LRRFIP1, a new FGFR1 partner gene associated with 8p11 myeloproliferative syndrome

Hematological malignancies associated with FGFR1 rearrangements (8p11-chromosome eighth-myeloproliferative syndrome (EMS) are rare atypical chronic myeloproliferative disorders that are in most cases associated with eosinophilia and T-cell proliferation. Clinically, EMS is an aggressive disease with a short chronic phase leading to rapid transformation into acute myeloid leukemia. The only effective treatment to date is allogenic stem cell transplantation. The genetic hallmark of this pathology is a recurrent chromosomal 8p11 rearrangement that fuses the tyrosine kinase domain of the fibroblast growth factor receptor 1 gene (FGFR1) to different gene partners. To date, nine FGFR1 partners have been identified in patients with EMS: ZMYM2/ZNF198 at 13q12, FGFR1OP/FOP at 6q27, CEP110/CEP1 at 9q33, BCR at 22q11, FGFR1OP2 at 12p11, TRIM24/TIF1 at 7q34, MYO18A at 17q23, CPSF6 at 12q15 and a human endogenous retroviral sequence, HERV-K at 19q13.3. Here, we report the identification of a tenth FGFR1 partner gene in a case of EMS associated with a t(2;8)(q37;p11) translocation.

The patient, an 82-year-old man, was referred to our department in May 2002 for pancytopenia with hemoglobin level of 10 g per 100 ml, a white blood cell count of 2.4 × 109/l and a platelet count of 40 × 109/l. Bone marrow aspiration showed a refractory anemia with an excess of blasts (15%). Cytogenetical analysis was not performed at that time. Between 2002 and 2007, the white blood cell count remained stable and the patient was occasionally treated with red blood cell transfusion depending on the hemoglobin levels (see Table 1 for evolution of blood counts). In September 2007, the white blood cell count increased to 20 × 109/l with 10% eosinophils, 2–4% myelocytes and metamyelocytes, and 8% circulating blasts. Bone marrow aspiration was hypocellular with moderate dysgranulopoiesis and 15% blasts. The presence of dacryocytes on blood smears suggested some degree of myelofibrosis. In May 2008, a new blood count showed a decrease in hemoglobin levels (8 g per 100 ml) and a stable white blood cell count with an increase in blasts (42%), attesting to the transformation into acute myeloid leukemia. Supportive care with red blood cell transfusions permitted outpatient care at first, but the patient died 1 year later.

Table 1 Patient blood counts during disease progress

The karyotype, as performed on bone marrow cells in September 2007, was interpreted as follows: 46,XY,t(2;8)(q37;p11)[20] (Figure 1a). Conventional cytogenetic analysis on patient blood lymphocytes revealed a normal karyotype, confirming that the t(2;8)(q37;p11) translocation was acquired, and suggesting the diagnosis of EMS. A fluorescence in situ hybridization experiment with BAC RP11-333B24 as a probe spanning the FGFR1 locus confirmed the expected gene rearrangement (Figure 1b). To identify the derivative chromosome der(2) breakpoint, fluorescence in situ hybridization experiments were performed with a series of BAC clones covering chromosomal band 2q37. A split signal was observed for the BAC RP11-497D24 indicating that it contained the chromosome 2 breakpoint (Figure 1b). Furthermore, retrospective analysis by fluorescence in situ hybridization using BACs RP11-333B24 and RP11-497D24 on bone marrow smears performed in 2002 revealed two normal signals for each BAC (data not shown), indicating that the t(2;8) translocation was a secondary event acquired at the time of transformation into acute myeloid leukemia.

Figure 1
figure1

Cytogenetic analysis of patient bone marrow cells. (a) Partial karyotype showing the derivative chromosomes 2 and 8. (b) Fluorescence in situ hybridization using the RP11-333B24 BAC spanning the FGFR1 locus (left) and the RP11-497D24 BAC (right). RP11-333B24 hybridization shows one normal signal on a chromosome 8 (arrow), one signal on chromosome der2 (arrow head) and one signal on chromosome der 8 (asterisk). RP11-497D24 hybridization gives one normal signal on a chromosome 2 (arrow), one signal on chromosome der2 (arrow head) and one signal on chromosome der8 (asterisk).

The 2q37 BAC RP11-497D24 harbors two genes in the same orientation as FGFR1 (5′ centromere—3′ telomere): LRRFIP1 and RAMP1. As all FGFR1 fusion genes reported to date share the feature of contributing their 5′ portion to the fusion joined by the 3′ of FGFR1, and because the LRRFIP1 5′ part was coding for an N-terminal coiled-coil domain, as in many FGFR1 partners, we hypothesized that LRRFIP1 was a more likely candidate partner for an in-frame fusion with FGFR1. Thus, we performed reverse transcription-PCR with specific primers designed to amplify a putative LRRFIP1–FGFR1 fusion transcript. Total RNA was extracted from patient bone marrow cells and reverse transcribed using random hexamers and Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA, USA). We obtained specific amplification of the putative fusion transcript from cDNA with the following design: as all FGFR1 fusions described show a genomic breakpoint close to exon 9, we performed reverse transcription-PCR using several forward primers located in the LRRFIP1 reference sequence (NM_004735.2) and one reverse primer located in exon 10 of FGFR1 (FGFR1ex10: 5′-IndexTermACAGCCACTTTGGTCACACGGT-3′). A specific fragment was detected using a forward primer located in exon 4 of LRRFIP1 (LRRex4: 5′-IndexTermAGACGAGCGCATGTCAGTGGGTAGT-3′), which was not amplified in U937 control cells (Figure 2a, lanes 1 and 4). Sequence analysis of the PCR product revealed the in-frame fusion of LRRFIP1 exon 9 with FGFR1 exon 9 (Figure 2b). As public sequence databases contain an alternative transcript for LRRFIP1 that has coding potential for a highly divergent protein (ENST00000308482, at the Ensembl Genome database), we performed reverse transcription-PCR using a primer that is located in this alternative sequence but is absent from the LRRFIP1 reference sequence (L-ex6: 5′-IndexTermTGAGGTCGCAGCCTGACTTGGAGTAT-3′). No reverse transcription-PCR product was amplified from this second reaction (Figure 2a, lanes 2 and 5), indicating that the LRRFIP1–FGFR1 fusion transcript above may be the single specific transcript produced by the translocation. A reciprocal FGFR1LRRFIP1 fusion transcript was also detected in patient cells using a forward primer located in FGFR1 exon 8 (5′-IndexTermTGTACCTGGAGATCATCATCTATTGCA-3′) and a reverse primer located in LRRFIP1 exon 11 (5′-IndexTermTCACCTCCACTTCACTGGCTCT-3′) (Figure 2a, lane 3). This product was sequenced and confirmed to be the in-frame fusion of exon 8 of FGFR1 with exon 10 of LRRFIP1 (Figure 2c).

Figure 2
figure2

Molecular analysis of the LRRFIP1–FGFR1 fusion. (a) Lanes 1 and 4: specific reverse transcription (RT)-PCR amplification of an LRRFIP1–FGFR1 fusion transcript from a patient sample. Lanes 2 and 5: no amplification was obtained with primer localized in the alternative transcript of LRRFIP1. Lanes 3 and 6: specific amplification of the reciprocal FGFR1LRRFIP1 fusion transcript in patient cells. RT-PCR using primers for ARNT serve as control. (b) Partial sequence of the LRRFIP1-FGFR1 fusion transcript. The exon 9 of FGFR1 is fused to LRRFIP1 exon 9 (bold). The amino-acid translation spanning the fusion is shown under the sequence. (c) Partial sequence of the reciprocal LRRFIP1–FGFR1 fusion cDNA with its amino-acid translation.

LRRFIP1 (Leucine-rich repeat Flightless-Interacting Protein 1) gene (also known as GCF2 for GC-binding factor 2 or TRIP for TAR RNA Interacting Protein) is thus the tenth FGFR1 gene partner. LRRFIP1 is ubiquitously expressed and encodes for a protein found in the nuclear and cytoplasmic compartments. The LRRFIP1 protein has multiple and complex functions. In the nucleus, it acts as a transcriptional repressor that decreases the expression of the epidermal growth factor receptor1 and of the platelet-derived growth factor α-chain.2 LRRFIP1 was also recently identified as the first repressor to directly regulate the tumor necrosis factor-α promoter.3 In the cytoplasm, through its N-terminal coiled-coil domain, LRRFIP1 interacts with the leucine-rich repeat (LRR) region of the Flightless-1 protein, which belongs to the gelsolin family of actin-binding proteins. Finally, LRRFIP1 contains a lysine-rich motif that binds to the TAR RNA element present at the 5′ end of human immunodeficiency virus-1 viral transcripts.

The LRRFIP1-FGFR1 transcript sequence encodes a predicted chimeric protein of 668 amino acids, containing the N-terminal coiled-coil domain of LRRFIP1 and the two tyrosine kinase domains of FGFR1 (Figure 3). The presence of the coiled-coil domain may induce the dimerization of LRRFIP1-FGFR1, thus leading to the constitutive activation of the kinase domains. By this mechanism, LRRFIP1-FGFR1 may promote cellular transformation as shown earlier for other FGFR1 fusions,4, 5 thus representing a possible specific target for therapy.

Figure 3
figure3

Schematic representation of FGFR1, LRRFIP1 and the predicted LRRFIP1–FGFR1 fusion protein. Relevant protein domains are shown. The breakpoints within proteins are indicated by arrows.

Conflict of interest

The authors declare no conflict of interest.

References

  1. 1

    Reed AL, Yamazaki H, Kaufman JD, Rubinstein Y, Murphy B, Johnson AC . Molecular cloning and characterization of a transcription regulator with homology to GC-binding factor. J Biol Chem 1998; 273: 21594–21602.

    CAS  Article  Google Scholar 

  2. 2

    Khachigian LM, Santiago FS, Rafty LA, Chan OL, Delbridge GJ, Bobik A et al. GC factor 2 represses platelet-derived growth factor A-chain gene transcription and is itself induced by arterial injury. Circ Res 1999; 84: 1258–1267.

    CAS  Article  Google Scholar 

  3. 3

    Suriano AR, Sanford AN, Kim N, Oh M, Kennedy S, Henderson MJ et al. GCF2/LRRFIP1 represses tumor necrosis factor alpha expression. Mol Cell Biol 2005; 25: 9073–9081.

    CAS  Article  Google Scholar 

  4. 4

    Guasch G, Delaval B, Arnoulet C, Xie MJ, Xerri L, Sainty D et al. FOP-FGFR1 tyrosine kinase, the product of a t(6;8) translocation, induces a fatal myeloproliferative disease in mice. Blood 2004; 103: 309–312.

    CAS  Article  Google Scholar 

  5. 5

    Ollendorff V, Guasch G, Isnardon D, Galindo R, Birnbaum D, Pebusque MJ . Characterization of FIM-FGFR1, the fusion product of the myeloproliferative disorder-associated t(8;13) translocation. J Biol Chem 1999; 274: 26922–26930.

    CAS  Article  Google Scholar 

Download references

Author information

Affiliations

Authors

Corresponding author

Correspondence to S P Romana.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Soler, G., Nusbaum, S., Varet, B. et al. LRRFIP1, a new FGFR1 partner gene associated with 8p11 myeloproliferative syndrome. Leukemia 23, 1359–1361 (2009). https://doi.org/10.1038/leu.2009.79

Download citation

Further reading

Search

Quick links