Mutations PTC and GAS1 Bladder Cancer and the ...

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Evidence for Two Tumor Suppressor Loci Associated with Proximal Chromosome 9p to q and Distal Chromosome 9q in Bladder Cancer and the Initial Screening for GAS1 and PTC Mutations Anne R. Simoneau, Charles H. Spruck III, Mirella Gonzalez-Zulueta, et al. Cancer Res 1996;56:5039-5043. Published online November 1, 1996.

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[CANCER RESEARCH56. 5039-5043. November 1, 19961

Evidence for Two Tumor Suppressor Loci Associated with Proximal Chromosome 9p to q and Distal Chromosome 9q in Bladder Cancer and the Initial Screening for GAS1 and FTC Mutations1 Anne R. Simoneau, Yvonne


C. Tsai, Michael

H. Spruck



Ill, Mirella Gonzalez-Zulueta, Steven,



Mark L. Gonzalgo,

and Peter


F. Chan,

A. Jones2

Urological Cancer Research Laboratory. University of Southern California/Norris Comprehensive Cancer Center, University of Southern California School of Medicine, Los Angeles, California 90033 [A. R. S., C. H. S., M. G-Z, M. L G., M. F. C.. Y. C. T., P. A. ii; Human Genetics Section. Laboratory of Viral Carcinogenesis, National Cancer Institute, Frederick Cancer Research and Development Center, [email protected] Maryland 21702 (M. DI; and Herlev Hospital. University of Copenhagen, DK-2730 Herlev, Denmark (KS.. T.H.J


ABSTRACT The most common genetic alteration identified to date in bladder cancer Is loss of heterozygosity

(LOH) of chromosome

9, suggesting


presence of possible tumor suppressor genes on this chromosome. We attempted to map the location of these genes by analyzing 69 primary transitional cell carcinomas of the bladder with a panel of microsatellite markers

for LOH on chromosome

9. Monosomy

Our understanding of the genetic alterations underlying TCC3 of the bladder has expanded significantly over the last decade. LOH on chromosome 9 is the most common genetic alteration identified in bladder tumors and is present in all stages and grades. Approximately

60%of superficialandinvasiveTCCsshowLOHof markersonthis

9 (defined by LOH of all

chromosome; thus, chromosome 9 allehic losses are considered to be informative markers analyzed on 9p and 9q) was detected in 26 of 69 early events in bladder tumorigenesis (1—8).In contrast, mutations in (38%) tumors, and 22 of 69 (32%) tumors showed subchromosomal the p53 tumor suppressor gene and LOH of chromosome lip occur in deletions. Twelve tumors (17%) demonstrated partial LOH of chromo more advanced and aggressive bladder tumors and are thought to be some 9 and indicated two distinct regions of LOH. Eight tumors showed secondary events important in progression (1—4,6, 9, 10). An excep distal allehic loss of 9q with a minimal region of common deletion flanked tion is primary carcinoma in situ, in which p53 mutations are frequent, proximally by marker GSN on 9q33. Six tumors showed proximal allele and LOH of chromosome 9 is rare (1 1). Bladder cancer progression loss of 9p and 9q with a minimal area of common deletion flanked by may therefore proceed through two molecular pathways to invasive markers D9S970 on 9p12 and D9S283 on 9q21. Two tumors showed loss TCC (1 1, 12). Squamous cell carcinomas of the bladder may have ofboth the distal region of9q and the proximal region of9p and 9q, which different molecular origins with p16 on 9p playing more prominent were separated by a possible 6—44 cM of retained genetic material. The roles ( 13) than in primary TCC (14). Other genetic alterations detected proximal minimal area ofcommon deletion excluded 9q22.3—q31to where in invasive TCCs include allelic losses of chromosomes 3, 5, 6, 8, 11, two putative tumor suppressor genes, the nevold basal cell carcinoma 13, and 18, which are thought to be important in progression (1, 4, 12, syndrome and multiple self-healing squamous epitheioma (ESSJ) genes, 15—18). have been [email protected] The growth arrest-specific gene (GASI), a candidate Mapping the precise location of the putative tumor suppressor tumor suppressor gene, was Included within the proximal minimal region. We evaluated the GASJ gene for its potential role in bladder cancer using gene(s) on chromosome 9 involved in bladder tumorigenesis has single-strand conformational polymorphism to screen for mutations in proven to be difficult because of the high frequency of monosomy 9, GAS1 In 10 bladder cancer cell lines and 14 primary bladder tumors. A infrequent partial chromosome 9 losses, and no clear familial form of polymorphism at codon 88 was noted In one primary bladder tumor, but bladder cancer for pedigree analysis. Several reports have presented no other abnormalities were found, suggesting that another potential evidence for at least two tumor suppressor genes based on LOH tumor suppressor gene Important to bladder cancer resides in these analysis of chromosome 9. One or more tumor suppressor genes are minimally deleted regions. Because the nevoid basal cell carcinoma syn believed to be located on 9p, with other genes possibly located on 9q drome gene has long been speculated to be a putative tumor suppressor (19, 20). The minimal area of deletion on 9p has been mapped to a geneInbladdercancerandthisgenehasrecentlybeencharacterized as 2-cM region between markers D9S171 and IFNA on 9p2l (2 1—23). the human homologue of the Drosophilapatched gene (Pit'), 20 primary This region includes the recently identified tumor suppressor gene p16 bladder tumors with chromosome 9q LOH were screened for mutations in (24); however, the locations of other putative suppressor gene(s) on PTc usingsingle-strandconformationalpolymorphismandheteroduplex chromosome 9 remain elusive. Most studies have localized the prox analysis. No alterations were found in any of the samples analyzed. Furthermore, 4 of37 noninvasive papifiary (Tg) tUInOSSdemonstrated loss imal marker of a common area of deletion to D9S18 on 9pl3—ql3 or of all 9q markers with retention of 9p, whereas no Ta tumor showed loss ASSP3 on 9q13. The distal marker varies from D9S22 on 9q22 to loss of 9p with retention of all 9q markers, suggesting that LOH of 9q is the of the entire q arm (4—7,19, 20, 25). Furthermore, because monosomy earlier event In bladder tumorigenesis.In summary, our results indicate is the most common alteration of chromosome 9, it has been difficult two tumor suppressor lod associated with proximal chromosome 9p to q to demonstrate which event occurs first, loss of 9p or loss of 9q. We have examined 69 primary TCCs for allelic loss of microsat and distal chromosome9q that may be Important in bladder cancer. GASJ ellite markers spanning chromosome 9 to further map the putative and PTC do not seem to be frequenfly mutated in bladder cancer. tumor suppressor gene(s) on this chromosome. Two putative tumor suppressor loci associated with chromosome 9 were identified. One Received6/20/96;accepted9/4/96. The costs of publication of this article were defrayed in part by the payment of page was mapped to a common minimally deleted area between D9S970 charges. This article must therefore be hereby marked advertisement in accordance with and D9S283 on 9pl2—q2l, and a second deleted region was observed 18 U.S.C. Section 1734 solely to indicate this fact. telomeric to 9q33. We also screened for mutations in two candidate I Supported by Orant R35 CA49758 from the National Cancer Institute. A. R. S. is supported in part by a fellowship awarded by the American Foundation for Urological tumor suppressor genes, GAS] and PTC, that have been suggested to Disease via funds contributed by Microvasive. M. G-Z. was a recipient of a fellowship from the Government of Navarra, Spain. 2 To





California/Norris Comprehensive Angeles, CA 90033.








3 The

Cancer Center, M/S 83 1441 Eastlake Avenue, Los


used are: TCC,





loss of heterozy

gosity; NBCCS, nevoid basal cell carcinoma syndrome; SSCP, single-strand conformation polymorphism.


Downloaded from on July 21, 2011 Copyright © 1996 American Association for Cancer Research


be of potential importance in bladder cancer. Evidence is also pre sented that suggests that the loss of 9q occurs earlier than the loss of 9p in the formation

of noninvasive



MATERIALS AND METHODS Mapping of Chromosome 9. Sixty-nine primarybladder TCCs of all stages and grades were analyzed.


T,/Pa (papillary


11 T1/P1 (lamina propria invasive), and 21 T2-T4/P2-P4(muscle invasive) tumors








embedded transurethral resections of bladder tumor specimens. Normal and tumor tissues were microdissected obtained

as described




(I 1). All attempts cells for analysis,

8—[email protected], and DNA were made to micmdissect



only the

these cells from the benign

muscle and connective tissues present on the same section. Additionally, DNA was extracted as described from 16 specimens obtained from radical cystec tomy (I). For these specimens, DNA from the patient's blood lymphocytes was used

as the normal






the University


Southern California/Norris Comprehensive Cancer Center (Los Angeles, CA) and the Herlev Hospital (University ofCopenhagen, Herlev, Denmark). Thirty two specimens had been used for previously reported studies involving LOH

of chromosomes 9, 11, and 17 and analysis of pitS (6, 11, 13, 14), and 37 specimens

were new cases.

The 69 tumors were examined using 5— 8 microsatellite markers on chro mosome 9 (D9S171 and D9S156 on 9p and D9S15, D9S153, D9S12, D9S176, GSN, and D9S63 on 9q). The results obtained with the D9S12 marker were

markers were used to better define a proximal region ofLOH on 9p21-9q2l-22 in six tumors (D9S169, D9S736, D9S1749, D9S1747, D9S1748, D9S200, D9S970, D9S55, and D9S942 on 9p and D9S166, D9S201, D9S739, and D9S283 on 9q; Ref. 26). Primer sequences were retrieved from the Genome Medical


The Johns Hopkins



MD). PCR amplifications were performed in 25-pAvolumes containing 200 p.M of dATP,

dGTP, dUP,

and dCTP;

I X buffer;

I @tMprimers; 0.2 MCi

[[email protected];and 0.625 unit of Taq DNA polymerase (Boebringer Mannheim, Indianapolis,

IN). Annealing temperatures

varied from 51—61°C,and cycle

number varied from 27—30,depending on primer sets. PCR products were resolved

on 5—8% pohyacrylamide/7


were assessed

M urea gels, depending


and informative

on allele size. The cases were scored


ahlehicloss when intensity of the signal for a tumor allele was significantly reduced


to the matched



SSCP Analysis of GASJ and PTC. Ten bladder cancer cell lines (J82, RT4,T24, TCCSUP,HT1376,andUM-UC-3fromtheAmericanTypeCulture Collection and LD71, LD137, LD583, and LD605 generated in our laboratory) were screened by SSCP for mutations in GASJ. Isolated total RNA (10 Mg) from these cell lines was used as a template for first-strand synthesis of cDNA. The GASJsequence (27) was divided into six fragmentsfor PCR amplifica t.ion. The 12 primer


and PCR conditions

are available

upon request.

PCR products were analyzed on 6% polyacrylamide gels containing 10% glycerol at 30 W constant power at room temperature. Fourteen primary bladder tumors that were known to have LOH of chro mosome 9 were screened for GAS! mutations by SSCP. Purified DNA from the primary specimen was used as the template for PCR amplification. Con ditions

were the same as for the cell lines. One primary

The results obtained from the chromosome 9 allelic loss analysis of 69 TCCs are shown in Table 1. Overall, 30% (21 of 69) of the tumors retained all informative markers on both chromosome arms. A total of 38% (26 of 69) of the tumors showed LOH of all informative markers examined, indicating monosomy 9. A total of 12% (8 of 69) of the tumors showed LOH of all informative markers on 9q with retention of markers on 9p, and 3% (2 of 69) had LOH of the 9p markers with retention of all informative 9q markers. A total of 17% (12 of 69) of the tumors showed partial LOH of chromosome 9, based on the informative loci. Table 1 further demonstrates the accumulation of chromosome 9 losses in more invasive tumors. The largest percentage of partial deletions (22%) was observed in noninvasive Ta tumors, which was greater than that observed in superficially invasive T1 tumors (18%) or muscle-invasive T2-T4 tumors (10%). Conversely, monosomy 9 was more common in the invasive tumors. Although Ta tumors represented the largest portion of TCCs examined (54%), none of the Ta tumors



Life Science,


OH) to confirm

8, 9, and 11 retained


the results


all 9p markers




in which



[email protected]' 15/37(41%) 2/11(18%)

lowed-up with sequencing of the PCR product.

Twenty primary bladder tumors that were known to have LOH of chromo some 9q were also screened for PTC mutations by SSCP and heteroduplex analysis using conditions and primers described previously (28—31).Primer sequences for PTC analysis can be obtained from M. D.4

9 monosomyc 9q 1055d 9p losse

10/37 (27%) (1 1%) 0/37 (0%)



5/1 1 (46%) 1/1 1 (9%) 1/1 1 (9%)

11/21 (52%) 3/21 (14%) 1/21 (5%)

Partial 9 losi 8/37 (22%) 2/11 (18%) 2/21 (10%) a Table is based on data from all informative markers. 9 retention represents no LOH of any markers on 9p or 9q. C







d 9q loss represents LOH of all informative e 4 E-mail:

for 12 tumors



Table 1 Sumtnary of allelic-loss analysis in 69 TCCs'@

and the sensitivity of the SSCP screening. One shift in the SSCP was fol @

of markers

(located 5 kb upstream from exon 1 of the pitS gene), suggesting that gross p16 deletion may not be present in these tumors. Tumor 11 showed retention of the majority of 9p with proximal and distal 9q losses (Fig. 2). Interestingly, tumors 10 and 11 demonstrated both proximal and distal areas of LOH while retaining markers between the two possible tumor suppressor loci. The putative tumor suppressor gene GAS], which lies within the proximal minimally deleted region, was screened for mutations by SSCP in a total of 10 bladder cancer cell lines and 14 primary tumors. The data for this SSCP screening are not shown, but a band shift was observed in only one primary bladder tumor. Sequencing of the

tumor with a wild-type its wild-type

on 9p with retention

LOH of 9q was detected. Fig. la shows data for eight tumors that lost distal 9q markers located on the tip of the q arm but retained one or more proximal 9q marker. Tumors 4, 11, and 15 showed LOH of D9S63 with retention of GSN; therefore, the common region of LOH for these tumors was OSN-telomeric. Fig. lb shows the results for six tumors that had LOH of proximal 9q markers with retention of distal 9q markers. Tumor 11 showed retention of D9S283, which narrowed the distal extent of LOH to 9q2l.3, and also excluded the region of 9q22.3 in which the putative tumor suppressor gene NBCCS resides. Tumors 8 and I 1 showed retention of D9S970 on 9pl2, which narrowed the proximal extent of LOH, and excluded the region of 9p2l in which p16 resides. Tumors

banding pattern was sequenced using the PCR template and Sequenase 2.0 kit (Amersham

of markers

on 9q, whereas two invasive tumors showed loss of only 9p. In contrast, four Ta tumors showed loss of markers on 9q with retention of the markers on 9p. Fig. 1, a and b, illustrates that of the eight Ta tumors with partial 9q deletions, four had concomitant LOH of 9p markers (tumors 4, 5, 6, and 10), and four did not (tumors 8, 11, 13, and 14). Fig. 1, a and b, shows

difficult to interpret due to an excessive number of stutter bands; thus, many tumors were not evaluated at this locus. Thirteen additional microsatellite

Data Base (Welch


loss represents


of informative





21/69(30%) 26/69(38%) 8/69(12%) 2/69 (3%)



9q markers with retention of 9p markers.

9p marker(s)



of all 9q markers.

1Partial 9 loss represents LOH of some 9 markers with retention of other 9 markers.

[email protected]


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Tumor #



Th 12 lii Th 1k 1k TI 1k

15 11



10 13 12 14

Tumor # 7




10 11

P1 1k P2 Pa 1k 1k


E[[email protected] ILJ]JILI ILIJIE[

[email protected] IEL:IEEJrn


rnLJDuUrn 9q



99nur Fig. 1. TCCs with partial LOH of markers on 9, demonstrating two regions for putative tumor suppressor genes for bladder cancer. Twelve tumors showed partial LOH of 9. a, eight tumors lost distal 9q but retained informative markers more proximally. Three of these tumors (4, 11, and 15) had LOH of D9S63 with retention of OSN on 9q33. evidence that a putativetumor suppressorgeneis telomericto GSN. b, six tumorslostproximal9q markersbut retainedinformativemarkersmoredistally.The commoninterstitialdeletionis flanked

by D9S970 on 9pl2 and 095283 on 9q2l. This is best illustrated by tumor II and demonstrates a second region for a putative tumor suppressor gene. 0, retained; U, LOH; speckled box, not informative; horizontal lines, not done.

fragment showed a bp change at codon 88: GCC —@ GCT (Ala ‘—p Ala) that was also present in the corresponding WBC DNA, suggesting that it was a polymorphism. Therefore, no acquired mutations in this gene were detected in the 24 samples examined. The PTC gene was also screened for mutations because it has long been speculated that the same gene responsible for NBCCS may be involved in bladder carcinogenesis although it maps to 9q22.3, which lies outside either region of minimal deletion. The PTC gene consists of at least 23 exons spanning approximately 34 kb (28, 32, 33). Twenty primary bladder tumors with chromosome 9q LOH were screened by SSCP and heteroduplex analysis; however, no alterations were found in any of the samples analyzed (data not shown). DISCUSSION Much recent research has focused on the role of 9p deletions and the p]6 gene in particular in bladder and other cancers, but 9q deletions have received less attention. Our focus was to further map a common minimal region of LOH on chromosome 9 that could lead to the localization of putative bladder tumor suppressor genes. Overall, 38% (26 of 69) of tumors showed a loss of all informative markers, classified as monosomy 9, and 32% (22 of 69) of tumors had sub chromosomal or partial deletions. These values are consistent with a published report on TCCs in which 32% (30 of 95) had probable monosomy 9, and 22% (19 of 95) had subchromosomal deletions by

microsatellite analysis (20). Perhaps the slightly higher percentage of partial LOH we report was due to our analysis of a larger proportion of Ta tumors (54%). A total of 17% of tumors (12 of 69) showed partial LOH of markers on chromosome 9, and two distinct regions of LOH were apparent. Eight tumors demonstrated LOH of marker D9S63 on 9q34, the most telomeric marker used in this study, and three of these tumors retained the adjacent marker GSN on 9q33 (Fig. Ia). Thus, GSN on 9q33 flanks a possible location for a putative tumor suppressor gene on the tip of 9q. Loss of the ABO antigen

had been suggested

as a marker


progressive bladder cancer (34). ABO is approximately 6 cM telo meric to D9S63 (26), and Orlow et a!. (8) also reported a high frequency of LOH (45%) associated with the 9q34. 1-2 band in human bladder tumors. The tuberous sclerosis gene (TSC]) has been consistently reported to reside distal to D9S149 and proximal to D951 14 around the ABO locus on 9q34 (26). The hereditary hemorrhagic telangiectasia gene (HHT) has been mapped between D956l and D9S63 on 9q33-34 (35), and both of these genes are involved in syndromes of angiodysgen esis. McDonald et a!. (35) speculated that HHT may be a tumor suppressor gene responsible for an antiangiogenesis factor. Tuberous sclerosis consists of angiofibromas and angiomyohipomas in 40—80% of patients and has a higher-than-average incidence of renal cell carcinoma (36). Angiogenesis is vital for solid tumor development,


Downloaded from on July 21, 2011 Copyright © 1996 American Association for Cancer Research




primary bladder tumors screened by SSCP is further supporting evi dence that these two tumor types do not share the same putative tumor suppressor gene. Other genes excluded include ESS] (multiple self healing squamous epithelioma), XPAC (xeroderma pigmentosum complementation group A), and FACC (Fanconi's anemia comple mentation group C (26). However, the exclusion of these genes, which have been considered possible candidate genes, is based on the anal ysis of one tumor, so further work is needed before this can be

@;I1I @;[WI


F conclusively shown.

Bernuéset a!. (42) have reported on a case of bladder cancer with a deletion of this proximal region, del (9) (ql lq2l.l), by karyotyping, and several candidate suppressor genes have been mapped to proximal 9q. We concentrated on the human growth arrest-specific gene, GAS], because it has been mapped to 9q2l.3— q22 and shown to be proximal to D9S12 (27, 43). GASJ is a putative tumor suppressor gene that may be involved in bladder carcinogenesis. In particular, GAS] has been shown to inhibit DNA synthesis (44), and levels of GAS] expression were found to increase in quiescent cells with a subsequent decrease during progression through the cell cycle (45). Furthermore, overexpres sion of GAS] had been reported to block cell proliferation in the bladder cancer cell line T24 but not in an osteosarcoma or adeno

I I I. C,,



z 1-'



—I I





virus type 5-transformed cell line (27). Importantly, it seems GAS] was responsible for the induction of the temporary resting state of cells that retain replicative capabilities and was not associated with the permanent exit from the cell cycle (46). Bladder cancer cell lines were screened by SSCP to determine if mutations in GAS] could be found, and RT-PCR analysis (data not shown) gave indirect evidence that GAS] was expressed in these cell lines. No mutations were detected by SSCP screening of I0 bladder cancer cell lines and 14 primary bladder tumors with chromosome 9 LOH. Although there are limitations to the ability of SSCP to detect all mutations, the method did successfully identify a naturally occur ring polymorphism. We conclude that GAS] does not seem to be a frequent target for mutation in bladder cancer. Two tumors demonstrated both regions of LOH on 9q separated by a retained area of possibly 6—44cM (Fig. I, a and b). Cairns et a!. (21) have reported that an area of retention flanked by LOH may not be a retained locus but may represent an area of homozygous deletion due to the presence of contaminating normal cells. However, that possibility seems unlikely in these two tumors because the region was so large. There are most likely multiple genes on 9q and 9p involved in bladder cancer. Keen and Knowles (20) have reported on two possible target sites on 9p based on one tumor, and our results indicate the existence of at least two further suppressor loci involving chromo some 9. It is interesting that the clinical scenarios associated with LOH of 9q involve epithelial tumors; NBCCS, ESS 1, XPAC, bladder cancer, and recently other epithelial tumors, squamous carcinomas of the head and neck, non-small cell carcinoma of the lung, and epithelial


Fig. 2. Microsatellite patterns for tumor I I demonstrating LOH of both proximal and

distal portions of chromosome 9. 09563 shows loss of the top allele, and D9S153 shows loss of the bottom allele. The other loci demonstrate retention of both alleles.

and angiogenesis as correlated by microvessel density has been shown to be an independent predictor of disease progression in bladder cancer (37); thus, it is possible that a gene regulating angiogenesis could be a tumor suppressor gene candidate. A pu tative cyclin-dependent kinase, PITALRE, has also been mapped to 9q34. 1 (38), although its role in controlling cell proliferation is not ovarian cancer have shown 9q losses in 35—54% of tumors analyzed yet clear. (47—50). A second location for a putative tumor suppressor gene in bladder It is still not clear which of the losses (9p or 9q) occurs first during cancer is defined by D9S970 on 9p12 and D9S283 on 9q21 . Six tumorigenesis. Our results suggest that papillary tumor formation may tumors demonstrated LOH of markers proximal to 9q2l with retention of one or more markers distal to 9q2l . This region excluded the occur through preferential LOH of 9q because there were no Ta previously defined locus at 9p21 where p]6 resides and has proven to tumors with LOH of only 9p, but four Ta tumors showed 9q loss with 9p retention. Thus, the inactivation of a gene located on 9q may be an be important in TCC (13, 14). Also excluded from this second region early event in bladder tumorigenesis, but the sample numbers are of LOH is the NBCCS gene, now identified as PTC. PTC has long small, and further analysis will be necessary to determine the order (if been considered a candidate tumor suppressor in bladder cancer (39—41).The PTC gene maps to 9q22.3 outside the defined area of any) in which these putative tumor suppressor genes may be macti proximal deletion, and the finding of no alterations of PTC in 20 vated. 5042

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Analysis of the p16 gene (CDKN2) as a candidate for the chromosome 9p melanoma susceptibility locus. Nat. Genet., 8: 22—26,1994.

We thank Bernard Gerrard for technical assistance with the PTC gene. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.

25. Cairns, P., Shaw, M. E., and Knowles, M. A. Preliminary mapping of the deleted region of chromosome 9 in bladder cancer. Cancer Res., 53: 1230—1232, 1993.

26. Pencak-Vance, M. A., Bale, A. E., Haines, J. L, Kwiatkowski, D. J., Pilz, A.,

Slaugenhaupt, S.,White,J. A.,Edwards,J. H.,Marchuk,D.,Olopade,0. 1.,Attwood,J., and Povey, S. Report on the Fourth internatiOnal Workshop on Chromosome 9 held at Williamsburg, Virginia, USA, April 23-25, 1995. Ann. Hum. Genet., 59: 347-365, 1995.

27. Del Sal, G., Collavin, L, Ruaro, M. E., Edomi. P., Saccone, S., Della Valle. G., and Schneider, C. Structure, function. and chromosome mapping of the growth-suppress

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