Absence of mutations in the key megakaryocyte transcriptionalregulator FOG-1 in patients with idiopathic myelofibrosis

Idiopathic myelofibrosis (IM) is a clonal myeloproliferative

disorder of unknown aetiology, characterized by progressive

bone marrow fibrosis and extramedullary haematopoiesis. A

cardinal feature of IM is the excessive proliferation and

abnormal maturation of megakaryocytes. Recently, abnormal-

ities in two critical transcription factors that regulate mega-

karyocyte differentiation and proliferation, GATA1 and its key

co-factor FOG-1, have been implicated in human megakaryo-

cyte disorders, including IM. In humans, germline structural

mutations in the GATA1 gene that weaken GATA1:Fog-1

interaction (Nichols et al, 2000; Freson et al, 2002) or alter

DNA binding (Yu et al, 2002) lead to excessive accumulation

of abnormally differentiating megakaryocytes. Moreover,

acquired GATA1 mutations in Down syndrome (DS) neonates

and children have been reported in the linked disorders of

Transient Myeloproliferative Disorder and DS-associated

Acute Megakaryocytic Leukaemia (reviewed in Gurbuxani

et al, 2003). Similarly, mice that lack megakaryocyte GATA1

expression (GATA1lo mice) develop similar phenotypic

abnormalities in megakaryocytopoeisis (Shivdasani et al,

1997; Vyas et al, 1999). More specifically with reference to

IM, over time, GATA-1lo mice develop a condition that closely

resembles IM (Vannucchi et al, 2002). Lastly, a recent report

(Martyre et al, 2003) showed that expression levels of the

megakaryocyte transcription regulator FOG-1 were increased

in peripheral blood CD34+ cells from 20 IM patients when

compared with normal controls. The combination of these

findings suggested that abnormal megakaryocyte differenti-

ation may, in part, be a primary abnormality in IM and that

searching for mutations in a pathway regulated by GATA1/

FOG-1 is warranted in patients with IM.

Given the above, particularly the finding of Martyre et al

(2003), we studied genomic DNA from peripheral blood

mononuclear cells from 18 adult patients (age 49–80 years)

with IM (after obtaining informed consent and ethical

approval from our institutions), looking for mutations in the

FOG-1 gene. Details of the mutation analysis are in Table I.

Two sequence abnormalities were detected that would be

Table I. Analysis of in FOG-1 exon sequence in patients with idiopathic myelofibrosis.

Patient no.

Abnormalities in DHPLC migration profiles or sequence

DHPLC analysis Sequence analysis

1. Fragment 5 Intron 4 88 337 661 (-A) heterozygous

Intron 4 88 337 665 (T > C) homozygous

2. Fragment 2 Exon 2: AGA > GGA substitution in nucleotide 88 299 313. Change in amino acid

22 R > G. Known SNP ID: rs3751673

3. Fragments 2 and 7 Exon 2: AGA > GGA in nucleotide 88 299 313. Change in amino acid

22 R > G. Known SNP ID: rs3751673

Exon 7: TGC > TGT heterozygous substitution in nucleotide 88 342 947 resulting

in a neutral change at amino acid 264

4. Fragments 7 and 8 Intron 8 88 343 126 (C > T) heterozygous

Exon 8: GGA > GGG substitution in nucleotide 88 343 480 resulting in a neutral

change at amino acid 316

5. Normal DHPLC profile

6. Fragment 7 Exon 7: TGC > TGT heterozygous substitution in nucleotide 88 342 947 resulting in a

neutral change at amino acid 264

7. Normal DHPLC profile Wild type sequence

8. Fragments 7 and 8 Intron 8 88 342 126 (C > T) heterozygous

Exon 8: GGA > GGG substitution in nucleotide 88 343 480 resulting in a neutral

change at amino acid 316

9. Fragments 2 and 5 Exon 2: AGA > GGA in nucleotide 22 299 313. Change in amino acid 22 R > G. Known

SNP ID: rs3751673

Intron 4 88 337 661 (-A) heterozygous

Intron 4 88 337 665 (T > C) homozygous

correspondence

doi:10.1111/j.1365-2141.2004.05100.x ª 2004 Blackwell Publishing Ltd, British Journal of Haematology, 126, 750–755

predicted to result in amino acid changes in the FOG-1

protein. In six of 19 patients there was a single nucleotide

change at position 386 (AGA > GGA) in exon 2 that would

result in a change in amino acid 22 (R > G). This nucleotide

change is likely to be a common polymorphism as it was seen

in 46 of 80 chromosomes in a control European population

and has been described in the National Center for Biotechno-

logy Information Single Nucleotide Polymorphism (NCBI

SNP) database (Build 121). In one patient there was an

addition of three nucleotides (AAG), at nucleotide 931 that

would result in addition of lysine at residue 204. This change

was not seen in 80 chromosomes from a Caucasian control

population. This lysine residue does not reside in any known

functional important motif in FOG-1. These findings suggest

that sequence changes in FOG-1 resulting in possible patho-

genetic mutations are not a common abnormality in IM.

However, our study does not rule out the possibility of

mutations in FOG-1 cis-regulatory elements leading to abnor-

mal FOG-1 expression in IM patients.

Chris Fisher1

David Steensma1

Riaz Janmohamed2

Richard Kaczmarski2

John T. Reilly3

Paresh Vyas1,4

1MRC Molecular Haematology Unit, Weatherall Institute of Molecular

Medicine, John Radcliffe Hospital, University of Oxford, Oxford,2Department of Haematology, Hillingdon Hospital, Uxbridge, Middlesex,3Department of Haematology, Royal Hallamshire Hospital, Sheffield, and4Department of Haematology, Weatherall Institute of Molecular

Medicine, John Radcliffe Hospital, University of Oxford, Oxford, UK.

E-mail: paresh.vyas@imm.ox.ac.uk

References

Freson, K., Matthijs, G., Thys, C., Marien, P., Hoylaerts, M.F.,

Vermylen, J. & Van Geet, C. (2002) Different substitutions at residue

Table I. Continued

Patient no.

Abnormalities in DHPLC migration profiles or sequence

DHPLC analysis Sequence analysis

10. Fragment 2 Exon 2: AGA > GGA in nucleotide 88 299 313. Change in amino acid 22 R > G. Known

SNP ID: rs3751673

11. Fragment 2 Exon 2: AGA > GGA in nucleotide 88 299 313. Change in amino acid 22 R > G. Known

SNP ID: rs3751673

12. Fragments 5 and 6 Intron 4 88 377 661 (-A) heterozygous

Intron 4 88 337 665 (T > C) homozygous

Exon 6: insertion of three nucleotides AAG, at nucleotide 88 339 000 resulting in the addition

of K at amino acid 204

13. Fragment 2 Exon 2: AGA > GGA in nucleotide 88 299 313. Change in amino acid 22 R > G. Known

SNP ID: rs3751673

14. Fragment 2 Exon 2: AGA > GGA in nucleotide 88 299 313. Change in amino acid 22 R > G. Known

SNP ID: rs3751673

15. Normal DHPLC profile

16. Fragment 4 Intron 3 88 327 704 (G > A) heterozygous

17. Fragment 2 and 7 Exon 2: AGA > GGA in nucleotide 88 299 313. Change in amino acid 22 R > G. Known

SNP ID: rs3751673

Exon 7: TGC > TGT heterozygous substitution in nucleotide 88 342 947 resulting in a

neutral change at amino acid 264

18. Fragments 2 and 4 Exon 2: AGA > GGA in nucleotide 88 299 313. Change in amino acid 22 R > G. Known

SNP ID: rs3751673

3Æ4 kb mRNA of the FOG-1 gene was divided into 10 exons. Genomic DNA from 18 patients was used as template to generate polymerase chain

reaction (PCR) fragments encompassing each exon and c. 50 nucleotides of flanking sequence intron on each side of the exon were isolated, with the

exception of large exon 10 (1Æ1 kb), which was divided into five fragments for analysis. All PCR fragments were subject to DHPLC (Denaturing High

Pressure Liquid Chromatography, Wave, Transgenomics, Omaha, NE, USA). If abnormal migration profiles were detected, the PCR products were

directly sequenced by bi-directional sequencing using the ABI BigDye terminator sequencing kit v3Æ1 (Perkin Elmer, Beaconsfield, UK) and an ABI

3100 Capillary Array sequencer (Perkin Elmer, Beaconsfield, UK). In four patients, all FOG-1 exons were completely sequenced. Sequences were

analysed using macvector (Accelrys, Cambridge, UK) and sequencher v3.1.1 (Genes Codes Corporation, Ann Arbor, MI, USA) software packages.

The nucleotide position of FOG-1 gene was obtained from ensembl (http://www.sanger.ac.uk/). The NCBI SNP (Build 121, http://

www.ncbi.nlm.nih.gov/SNP/) database was screened to determine if sequence changes detected were previously known polymorphisms. Details of

PCR primers and conditions and parameters for DHPLC analysis are available on request.

Correspondence

ª 2004 Blackwell Publishing Ltd, British Journal of Haematology, 126, 750–755 751

D218 of the X-linked transcription factor GATA1 lead to altered

clinical severity of macrothrombocytopenia and anemia and are

associated with variable skewed X inactivation. Human Molecular

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mechanisms of myeloid leukemogenesis in Down syndrome. Blood,

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Martyre, M.C., Steunou, V., LeBousse-Kerdiles, M.C. & Wietzerbin, J.

(2003) Lack of alteration in GATA-1 expression in CD34+ hema-

topoietic progenitors from patients with idiopathic myelofibrosis.

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Nichols, K.E., Crispino, J.D., Poncz, M., White, J.G., Orkin, S.H.,

Maris, J.M. & Weiss, M.J. (2000) Familial dyserythropoietic anaemia

and thrombocytopenia due to an inherited mutation in GATA1.

Nature Genetics, 24, 266–270.

Shivdasani, R.A., Fujiwara, Y., McDevitt, M.A. & Orkin, S.H. (1997)

A lineage-selective knockout establishes the critical role of

transcription factor GATA-1 in megakaryocyte growth and platelet

development. EMBO Journal, 16, 3965–3973.

Vannucchi, A.M., Bianchi, L., Cellai, C., Paoletti, F., Rana, R.A.,

Lorenzini, R., Migliaccio, G. & Migliaccio, A.R. (2002) Development

of myelofibrosis in mice genetically impaired for GATA-1 expression

(GATA-1(low) mice). Blood, 100, 1123–1132.

Vyas, P., Ault, K., Jackson, C.W., Orkin, S.H. & Shivdasani, R.A.

(1999) Consequences of GATA-1 deficiency in megakaryocytes and

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Keywords: myleofibrosis, megakaryopoiesis, GATA1,

FOG-1, transcription factors.

Insertion of a genomic fragment of chromosome 19 betweenBCR intron 19 and ABL intron 1a in a chronic myeloid leukaemiapatient with l-BCR-ABL (e19a2) transcript

The l-BCR-ABL type of chronic myeloid leukaemia (CML),

where the BCR exon 19 fuses to ABL (Saglio et al, 1990) is very

rare. To date, over 20 patients with l-BCR-ABL type have been

reported, although not all of them were diagnosed with CML

and the clinical features of l-BCR-ABL-type CML have not yet

been well characterized yet. We analysed 226 Philadelphia

chromosome-positive CML patients. In 126 patients, reverse-

transcription polymerase chain reaction (RT-PCR) was per-

formed for both M-BCR-ABL and l-BCR-ABL. The frequencyof each type was e14a2 67.5% (85/126), e13a2 30.2% (38/126),

rare e13a3 0.8% (1/126) (Liu et al, 2003), and e19a2 1.6%

(2/126). Southern blotting was performed (Tanaka et al, 1993)

in the remaining 100 patients; one patient was identified as

l-BCR-ABL-type and confirmed by RT-PCR. Altogether, we

found three l-BCR-ABL-type CML patients of 226 (frequency

1.3%), which were similar to other reports, i.e. less than 0.8%

(2/250) (Saglio et al, 1990) and 1.6% (4/250) (Arana-

Trejo et al, 2002). Patient 1 was a 72-year-old woman.

Her white blood cell (WBC) count was 10.0 · 109/L and

the platelet count was 799 · 109/L. 9.5% of blasts were seen

in the bone marrow. Chromosomal analysis showed

47,XX,+8,t(9;22)(q34;q11)[13/14], indicating CML in acceler-

ated phase. She refused therapy. Myeloid-type blastic crisis

(BC) occurred after 1 year, with additional chromosomal

abnormalities [+8, i(17)(q10)], which then evolved to +8,

i(17)(q10), +i(17)(q10) 5 months after BC. Patient 2 was a

59-year-old woman with a WBC count of 12.0 · 109/L and a

platelet count of 1050 · 109/L. She was treated with interferon-

a and achieved a complete cytogenetic response. Patient 3 was

an 86-year-old woman. Her WBC count was 48.3 · 109/L and

the platelet count was as high as 2094 · 109/L. She was treated

with imatinib mesylate. Chromosomal analysis 9 months after

the therapy was 46,XX,t(9;22)(q34;q11)[18/20], 47,XX,+8,t

(9;22)(q34;q11)[1/20], 46,XX[1/20], indicating a minor cyto-

genetic response with an additional chromosomal abnormality.

In addition to the normal e19a2 fusion transcript, patient 3

had a larger transcript (Fig 1A), where 82 bp of BCR intron 19

was transcribed after exon 19, and then fused to ABL exon a2.

We tried to determine the DNA breakpoints by PCR using

genomic DNA as templates. Sequencing of the PCR product

(1031 bp) from patient 3 (Fig 1B) showed that the DNA

breakpoint of BCR was 82 bp downstream of the start of intron

19, and that of ABL was 416 bp upstream of the end of ABL

intron 1a (Fig 1C). Amazingly, a partial genomic fragment of

chromosome 19 clone CTC-273B12 (Genbank AC008403,

bases 4728–5199) was inserted between these breakpoints. No

mutation was found at the splicing-donor site of BCR intron

19. However, a sequence GTACCA was found in the inserted

fragment, which seems to have been recognized as a splicing-

donor site for the aberrant transcript, though it did not

completely match the consensus sequence.

In the literature, at least five l-BCR-ABL-type CML patients

have been reported who developed blastic transformation with

additional chromosomal aberrations, such as i(17)(q10), +8,

+Ph (Verstovsek et al, 2002). These reports and our two cases

indicate that l-BCR-ABL-type CMLmay not differ significantly

from M-BCR-ABL type in terms of clonal evolution and

chromosomal aberration at BC. Aberrant transcripts and

Correspondence

752 ª 2004 Blackwell Publishing Ltd, British Journal of Haematology, 126, 750–755