Gene panel sequencing improves the diagnostic work-up of patients with idiopathic erythrocytosis and identifies new mutations

Abstract

Erythrocytosis is a rare disorder characterized by increased red cell mass and elevated hemoglobin concentration and hematocrit. Several genetic variants have been identified as causes for erythrocytosis in genes belonging to different pathways including oxygen sensing, erythropoiesis and oxygen transport. However, despite clinical investigation and screening for these mutations, the cause of disease cannot be found in a considerable number of patients, who are classified as having idiopathic erythrocytosis. In this study, we developed a targeted next-generation sequencing panel encompassing the exonic regions of 21 genes from relevant pathways (~79 Kb) and sequenced 125 patients with idiopathic erythrocytosis. The panel effectively screened 97% of coding regions of these genes, with an average coverage of 450×. It identified 51 different rare variants, all leading to alterations of protein sequence, with 57 out of 125 cases (45.6%) having at least one of these variants. Ten of these were known erythrocytosis-causing variants, which had been missed following existing diagnostic algorithms. Twenty-two were novel variants in erythrocytosis-associated genes (EGLN1, EPAS1, VHL, BPGM, JAK2, SH2B3) and in novel genes included in the panel (e.g. EPO, EGLN2, HIF3A, OS9), some with a high likelihood of functionality, for which future segregation, functional and replication studies will be useful to provide further evidence for causality. The rest were classified as polymorphisms. Overall, these results demonstrate the benefits of using a gene panel rather than existing methods in which focused genetic screening is performed depending on biochemical measurements: the gene panel improves diagnostic accuracy and provides the opportunity for discovery of novel variants.

Figure 1.

Classification and pathogenesis of erythrocytosis. (A) Causes of erythrocytosis. Erythrocytosis can be congenital or acquired. It is classified as primary, when there is an intrinsic defect in erythropoietic cells and erythropoietin (Epo) levels are low, or secondary, when the increased red cell production is externally driven through increased EPO production and EPO levels are high or inappropriately normal. Note: in this article, the term erythrocytosis rather than polycythemia is used consistently throughout (B) Pathways involved in the pathogenesis of erythrocytosis. (i) Hypoxia inducible factor (HIF) oxygen sensing pathway in renal EPO-producing cells. HIF are dimeric transcription factors composed of one α- and one β- subunit. In normoxia, HIFα subunits are hydroxylated by oxygen-dependent prolyl-hydroxylases (PHD) and asparaginyl hydroxylase (HIF1AN). The hydroxylated prolines (P) are recognized by VHL, which mediates the ubiquitination and proteasomal degradation of HIFα. The hydroxylated asparagine (N) compromises the interaction of HIFα with cofactors necessary for transcriptional activity (p300/CBP). In hypoxia, PHD and HIF1AN are less active, HIFα subunits stabilize and translocate into the nucleus where they interact with the HIFβ subunit and cofactors and initiate transcription of target genes, including EPO (ii) Erythropoiesis in the bone marrow. This is triggered by the binding of EPO to the EPO receptor (EPOR) located on the surface of erythroid progenitor cells and subsequent activation of the JAK2-signaling cascade. The process is inhibited by the interaction of SH2B3 and JAK2. (iii) Hemoglobin (Hb) synthesis and oxygen transport. BPGM produces 2,3-BPG, which promotes the release of oxygen to local tissues by decreasing the affinity of deoxygenated Hb to oxygen. Alterations in the Hb chains (Hb-α and Hb-β) or BPGM could shift the Hb-oxygen dissociation curve and alter oxygen levels, which directly influence EPO production. (PV, polycythemia vera; ECYT 1–4, erythrocytosis type 1-4; Hb, hemoglobin; O₂, oxygen; 2,3-BPG, 2,3-bisphosphoglycerate; RBC, red blood cells; EPO, erythropoietin; PHDs, prolyl hydroxylases). PHDs: PHD1 (EGLN2), PHD2 (EGLN1) and PHD3 (EGLN3).

Figure 2.

Coverage of the amplicons generated by the erythrocytosis gene panel across 135 samples. (A) Each boxplot represents the distribution of the number of reads obtained for all the amplicons generated by the panel within each sample. The horizontal line across the plot shows the average coverage (450×). (B) Each dot represents the percentage of amplicons with coverage over 20× within each sample.

Figure 3.

Overview of the exonic variants detected with Ion Torrent sequencing among 125 patients with erythrocytosis, their validation and further classification.

Figure 4.

Proposed use of a gene panel in the investigation of erythrocytosis. A gene panel would make genetic testing more efficient and streamlined. It enables the simultaneous survey of the full length of 21 candidate genes, in a systematic and unbiased manner, allowing the detection of known causal variants as well as novel variants in known and novel genes.