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

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

doi:10.1111/j.1365-2141.2010.08551.x

Cited by 17 publications

(10 citation statements)

References 10 publications

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“…All the three new erythrocytosis-associated PHD2 mutations here reported involve the catalytic domain of the protein and add to those already known (Figure 2A-B). [16][17][18][19] We previously described as mutation a germ-line missense substitution, 20 Q157H, found in association with JAK2 V617F in a propositus with polycythemia vera and found in an isolated form also in his son, presenting only with mild erythrocytosis. Q157H has been reported also as SNP (NCBI entry, rs61750991) with a frequency of around 2% in normal subjects but with a much higher frequency in cancer patients.…”

Section: Resultsmentioning

confidence: 99%

Isolated erythrocytosis: study of 67 patients and identification of three novel germ-line mutations in the prolyl hydroxylase domain protein 2 (PHD2) gene

Albiero¹,

Ruggeri²,

Fortuna³

et al. 2011

Haematologica

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The oxygen sensing pathway modulates erythropoietin expression. In normal cells, intracellular oxygen tensions are directly sensed by prolyl hydroxylase domain (PHD)-containing proteins. PHD2 isozyme has a key role in tagging hypoxia-inducible factor (HIF)-α subunits for polyubiquitination and proteasomal degradation. Erythrocytosis-associated PHD2 mutations reduce hydroxylation of HIF-α. The investigation of 67 patients with isolated erythrocytosis, either sporadic or familial, allowed the identification of three novel mutations in the catalytic domain of the PHD2 protein. All new mutations are germ-line, heterozygous and missense, and code for a predicted full length mutant PHD2 protein. Identification of the disease-causing genes will be of critical importance for a better classification of familial and acquired erythrocytosis, offering additional insight into the erythropoietin regulating oxygen sensing pathway. Sequencing analysisThe 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, Foster City, CA, USA). Electropherograms were compared with the PHD2 (NCBI GenBank accession n. NM_022051), VHL (NCBI GenBank accession n. NM_000551) and HIF2A (NCBI GenBank accession n. NC_000002) wild-type sequences. Mutations were named in accordance with the standard international nomenclature guidelines recommended by the Human Genome Variation Society (HGVS, http://www.hgvs.org/mutnomen/). Characterization of the new PHD2 mutationsTo confirm the identity of the newly identified mutations, allele-specific PCR (AS-PCR) was performed for each allelic variant ( Figure 1B). Reaction mixtures were run on a GeneAmp ® PCR System 2700 at standard conditions and PCR fragments were analyzed by agarose gel. Primer sequences and PCR fragment lengths are listed in Online Supplementary Table S1.The germ-line origin of these new genetic lesions was confirmed by their identification on DNA obtained from epithelial cells (buccal cotton-swab sampling) of the probands.DNA samples from peripheral blood mononuclear cells from 100 normal controls with the same ethnic background were also screened for the novel mutations, by means of AS-PCR. Control DNA was obtained from the DNA samples stored in a biobank of healthy subjects at San Bortolo Hospital. Informed consent from the donors to the biobank had already been obtained for research purposes. Results and DiscussionIn this study, three different new germ-line heterozygous mutations of PHD2 were found in a cohort of 67 patients with IE, including 14 and 5 cases subsequently reclassified as PV on the basis of JAK2 V617F or exon 12 mutations. None of the PHD2 mutated subjects was JAK2 V617F positive while one co-harbored JAK2-exon 12 mutation. Main clinical and laboratory features of mutated patients are listed in Table 1.In Patient 1, molecular studies revealed a C>G missense mutati...

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Section: Resultsmentioning

confidence: 99%

Isolated erythrocytosis: study of 67 patients and identification of three novel germ-line mutations in the prolyl hydroxylase domain protein 2 (PHD2) gene

Albiero¹,

Ruggeri²,

Fortuna³

et al. 2011

Haematologica

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“…In addition, frame-shift (M202IfsX72, R227AfsX20 [Bento and Almeida, unpublished data, 2014], L279TfsX43, R281TfsX3, p.V338GfsX18) and nonsense mutations producing predicted C-terminally truncated proteins (Q221X [Lambert, unpublished data, 2013], Q239X [Bento and Almeida, unpublished data, 2014], Q377X, R398X) have been identified, 40 , 52 , 54 , 55 and two polymorphisms described in the normal population have been identified in patients with erythrocytosis (PHD2-C127S and PHD2-Q157H). 50 , 52 , 56 These studies identify a total of 37 patients with erythrocytosis who carry 24 different EGLN1 mutations. The sex ratio is unbalanced, with only six women versus 31 men.…”

Section: Erythrocytoses Described In Patients With Egln1mentioning

confidence: 99%

“…Defining the contribution of the germline EGLN1 mutations to the occurrence of the acquired JAK2 mutation and the myeloproliferative neoplasms requires more extensive studies. 49 , 56 …”

Section: Erythrocytoses Described In Patients With Egln1mentioning

confidence: 99%

The role of PHD2 mutations in the pathogenesis of erythrocytosis

Gardie¹,

Percy²,

Hoogewijs³

et al. 2014

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The transcription of the erythropoietin (EPO) gene is tightly regulated by the hypoxia response pathway to maintain oxygen homeostasis. Elevations in serum EPO level may be reflected in an augmentation in the red cell mass, thereby causing erythrocytosis. Studies on erythrocytosis have provided insights into the function of the oxygen-sensing pathway and the critical proteins involved in the regulation of EPO transcription. The α subunits of the hypoxia-inducible transcription factor are hydroxylated by three prolyl hydroxylase domain (PHD) enzymes, which belong to the iron and 2-oxoglutarate-dependent oxygenase superfamily. Sequence analysis of the genes encoding the PHDs in patients with erythrocytosis has revealed heterozygous germline mutations only occurring in Egl nine homolog 1 (EGLN1, also known as PHD2), the gene that encodes PHD2. To date, 24 different EGLN1 mutations comprising missense, frameshift, and nonsense mutations have been described. The phenotypes associated with the patients carrying these mutations are fairly homogeneous and typically limited to erythrocytosis with normal to elevated EPO. However, exceptions exist; for example, there is one case with development of concurrent paraganglioma (PHD2-H374R). Analysis of the erythrocytosis-associated PHD2 missense mutations has shown heterogeneous results. Structural studies reveal that mutations can affect different domains of PHD2. Some are close to the hypoxia-inducible transcription factor α/2-oxoglutarate or the iron binding sites for PHD2. In silico studies demonstrate that the mutations do not always affect fully conserved residues. In vitro and in cellulo studies showed varying effects of the mutations, ranging from mild effects to severe loss of function. The exact mechanism of a potential tumor-suppressor role for PHD2 still needs to be elucidated. A knockin mouse model expressing the first reported PHD2-P317R mutation recapitulates the phenotype observed in humans (erythrocytosis with inappropriately normal serum EPO levels) and demonstrates that haploinsufficiency and partial deregulation of PHD2 is sufficient to cause erythrocytosis.

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“…19,26 EGLN1 mutation is usually found in familial non-PV/nonneoplastic erythrocytosis but can also be present in familial JAK2 V617F PV. 15 JAK2 and TET2 mutations are not specific for PV, neither in general nor for sporadic and familial cases. These mutations can be detected in other MPN (50-60% of essential thrombocythemia and primary myelofibrosis cases are JAK2 V617F-positive), myelodysplastic syndromes (particularly TET2 mutations), MPN, unclassifiable and myelodysplastic/myeloproliferative neoplasms (both JAK2 and TET2 mutations).…”

Section: Endogenous Erythroid Colony Formation In Vitromentioning

confidence: 91%

“…The risk to develop a familial PV is undetermined if EGLN1 is found to be mutated. 15 TET2 and cytogenetic aberrations alone are too unspecific (occur also in other myeloid neoplasms) for determination of a positive clinical predictive value.…”

Section: Positive Clinical Predictive Value (Life-time Risk Of Develomentioning

confidence: 99%

Clinical utility gene card for: familial polycythaemia vera

et al. 2012

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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

Cited by 17 publications

References 10 publications

Isolated erythrocytosis: study of 67 patients and identification of three novel germ-line mutations in the prolyl hydroxylase domain protein 2 (PHD2) gene

Isolated erythrocytosis: study of 67 patients and identification of three novel germ-line mutations in the prolyl hydroxylase domain protein 2 (PHD2) gene

The role of PHD2 mutations in the pathogenesis of erythrocytosis

Clinical utility gene card for: familial polycythaemia vera

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