Letters to the Editor 146

Figure 1 The hRap1B-retro is localized on 5 q13.3. Upper part: summary of differences in the sequence alignment between hRap1B-retro and hRap1B cDNAs and the corresponding genomic regions on the 5th and 12th . In addition to the three nucleotide substitutions in the open reading frame (ORF), seven more differences between both are present in the untranslated regions (UTRs). The coordinates above the sequences are relative to the start codon ATG in hRap1B cDNA (GI: 58219793). hRap1B-r_Ram1-3: full-length cDNAs of hRap1B-retro from Ramos cell line (GIs: 50475494, 50474682, 50470789); hRap1B-r_Neur: full-length cDNA of hRap1B-retro from neuroblastoma (GI: 50498405); hRap1B-r_Plac: full-length cDNA of hRap1B-retro from placenta (GI: 50492447). 5chr: genomic DNA sequence from chromosome 5 (GI: 51465008: 26064529–26060269), 12chr: genomic DNA sequence from (GI: 29803948: 31147958– 31197631). Lower part: exonic structure of the hRap1B-retro and hRap1B mother genes mapped onto their cDNAs. Numbering stands for exon numbers.

shown to transform NIH 3T3 cells oncogenically.2 In this light, a 4Department of Physiological Chemistry and the Centre for better expression profiling of the hRap1B-retro gene identified Biomedical Genetics, University Medical Centre Utrecht, by us will help to validate whether it defines a novel drug target. CG Utrecht, The Netherlands E-mail: [email protected]

Acknowledgements

TZ thanks Professor Martin Vingron for the helpful discussions and References support. TZ acknowledges support received from the BioSapiens Network of Excellence, funded by the European Commission 1 Gyan E, Frew M, Bowen D, Beldjord C, Preudhomme C, Lacombe C within its FP6 Programme, under the thematic area ‘Life sciences, et al. Mutation in is a rare event in myelodysplastic syndromes. Leukemia 2005; 19: 1678–1680. genomics and biotechnology for health’, contract number LHSG- 2 Takahashi K, Mitsui K, Yamanaka S. Role of ERas in promoting CT-2003-503265. tumour-like properties in mouse embryonic stem cells. Nature 1,2 2,3 2 2003; 423: 541–545. T Zemojtel , T Penzkofer , M Duchniewicz and 3 Penzkofer T, Dandekar T, Zemojtel T. L1Base: from functional FJT Zwartkruis4 1 annotation to prediction of active LINE-1 elements. Nucleic Acids Department of Computational Molecular Biology, Max Res 2005; 33: D498–D500. Planck Institute for Molecular Genetics, Berlin, Germany; 4 Cost GJ, Boeke JD. Targeting of human retrotransposon integration 2 In Silico Miners, Berlin, Germany; is directed by the specificity of the L1 endonuclease for regions of 3Biozentrum, Am Hubland, Wuerzburg, Germany and unusual DNA structure. Biochemistry 1998; 37: 18081–18093.

Supplementary Information accompanies the paper on the Leukemia website (http://www.nature.com/leu)

Reply to ‘hRap1B-retro: a novel human processed Rap1B gene blurs the picture?’ by Zemojtel et al

Leukemia (2006) 20, 146–147. doi:10.1038/sj.leu.2404042; published in the September issue of Leukemia,2 we initially used published online 24 November 2005 previously published oligonucleotides3 to analyze rap1b tran- script in bone marrow samples from 27 patients with myelo- We agree with the comments made by Zemojtel et al.1 and we dysplastic syndromes (MDS) and two secondary acute myeloid thank them very much for the proposed hypothesis. In our work leukemias (sAML). In one patient with RARS and abnormal

Leukemia Letters to the Editor 147 karyotype (45, XX, À7/46, XX (19/1)), we observed two 5q13.3 (hrap1b-retro) regions. FISH analysis of these loci is substitutions as compared to rap1b cDNA published sequence, ongoing in three of the four patients. which we interpreted as two amino-acid substitutions in G12R If the hrap1b-retro pseudogene is transcribed, the biological and K42E in Rap1B . We then analyzed rap1b genomic consequences may be equivalent to that of an activating DNA by using denaturating gradient gel electrophoresis. At that mutation in the mother gene. Indeed, the G12R substitution time, we were aware of the presence of a sequence highly involves an amino acid that is crucial for the regulation of the homologous to rap1b corresponding to a pseudogene located on GTP loading of small G of the Ras superfamily. Thus, chromosome 5q13.3 and, in order to avoid the amplification of there is a possibility that the corresponding protein is converted this pseudogene, we designed oligonucleotides located in the into an active state. During the course of our study, we tried to intronic sequences of rap1b on chromosome 12, including one detect Rap1-GTP in bone marrow mononuclear cells from MDS pair specific for exon 2 and another one to amplify exon 3. patients using a RalGDS Ras-binding domain, as previously Using this strategy, we did not identify any rap1b gene mutation described.4 Although a positive control was easily obtained after in genomic DNA of 56 MDS patients tested. Unfortunately, activation of normal platelets, we were unable to detect any DNA from the patient in whom we had identified substitutions activation of Rap1 in MDS cells. in cDNA was not available for genomic analysis. In conclusion, it should be interesting to screen MDS patients The rap1b pseudogene on chromosome 5q13.3 is a retro- for the expression of the hrap1b-retro transcript coupled to FISH transposon (hRap1B-retro) with two exons expected to lack analysis of the 12q14 and 5q13.3 loci. Evidence for a recurrent intronic sequences. As indicated by Zemojtel and co-workers, transcription of the pseudogene could lead to a novel therapeutic an intronic sequence is actually created by insertion of a SVA strategy targeting the anchorage of Rap1 to the plasma sequence in the retrogene. Evidence for the transcription of this membrane by using geranylgeranyltransferase inhibitors.5 Farne- retrogene is provided by the fact that the corresponding cDNAs syltransferase inhibitors of Ras have already been used in patients in the database are devoid of the intronic sequence. At the time with high grade MDS, because of recurrent activating mutations we prepared our manuscript, we missed this information. of N-ras. However, sensitive tools for evaluating the efficacy of We would like to provide additional data that may reinforce inhibitors on GTP loading of Ras and Rap1 are needed. the discussion on the role of rap1 gene products in leukemogen- esis. We recently analyzed additional blast cell cDNAs from patients with sAML and de novo AML (n ¼ 8). In three of these Acknowledgements samples, including two with normal karytotype and one with trisomy 8, we again identified the changes that could indicate We thank Dr J. Andrieux, CHRU Lille, who provided 12q14 BAC amino-acid substitutions in G12R and K42E in Rap1B protein. In and Dr F. Viguie´ from Hotel-Dieu (Paris) for cytogenetic analysis. addition, we observed a third substitution suggesting a G77G mutation when compared to the published sequence of rap1b No further reply from Zemojtel et al cDNA. Considering the similarity of nucleotide substitutions E Gyan1, F Porteu1 and M Fontenay1,2 between the cDNAs that we have sequenced and the hRap1B- 1Departement d’Hematologie, Institut Cochin INSERM U567, retro cDNA described by Zemojtel, we could have amplified CNRS UMR 8104, Universite Rene-Descartes, hRap1B-retro cDNA encoded by the gene located on chromo- Paris, France and some 5q13.3. However, we remain puzzled by the fact that the 2Service d’He´matologie Biologique, Hoˆpital Cochin, AP-HP, identified pattern of substitutions demonstrates a homozygous Paris, France pattern. As using the same set of oligonucleotides unable to E-mail: [email protected] distinguish between rap1b and hrap1b-retro in the same PCR conditions, why did we detect the hrap1b-retro transcript in some cases whereas the rap1b transcript was amplified in the References others? Because the set of oligonucleotides used for amplifica- tion of rap1b cDNA was also able to amplify hrap1b-retro 1 Zemojtel T, Penzkofer T, Duchniewicz M, Zwartkruis FJT. transcript and genomic sequence, we first hypothesized that hRap12B-retro: A novel human processed Rap1B gene blurs the cDNA samples could be contaminated by genomic DNA. To picture? Leukemia 2005. test this hypothesis, we designed a genomic PCR that 2 Gyan E, Frew M, Bowen D, Beldjord C, Preudhomme C, Lacombe C specifically amplified the hrap1b-retro gene as a 1350 bp et al. Mutation in RAP1 is a rare event in myelodysplastic fragment using a 50oligonucleotide located in exon 1 and a syndromes. Leukemia 2005; 19: 1678–1680. 30oligonucleotide located in the intronic region. The expected 3 Vanvooren V, Allgeier A, Nguyen M, Massart C, Parma J, Dumont JE et al. Mutation analysis of the Epac-Rap1 signaling pathway in cold product was easily identified in the genomic DNA of controls thyroid follicular adenomas. Eur J Endocrinol 2001; 144: and of the two sAML with amino-acid substitutions, but not in 605–610. the corresponding cDNAs. These results confirmed that cDNA 4 M’Rabet L, Coffer P, Zwartkruis F, Franke B, Segal AW, Koenderman samples were not contaminated with genomic DNA. We could L et al. Activation of the small GTPase rap1 in human neutrophils. also hypothesize that we preferentially amplified the hrap1b- Blood 1998; 92: 2133–2140. retro because of either a deletion of the mother gene or the 5 Di Paolo A, Danesi R, Caputo S, Macchia M, Lastella M, Boggi U et al. Inhibition of protein farnesylation enhances the chemother- amplification of the pseudogene locus. As mentioned pre- apeutic efficacy of the novel geranylgeranyltransferase inhibitor viously, cytogenetic analysis (R-banding) of the reported patients BAL9611 in human colon cancer cells. Br J Cancer 2001; 84: did not show any abnormality of the 12q14 (rap1b) or the 1535–1543.

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