Characterization of genetic variants in the EGLN1/PHD2 gene identified in a European collection of patients with erythrocytosis

Abstract

Hereditary erythrocytosis is a rare hematologic disorder characterized by an excess of red blood cell production. Here we describe a European collaborative study involving a collection of 2,160 patients with erythrocytosis sequenced in ten different laboratories. We focused our study on the EGLN1 gene and identified 39 germline missense variants including one gene deletion in 47 probands. EGLN1 encodes the PHD2 prolyl 4-hydroxylase, a major inhibitor of hypoxia-inducible factor. We performed a comprehensive study to evaluate the causal role of the identified PHD2 variants: (i) in silico studies of localization, conservation, and deleterious effects; (ii) analysis of hematologic parameters of carriers identified in the UK Biobank; (iii) functional studies of the protein activity and stability; and (iv) a comprehensive study of PHD2 splicing. Altogether, these studies allowed the classification of 16 pathogenic or likely pathogenic mutants in a total of 48 patients and relatives. The in silico studies extended to the variants described in the literature showed that a minority of PHD2 variants can be classified as pathogenic (36/96), without any differences from the variants of unknown significance regarding the severity of the developed disease (hematologic parameters and complications). Here, we demonstrated the great value of federating laboratories working on such rare disorders in order to implement the criteria required for genetic classification, a strategy that should be extended to all hereditary hematologic diseases.

Figure 1.

Diagnostic flow chart for patients presenting with erythrocytosis. EPO: erythropoietin; EPOR: erythropoietin receptor, CO: carbon monoxide; Hb: hemoglobin; P50: partial pressure; O₂: oxygen; MCHC: mean corpuscular hemoglobin concentration; RCM: red cell mass; NGS: next-generation sequencing.

Figure 2.

Pedigrees of families carrying genetic variants in EGLN1 and localization of the mutated amino acids on the PHD2 protein. (A) Pedigrees of two families carrying genetic variants in EGLN1. (Left) The pedigree of patient #34 carrying the variant c.1000T>C, p.Trp334Arg. (Right) The pedigree of patient #42 carrying the variant c.1165T>C, p. Trp389Arg. +: carrier of the genetic variant. The arrow indicates the proband. (B) Localization of the targeted amino acids on the three-dimensional structural prediction of PHD2 obtained from the AlphaFold Protein Structure Database via the MobiDetails website. The regions of the structure modeled with high, medium, low, or very low confidence are colored blue, light blue, yellow, and orange, respectively. The prediction indicates that the relative positions of the two folded domains are not reliably modeled.

Figure 3.

Scores from single and meta-predictors of studied variants and analysis of the UK Biobank data. (A) Representation of scores obtained by the in silico predictors analyzed by the MobiDetails annotation platform. Values are normalized (0-1), 0 being the least damaging and 1 the most for each predictor. The graph shows mean normalized scores obtained by single predictors (SIFT, Polyphen 2 HumDiv and HumVar) and meta-predictors (Fathmm, REVEL, ClinPred, Meta SVM, Meta LR, Mistic). For each variant, we analyzed the scores obtained with single and meta-predictors and classified the variants as benign when both scores were <0.4, and as deleterious when both scores were >0.6. (B) Analysis of hemoglobin and hematocrit levels in male carriers of PHD2 variants identified in the UK Biobank. The colored bars represent cases. The red dotted lines represent the 90^th and 99^th percentile values of the control population. *Variants present in the UK Biobank also identified in the present study. n: number of cases.

Figure 4.

Functional study of PHD2 mutants using luciferase reporter assays. (A) Functional study of PHD2 mutants using end-point luciferase reporter assays. Cells were co-transfected with various amounts of PHD2 expression vectors (to enable the expression of the same amount of PHD2 proteins) in addition to HIF-2a expression vector, firefly luciferase reporter plasmid driven by hypoxia responsive elements and Renilla luciferase plasmid as a control of transfection efficiency Luciferase activity was measured 24 h after transfection. Results are given as percentage of firefly luciferase activity normalized to Renilla luciferase activity. The amount of HA-PHD2 transfected (PHD2) was quantified by immunoblotting using anti-HA antibody. Results are mean values of experiments performed in triplicate. (B) Functional study of PHD2 mutants using real time luciferase reporter assays. Cells were co-transfected with various amounts of PHD2 expression vectors in addition to HIF-2a expression vector and firefly luciferase reporter plasmids driven by hypoxia-responsive elements. Cells were incubated for 24 h in the bioluminometer Kronos HT® (ATTO) and luciferase activity was measured during 10 sec every 30 min. Results are given in relative light units (counts/10 sec). The amount of HA-PHD2 transfected was quantified by immunoblotting using an anti-HA antibody. Results are mean values of experiments performed in triplicate. FL: firefly luciferase; RL: Renilla luciferase; R.L.U.: relative light unit; IB PHD2: immunoblot of transfected HA-PHD2 by using anti-HA antibody; WT: wild-type; 0: cells transfected with an empty pcDNA3 vector.

Figure 5.

Study of PHD2 protein stability by the cycloheximide chase assay. The graph shows the amount of transfected HA-PHD2 protein after treatment with cycloheximide, reported as a percentage of the initial HA-PHD2 protein level (100% at 0 h of cycloheximide treatment) normalized to the intensity of actin. Data are shown as mean ± standard error of the mean of three independent experiments. Two-way analysis of variance was used for statistics (****P ≤0.0001). WT: wild-type.

Figure 6.

Functional studies of splicing variants. (A) Schematic representation of the splicing reporter assay (minigene experiment). The PHD2 exon of interest and flanking intronic sequences were cloned in a minigene pCAS2 plasmid between the SERPING1 exons (named A and B) flanked by short intronic sequences and conserved consensus splicing sequences. Mature and spliced mRNA were studied by reverse transcriptase polymerase chain reaction (RT-PCR) using primers located in exons A and B (schematized by arrows). (B) Characterization of PHD2-exon 3 splicing by a minigene experiment. RT-PCR was performed on mRNA obtained from HEK293T cells transfected with a minigene construct containing PHD2-exon 3 (wild-type or mutated) cloned in pCAS2. The pCAS2 plasmids were transfected, and the expression of the spliced chimeric transcripts was analyzed. Bands corresponding to exon A [A] and exon B [B] spliced together or with PHD2 exon 3 (Ex3) are indicated on the right (representative picture of agarose gel; N=3). (C) Characterization of PHD2-exon 4 splicing by the minigene experiments. RT-PCR was performed on mRNA obtained from cell lines (HEK293T, Hep3B or UT-7) transfected with a minigene construct containing PHD2-exon 4 (wild-type or mutated) flanked by intronic sequences cloned into pCas2 plasmids. The plasmids were transfected, and the expression of the spliced chimeric transcripts was analyzed. Bands corresponding to exon A and exon B spliced together or with PHD2-exon 4 (Ex4) are indicated on the left (representative picture of agarose gel, N=3). (D) Study of endogenous PHD2 splicing in a patient’s cells. RT-PCR using primers located in exons 3 and 5 of the PHD2 gene was performed on mRNA extracted from whole blood cells of the patient carrying the c.1152C>T, p.Y384Y variant collected into Paxgene® tubes. The lower band was purified and sequenced. The sequencing chromatogram is presented and shows the sequence of a transcript containing the exon 3 spliced with exon 5. (E) RT-PCR was performed on PHD2 mRNA (exons 3-5) extracted from peripheral blood mononuclear cells of the patient carrying the c.1216+1G>T variant. (F) RT-PCR was performed on PHD2 mRNA (exons 3-5) extracted from lymphoblastoid cell lines established from different patients. The cells were cultured in the absence (-) or presence (+) of puromycin, an inhibitor of nonsense-mediated mRNA decay mechanisms (representative picture of agarose gel, N=3). (G) Study of human induced pluripotent stem cells established from the patient carrying the c.1152C>T, p.Y384Y variant and differentiated into the hepatocyte-like cells. A representative gel of RT-PCR performed on PHD2 mRNA (exons 3-5) is shown on the left. The quantification of the percentage of PHD2-exon 4 skipping in all replicates was performed using the TapeStation® migration system and the results are shown on the right. Each column represents the mean ± standard error of the mean of independent experiments (see details in Online Supplementary Figure S8B). Two-way analysis of variance was used for statistics (**P≤0.01, ****P≤0.0001). M: molecular-weight size marker; WT: wild-type.