Blood, Vol. 92 No. 9 (November 1), 1998:
pp. 3481-3484
CORRESPONDENCE
EHT, a New Member of the MTG8/ETO Gene Family, Maps on 20q11
Region and Is Deleted in Acute Myeloid Leukemias
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LETTER |
To the Editor:
In an attempt to identify potential new genes homologous to
ETO,1,2 we screened the dbEST database
(http://www.ncbi.nlm.nih.gov./dbEST/index.html) using the entire ETO
cDNA sequence as a probe (EMBL accession no. X79990). Among the ESTs
identified, we selected two overlapping clones (clone IDs 43629 and
274508) and sequenced them to completion. A putative translation
initiation site was identified by the presence of a strong Kozak
consensus sequence,3 followed by a 1,725-bp open reading
frame (ORF) coding for a putative protein of 575 amino acids (aa)
(GenBank accession no. AF068266). We named this gene EHT
(ETO Homologous on chromosome Twenty; see mapping data
below). The putative EHT protein is closely homologous to ETO/MTG8 (
65% identity) and Nervy, an ETO Drosophila
homolog (24% identity) showing the four conserved domains found in
MTG8 and Nervy.4 To find EST clones that could
represent the EHT 5
UTR, we screened the dbEST data bank with the 5
end of clone 274508, and identified an EST clone (GenBank accession no.
AA635096) encompassing the EHT ATG codon, preceded by an in-frame stop
codon 129 bp upstream (Fig 1, top).
Therefore, we considered this sequence the putative 5UTR of EHT gene.
While this work was in progress, the cloning of a similar ORF, named
MTGR1 (myeloid translocation gene-related protein 1) was
reported5; it presents an ATG codon, immediately preceded
by a stop codon and with no clear Kozak sequence, upstream to and
in-frame with the EHT ATG (see Fig 1, top), leading to the coding of
additional 29 aa with respect to EHT ORF. Interestingly, Calabi and
Cilli have recently deposited in the dbEST data bank a 5 UTR MTGR1 sequence (accession no. AF052212) that is identical to the putative EHT
5 UTR (GenBank clone AA635096). Overall, these data suggest the
presence of two alternative 5 ends in the MTGR1/EHT gene, similar to
ETO/MTG8 gene.1,2 The existence in nature of EHT ORF was
confirmed by direct sequencing of a reverse transcriptase-polymerase chain reaction (RT-PCR) amplified fragment encompassing the
coding sequence from HL60 mRNA (data not shown).
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| Fig 1.
(Top) Sequence comparison between the 5 end of EHT and
MTGR1 (italics) cDNAs. Translated sequences are shown in capital
letters; a 27-bp nucleotide stretch upstream of the EHT ATG which is
similar to the MTGR1 is underlined. The stop codons upstream of ATGs
are in bold letters. (Bottom) Mapping by FISH of EHT gene on chromosome
20q11.
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To map the EHT gene, we generated by PCR a probe from the specific 3
UTR of the EHT gene and screened a human placenta cosmid library
(Clontech, Palo Alto, CA), isolating a specific cosmid clone
subsequently used as probe in fluorescence in situ hybridization (FISH)
analysis on normal human metaphase spreads. By FISH we mapped the gene
on chromosome 20q11 (Fig 1, bottom); the mapping was further refined by
PCR of DNA from somatic cell hybrids (kindly provided by Mariano
Rocchi, University of Bari, Italy) on 20q11.2-20q11.3 (data not shown).
Cytogenetic studies have shown that this region is deleted in 10%
cases of polycythemia vera (PV),6,7 5% myelodysplastic syndromes (MDS), and in 3% of acute myeloid leukemias
(AML).8-11 We performed Southern blot analysis on 40 cases
of AML (9 M0, 8 M1, 6 M2, 9 M4, 8 M5) and found gene deletion
apparently in four cases (1 M0, 1 M1, 2 M4) (10%) (Fig
2A), a frequency higher than that found by
conventional cytogenetics. However, the assessment of the deletion
frequency needs the analysis of a more representative series.
Furthermore, as no RNA was available from hemizygous deleted patients,
we were not able to test whether EHT may be a candidate tumor
suppressor gene of the region by mutation analysis of the retained
allele.12
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| Fig 2.
(A) Analysis of EHT gene in AML cases by quantitative
Southern blot: cases AML11, 12, 22, and 24 show a loss of signal with
respect to the control probe (PFL-1 probe for Bcl-2 gene). Because the
percentage of leukemia cells in all cases is greater than 70%, this
finding is consistent with an homozygous deletion. The probe (EHT3)
was specific for the 3 UTR of EHT. (B) EHT gene expression analysis.
Poly-A+ Northern blot filters (Clontech), were hybridized
with the EHT3 probe. The two dashes indicate the molecular weight (9.5 and 7.5 kb). The two transcripts were not represented in all of the
tissues at the same level; in the testis, heart, brain, and skeletal
muscle, the lower transcript was particularly expressed.
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We investigated the expression pattern of EHT by means of Northern blot
analysis using the 3 UTR specific probe. Two transcripts of 9.5 and
7.5 kb (Fig 2B) were detected; the difference in size of the two
transcripts may be due to the use of different polyadenylation signals
and/or alternative 5UTR regions, as in the case of the MTGR8
gene.13 In normal fetal and adult human tissues, the two transcripts were expressed ubiquitously, although at different levels
(Fig 2B); in tumoral cell lines, low levels of expression were detected
in hematopoietic cell lines of lymphoid and myeloid origin and melanoma
cells, while higher levels of expression were found in the SW480
carcinoma cell line, but apparently absent in the A549 lung
carcinoma cell line (data not shown).
As far as normal MTGR1/EHT function is concerned, Kitabayashi et
al5 showed the direct interaction of MTGR1 and the
AML1-MTG8 fusion protein, leading to an enhancement of cell
proliferation mediated by granulocyte colony-stimulating factor (G-CSF)
in a murine myeloid model (L-G cell line). This suggests that MTGR1 has
an oncogenic rather than a tumor suppressor activity. Nevertheless, when MTGR1 is transfected alone in L-G cells, the proliferative response to G-CSF was lower than in the normal control, thus suggesting a possible negative growth-control in normal cells.
In conclusion, the data presented here and those previously reported
suggest that the MTGR1/EHT gene may represent a new candidate for the
tumor suppressor gene supposed to be involved in the deletion of the
20q11 region in myeloid tumors. Further studies are necessary to rule
out this hypothesis.
Nicola Stefano Fracchiolla
Gualtiero Colombo
Palma Finelli
Anna Teresa Maiolo
Antonino Neri
Laboratorio di Ematologia Sperimentale e Genetica
Molecolare
Servizio di Ematologia and Dipartimento di Medicina
Interna
Università di Milano, Ospedale Maggiore
I.R.C.C.S.
Milano, Italy
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ACKNOWLEDGMENT |
A partial cDNA sequence of the EHT gene was deposited by us in the
GenBank database under accession number AF039200, on 18-DEC-1997,
before the MTGR1 delivery date (January 22, 1998). This work was
supported by a grant from Associazione Italiana Ricerca sul Cancro
(AIRC) to A.N.
| |
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