A novel erythrocytosis-associated PHD2 mutation suggests the location of a HIF binding groove

Percy, Melanie J.; Furlow, Paul W.; Beer, Philip; Lappin, Terence; McMullin, Mary Frances; Lee, Frank S.

doi:10.1182/blood-2007-04-084434

Cited by 139 publications

(110 citation statements)

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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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“…17,22 EpoR mutations are also rare and account for the erythrocytosis in only a minority of cases. Studies that have included cases of familial erythrocytosis have suggested a prevalence of 12-15%, [23][24][25] whereas another study found only one EpoR mutation in 100 IE cases.…”

Section: Discussionmentioning

confidence: 99%

The frequency of JAK2 exon 12 mutations in idiopathic erythrocytosis patients with low serum erythropoietin levels

Percy¹,

Scott²,

Erber³

et al. 2007

Haematologica

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“…pFastBac-HT-HA-p23 was constructed by subcloning the 0.5-kb BamHI/XhoI fragment of pcDNA5/ FRT/TO-3ϫFlag-p23 into the BamHI/XhoI site of pFastBac-HT-HA-VHL. The sources of pGEX-HIF-1␣ (531-575), pGEX-HIF-2␣ (516 -549), and all other plasmids have been described (19,26).…”

Section: Methodsmentioning

confidence: 99%

Defective Tibetan PHD2 Binding to p23 Links High Altitude Adaption to Altered Oxygen Sensing

Song

Arsenault

et al. 2014

Journal of Biological Chemistry

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A novel erythrocytosis-associated PHD2 mutation suggests the location of a HIF binding groove

Cited by 139 publications

References 19 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 frequency of JAK2 exon 12 mutations in idiopathic erythrocytosis patients with low serum erythropoietin levels

Defective Tibetan PHD2 Binding to p23 Links High Altitude Adaption to Altered Oxygen Sensing

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