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Analysis of the oxygen sensing pathway genes in familial chronic myeloproliferative neoplasms and identification of a novel EGLN1 germ‐line mutation

2011; Wiley; Volume: 153; Issue: 3 Linguagem: Inglês

10.1111/j.1365-2141.2010.08551.x

1365-2141

Elena Albiero, Marco Ruggeri, Stefania Fortuna, M. Bernardi, Silvia Finotto, Domenico Madeo, Francesco Rodeghiero,

Acute Myeloid Leukemia Research

Ph-negative chronic myeloproliferative neoplasms (MPNs) include polycythaemia vera (PV), essential thrombocythaemia (ET) and primary myelofibrosis (PMF) (Swerdlow et al, 2008). Despite the discovery of increasing numbers of genetic aberrations in MPNs (Oh et al, 2010; Tefferi, 2010), the oncogenic causes of this group of clonal hematologic neoplasms are complex and largely unknown. In addition to JAK2V617F, found in most PV patients and in about half of the patients with ET or PMF (Baxter et al, 2005), JAK2-exon 12 mutations have also been found in patients with isolated erythrocytosis (IE) or in JAK2V617F-negative PV cases (Bernardi et al, 2009). Familial clustering of MPNs supports the evidence that pathological phenotype is driven by yet undefined susceptibility genes (Bellanné-Chantelot et al, 2006; Landgren et al, 2008) and that multiple genetic lesions are involved in the pleiotropy of these neoplasms, including the existence of a pre-JAK2 mutated clone (Jäger & Kralovics, 2010). Recent insights of the genetic causes of IE came from the functional and biochemical investigation of the oxygen-sensing pathway, which includes egl nine homolog 1 (EGLN1) (also known as prolyl hydroxylase domain protein 2), von Hippel-Lindau tumour suppressor (VHL) protein and α-subunit of the hypoxia-inducible factor (HIF-α) protein. Heterozygous mutations in EGLN1 have been associated with both familial and sporadic IE (Lee, 2008). We investigated the oxygen-sensing pathway genes in 26 patients from 12 families with at least two cases of MPNs, including the proband and his/her first-degree relatives, to investigate the possible contribution of disease alleles other than JAK2 in the pathogenesis or in phenotype modulation of these disorders. Patients were identified from family investigation of all consecutive MPN patients diagnosed at our centre between 1985 and 2008. Patients met the World Health Organisation criteria for PV, ET or PMF. JAK2 (V617F and exon 12) and MPL (W515L/K) mutational status had been determined in all cases or in ET affected subjects, respectively. Peripheral blood samples at diagnosis and epithelial cells from the mutated patients were collected after informed consent and approval of the local Ethical Committee. Genomic DNA was extracted from whole blood with Blood Core kit B (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Amplifications of the EGLN1 coding region (exon 1–5) and of the intron/exon boundaries were performed under standard conditions by polymerase chain reaction (PCR) using FastStart Taq DNA Polymerase (Roche, Nutley NJ, USA), in a GeneAmp® PCR system 2700 (Applied Biosystems, Foster City, CA, USA). The three VHL exons and EPAS1-exon 12 were also analyzed, according to a similar procedure (PCR conditions are available upon request). Primers sequences (obtained from Primer3 software v4.0, http://frodo.wi.mit.edu/primer3/input.htm) and PCR fragments lengths are given in Table I. The PCR products were purified. Sequencing reactions were carried out using Big Dye® sequencing kit (Applied Biosystems). Direct sequencing was performed in both directions for all samples on an automated sequencer ABI Prism® 3130 Genetic Analyzer (Applied Biosystems). Electropherograms were compared with the EGLN1 (National Center for Biotechnology Information, NCBI RefSeq NM_022051), VHL (NCBI RefSeq NM_000551) and EPAS1 (NCBI RefSeq NC_000002) wild-type genes sequences. Allele-specific PCR (AS-PCR) was performed to confirm the identity of the newly identified mutation. Reaction mixtures were run on a GeneAmp® PCR System 2700 at standard conditions and PCR fragments were detected on agarose gel (Fig 1B). Molecular analyses results. (A) Electropherograms showing the presence of mutated allele against the wild-type (wt) control. Nucleotide position, nucleotide change and corresponding amino acid change are indicated below. (B) Allele-specific polymerase chain reaction. Lane 1: molecular weight marker 100 bp; Lane 2: proband DNA amplified with mutation-specific primer; Lane 3: DNA from probands' son amplified with mutation-specific primer; Lane 4: control DNA (wt) amplified with mutation-specific primer; Lane 5: reaction control (no template). (C) Pedigree of the family with MPD. Squares represent males, circles females, affected individuals are indicated in black and slashes indicate deceased members. An asterisk indicates the genetically tested individuals. (D) Schematic diagram representing the human EGLN1 protein. Znf_MYND: Zinc finger MYND-like domain. Black diamonds indicate the location of the erythrocytosis-associated EGLN1 mutations (Lee, 2008). Thick arrow shows the novel genetic variation described in this study. Numbers indicate amino acid residue positions. In one patient, molecular biology studies revealed a G>C missense heterozygous mutation at c.471 that is predicted to result in a p.Gln157His replacement in the EGLN1 amino acid sequence (Fig 1A). The novel mutation was named in accordance with the standard international nomenclature guidelines of the Human Genome Variation Society (HGVS, http://www.hgvs.org/mutnomen/). The patient with the new mutation, a male aged 65 years, was referred on January 2003 for PV. At diagnosis haemoglobin and haematocrit were increased (171 g/l and 55·2% respectively) in the presence of leucocytosis (21·8 × 109/l) and thrombocytosis (703 × 109/l). Physical examination was negative. Serum erythropoietin level was within normal range (29 iu/ml, NR 5–30). A bone marrow biopsy showed hyperplasia of all myeloid lineages (erythropoietic, megakaryocytic and granulocytic cells). The subject underwent regular repeated phlebotomies to lower the haematocrit; cytoreduction with hydroxycarbamide was prescribed to normalize the platelet count over a follow-up period of 6 years; moreover, he received warfarin for atrial fibrillation. In May 2006 the patient was found to be homozygous for JAK2V617F. At last follow-up (May 2009) the subject remained JAK2V617F positive and erythropoietin-independent endogenous erythroid colonies were obtained. The patient showed a normal haematocrit (46%), haemoglobin level (139 g/l), platelet count (201 × 109/l) and increased white blood cell count (23 × 109/l). Splenomegaly developed during follow-up, but no progression to myelofibrosis was observed; no thrombosis or haemorrhage was recorded during follow-up. The germ-line nature of the EGLN1 mutation was confirmed by comparison with non-hematopoietic tissue, and genomic DNA from oral epithelial cells of the index case (buccal cotton-swab sampling) was used as germ-line control. The patient's son (aged 40 years) was also affected by mild erythrocytosis, was heterozygous for the p.Gln157His variation, but was JAK2V617F-negative. Haemoglobin was slightly increased (170 g/l) with normal haematocrit (50%) and leucocyte (7·4 × 109/l) and platelet (211 × 109/l) counts. Serum erythropoietin level was low (8 iu/ml). The patient had no splenomegaly. The proband's sister was also affected by JAK2V617F positive PV as well, but was already deceased at the time of study. Haematological data for the older generation (Fig 1C) is not available. The two subjects with the novel mutation did not carry VHL, EPAS1-exon 12, MPL or JAK2-exon 12 mutations. p.Gln157His is the most N-terminal mutation reported to date in the EGLN1 protein (Fig 1D), lying between the N-terminal MYND zinc finger-like domain (amino acids 21–58) and the highly conserved C-terminal catalytic domain (amino acids 181–426). This amino acid variation does not directly involve the catalytic domain; it introduces a positively charged histidine, with a bulky and reactive side chain, instead of an electrically neutral glutamine. A recent report (Villar et al, 2007) showed that a truncated version of EGLN1, lacking residues 76–177 from its N-terminal unique region, behaved as full-length EGLN1, but further studies will be required to establish the function of this EGLN1 unique region. In conclusion, a novel germ-line EGLN1 mutation outside the catalytic domain was detected in two members of a family with MPN. Mutation p.Gln157His, reported here for the first time, is the first published germ-line EGLN1 mutation associated with PV. The co-presence of EGLN1 and JAK2V617F genetic lesions in the proband supports growing evidence that JAK2 mutation is a secondary, not a disease-initiating, molecular event. Further functional studies will be required to demonstrate that this EGLN1 mutation is the primary cause of MPN in our proband. The authors wish to thank the patients and the families who participated in this study. This work was supported by the Associazione Vicentina per le Leucemie, i Linfomi e il Mieloma (AViLL-AIL). EA and MB were recipient of a grant from the 'Fondazione Progetto Ematologia', Vicenza.