8 chromosome translocation breakpoint

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of one or more 3p tumor suppressor genes. ... renal carcinoma due to a reciprocal 3;8 chromosomal trans- ..... (1989) Genes Chromosomes Cancer 1, 95-105. 9.
Proc. Natl. Acad. Sci. USA Vol. 90, pp. 8509-8513, September 1993 Genetics

Positional cloning of the hereditary renal carcinoma 3;8 chromosome translocation breakpoint (suppresor gene/fagile site/polycystic kidney diease/lung cancer/thyroid cancer)

FERENC L. BOLDOG*, ROBERT M. GEMMILLt, CHARLES M. WILKES, THOMAS W. GLOVER*, ANN-SOFIE NILSSON*, SETTARA C. CHANDRASEKHARAPPA§, ROBERT S. BROWN¶, FREDERICK P. Lill, AND HARRY A. DRABKIN*t *The University of Colorado Cancer Center, Division of Medical Oncology, B171, 4200 East Ninth Avenue, Denver, CO 80262; tThe Eleanor Roosevelt Institute, 1899 Gaylord, Denver, CO 80206; *Departments of Pediatrics and Human Genetics,and of §Human Genetics and Human Genome C-nter, University of Michigan, Ann Arbor, MI 48109; IBeth Israel Hospital, Harvard Medical School, Boston, MA 02215; and 'iDana-Farber Cancer Institute and Harvard School of Public Health, 44 Binney Street, Boston, MA 02215

Communicated by Theodore T. Puck, June 21, 1993

at the genomic level has been detected in studies that included a pulsed-field analysis extending 1 Mb in the direction of the translocation breakpoint (20). The 3p14.2 region is felt to be the critical component of the 3;8 translocation based on several observations (see Discussion) and might be expected to contain a tumor suppressor gene. In this report, we describe the positional cloning of the 3p14.2 breakpoint and the isolation of cDNA clones adjacent to the site of rearrangement. The translocation also occurs in close proximity to an aphidicolin-induced 3pl4 breakpoint.

The chromosome (p14.2;q24.1) translocation ABSTRACT t(3;8)has been ated with hereditary renal cancer In one family. Based on cytogenetic analyses and loss-of-heterozygodty experiments, the 3pl4 region has been independently implicated as harboring a tumor suppressor gene critical to kidney and lung cancer development. The 3p14.2 region also contains FRA3B, the most senitive fragile site induced by aphidicolin. A chromosome 3 probe, R7K145, derived from a radiation-reduced hybrid was poitioned between the t(3;8) breakpoint and an aphidicolin-induced 3pl4 breakpoint. A yeast artificial chromosome (YAC) contig containing R7K145 was developed that crossed the aphldlcoin-induced breakpoint on its telomeric side. A subsquent chromosome walk identified a YAC that crossed the 3;8 translocation breakpoint. A A sublibrary allowed Isolation of clones snninag the rearrangement. Unique and evolutionarily conserved DNA sequences were used to screen a kidney cDNA library. We have identified a gene, referred to asHRCAI (hereditary renal cancer associated 1), that maps immediately adjacent to the breakpoint. On the basis of its chromosomal position, HRCAI may be a candidate tumor suppressor gene.

MATERIALS AND METHODS Cell Lines. Somatic cell hybrids containing the der(3) and der(8) chromosomes from the 3;8 translocation have been described (19). Hybrid TL12-8 contains the der(3) chromosome (8qter-8q24.1: :3p14.2-3qter) as its only cytogenetically identifiable human material, whereas hybrid 3;8/4-1 contains the der(8) chromosome (8pter-8q24.1::3p14.2-3pter) in the absence of the normal 3, 8, or der(3) chromosome. Prior to DNA preparations, the hybrids were subcloned and examined cytogenetically. Hybrid A5 contains an aphidicolininduced terminal deletion of 3p with a breakpoint at approximately 3pl4 (21). This hybrid was derived from UCTP2A3, which contains an intact human chromosome 3 as its only identified human material (22). Routine subculturing of A5 cells resulted in two clones that were used in subsequent experiments. Clone A5-4 appeared identical to the original A5 hybrid. Clone A5-5 contained CHO chromosomal material spontaneously translocated onto the end of the deleted human 3p arm. No other changes to the human chromosome 3 were cytogenetically apparent. However, molecular analysis of clone A5-5 showed that some 3p material had been lost. The hybrid 3 ;6/UC2 retains the der(3) chromosome (6pter-6p11::3p14.3-3qter) present in a constitutional translocation associated with hereditary hematological malignancies (23). The kidney-derived cell lines shown in Fig. S were obtained from the American Type Culture Collection. DNA Probes. The development of a chromosome 3 somatic cell hybrid mapping panel and a large series of localized probes has been described (22). Probe R7K145 was isolated from a partial Mbo I digest genomic library constructed in EMBL3 by using DNA from a radiation-reduced hybrid, R7K1-6. This hybrid was found to contain the markers MS156 and MS453 (22), which mapped to the general region of the 3;8 translocation. Genomic probes derived from the 850A6 yeast artificial chromosome (YAC) were isolated from partial Mbo I digest libraries using two separate vectors, A

Rearrangements or loss of heterozygosity (LOH) involving the short arm of chromosome 3 have been frequently identified in several malignant diseases, suggesting the presence of one or more 3p tumor suppressor genes. Tumors having the highest reported loss include renal, lung, and cervical carcinomas. In renal carcinoma, 3p involvement approaches 90o in the clear cell histologic variety (1-4), while in smallcell lung cancer the loss is universal (5, 6). In non-small-cell lung cancer, which includes squamous, adeno, and large cell histologies, 3p loss occurs in up to 75% of cases (7, 8). In addition, 3p loss has been reported in 90-100%o of cervical cancers (9, 10) and other frequent 3p losses have been described in thyroid, head and neck, breast, and testicular cancers (11-15). In most cases, the amount of chromosome 3 material lost has been extensive. A chromosomal translocation or small deletion involving a suspected tumor suppressor locus would greatly facilitate a positional cloning approach to isolate such genes. In 1979, Cohen et al. (16) described a family with hereditary renal carcinoma due to a reciprocal 3;8 chromosomal translocation. The reported breakpoint at 3p21 was later shown by

high-resolution cytogenetic analysis to occur at 3p14.2 (17),

which coincides with the most sensitive common fragile site in the genome (18). The chromosome 8 breakpoint in the 3;8 translocation occurred at band 8q24.1, the site of the MYC oncogene. While MYC is translocated (19), no rearrangement The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Abbreviations: YAC, yeast artificial chromosome; LOH, loss of

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FIX II (Stratagene) and EMBL3cos-Not (kindly provided by Noreen Murray, University of Edinburgh, U.K.). The libraries were constructed by standard procedures (24). YAC and cDNA Library Screening. YAC clones were isolated from the Centre d'Etude du Polymorphisme Humain human YAC library prepared in the vector pYAC4 (25) and from additions to this library containing megabase-sized inserts. YACs were isolated by conventional hybridization and PCR screening approaches. Restriction maps of YACs were constructed by using partial digests and hybridization to pYAC4 end probes. A phage subclones of the 850A6 YAC were used as probes to screen an adult human kidney cDNA library (Clontech). All Southern blot hybridizations were performed by using a charged nylon membrane (Oncor) in a buffer described by Amasino (26). The RNA isolation and Northern blot analysis were according to standard protocols

(24). RESULTS Identication f a Chromosome 3 Marker In Cbse Proximity to the t(3;8) Breakpoint. We previously described the development of a somatic cell hybrid mapping panel and nearly 500 regionally localized DNA probes for chromosome 3 (22). In addition to hybrids containing the der(3) and der(8) chromosomes from the t(3;8) family, three additional hybrids were used to identify a subset of markers in close proximity to the translocation breakpoint. These breakpoints are shown in Fig. 1. DNA probes mapping between the 3;6 and 3;8 translocation breakpoints were tested against DNA from the A5-4 hybrid, which contains an aphidicolin-induced breakpoint in 3p14. As shown schematically in Fig. 1, of 14 markers only 1, R7KAC1-6#145, was retained in hybrid A54. This suggested that the distance between the 3;8 and A5-4 breakpoints was small. Hybrid AS-S allowed definition of a subset of markers immediately centromeric to the 3;8 translocation. By molecular analysis, R7KAC1-6#145 had been lost along with m25% of probes previously mapped to the 3p13-p14.2 region (22). This defined a subset of markers adjacent to the 3;8 translocation on the centromeric side and reinforced the placement of R7KAC1-6#145.

Proc. NatL Acad. Sci. USA 90 (1993)

Moleculr Cloning of the Trandocation Breakpoint Region. Chromosomal walking was initiated on both sides of the breakpoint using corresponding YACs isolated from the Centre d'Etudes du Polymorphisme Humain library (27). Three YACs were isolated by using probe R7KAC1-6#145 and a contig was established on the basis ofend clone analysis (Fig. 2A). The left end (TRP1) of YAC 74B2 was found to cross the aphidicolin-induced breakpoint present in hybrid A5-4 (Fig. 3), thus establishing the orientation of the contig. A probe derived from the right end of YAC 65E7, located between the 3;8 and A54 breakpoints, detected an altered fragment in an Mlu I digest of DNA bearing the 3;8 translocation (Fig. 3C). The differences observed between the sizes of the Mlu I fragments detected in YAC DNA (shown in Fig. 2A) and the human DNA (shown in Fig. 3C) are due to methylation involving the two Mlu I sites on the centromeric side of the translocation breakpoint. A subsequent walking step in the centromeric direction was performed. YAC 850A6 (Fig. 2A) was found to cross the 3;8 breakpoint by both end clone analysis and fluorescence in situ hybridization (data not shown). A A phage library was constructed from the 1.3-Mb 850A6 YAC and m7 equivalents of human recombinants were identified and placed onto grids. Random human clones were mapped with respect to the 3;8 translocation breakpoint using the der(3) and der(8) somatic cell hybrids and positioned on the Mlu I map of the 850A6 YAC shown in Fig. 2A. This allowed localization of the breakpoint to a 185-kb Mlu I fragment. This fragment was gel A

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FiG. 1. Location of markers surrounding the t(3;8) breakpoint. A diagram of 3p is shown along with the cytogenetic locations of four breakpoints defining the t(3;8) region. The AS4 breakpoint is telomeric to the t(3;8) breakpoint and separated from it by S500 kb. The cloned DNA markers mapped into each interval are listed along with R7KAC1-6#145, the only marker to map between AS-4 and t(3;8).

FIG. 2. Physical map of the t(3;8) breakpoint region. (A) The YAC contig isolated using R7KAC1-6#145 is shown along with the locations of all cleavage sites for Mlu I (M) and some for Xho I (X). Note that these sites have been determined on unmethylated DNA. The positions of both the t(3;8) and aphidicolin-induced A5-4 breakpoints are indicated by the vertical hatched lines, while the orientation of the contig along chromosome arm 3p is designated centromere to telomere. (R) and (L) indicate the left (CEN, TRP1) and right (URA3) ends of these pYAC4-derived YACs. The 189B12 YAC is known to be chimeric (indicated by horizontal hatched line) but the extent of the chromosome 3 portion is unknown. YAC sizes are indicated in kb. (B) A phage contig situated between the two indicated Mlu I sites is shown. Clones 1023 and 3068 cross the t(3;8) breakpoint. Clones 2071, 4004, and 3111 are detected by the right end of YAC 74B2. (C) Positions of EcoRI cleavage sites in genomic DNA are shown for the region immediately surrounding the t(3;8) breakpoint. Solid box indicates position of the HRCAI gene.

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FIG. 3. Demonstration that R7K145 YAC contig crosses the A5-4 breakpoint and identification of the t(3;8) breakpoint by pulsed-field gel analysis. (A and B) End probes developed from YAC clones shown in Fig. 2 were used as hybridization probes against the A5-4 and A5-5 hybrid DNAs and controls. EcoRI-digested DNA samples included normal human (lane a), A5-4 (ane b), A5-5 Oane c), and UCTP2A3 (lane d). Hybridization with 65E7(L) is shown in A, while 74B2(L) is shown in B. The 65E7(L) probe is present in A54 but missing in A5-5, while the 74B2(L) probe is missing from both. This establishes the orientation of the YAC contig as drawn in Fig. 2. (C) Mlu I-digested DNA samples are from a normal human lymphoblastoid cell line (lane a) and a lymphoblastoid cell line containing the 3;8 translocation (ane b). After separation on a pulsed-field gel, the resulting filter was hybridized with probe 65E7(R). The normal DNA fragment of 550 kb was reduced to 250 kb in the 3;8 translocation cells.

purified and used as a hybridization probe to identify the corresponding subset of human A clones. End probes were isolated by EcoRI/Sal I double digestions and used to establish the phage contig shown in Fig. 2B. Clones 1023 and 3068 were found to span the breakpoint. Fig. 4A shows an Xba I digest in which probe 1023 detects a rearrangement on the centromeric side of the breakpoint. Germ-line fragments of approximately 8.0, 5.0, and 3.0 kb are seen in the normal chromosome 3 hybrid (lane b). The 5.0-kb fragment is present in the der(8) hybrid (lane c), while the 3.0-kb fragment and a rearranged band of lS5.0 kb are present in the der(3) hybrid (lane a). In Fig. 4B, germ-line EcoRI bands of approximately a

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12.0, 7.0, and 2.5 kb were detected by using probe 1023 in total human DNA (lane a) and in the somatic cell hybrid containing a normal chromosome 3 (lane c). In contrast, only the 2.5- and 7.0-kb bands were present in the der(3)containing hybrid (lane b), while an altered fragment of :20 kb on the telomeric side of the translocation could be seen in the der(8) hybrid (lane d). Isolation of a cDNA Clone Adjacent to the Translocation Breakpoint. Individual A clones shown in Fig. 2B were tested for both unique and evolutionarily conserved DNA sequences and used to screen an adult kidney cDNA library. Three isolates were identified from %600,000 plaques by a bc d

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FIG. 4. Detection of rearranged DNA fragments at the t(3;8) breakpoint using both genomic and cDNA probes. (A) DNA samples were digested with Xba I. DNA samples included TL12-8 (lane a), UCTP2A3 (lane b), and 3;8/4-1 (lane c). Hybridization with A clone 1023 revealed rearranged bands as discussed in the text. (B and C) DNA samples were digested with EcoRI and hybridized with A clone 1023 (B) or the cDNA clone K7 from the HRC1 locus (C). DNA samples are normal human (lane a), TL12-8 (lane b), UCTP2A3 (lane c), and 3;8/4-1 (lane d). Molecular size markers (m) are indicated in kb.

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using a fragment from A clone 4040, which is adjacent to the breakpoint on the telomeric side. The largest of these cDNA clones, K7, contains a 1.7-kb insert with a poly(A) tract. On Southern hybridization, the K7 probe detects the 3;8 translocation breakpoint on EcoRI digestion of DNA from the der(8) hybrid. This is shown in Fig. 4C. The band sizes correspond to those seen with the genomic probe 1023, indicating that all the K7 cDNA sequences are contained within the 12.0-kb EcoRI genomic fragment. A second gene, represented by cDNA clone K50, was identified by using the same kidney cDNA library by an evolutionarily conserved fragment from the A clone 4082 located -50 kb centromeric to the 3;8 breakpoint. This cDNA does not detect a rearrangement in the 3;8 translocation. The genes represented by the K7 and K50 cDNAs appear distinct. An %2.0-kb transcript was identified in various kidney-derived cell lines by Northern blot analysis using the K50 probe (Fig. 5), while no transcript was detected when the same filter was rehybridized with the K7 cDNA (data not shown). This suggests that the gene represented by the K7 cDNA is transcribed at a low level. This is in agreement with the low frequency (1/200,000) of K7-related clones in the adult kidney cDNA library.

DISCUSSION LOH studies in several malignant diseases suggest that there are three separate regions on 3p, p13-14.3, 3p21.3, and 3p25 that may contain tumor suppressor genes (6, 28-30). The site of the 3;8 translocation breakpoint in 3p14.2 may correspond to the most proximal of these loci. Members of the t(3;8) family who inherit the translocation have a high (79%o) risk of developing renal carcinoma with the associated familial characteristics of bilaterality and an earlier age of onset than occurs in the general population (16, 31). These individuals also appear to be at risk for the development of thyroid cancer, which is associated with LOH involving 3p (11). The 3p14.2 breakpoint is believed to contain the critical gene in the 3;8 translocation based on the involvement of this region in other sporadic tumors (28, 30). Although the chromosome 8 breakpoint region, 8q24.1, contains the MYC za h)

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oncogene, which is translocated to the der(3) chromosome (19), a pulsed-field analysis indicated the breakpoint was >1 Mb away (20). Similarly, expression studies have failed to demonstrate any abnormalities in MYC (F.L.B. and H.A.D., unpublished observations) and the 8q24.1 region has not been implicated by LOH or cytogenetic alterations in other renal tumors. We have identified a gene immediately adjacent to the 3;8 translocation breakpoint, which we refer to as hereditary renal cancer-associated gene 1, HRCA1. We have not detected expression in kidney-derived cell lines by Northern blot analysis, suggesting that HRCAJ is expressed at a low level. Exon-trapping experiments have resulted in two clones, which, by DNA sequence analysis, are contained within the K7 cDNA. These results, in addition to the poly(A) tract present in the K7 clone, confirm that HRCAI is a gene. DNA sequence analysis of HRCAI, although incomplete, has not yielded any significant similarities in a BLAST search of GenBank. A cytogenetic examination of a renal carcinoma from the 3;8 translocation family demonstrated loss of the der(8) chromosome, which contains the p14.2-pter region of chromosome 3 (31). This suggests that an additional critical gene located distal to the 3;8 breakpoint was lost. However, another report containing an identical appearing 3;8 translocation demonstrated a 3p14-p21 deletion in the normal chromosome 3 allele while retaining the der(8) chromosome (case 17 in ref. 32). There is precedent, in the case of Wilms tumor, for the independent loss of two distinct chromosome 11 loci (33, 34). On chromosome 3, the recently cloned von Hippel-Lindau gene located in 3p25 (35) may represent a separate suppressor locus. The relative importance of each locus in the development of spontaneous renal carcinomas remains unknown. An additional putative proximal 3p suppressor locus has been suggested by the finding of a submicroscopic homozygous deletion involving the 3p13(p14) region in the small-cell lung cancer cell line U2020 (36, 37). The U2020 deletion and the 3;8 translocation breakpoint are nonoverlapping. None of the probes we have identified as being immediately proximal to the 3;8 breakpoint is deleted in the U2020 cell line (37). We have developed a YAC contig and pulsed-field map for this set of probes, which covers -5 Mb of DNA (unpublished data). Thus, the 3;8 translocation breakpoint and the U2020 deletion are separated by at least this distance. Renal cysts are associated with the development of renal carcinoma in the von Hippel-Lindau syndrome (38) and acquired cystic disease due to chronic renal failure and dialysis (39). It is of interest that a mouse autosomal recessive polycystic kidney mutation, pcy, has been linked to the dilute and transferrin loci on mouse chromosome 9 (40, 41). Since the von Hippel-Lindau locus in 3p25 is believed to be syntenic with mouse chromosome 6 (42), the site of the 3;8 translocation should be examined as a candidate for the mouse pcy mutation. We gratefully acknowledge the technical assistance of Jan Jacobson, Richard Bolin, Steve Johnson, and Efang Li for cell culture and YAC isolations as well as Bryan Hall for fluorescence in situ hybridization analysis. We thank Dr. Daniel Cohen and Dr. Denis Le Paslierfor providing the Centre d'Etudes du Polymorphisme Humain YACs. This work was supported by National Institutes of Health Awards HD23826 (H.A.D.), HG00358 (H.A.D. and R.M.G.), CA43222 (T.W.G.), and NCHGR HG00209 (which supported screening of the megabase YAC library).

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FIG. 5. Northern blot analysis of various kidney-derived cell lines. Lanes contain 20 Mg of total RNA isolated from 293 (lane a), KRC/Y (lane b), KV-6 (lane c), CAKI-1 (lane d), CAKI-2 (lane e), or MDBK (lane f) cells. Probes were K50 in A and actin in B.

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