell clones with normalkaryotype over those with monosomy 22 or overgrowth ofnontumor cells, such as fibroblasts under in vitro cell cultureconditions prior to karyotyping. Alternatively, these twomeningiomas might well represent cases with small deletionson chromosome 22, undetectable by cytogenetic analysis.The application of additional high-quality DNA markers frommany different regions on chromosome 22 will facilitate thedetection of such tumors and help to define the limits of thedeleted regions.The human genome clearly contains a number of loci where

mutations or deletions predispose to the development ofcertain sets of tumors (41). They have been termed "reces-sive oncogenes" or "anti-oncogenes" (41). For example, theRBI gene on chromosome 13q is probably not only importantfor tumorigenesis of retinoblastoma but also for that ofosteosarcoma, which commonly arises as a second cancer inchildren who survive retinoblastoma (42, 43). Three embry-onal cancers, Wilms' tumor, rhabdomyosarcoma, and hepato-blastoma have been shown to cluster in the Beckwith-Wiede-man syndrome and were found to be associated with loss ofgenes on chromosome 11p, suggesting a common pathogenicmechanism (44). However, molecular genetic studies withadditional markers have revealed (45) that the deleted regionson chromosome 11p in rhabdomyosarcoma are in closeproximity, but not identical, to the Wilms' tumor locus.Despite their strikingly frequent clinical association inBANF, it is not yet clear whether meningioma and acousticneuroma arise from a mutation or deletion at the same locuson chromosome 22 or whether they result from alterations attwo distinct loci. Fine structure molecular genetic analysis ofchromosome 22 deletions in both tumor types will be neededto settle this issue. Such studies could ultimately lead to theidentification of the specific gene or genes responsible fordevelopment of these tumors, thereby providing a window onthe fundamental genetic mechanisms controlling both normaland abnormal development of the human nervous system.

We thank Dr. N. T. Zervas, Dr. R. 6. Ojemann, and theNeurosurgical Staff for providing specimens; and A. Lane, K. Riley,K. Dashner, and J. Logan for technical assistance. We are gratefulto the following scientists for providing DNA probes: Drs. A.Ullrich, J. Habener, T. Dryja, G. Bell, C. Shih, R. Weinberg, R.White, B. White, H. Goodman, J. L. Mandel, G. Fey, G. Stewart,M. Litt, and P. Leder. B.R.S. is supported by a fellowship and a grantfrom the National Neurofibromatosis Foundation. J.F.G. is a SearleScholar of the Chicago Community Trust. R.L.M. is recipient of theNational Institute of Neurological and Communicative Disorders andStroke Teacher-investigator Career Development Award NS00654.Funds for this work were provided by Grants NS00654, NS22224,and NS20025 from the National Institute of Neurological andCommunicative Disorders and Stroke and by a grant from theMcKnight Foundation.

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