SHORT REPORT

FLRG (follistatin-related gene), a new target of chromosomalrearrangement in malignant blood disorders

Sandrine Hayette1, MyleÁ ne Gadoux1, Sylvie Martel2, Suzanne Bertrand2, Isabelle Tigaud1,Jean-Pierre Magaud1,2 and Ruth Rimokh2

1Laboratoire de CytogeÂneÂtique MoleÂculaire, Hopital Edouard Herriot, 69437 Lyon; 2Unite lNSERM U453, Centre LeÂon BeÂrard,69373 Lyon cedex 08, France

We report here the molecular study of at(11;19)(q13;p13) translocation observed in a case of B-cell chronic lymphocytic leukemia. This translocationleads to the juxtaposition of the CCND1 gene onchromosome 11 to a new transcriptionnal unit onchromosome 19. The cDNA of this new evolutionarilyconserved gene (named FLRG for Follistatin-RelatedGene) codes for a secreted glycoprotein of the follistatin-module-protein family. FLRG is expressed in a widerange of human and murine adult tissues and itsexpression seems to be tightly regulated during murineembryogenesis. Its transcripts could not be detected inhematopoietic cells from all lineages and in particular incells from lymphoid B and T lineage except in thet(11;19)-carrying leukemia described here. A greatvariability of expression is observed among the othertumoral cell lines analysed. Besides the t(11;19)-carryingleukemia described in this work, structural rearrange-ments of the FLRG locus have been found in a non-Hodgkin lymphoma, suggesting that it may play a role inleukemogenesis.

Keywords: FLRG; follistatin; chromosomal transloca-tion; carcinogenesis

A majority of human hematopoietic malignancies carrynonrandom chromosomal alterations; experimentalevidence indicates that genes located at recurringchromosomal breakpoints are directly involved intumor pathogenesis (Rabbitts, 1994). In a previouswork, we have reported the molecular study of at(11;19)(q13;p13) chromosomal translocation observedin a B-cell chronic lymphocytic leukemia (Rimokh etal., 1993). Cytogenetic analysis of the fresh leukemiccells and of the corresponding cell line revealed, in allthe metaphases analysed, the following karyotype: 46,XX, t(l;X)(p21;q27), t(8;9)(q24;p21), t(11;19)(q13;p13).The cell line established from these cells (GOB cell line)shows the same immunophenotype and the samekaryotypic anomalies as the parental cells. Thistranslocation involved CCND1 locus on chromosome11q13 and resulted in the production of an aberrantCCND1 transcript where the 3' UTR of CCND1 wasfused to a member of the MER11 sequence family(Medium reiteration frequency repetitive sequences)

(Rimokh et al., 1994). In the present study we reportthe molecular cloning of the t(11;19) chromosomalbreakpoints and show that the CCND1 gene isjuxtaposed to a chromosome 19 coding region(named FLRG for Follistatin-Related Gene) whoseprotein product is homologous to members of aprotein family known as the follistatin-module-proteinfamily. These proteins act as growth/di�erentiationfactors through paracrine control during embryogen-esis and adult life (Besecke et al., 1996; Graham andLunsden, 1996).Previous analysis (Rimokh et al., 1994) of the

aberrant CCND1 mRNA expressed in this t(11;19)-carrying leukemia had allowed us to locate thebreakpoint on the der(11) chromosome at nucleotide2276 of the cDNA CCND1 sequence (Motokura et al.,1991), in the 3' untranslated region. To analyse thisrearrangement, a genomic phage library prepared fromGOB cells was screened with the 35PS and 14EXprobes which surround the chromosomal breakpointson the der(11) chromosome (Figure 1a). In this way,we were able after restriction mapping analysis toisolate recombinant bacteriophages containing insertsrepresentative of the normal chromosome 11, of theder(11) and der(19) chromosomes. The O2BS probeoriginating from the der(19) chromosome was thenused to screen a placental genomic DNA library andallowed us to clone recombinant phages correspondingto the normal chromosome 19 counterpart (Figure 1a).Southern blot analysis of interspecies somatic hybridsDNA and FISH analysis con®rmed the location ofFLRG on chromosome 19 at band 19p13 (data notshown). Finally, the O2BS probe derived from theder(19) chromosome and the chromosome 11 14EXprobe detected the same rearranged band in BglII andBamHI-cleaved GOB DNA as expected in this t(11;19)-carrying leukemia (Figure 1b).We next determined the nucleotide sequence

spanning the breakpoints on both parental andtranslocated chromosomes. On chromosome 11, thebreak occurred at nucleotide 2276 in the 3' untrans-lated region of the last CCND1 exon. Two extra-nucleotides (AA) are present at the chromosomaljunction on the der(11) chromosome and ®ve(GAAAG) at the chromosomal junction on theder(19) chromosome. Furthermore, deletion of shortstretches of nucleotides occurred on both parentalchromosomes during the translocation process (Figure1c). No recombination signal sequences were observedin the vicinity of the breakpoints.In order to identify a transcriptional unit on 19p13,

unique fragments that spanned 19pl3 sequences were

Correspondence: R RimokhReceived 15 July 1997; revised 29 December 1997; accepted 29December 1997

Oncogene (1998) 16, 2949 ± 2954 1998 Stockton Press All rights reserved 0950 ± 9232/98 $12.00

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used as probes against Northern blots containing RNAfrom GOB cells and from di�erent normal and tumoralhuman tissues and from cell lines of various origin. The1SB probe (Figure 1a) hybridized to a transcript ofapproximately 2.5 kb present in total RNA extractedfrom the leukemic GOB cells and from normal humantissues. Then, this probe was used to screen a humanplacenta cDNA library (lDR2 phage, ClontechLaboratories, CA, USA). Seven positive clones wereisolated, their restriction mapping and their partialsequence showed that they all coded for the sameprotein. The sequence of the longest clone (Genbankaccession no. U76702) exhibits an open reading framecoding for a peptide of 263 amino acids, predictedmolecular weight 27.6 kDa, and a 1700 nt long 3'untranslated region. The longest open reading framecontains two possible ATG initiation codons (nucleo-tide 8 and 86); the second has the better initiationsequence consensus: CCATGG (Kozac, 1986). Aconsensus polyadenylation site AATAAA is found1666 bases 3' of the stop codon. Comparison of thehuman cDNA and genomic DNA sequences revealedthat the FLRG gene extends over 7 kbp and contains®ve exons (Figure 1a) with typical splice donor andacceptor sites at the intron-exon junctions (data notshown). It must be pointed out that the chromosomalbreakpoint of the t(11;19) observed in GOB cellsoccurred about 7 kbp upstream of FLRG and thus,that all the coding region is maintained on thederivative chromosome 19.

Northern blot analysis con®rmed that FRLG ismainly transcribed as a 2.5 kb RNA and showed thatit is expressed in a wide spectrum of normal andtumoral tissues (Figure 2). In normal human tissues(Figure 2b), the highest steady-state levels of the 2.5 kbtranscripts were observed in placenta, amniocytes,chorionic villosities and bone marrow stromal cells. Aweaker expression was observed in ®broblasts,keratinocytes and endothelial cells. It is noteworthythat none of the hematopoietic tissues (tonsil, spleen,lymph node, bone marrow cells) and tumoral cell linestested expressed the FLRG transcript. Among thetumoral cell lines tested, FLRG was found to bevariably expressed as shown in Figure 2a. Northernblot analysis shows that a small 1.3 kb FLRGtranscript is detected in some human tissues (Figures2a and b). This transcript is likely to correspond to ashorter form of the 2.5 kb RNA as a result of the useof a di�erent polyadenylation site since it cannot bedetected with a probe speci®c of the 3' untranslatedregion of the FLRG RNA (data not shown). Zoo-blotanalysis under high-stringency conditions using aFLRG cDNA probe showed that sequences homo-logous to FLRG can be detected in the chicken andmouse genome (data not shown). It must also bepointed out that a 2.2 kb transcript is observed in somemurine tissues when using a human FLRG cDNAprobe (Figure 2d). Northern blot analysis of murinetissues revealed that FLRG transcripts are present intestis, kidney, muscle, lung and heart. During mouse

G G G GT T T T— 23.1

— 9.4

— 6.7

— 4.3

Bg Ba Bg Ba

02BS 14EX

a

c

b

Figure 1 (a) Restriction map of the normal CCND1 allele, the t(11;19) translocation breakpoint and the normal FLRG allele. Blackbars, chromosome 11 DNA regions, grey bars, chromosome 19 regions. Sided-horizontal lines under the map show the location ofthe probes used in this study. Exons are indicated by boxes. CEN, centromere; TEL, telomere; E, EcoRI; Ba, BamHI; X, XbaI; Bg,BglII; H, HindlII. For the genomic phage library, GOB cell line DNA was partially digested with MboI restriction endonuclease.DNA fragments averaging 15 ± 20 kbp in length were selected by sucrose gradient centrifugation and ligated to BamHI arms of theEMBL3 phage (Stratagene Lab., CA, USA). A human placental DNA library was constructed using the same procedure in order toclone the chromosome 19 normal counterpart. (b) The t(11;19) translocation results in the juxtaposition of the CCND1 gene tosequence originating from chromosome 19. Ten micrograms of genomic DNA extracted from GOB cells (G) and normal peripheralblood lymphocyte (T) were digested with BglII (Bg) and BamHI (Ba) electrophoresed through a 0.8% agarose gel and blotted ontoa nylon membrane. The ®lter was sequentially assayed for hybridization to 02BS and 14EX probes (a). The scale is in kilo-bases. (c)Nucleotide sequence of the breakpoints of the t(11;19) and corresponding normal chromosome 11 and 19. Chromosome 11 sequenceare in normal type, chromosome 19 sequence are in italics. Gaps have been introduced for optimal alignment. Additionalnucleotides are double underlined. Deletion of short stretches of nucleotides are underlined.

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embryogenesis, FLRG is strongly expressed at the 7thdays of developement which corresponds to thegastrulation stage; then, its transcription seems to bedownregulated (Figure 2c).The predicted amino acid sequence of the largest

open reading frame was determined from the nucleo-tide sequence. Sequence analysis of the putative FLRGprotein showed the existence of two closely relateddomains coded by exons 3 and 4 (domain I: aminoacids 97 ± 168 and domain II: amino acids 169 ± 244)which are characterized by their richness in cysteineresidues. These domains encompass about 55% of theprotein. Exon 5 encodes the short 19 amino acidscarboxy-terminal part of the molecule and exons 1 ± 2code for the leader sequence and the N-terminus of theprotein. Comparison of the cDNA sequence with thosein the EMBL bank and in the GenBank and analysisof the intron-exon boundaries revealed that the twocysteine-rich repeats present in the FLRG protein areclosely related to the class 1-1 follistatin modules(Shimazaki et al., 1988; Patthy and Nikolics, 1993) (thesplice junctions occur between the ®rst and secondnucleotide of the respective codon) of follistatin, agrin,and SPARC/osteonectin. Apart from these modules,the homology between FLRG and the other membersof the follistatin-related protein family (follistatin,agrin, SPARC, SC1, QR1) is not signi®cant. Align-

ment and comparison of the follistatin modules whichcharacterize this protein family shows that the higherhomology is observed between follistatin and FLRG(Figure 3a).Computerized analysis of the amino acid sequence

(PROSITE program) showed that the FLRG proteincontains two potential N-glycosylation site on Aspar-agin 73 and 215. The hydropathic pro®le of FLRGobtained by the Kyte and Doolittle program showed aunique region of hydrophobicity at the N-terminus(amino acids 10 ± 30) of the protein. Although theredoes not exist any potential cleavage site, wehypothesized that this hydrophobic domain corre-sponds to a signal peptide and that the FLRG proteinmight be secreted. These predictions are borne out byCOS-7 cells expression studies shown in Figure 3b. TheFLRG cDNA was subcloned in the expression vectorpME18S and transfected into COS-7 by electropora-tion. Western blot analyses using a polyclonal rabbitanti-GST-FLRG antibody demonstrated the presenceof a 33 kDa protein in cell supernatant of transfectedCOS-7 and in the supernatants of the LOVO, JAR andHELA tumoral cell lines. In GOB cell supernatant theprotein is weakly detectable probably as a result of thelow transcription rate of FLRG in these cells.Glycosylation causes the increased size of the

33 kDa protein. Western blot analysis of the trans-

LOVO (colon)

JAR (choriocarcinoma)

HBL (breast)

TE85 (osteosarcoma)

CACO (colon)

EJ (bladder)

HELA (cervix)

IMR32 (neuroblastoma)

T47D (breast)

MCF7 (breast)

MB436 (breast)

HUH7 (liver)

HEPG2 (liver)

MALAHVU (liver)

COLO320 (neuroendocrin)

GOB t(11;19)

1.3 2.5 kb

7-day

11-day15-day

17-day

S26 S262.2 kb 2.2 kb

tonsil

amniocytes

chorionic villosities

placenta

bone marrow

bone marrow stromal cells

fibroblasts

endothelial cells

keratinocytes

1.3 2.5 kb

heartbrain

spleen

lung

liverskeletal muscle

kidney

testis

a

c

b

d

Figure 2 Pattern of expression of FLRG. Total RNA were extracted from the indicated normal human tissues (b) or humantumoral cell lines (a), size-fractionated in formaldehyde-1.2% gels, transferred onto nylon ®lters and hybridized with an FLRGcDNA probe. Northern blots of poly (A)+ RNA (premade Northern blots, Clontech Laboratories CA, USA) from murine embryoat day 7, 11, 15 and 17 (c) and from di�erent murine tissues (d) were sequentially assayed for hybridization to FLRG cDNA andS26 (rRNA) probes.

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fected COS-7 cell supernatant, following Peptide N-glycosidase F digestion, reveals an accumulation of27 kDa material with concommitant loss of 33 kDamaterial (Figure 3b). 27 kDa is in fact the predictedsize of the unmodi®ed core protein. This demonstratesthe presence of a sizeable amount of N-linkedpolysaccharide. Like other members of the follistatin-related protein family, FLRG is a secreted glycopro-tein.Besides this t(11;19)-bearing leukemia, structural

rearrangement of FLRG locus was observed in a caseof B-cell Non-Hodgkin Lymphoma (NHL). Southernblot analysis of other LNH (eight mantle zonelymphomas, 18 follicular lymphomas and 10 di�uselymphomas) allowed us to show rearrangement of theFLRG locus in one case of B-cell Mantle ZoneLymphoma (Figure 4). Cytogenetic analysis was notperformed in this tumor. Rerrangement was identi®edwith three restriction endonucleases and restrictionmapping analysis showed that the break occurreddownstream of the gene, leaving intact its codingregion and its 3' untranslated region. Unfortunately, no

1 2 3 4 5 6 7 8 9

— 41.8

— 30.6

a

b

Figure 3 (a) Comparison of the amino acid sequences of the predicted FLRG and follistatin proteins. The amino acid sequencesare aligned for optimal homology. Shaded sections represent the three modules (cystein-rich) of follistatin protein. FLRG proteincontains 2 of these modules which are 55% identical in amino acid sequence with the ®rst and the second module of the follistatinprotein. *, identical residue; ., conservative substitution. (Nucleotide sequence of the FLRG cDNA has been deposited in Genbank,accession no. U76702). (b) FLRG is a secretory glycoprotein. Western blot analysis using a polyclonal rabbit anti-GST-FLRGantibody demonstrated the presence of a 33 kDa protein in cell supernatants of COS-7 cells transfected with FLRG and of theLOVO, JAR, HELA and GOB tumoral cell lines. Digestion of the COS-7 cell supernatant with Peptide N-glycosidase F (PNGase,New England Biolabs) removes most of the 33 kDa material and causes an accumulation of 27 kDa material. The scale is in kDa.Cell supernatants of HELA (1), HepG2 (2), LOVO (3), JAR (4), T47D (5), GOB (6), COS-7 transfected by wild type vectorPME18S (7), COS-7 transfected by FLRG (8) and COS-7 transfected by FLRG digested by PNGase (9). The polyclonal antiseraagainst FLRG were produced in rabbits by standard methods. A 940 bp BamHI cDNA fragment containing the whole FLRGcoding sequence was inserted in frame into the pGEX expression vector generating a GST (Gluthathion-S-Transferase) fusionprotein (designated GST±FLRG). Fusion protein extracts were then prepared and puri®ed by preparative SDS±PAGE. The regionof the gel containing the GST±FLRG fusion protein was excised, and polyclonal antisera against this protein were produced inrabbits. For Western blot analysis, culture supernatants were separated by SDS-12% polyacrylamide slab gel electrophoresis. Forimmunodetection, the ®lters were incubated with 1/2000 dilution of the rabbit anti-GST±FLRG antibody. Fixation of the antibodywas revealed by using an ECL detection kit for rabbit antibodies (Amersham Uppsala, Sweden). A pre-immune serum was used asnegative control

B B BT T T

Xbal Hindlll Xhol

— 23.1

— 9.4

— 6.7

— 4.3

Figure 4 Rearrangement of the FLRG locus in a case of B-cellmantle zone lymphoma. Ten micrograms of genomic DNAextracted from a B-cell NHL (B) and normal peripheral bloodlymphocyte (T) were digested with XbaI, HindIII and XhoI,electrophoresed through a 0.8% agarose gel, blotted onto a nylonmembrane and hybridized to 1SB probe (Figure 1a). The scale isin kilo-bases

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mRNA was available and Northern blot could not becarried out.The molecular cloning of this t(11;19) chromosomal

translocation led us to identify a new gene onchromosome 19, the FLRG gene, which codes for aprotein homologous to members of the follistatin-module-protein family. Follistatin related proteinsconstitute a family of extracellular matrix-associatedglycoproteins which bind morphogens or growth/di�erentiation factors and regulate their activityduring human and animal development. More than®ve members of this family have been identi®ed so far.Follistatin was the ®rst member of this family to befully characterized. It is a monomeric protein originallyisolated from ovarian ¯uid by virtue of its ability tosuppress follicle-stimulating hormone from the pitui-tary (Ueno et al., 1987). Actually, further studies haverevealed that this molecule displays more pleiotropice�ects that can be explained by its capability to bind toand inactivate activin, a member of the transforminggrowth factor b (TGF-b) superfamily (Nakamura etal., 1990; Michel et al., 1993). Binding of activin byfollistatin inhibits activin function on pituitary cells,granulosa cells, embryonal carcinoma cells, osteoblasts,embryogenesis, hepatocytes and erythroid cells (Math-ews, 1994). It has also been demonstrated thatfollistatin can associate with cell surface proteoglycan,and in this way might modulate activin binding to itsreceptors (Nakamura et al., 1991).SPARC/osteonectin is another important follistatin-

module protein. It is a collagen-binding glycoproteinexpressed in a variety of tissues during embryogenesisand repair (Lane and Sage, 1994). It has been shown toinduce changes in cell shape, to modulate cell cycleprogression and to be associated with cellular eventsrequired in tissue remodelling, cell movement and/orproliferation. Agrin, QR1 and SC1 are other follistatin-related proteins involved in the formation of thevertebrate nervous system (Hoch et al., 1994; Pieraniet al., 1995; Mendis et al., 1996).As the other members of this gene family, FLRG

encodes a secreted glycoprotein and is highly conservedin evolution, a feature that indicates a large selectivepressure to conserve speci®c structural and functionalcharacteristics of the protein. Sequences homologous toFLRG can be detected in lower species such as chickenand mouse; partial sequencing of the mouse FLRG(exons 2, 3 and 4) showed an 85% identity at theamino acids level (data not shown). FLRG is expressedin a wide range of human and murine adult andembryonic tissues but its transcripts could not bedetected in hematopoietic cells from all lineages and inparticular in cells from lymphoid B and T lineageexcept in the t(11;19)-carrying leukemia described here.With regards to their wide-ranging e�ects on cell

di�erentiation, proliferation and organization, one canassume that deregulated expression of follistatin relatedgenes could participate in cell transformation. The datapresented in this paper constitute an argument in favorof this likelihood. In addition to being involved in at(11;19)-bearing leukemia, the FLRG locus was foundto be rearranged in one case of B-cell NHL. Thetranslocation described here leads to an ectopicexpression of FLRG, probably as a result of aconsecutive alteration of upstream cis-regulatingsequences. Interestingly, the B-cell NHL showing

rearrangement of the FLRG locus was also chraracter-ized by rearrangement of the CCND1 locus. Analysesof larger series of tumors with CCND1 rearrangementsare indeed necessary to determine if this association issigni®cant or not. In the same way, further studies areneeded to determine if the variability of FLRGexpression among the tumoral cells analysed, is dueto a di�erence of expression in the correspondingnormal tissue or is a consequence of speci®c geneticalterations involved in the tumoral process.Rearrangement of the short arm of chromosome 19

is a common event in malignant blood disorders butdoes not seem to be associated with a particular typeof leukemia or lymphoma. Anomalies concerningchromosome 19p mainly consist of translocationsinvolving di�erent partner chromosomes. It isnoteworthy that a recent work has reported asigni®cant frequency of t(11;19)(q13;p13) in multiplemyeloma (Taniwaki et al., 1994). Neither of the twocases that were made available for us by our Japanesecolleagues demonstrated rearrangement of the FLRGlocus by Southern blot analysis. This does not ruleout the possibility that, in these cases, the chromoso-mal breakpoints on chromosome 19p might haveseparated the FLRG coding sequence from distant cis-regulating sequences.The high homology between FLRG and follistatin

suggests that these two proteins could be functionallyredundant, being able, for example, to bind and tomodulate the activity of activin or other members ofthe TGFb superfamily. It is also to be noted that thedistribution pattern of these two proteins is similarduring embryogenesis and adult life. This redundancymay explain that, if follistatin is considered as a directneural inducer in Xenopus (Hemmati-Brivanlou et al.,1994), its absence during embryogenesis of follistatinnul mice is compatible with the development of anormal peripheral and central nervous system (Matzuket al., 1995). In the same way, expression of FLRG bybone marrow stromal cells suggests that FLRG may beimplicated in the modulation of hematopoiesis througha paracrine control in the bone marrow microenviron-ment. In this respect, its biological activity could becompared with that of follistatin which inhibits theerythoid di�erentiation activity of activin (also namedErythroid Di�erentiation Factor, EDF) in human andmouse (Hino et al., 1995; Yamashita et al., 1992).In conclusion, we report here the characterization of

a new member of the follistatin-module-protein familythat might be involved in the leukemogenesis process.Furthermore, this gene could be of interest in other®elds of human biology because it belongs to a genefamily whose members have been demonstrated to playa major role in the maintenance of tissue homeostasis.Goals for future studies include functional study of thisnew protein in order to de®ne its role in tumorogenesisand in physiology of normal cells.

AcknowledgementsThis work was supported by grants from FEGEFLUC, laLigue Contre le Cancer (ligue nationale, Comite de parte-mental du Rhoà ne) and ARC. We are grateful to PhilippeFort for providing S26 probe. FLRG cDNA sequence hasbeen deposited in Genbank, accession no. U76702.

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