Endogenous retroviral sequence is fused to FGFR1 kinase in the 8p12 stem-cell myeloproliferative disorder with t(8;19)(p12;q13.3)

Neoplastic disorders arise because of acquired, and sometimes inherited, genetic abnormalities.1 Recurrent translocations in malignancy have served as signposts to identify genes critical to the molecular pathogenesis of the disease. In the stem-cell myeloproliferative disorder (MPD) linked to the 8p12 chromosomal region, the recurrent translocations involve the FGFR1 gene, which encodes one of the tyrosine kinase receptors for fibroblast growth factors.2 This syndrome is characterized by myeloid hyperplasia with frequent peripheral blood eosinophilia and B- or T-cell lymphoblastic leukemia/lymphoma; it generally progresses to acute myeloid leukemia.3 To date, 3 FGFR1partner genes have been cloned, FOP at 6q27,4,CEP110 at 9q33,5 andFIM/ZNF198 at 13q12.6-9 Four other translocations have been identified in myeloid disorders.10,FGFR1 is also disrupted and fused to BCR in patients with chronic myeloid leukemia associated with the t(8;22).11,12 We recently identified the t(8;19)(p13;q13.3) as a novel translocation involving theFGFR1 gene in a patient suffering from an atypical myeloproliferative syndrome.13 We report here the cloning of this translocation breakpoint that fuses a sequence from a human endogenous retrovirus (HERV) gene of the HERV-K3 family toFGFR1 sequences encoding its entire tyrosine kinase domain.

The clinical and cytogenetic data of the patient's case have been previously described.13 

5′ Rapid amplification of cDNA ends polymerase chain reaction (RACE-PCR) was done as previously described.5 Half of the PCR products were resolved on 2% agarose gels, purified by means of a NucleoSpin extract kit (Macherey-Nagel, Düren, Germany), subcloned into pGEM-T vector (Promega, Madison, WI), and sequenced by Génome Express (Meylan, France). The other half of the PCR products were separated by electrophoresis on 2% agarose gel and transferred to Hybond N⁺ membrane (Amersham Pharmacia, Orsay, France). A specific primer spanning the t(8;19) breakpoint (5′-TCCTGTGCGGTGTCTGCTGA-3′) was used for hybridization.

Two-color fluorescence in situ hybridization (FISH) experiments were done on metaphase chromosome spreads from normal lymphocytes and patient's bone marrow cells as previously described.14DNA from BAC clone RP11-706G10 (BACPAC Resources, Children's Hospital, Oakland, CA) specific to the currently identified HERV-K sequences was purified by means of a Nucleobond BAC 100 kit (Macherey-Nagel, Hoerdt, France).

Reverse transcriptase (RT) reactions were done with 2 μg polyadenylated RNA from the patient's bone marrow cells or 5 μg of total RNA from several human tissues. Each resulting complementary DNA (or control lacking RT) was used as a template for PCR as previously described.4 The following primers were used:FGFR1-specific primers: 5′-ATCATCTATTGCACAGGGGCC-3′ (FGFR1-sense) and 5′-CATACTCAGAGACCCCTGCTAGC-3′ (FGFR1-antisense); XM_054197-specific primers: 5′-TCAAGAAAACGACACAAGAAGC-3′ (HERV-K-sense) and 5′-CCTCCAGTGGTATACTGAGTTGG-3′ (HERV-K-antisense). Products were subcloned into pGEM-T vector (Promega), and sequenced by Génome Express.

In a previous study, we identified FGFR1 as the chromosome 8 breakpoint gene by FISH experiments in patient's hematopoietic cells harboring t(8;19)(p12;q13.3) translocation.13 In the present study, the chromosome 19 partner was identified by means of anchored PCR with FGFR1primers to amplify the fusion transcript from the patient's bone marrow cDNA. Analysis of cDNA clones showed a 209-bp non-FGFR1 sequence encoding an open reading frame fused to part of the juxtamembrane domain and the tyrosine kinase–encoding regions of the FGFR1 gene. This non-FGFR1 sequence was localized on the LLNLR-245B6 clone located on human chromosome 19 that has been recently annotated asHomo sapiens hypothetical gene LOC113386 (GenBank accession no. XM_054197; Figure 1A). Sequence analysis revealed nucleotide similarities with sequences from human endogenous retroviruses type K (HERV-K)15 and HERV-K113.16 RepeatMasker analysis demonstrated the presence of two 5′ long terminal repeat (LTR) sequences (exons 2 and 4), two 3′ LTR fragments (exon 3), and one long interspersed repeated (LINE) sequence (exon 4). An open reading frame (ORF) of 225 bp was identified in the first 3′ LTR and showed similarities with retroviral envelope protein (ORF ENV-Like; Figure1A). This peculiar structure is characteristic of a defective retrovirus.17 18 The 5′ sequence of this ORF (150 bp) is fused in frame to FGFR1 sequences at nucleotide 1273, which corresponds to the beginning of exon 9 (accession no.M34185; Figure 1B).

FISH analyses on normal metaphase cells showed that the HERV-K–containing BAC RP11-706G10 (AC023149) mapped to chromosome 19q13.3 (Figure 2A). On patient's cells, the HERV-K sequence hybridized to the normal 19, der 8, and der 19 chromosomes, confirming its 19q13.3 position and involvement in the t(8;19)(p12;q13.3) (Figure 2B).

Expression of the HERV-K-FGFR1 fusion transcript was detected in the patient's peripheral blood cells but not in the healthy control cells (Figure 3A). Expression of the reciprocal transcript was not detected, in contrast to the results we obtained in patients harboring the other gene rearrangements involved in the 8p12 myeloproliferative syndrome (ie,FOP/FGFR1, FIM/FGFR1, andCEP110/FGFR1).4-6 These results were confirmed by Southern blot analysis of the RT-PCR products in which we used a ³²P-labeled oligonucleotide probe specific to the fusion junction (Figure 3B). Sequence analysis demonstrated that the unique PCR product contained a fusion of HERV-K and FGFR1sequences as expected.

The novel HERV-K we cloned and assigned to chromosome 19q13.3 is ubiquitously expressed, as revealed by RT-PCR done on various tissues (Figure 3C). HERV-Ks are human specific19 and are characterized by their similarity to the B-type mouse mammary tumor virus (MMTV) and Syrian hamster intracisternal A-type particles (IAPs).20 The human genome harbors 25 to 50 copies of endogenous retrovirus type K, some of which code for the characteristic retroviral proteins (for a review, see Urnovitz and Murphy17). The endogenous retroviral sequences found in the human genome are a heterogeneous group of retroelements that were presumably derived from ancient germ-line infections of exogenous retrovirus that became fixed in the species. The HERV-K subgroup of retroviruses have frequently been proposed as etiological cofactors in chronic diseases such as cancer, autoimmune diseases, and neuronal disease.21 Much of the evidence that links HERVs to disease comes from the detection of expressed retroviral sequences in tissues from patients with multiple sclerosis or schizophrenia and in teratocarcinoma-derived cell lines (for a review, see Griffiths18). The high copy number of HERV-K–related elements in the genomes of humans and monkeys indicates that these sequences have been mobile within the primate genome over a long period of time and may still be actively transposed. In cancers, these biologically active elements have been shown to disrupt tumor suppressor22 and/or DNA repair23 genes by recombination events mediated by direct retroposition or by repeat elements generated by the process. The induction and progression of rodent erythroleukemias by Friend spleen-forming virus is due mainly to the ability of proviruses to activate cellular oncogenes or inactivate tumor suppressor genes. Moreover, it has been shown that DNA-damaging agents often activate transposable elements in genomes and induce gene rearrangement in leukemias.24 

We report here that in the t(8;19)(p12;q13.3) that leads to atypical myeloproliferative disorder linked to 8p12, the translocation breakpoint on chromosome 19 involved an endogenous retrovirus element. As a consequence of 8p12 chromosomal translocation with a promiscuous (heterogeneous) array of partner chromosomes, oncogenic fusion proteins are formed that replace the FGFR1 dimerization domain with a variety of protein domains. The characterization of self-association of truncated forms of HIV-1 gp120, as suggested by Malvoisin et al,25strongly suggests that dimerization of the envelopelike sequences may induce constitutive activation of FGFR1 kinase, as has been demonstrated for another FGFR1 partner.26 The strong promoter activity of the endogenous retroviral LTR we described may contribute to the high level of transcription of the fusion gene.