Can You Trace the Inheritance of the Epas1 Alleles in the Family Shown in This Pedigree?

J Clin Lab Anal. 2021 Apr; 35(4): e23715.

Genetic analysis of 39 erythrocytosis and hereditary hemochromatosis‐associated genes in the Slovenian family with idiopathic erythrocytosis

Aleša Kristan, ^one Jernej Gašperšič, ¹ Tadeja Režen, ² Tanja Kunej, ³ Rok Količ, ^four Andrej Vuga, ⁴ Martina Fink, ^five Špela Žula, ⁵ Saša Anžej Doma, ⁵ Irena Preložnik Zupan, ^{5 , 6} Tadej Pajič, ^{five , 7} Helena Podgornik, ^{5 , 8} and Nataša Debeljak ¹

Aleša Kristan

^one Medical Heart for Molecular Biology, Faculty of Medicine, Institute of Biochemistry and Molecular Genetics, University of Ljubljana, Ljubljana Slovenia,

Jernej Gašperšič

¹ Medical Centre for Molecular Biology, Kinesthesia of Medicine, Establish of Biochemistry and Molecular Genetics, University of Ljubljana, Ljubljana Slovenia,

Tadeja Režen

² Centre for Functional Genomics and Bio‐Chips, Faculty of Medicine, Institute of Biochemistry and Molecular Genetics, University of Ljubljana, Ljubljana Slovenia,

Tanja Kunej

^three Section of Animal Science, Biotechnical Faculty, University of Ljubljana, Ljubljana Slovenia,

Rok Količ

^iv Kemomed Research and Development, Kemomed Ltd, Kranj Slovenia,

Andrej Vuga

⁴ Kemomed Research and Development, Kemomed Ltd, Kranj Slovenia,

Martina Fink

⁵ Clinical Department of Haematology, University Medical Centre Ljubljana, Ljubljana Slovenia,

Špela Žula

⁵ Clinical Department of Haematology, University Medical Middle Ljubljana, Ljubljana Slovenia,

Saša Anžej Doma

⁵ Clinical Department of Haematology, University Medical Heart Ljubljana, Ljubljana Slovenia,

Irena Preložnik Zupan

⁵ Clinical Section of Haematology, University Medical Centre Ljubljana, Ljubljana Slovenia,

⁶ Department of Internal Medicine, Kinesthesia of Medicine, University of Ljubljana, Ljubljana Slovenia,

Tadej Pajič

^v Clinical Department of Haematology, University Medical Centre Ljubljana, Ljubljana Slovenia,

⁷ Clinical Institute of Genomic Medicine, University Medical Middle Ljubljana, Ljubljana Slovenia,

Helena Podgornik

⁵ Clinical Department of Haematology, University Medical Center Ljubljana, Ljubljana Slovenia,

^eight Chair of Clinical Biochemistry, Kinesthesia of Pharmacy, University of Ljubljana, Ljubljana Slovenia,

Nataša Debeljak

¹ Medical Eye for Molecular Biology, Faculty of Medicine, Institute of Biochemistry and Molecular Genetics, Academy of Ljubljana, Ljubljana Slovenia,

Received 2020 Aug 31; Revised 2020 Dec 10; Accustomed 2021 Jan xv.

Abstruse

Groundwork

Erythrocytosis is a status with an excessive number of erythrocytes, accompanied past an elevated haemoglobin and/or haematocrit value. Congenital erythrocytosis has a diverse genetic groundwork with several genes involved in erythropoiesis. In clinical practice, nine genes are usually examined, but in approximately 70% of patients, no causative mutation can be identified. In this study, we screened 39 genes, aiming to place potential disease‐driving variants in the family unit with erythrocytosis of unknown cause.

Patients and Methods

Two affected family unit members with elevated haemoglobin and/or haematocrit and negative for acquired causes and one good for you relative from the aforementioned family unit were selected for molecular‐genetic analysis of 24 erythrocytosis and 15 hereditary haemochromatosis‐associated genes with targeted NGS. The identified variants were further analysed for pathogenicity using various bioinformatic tools and review of the literature.

Conclusion

For the first time, we included 39 genes in the erythrocytosis clinical panel and identified two potential disease‐driving variants in the Slovene family studied. Based on the reported functional in vitro studies combined with our bioinformatics assay, we suggest farther functional analysis of variant in the JAK2 cistron and evaluation of a cumulative upshot of both variants.

Keywords: DNA, erythrocytosis, genetic variation, haemochromatosis, sequence analysis

Abstract

The genetic analysis was conducted on family with idiopathic erythrocytosis (IE), that is, erythrocytosis of unknown crusade. Afflicted family members were included in the report based on the inclusion criteria for congenital erythrocytosis (eg elevated haemoglobin/haematocrit, absence of variants in some other myeloproliferative tumour genes and absenteeism of secondary erythrocytosis). For the first, we included 39 erythrocytosis and haemochromatosis‐associated genes in genetic assay with targeted NGS. Based on the inheritance pattern and selection of variants with modest allele frequency below 0.05, 1 missense variant in the EGLN1 factor and 1 intron variant in the JAK2 factor were identified in the affected family members.

1. INTRODUCTION

Erythrocytosis is a heterogeneous group of disorders that occur when there is an increase in the ruby-red blood prison cell (RBC) mass by more than 125% of the predicted value for specific torso height and weight, which defines increased haemoglobin levels and/or haematocrit. 1 , 2 Erythrocytosis is classified according to its cause, as primary or secondary, and as congenital or acquired. Principal erythrocytosis occurs when there is an intrinsic defect in the erythroid progenitor cells that is associated with expanded proliferation of RBCs, with erythropoietin (EPO) levels remaining below normal. On the contrary, secondary erythrocytosis occurs when the defect is extrinsic to the erythroid compartment and is instead associated with normal or inappropriately high EPO levels. 3 Nearly cases of erythrocytosis are acquired, which defines erythrocytosis secondary to various cardiac, pulmonary or renal diseases, or to external hypoxia, or where erythrocytosis is the consequence of somatic factor variants. Polycythaemia vera is the virtually mutual primary caused erythrocytosis, which is characterized by the somatic variant p.Val617Phe or variants in exon 12 of the Janus kinase 2 (JAK2) gene. four The JAK2 protein is a tyrosine kinase that has important roles in the signal transduction for proliferation and differentiation of myeloid cells. JAK2 binds to the intracellular domains of the cytokine receptors that lack catalytic activeness, including the EPO receptor (EPOR). When ligands such as the cytokine EPO demark to their receptors, they induce conformational changes and phosphorylation events that activate JAK2, with the consequent signal transduction via the downstream JAK2/STAT5, MAPK/ERK and PI3K/AKT pathways. In haematopoiesis, JAK2 is a critical mediator of an effective erythropoiesis. five

Congenital erythrocytosis, which is likewise known as familial erythrocytosis, is a rare clinical condition that is present from birth due to a germline defect in ane of the various genes involved in the regulation of oxygen homeostasis. 4 , half dozen For males, the haemoglobin levels are unremarkably >185 g/Fifty (with haematocrit >0.52), and a petty lower for females, at >165 yard/Fifty (for haematocrit >0.48). 1 , ii Eight types of congenital erythrocytosis have been classified according to Online Mendelian Inheritance in Man (ECYT1‐viii). seven Principal congenital erythrocytosis is defined every bit blazon 1 (ECYT1), and this is caused by variants in the EPOR gene. Secondary built erythrocytosis is classified into remaining seven types (ECYT2‐8), and these are caused past variants in genes involved in the oxygen‐sensing pathway (VHL, EGLN1, EPAS1, EPO) or variants that affect the haemoglobin oxygen affinity (HBB, HBA1, HBA2, BPGM) (Table1). 6 , 7 , 8 , ix Oxygen homeostasis is regulated through a circuitous pathway that is mediated by the essential transcription factors known every bit hypoxia‐inducible factors (HIFs). HIFs can upward‐regulate a number of target genes involved in maintenance of sufficient tissue oxygenation, including the hormone EPO, which regulates proliferation and differentiation of erythroid progenitors. Transcription circuitous HIF is a dimer, regulated through oxygen‐dependent regulation of blastoff subunit. In humans, three isoforms of blastoff subunit are known, with HIF2α [officially termed endothelial PAS domain protein 1, EPAS1] as major isoform involved in erythropoiesis. At normal oxygen concentrations (ie, normoxia), the HIF‐α subunit is hydroxylated by HIF‐prolyl hydroxylase 2 (PHD2) [officially termed EGLN1] at prolyl residues in its oxygen‐dependent deposition domain. Mail service‐translational hydroxylation regulates the oxygen‐dependent stability of EPAS1, as it allows binding of the von Hippel‐Lindau tumor suppressor (VHL) , which results in ubiquitination, followed past deposition by proteasomes. Nether hypoxic conditions (eg i% O₂), oxygen availability is limited, and thus, this hydroxylation is diminished, which prevent interactions with the VHL protein. This results in stabilization of HIF2α and germination of the stable HIF dimer complex that is responsible for the subsequent transcription activation of the targeted genes. x

Tabular array 1

Nomenclature of congenital erythrocytosis.

Type	OMIM #	Inheritance a	Gene	Gene location	Protein (synonym)
ECYT1	1333100	AD	EPOR	19p13.2	Erythropoietin receptor
ECYT2	263400	AR	VHL	3p25.three	von Hippel‐Lindau tumor suppressor
ECYT3	609820	AD	EGLN1	1q42.ii	Egl−9 family hypoxia‐inducible factor ane (HIF‐prolyl hydroxylase 2, PHD2)
ECYT4	611783	AD	EPAS1	2p21	Endothelial PAS domain protein 1 (hypoxia‐inducible factor 2 subunit alpha, HIF2α)
ECYT5	617907	AD	EPO	7q22.i	Erythropoietin
ECYT6	617980	AD	HBB	11p15.4	Haemoglobin subunit beta
ECYT7	617981	Advert	HBA1, HBA2	16p13.iii	Haemoglobin subunit alpha
ECYT8	222800	AR	BPGM	7q33	Bisphosphoglycerate mutase

The majority of the variants associated with ECYT1‐8 accept been collected in the Global Variome shared Leiden Open Variation Database (LOVD), which is supported past the LOVD iii.10 software. 11 Along with the nine genes responsible for ECYT1‐8, other central genes are involved in the erythropoiesis pathway and are hence plausible target genes for the development of erythrocytosis. 12 Because of heterogenic genetic groundwork, the cause for erythrocytosis remains unknown in 70% of patients with indication of congenital erythrocytosis that take been screened for known pathogenic variants in genes causative for ECYT1‐eight. Those patients are therefore diagnosed with erythrocytosis of unknown cause, so‐chosen idiopathic erythrocytosis. No proper diagnosis and prognosis can be made past clinicians, and farther genetic studies are needed for patients with idiopathic erythrocytosis. 12 , 13

Enhanced erythropoiesis can exist too induced by atomic number 26 overload, as fe is necessary for the synthesis of haemoglobin. xiv An excess iron in the blood could be a sign of hereditary haemochromatosis, a well‐known disorder caused by defects in genes that are involved in iron metabolism, ship and balance. The virtually common hereditary haemochromatosis is type 1, associated with homozygous or compound heterozygous variants in homeostatic iron regulator (HFE) gene. xv , sixteen Some authors take observed loftier frequency of heterozygous HFE variants amid patients with idiopathic erythrocytosis, indicating the interest of haemochromatosis genes in the evolution of erythrocytosis. 15

The aim of the present report was to investigate the hereditary gene variants of multiple genes involved in erythropoiesis, in a family with indication of congenital erythrocytosis. An expanded selection of 24 erythrocytosis and 15 hereditary haemochromatosis‐associated genes was included in the targeted molecular‐genetic analysis with next‐generation sequencing (NGS). Hither, we written report on these NGS screening results where we draw variants with plausible causative furnishings on increased erythropoiesis.

2. MATERIALS AND METHODS

2.1. Patients

The alphabetize patient (Figure1, private I:two) was selected from the individuals followed at the University Medical Center Ljubljana (UMC) over an eight‐year menses according to the diagnostic algorithm for erythrocytosis. 17 The inclusion criteria were as follows: (a) haemoglobin and/or haematocrit above reference values at least twice over two months; (b) absence of variants JAK2 p.Val617Phe and JAK2 exon 12; (c) absence of any defined cause of secondary acquired erythrocytosis; and (d) absence of variants in genes for the thrombopoietin receptor (MPL), calreticulin (CALR) and receptor tyrosine kinase (KIT). 17 The alphabetize patient was a 68‐year‐onetime male person who was referred to the Clinical Section of Haematology at UMC Ljubljana in 2013, due to his high RBC count of six.36 x 10¹² cells/L, high haemoglobin of 221 k/L and loftier haematocrit of 0.650. His EPO level was in the normal range (11.iv IU/Fifty). The patient as well had elevated blood pressure level and middle rate. The patient was prescribed for therapeutic phlebotomy, and at the last follow‐up in 2019, he had a slightly increased haemoglobin of 171 g/L. Upon medical history revision, he reported that his son also had clinical signs of erythrocytosis.

Identification of a heterozygous nucleotide variants in the EGLN1 and JAK2 genes (A) Family pedigree and segregation of the variants identified inside the family. (B) Next‐generation sequencing (NGS) and (C) Sanger sequencing, showing heterozygous EGLN1 variant in the two afflicted patients , just not in the unaffected family member. (D) Results of NGS showing intron variant in the JAK2 factor in both of the affected family members, but not in the unaffected family member. Squares, circles, represent males, females, respectively. Filled symbols, patients with clinical diagnosis of erythrocytosis; open symbols, unaffected family members; slashed symbol, subject is deceased. Carriers of heterozygous EGLN1 and JAK2 variants are indicated as ‐/M. Arrow indicates mutation point

The son (Figureone, individual 2:1) of the alphabetize patient was 41 years old, and at his first appointment in 2015, he presented with a RBC count of 6.29 × 10¹² cells/Fifty, loftier haemoglobin of 192 thou/L and loftier haematocrit of 0.552, while his EPO level was normal (eight.3 IU/L). The son was a regular blood donor, and so he was not referred for a phlebotomy. These two patients were tested for polycythaemia vera and hereditary haemochromatosis and were negative for variants p.Val617Phe and exon 12 in the JAK2 gene, and also for variants p.Cys282Tyr, p.His63Asp and p.Ser65Asp in the gene for homostatic iron regulator (HFE). Neither of these 2 patients showed any pulmonary, cardiac or renal abnormalities.

The wife and mother of these elder and younger patients, respectively (Figure1, individual I:i), is deceased, but had had normal RBC counts, haemoglobin levels and haematocrit, and no signs of erythrocytosis.

All three afflicted and healthy family unit members were, together with a reference Dna control NA12878, included in the NGS genetic testing for congenital erythrocytosis. The study was canonical by the Slovenian Upstanding Committee, No. KME 115/07/15.

2.ii. Genetic analysis

Patient's peripheral blood was nerveless for genetic analysis, together with written informed consent signed by all patients. Granulocytes were isolated from collected peripheral blood, and the genomic DNA was extracted from 1 to 2 × ten⁷ cells using QIAamp Dna mini kits (Qiagen). The patients underwent targeted NGS, with a custom gene panel that covered the target regions of 39 selected genes: 21 genes previously associated with congenital erythrocytosis and polycythaemia vera, 12 3 additional erythrocytosis‐associated genes (PKLR, TET2 and GATA); and fifteen genes previously associated with hereditary haemochromatosis. 18 All of the selected genes were targeted for exon regions, while EPO, VHL and some of the other genes had the first intron, promoter and enhancer regions as well included xix , 20 (Table2).

Table 2

Listing of sequenced genes and regions

Association	Exon	Intron 1	Promotor and enhancer
Erythrocytosis‐associated genes a	BHLHE41, BPGM, EGLN1, EGLN2, EGLN3, EPAS1, EPO, EPOR, GFI1B, HBA1, HBA2, HBB, HIF1A, HIF1AN, HIF3A, JAK2, KDM6A, OS9, PKLR, SH2B3, VHL, ZNF197, GATA1 and TET2	VHL, EPO, EPOR, HBB, HBA1, HBA2	EPO
Hereditary haemochromatosis‐associated genes b	HFE, HJV, HAMP, TFR2, SLC40A1, FTH1, TF, B2 K, CP, FTL, CDAN1, SEC23B, SLC25A38, STEAP3 and ALAS2	‐	‐

Libraries were prepared using Nextera Dna library preparation kits (Illumina), with enrichment performed by probe hybridization approach using custom gene panel (Integrated Dna Technologies) followed by sequencing (MiniSeq, Illumina). The disease‐run a risk variants identified were validated by Sanger sequencing (GATC Biotech). Sanger sequencing and prior PCR distension were performed with custom‐designed primers (Integrated DNA Technologies; bachelor upon request).

2.3. Bioinformatics analysis

The sequencing analysis was performed with built‐in bioinformatics tools (Illumina) and variant notation with an online tool (Variant Interpreter Illumina). The sequences were aligned to reference genome hg19 (GRCh37). To remove variants with depression sequencing quality, the following filters were used within the variant caller: genotype quality (ie GQX value) <30 or not present; quality by depth <2; root hateful foursquare mapping quality <20; strand bias > −x; and read depth <1. The variants were showtime selected based on the human relationship between genotypes and phenotypes; this selection was for variants identified every bit heterozygous in the affected family unit members and not in the good for you family members, or identified every bit homozygous in the affected family members and equally heterozygous in the healthy family members. For the final option, the variants with minor allele frequencies (MAFs) <0.05 in the European population were filtered, as MAF <0.05 is singled-out for depression‐frequency variants. 21 We selected data from a European Non‐Finnish population managed by the GnomAD genome and GnomAD exome databases, and data from a European population managed by the g Genomes database. Before filtering, the presence of high‐frequency variants in the JAK2 and HFE genes that cause polycythaemia vera and hereditary haemochromatosis was also assessed. The focus of our assay was on minor variants that involved one or a few nucleotides, such as single nucleotide variants (SNVs) and small insertions/deletions (INDELs).

With the aim to make up one's mind the degree of variant position conservation during development, conservation analysis was performed with the ConSurf server (https://consurf.tau.ac.il/). 22 The ConSurf server predicts the evolutionary conservation of amino acids or nucleotides based on multiple alignment and phylogenetic relations of homologous sequences that issue in position‐specific conservation scores. Continuous conservation scores are divided into scale of 9 colour grades for visualization, from the most variable position (grade one), through intermediately conserved position (class 5), to the most conserved position (course 9). To each colour grade, a confidence interval is assigned to each conservation score. If the interval spans four or more color grades, less than six homologous sequences are aligned and the conservation score is unreliable. 22 The parameters for the conservation analysis of protein sequences were as follows: CSI‐BLAST search algorithm; 3 iterations; E‐value cut‐off 0.0001; protein sequence database UNIREF90; number of analysed homologues 150; minimal 35% identity between homologues; maximal 95% identity between homologues; multiple sequence alignment algorithm ClustalW; and method for calculation of the evolution rate Bayesian paradigm. For conservation analysis of nucleotide sequences, we manually searched for homologous sequences using Basic Local Alignment Search Tool (BLAST) program BLASTN (https://blast.ncbi.nlm.nih.gov/Blast.cgi) 23 and perform multiple sequence alignment with ClustalW (https://www.ebi.ac.uk/Tools/msa/clustalo/). 24 For calculation of evolution rate in ConSurf server, we selected Bayesian image.

The pathogenicity of variants in coding regions was assessed using in silico prediction tools CADD score (https://cadd.gs.washington.edu/snv), 25 PolyPhen‐2 (http://genetics.bwh.harvard.edu/pph2/), 26 SIFT (https://sift.bii.a‐star.edu.sg/), 27 MutPred2 (http://mutpred.mutdb.org/index.html), 28 SNPs&Get (https://snps.biofold.org/snps‐and‐go/snps‐and‐go.html), 29 PANTHER (http://www.pantherdb.org/tools/csnpScoreForm.jsp), 30 PhD‐SNP (https://snps.biofold.org/phd‐snp/phd‐snp.html), 31 PROVEAN (http://provean.jcvi.org/index.php) 32 and Mutation Taster two (http://world wide web.mutationtaster.org/) 33 as described by Schiemann and Stowell, 2016, 34 Bris et al. 2018, 35 Wang et al. 2020. 36 The pathogenicity prediction of intron variants and impact on splicing features were analysed using tools CADD score, 25 the RegSNP‐intron (https://regsnps‐intron.ccbb.iupui.edu/), Human Splicing Finder (http://www.umd.be/HSF/), 37 IntSplice (https://www.med.nagoya‐u.ac.jp/neurogenetics/IntSplice/index.html) 38 as reviewed in Ohno et al. 2018 39 and Lin et. al. 2019. 40

With the CADD (Combined Annotation Dependent Depletion) tool, the prediction of the deleteriousness of variants results in 'raw' score and 'PHRED‐scaled' score. A PHRED‐scaled score expresses the rank of variant pathogenicity; for example, a score of 10 indicates that the variant is predicted to exist in the 10% of the most deleterious substitutions and a score of 20 betwixt the one% virtually deleterious. The suggested threshold is betwixt 10 and xx, and nosotros gear up the cut‐off score at >15. 25 The PolyPhen‐two (Polymorphism Phenotyping vs.‐2.0) predicts the effect of an amino acid change with classifiers 'beneficial', 'perhaps dissentious' and 'probably damaging' and scores from 0.0 to 1.0: variants with values closer to 0.0 are classified as benign and variants with values closer to one.0 are more confidently predicted as probably dissentious. Variants with score over 0.fifty were predicted to exist pathogenic. In bioinformatics assay with PolyPhen‐2, we nowadays values obtained with HumVar prediction model, which is preferred model for diagnostics of Mendelian diseases. 26 The prediction tool SIFT (Sorting Intolerant From Tolerant) categorizes impact of amino acid change on protein function based on SIFT score ranging from 0 to i: <0.05 is classified equally dissentious and >0.05 equally tolerated. 27 The output of MutPred2 (Mutation prediction vs.‐2.0) consists of a general score that ranges between 0.0 and ane.0, with a higher score indicating greater probability to exist pathogenic. The cut‐off general score >0.v was considered to be pathogenic. 28 The prediction tool SNPs&Go uses the reliability index (RI) to evaluate how reliable is the prediction, with 0 existence the most unreliable and 10 being the virtually reliable. Variants are predicted as neutral polymorphism (neutral) or disease related (illness) when the probability score is >0.5. 29 PANTHER (Protein Analysis Through Evolutionary Relationships) estimates the probability of a variant to touch protein function based on evolutionary preservation of a position in poly peptide. The likelihood of deleterious result increases with the longer preservation time. The thresholds values are >450 meg years (my) for 'probably dissentious' prediction, betwixt 200 my and 450 my for 'possibly dissentious' and <200 my for 'probably benign' prediction. thirty The prediction tool PhD‐SNP (Predictor of Human Deleterious SNP) classifies variants into 'neutral polymorphism' or 'disease related' with values from 0 to 1 and the decision threshold for disease causing is >0.5. It as well uses reliability alphabetize (RI) ranges from 0 to 10, from the most unreliable to the well-nigh reliable prediction. Prediction was used with 20‐fold cantankerous‐validation. 31 PROVEAN (Protein Variant Effect Analyzer) uses PROVEAN score for binary classification of variants into either deleterious or neutral. The threshold is set to > −2.5 for neutral predictions and < −two.5 for deleterious predictions. 32 The prediction tool Mutation Taster 2 employs a Bayes classifier to predict a variant as one of the four possible types: 'disease causing', that is probably deleterious, 'illness‐causing automatic', that is known to exist deleterious, 'polymorphism', that is probably harmless and 'polymorphism automatic', that is known to be harmless. It also uses a probability values of the predictions ranging betwixt 0 and ane, with values closer to 1 every bit more reliable prediction. 33 The RegSNP‐intron is a tool that predicts the pathogenicity of intron variants with probability scores 0.00 −0.36 for benign variants; 0.36–0.45 for perhaps damaging variants and 0.45–1.00 for dissentious variants. 40 The HSF (Human being Splicing Finder) allows the identification of all splicing features through multiple algorithms and besides the prediction of the impact of intron variants on these features. 37 IntSplice is a tool that predicts a splicing consequence of intron variant shut to the three' finish of an intron, and the consequence is either an aberrant or normal splicing. 38

The variants were named in understanding with the standard international nomenclature guidelines of the Human Genome Variation Society. 41

3. RESULTS

3.1. Genetic assay

Next‐generation sequencing revealed 74, 76 and 79 small-scale variants in individuals 2:1, I:2 and I:1, respectively. Twelve variants identified in genes associated with erythrocytosis and hereditary haemochromatosis were consistent with autosomal‐ascendant or autosomal‐recessive disease inheritance patterns in the family. Three variants in the EPAS1 and VHL genes were also identified in a reference Dna control NA12878, while the remaining nine variants in the CDAN1, EGLN1 and JAK2 genes were present just in family members (Table3). After filtering, iii variants in the VHL, JAK2 and EGLN1 genes were recognized as low‐frequency variants (MAF < 0.05) with an autosomal‐dominant inheritance pattern. Variant c.341‐325_341‐324delTT ({"blazon":"entrez-nucleotide","attrs":{"text":"NM_000551.three","term_id":"319655736","term_text":"NM_000551.3"}}NM_000551.three) in the VHL gene will non be discussed further here, as it is located in a office of the sequence that is rich in thymine (ie the T nucleotide), and therefore, the deletion of two T nucleotides might be due to sequencer fault. Next‐generation sequencing confirmed the absenteeism of polycythaemia vera‐causing variant p.Val617Phe and exon 12 variants in the JAK2 gene and hereditary haemochromatosis‐causing variants p.Cys282Tyr, p.His63Asp and p.Ser65Asp variants in the HFE gene in both of these patients and in healthy family member.

TABLE 3

List of all small variants in the analysed family trio consistent with autosomal‐dominant or autosomal‐recessive inheritance patterns

Genomic location (hg19)	Gene	Position	HGVS coding DNA/ HGVS protein	SNV ID	MAF a	Average DP	Inheritance in family b
chr. fifteen:43017426	CDAN1	Exon 27	{"type":"entrez-nucleotide","attrs":{"text":"NM_138477.two","term_id":"57222569","term_text":"NM_138477.ii"}}NM_138477.2:c.3474A>C / p.(Leu1158=)	rs16957091	0.23	288	Ad
chr. xv:43018486	CDAN1	Intron 24	{"type":"entrez-nucleotide","attrs":{"text":"NM_138477.2","term_id":"57222569","term_text":"NM_138477.2"}}NM_138477.two:c.3204+22C>T	rs2305085	0.nineteen	101	Advertisement
chr. fifteen:43020983	CDAN1	Exon 20	{"type":"entrez-nucleotide","attrs":{"text":"NM_138477.2","term_id":"57222569","term_text":"NM_138477.ii"}}NM_138477.2:c.2671C>T / p.(Arg891Cys)	rs8023524	0.19	421	AD
chr. 15:43021563	CDAN1	Intron 17/ splice region	{"type":"entrez-nucleotide","attrs":{"text":"NM_138477.2","term_id":"57222569","term_text":"NM_138477.2"}}NM_138477.2:c.2408‐3C>T	rs12905385	0.nineteen	348	AD
chr. 15:43021986	CDAN1	Intron 16/ splice region	{"type":"entrez-nucleotide","attrs":{"text":"NM_138477.2","term_id":"57222569","term_text":"NM_138477.ii"}}NM_138477.two:c.2352+8C>T	rs12594483	0.12	231	AD
chr. 1:231557164	EGLN1	Exon 1	{"type":"entrez-nucleotide","attrs":{"text":"NM_022051.2","term_id":"237649101","term_text":"NM_022051.2"}}NM_022051.ii:c.471G>C / p.(Gln157His)	rs61750991	0.03	93	AD
chr. 2:46603671c	EPAS1	Intron eight/ splice region	{"type":"entrez-nucleotide","attrs":{"text":"NM_001430.iv","term_id":"262527236","term_text":"NM_001430.4"}}NM_001430.4:c.1035‐7C>G	rs7557402	0.47	184	AR
chr. two:46605954 c	EPAS1	Intron 11	{"type":"entrez-nucleotide","attrs":{"text":"NM_001430.four","term_id":"262527236","term_text":"NM_001430.4"}}NM_001430.4:c.1554+48G>C	rs7598371	0.46	91	AR
chr. 9:5050706	JAK2	Exon vi	{"blazon":"entrez-nucleotide","attrs":{"text":"NM_004972.three","term_id":"223671934","term_text":"NM_004972.3"}}NM_004972.3:c.489C>T / p.(His163=)	rs2230722	0.31	297	Advert
chr. 9:5089661	JAK2	Intron 19	{"type":"entrez-nucleotide","attrs":{"text":"NM_004972.iii","term_id":"223671934","term_text":"NM_004972.3"}}NM_004972.three:c.2572‐13A>G	rs780797578	0.0001	140	Advertizement
chr. 9:5090934	JAK2	Intron 22	{"type":"entrez-nucleotide","attrs":{"text":"NM_004972.three","term_id":"223671934","term_text":"NM_004972.3"}}NM_004972.3:c.3059+23A>T	rs2274649	0.29	104	Advertising
chr. 3:10187858 c	VHL	Intron 1	{"type":"entrez-nucleotide","attrs":{"text":"NM_000551.3","term_id":"319655736","term_text":"NM_000551.3"}}NM_000551.3:c.341‐325_341‐324delTT	‐	0.0004	131	Advertizement

Both affected family members (father I:2, son Two:ane) were heterozygous for two SNVs in the JAK2 and EGLN1 genes, while unaffected family member (individual I:1) did non show either SNV (Tabular arrayiv; Figure1A). A missense variant in exon 1 of the EGLN1 factor was a heterozygous G > C substitution at nt. c.471, which caused an amino acid change at rest 157 from glutamine to histidine (Figure1B,C). This is a known variant, designated with SNV ID number rs61750991. Co-ordinate to the ClinVar online database, this variant is classified in association with congenital erythrocytosis type 3 (ECYT3) as benign. 42 A not‐coding variant in intron 19 of the JAK2 gene (rs780797578) is located 13 base of operations pairs from the exon 20 (c.2572‐13A>One thousand), in proximity to the splicing region (Figure1D).

TABLE 4

Ii single nucleotide variants consistent with inheritance design and with minor allele frequency beneath 0.05 in the Slovenian family studied

Genomic location (hg19)	Gene	HGVS coding Dna / HGVS protein	SNV ID	Location	MAF (database)	Functional predictions	Patient	Genotype	VRF
chr. 1:231557164	EGLN1	c.471G>C /p.(Gln157His)	rs61750991	Exon ane	0.03 (GnomAD) 0.03 (GnomAD Exome) 0.03 (thousand Genomes)	CADD: low pathogenicity PolyPhen−2: benign SIFT: tolerated MutPred2: low pathogenicity SNPs&GO: neutral PANTHER: probably benign PhD‐SNP: neutral PROVEAN: neutral Mutation Taster 2: polymorphism ClinVar: beneficial	I:2 Two:i I:1	Heterozygous Heterozygous WT	0.5 0.5 ‐
chr. ix:5089661	JAK2	c.2572‐13A>One thousand	rs780797578	Intron 19	0.0001 (GnomAD) 0.00003 (GnomAD Exome)	CADD: low pathogenicity RegSNP‐intron: benign HSF: potential alteration on splicing IntSplice: normal splicing	I:2 2:ane I:ane	Heterozygous Heterozygous WT	0.5 0.5 ‐

iii.two. Bioinformatics analysis

A conservation analysis of amino acids in EGLN1 protein using ConSurf server showed that Gln157 is a low conserved amino acrid, indicated every bit a variable position (grade 3), which rapidly evolved. The confidence interval was (four, 2) (Supplementary Figure S1). An amino acid modify in p.Gln157His was predicted to accept balmy furnishings on protein function according to all nine prediction algorithms (Tableiv; Supplementary Tabular array S1).

The ConSurf conservation analysis of 132 nucleotides at the 3′end of JAK2 intron 19 showed that nucleotide position c.2572‐13A is intermediately conserved (grade six). The confidence interval was (eight, 5) (Supplementary Effigy S2). The pathogenicity predictions of JAK2 variant c.2572‐13A>G using CADD score and RegSNP‐intron tools showed that variant has low deleterious consequence on protein function. Similarly, the prediction with IntSplice indicated that variant has no potential impact on splicing. However, the analysis with splice prediction tool HSF revealed that variant c.2572‐13A>One thousand could have an effect on splicing, since potential new donor site was identified because of nucleotide modify.

4. DISCUSSION

The aim of this study was to identify causative or disease‐associated variants in patients with erythrocytosis of unknown cause, in a family with obvious claret parameters and other signs for built erythrocytosis. The total number of full SNVs and small insertion/deletions plant in the target regions of 39 genes in ii affected and i healthy family member varied from 74 to 79. The called genotypes were consequent with autosomal‐dominant or autosomal‐recessive inheritance patterns of the illness at only 12 variant sites (approximately xv%) (Table3). As expected, only a few were missense variants located in coding regions, while the bulk of variants were in introns (Table3). Through evolution, introns accept been recognized as a much less conserved parts of sequences and their potential variants have mainly had indirect impacts on structural changes to proteins. Our sequencing data show that a large function of the variants identified were common variants, with MAF >0.05 in European populations, while only three variants identified within the family had frequencies <0.05. As congenital erythrocytosis is a rare clinical disorder, we focused only on the depression‐frequency variants. Overall, i SNV in the coding region of the EGLN1 gene and one SNV in the intron region of the JAK2 factor were examined in detail.

The EGLN1 missense variant c.471G>C/ p.(Gln157His) was already identified in previous studies. 43 , 44 Albiero et al. 43 identified this variant in ii family members (father and son) with increased haemoglobin, where the father was also positive for JAK2 p.Val617Phe. 43 Also Ladroue et al. 44 reported this variant in a patient with erythrocytosis. 44 Potential germline mutations in EGLN1 lead to stabilization of EPAS1 under normoxic atmospheric condition, which results in abnormal EPO levels and secondary erythrocytosis. 45 To appointment, a full of 39 different EGLN1 variants accept been identified in patients with erythrocytosis, which comprise heterozygous missense, frameshifts and nonsense variants. eleven The patients carrying these variants had predominantly normal to elevated EPO levels, like to the phenotypes of the afflicted father and son from the present study, who had EPO levels in the normal range. The variant c.471G>C segregated in the family with an autosomal‐dominant inheritance pattern (Figure1A–C), which is consistent with an autosomal‐dominant inheritance mode that was recorded for the variants in the EGLN1 factor, that are causative for the ECYT3 (Tableane). The majority of variants reported in previous studies lie between or near the C‐terminal catalytic domain, which is responsible for the HIFα/ii‐oxoglutarate or ferrous iron bounden. Nonsense and frameshift variants tin can have substantial effects on the EGLN1 protein in terms of impaired part, as these tin can produce a protein truncated in its C‐terminal region; in dissimilarity, missense substitutions have less clear effects. 45 Variant p.(Gln157His) is positioned in the first exon of the EGLN1 cistron, approximately 100 amino acids from the MYND zing‐finger domain at the N‐terminus (amino acids 21–58) and almost equally distant from the β substrate recognition loop at the C‐terminus (amino acids 241–251). 46 The conservation assay showed depression evolutionary conservation of residue p.Gln157 (Supplementary Figure S1), although not all causative variants are positioned at fully conserved residues (eg p.Asn203Lys, p.Lys204Glu, p.Gly285Arg, p.Lys291Ile). 45 Co-ordinate to in silico pathogenicity predictions, substitution of glutamine with histidine at residue 157 volition have a balmy effect on the poly peptide construction and function (Tablefour, Supplementary Tabular array S1), which corresponds to a benign estimation in the ClinVar database. 42 Ladroue et al. 44 also performed in vitro hydroxylation assays and showed that this variant has no functional issue on the protein. 44

Variant c.2572‐13A>Thou is located in intron 19 of the JAK2 cistron (Table4; FigureaneD). Somatic JAK2 variants are affliction‐causing events in patients with polycythaemia vera, as types of myeloproliferative neoplasms. On the other hand, JAK2 germline variants rarely correlate with haematological disorders. They accept been described, nonetheless, in patients with polycythaemia vera and other myeloproliferative neoplasms, such as essential thrombocythemia and primary myelofibrosis. 47 , 48 , 49 Kapralova et al. 50 reported on two heterozygous missense germline variants p.Glu846Asp and p.Arg1063His that they identified in a patient with erythrocytosis and aberrant megakaryopoiesis of the os marrow. 50 They showed that both of these variants lead to erythrocytosis with megakaryocyte abnormalities through hyper‐activation of the JAK2/STAT5 signalling pathway via EPOR. 50 Similarly, they suggested that combined impact of two variants, p.Glu846Asp in theJAK2 gene and p.Gln157His in the EGLN1 cistron, resulted in erythrocytosis phenotype in 1 patient. 51 Interestingly, we found the same EGLN1 variant in our studied family, which could imply on the cumulative effect of the ii identified variants in JAK2 and EGLN1 gene.

The JAK2 protein has four of import domains: the FERM domain (amino acids 37–380) and the SH2 domain (amino acids 401–482) accept roles in receptor and ligand binding; the JAK homology 2 (JH2) pseudo‐kinase domain (amino acids 545–809) acts as a negative regulator of the adjacent JAK2 homology one (JH1) kinase domain (amino acids 849–1124). 5 , 46 The variants associated with haematopoietic disorders lie predominantly in the pseudo‐kinase and kinase domains; for instance, the most prevalent somatic variant p.Val617Phe lies in the JH2 domain and disrupts the JH2‐JH1 machine‐inhibitory interaction, which leads to increased JAK2 kinase activity and activation of its downstream effectors, contained of cytokine binding. 5 Variant c.2572‐13A>Chiliad identified in the present study is located in intron xix, which is positioned betwixt exons 19 and 20, and these code for the JH1 domain. Previous in silico models have shown that variants in the JH1 domain can have effects on prolonged activation of STAT5 in a depression‐cytokine environment. 50 The bioinformatic analysis with three prediction tools showed that variant has no impact on splicing or protein function, while the algorithm of Human Splicing Finder pointed to a potential alteration of splicing event (Tabular array4, Supplementary Table S1). The effect of variant c.2572‐13A>Chiliad should be farther tested in vitro. In accordance with possible impact of variant on protein part were also results from the ConSurf server, every bit the conservation analysis showed that nucleotide position c.2572–13 was intermediately conserved through development (Supplementary Figure S2). Intron variant c.2572‐13A>G is a known variant, as it has already been assigned to the SNV ID number rs780797578, although this variant has not been cited in any publication, and as well no clinical significance is reported in the ClinVar database. 42

To study the development of erythrocytosis in the nowadays family, the cumulative effects of both identified variants demand to be assessed. In addition, the cumulative effects of mutual variants (ie MAF >0.05) should not exist neglected, equally minor contributions of common variants tin explain large proportions of those at illness risk in the population described by an minute model. 21 It has been reported that inherited common genetic variants tin establish the background for a rare affliction, or even rare disorders that are typically considered to exist monogenic. 52 , 53

five. CONCLUSIONS

In the 39 sequenced erythrocytosis and haemochromatosis‐associated genes, we identified two variants in one Slovene family with suspicion of congenital erythrocytosis: c.471G>C/ p.(Gln157His) in the EGLN1 gene and c.2572‐13A>Grand in the JAK2 gene. Hitherto, neither of these variants tin be interpreted as solely causative for the development of erythrocytosis and the cumulative effects of both variants and besides between other variants should be assessed. The affect of variant p.(Gln157His) on EGLN1 function was well described by Ladroue et al. 44 in which the functional analysis showed no damage of hydroxylation of EPAS1. 44 On the opposite, further functional assays for variant c.2572‐13A>Grand in the JAK2 factor are necessary to ameliorate empathize the bear on of this intron variant on the regulation of protein part.

In the current study, nosotros targeted exon regions and some regulatory regions of 39 genes involved in erythropoiesis, which are not all part of commercially available gene panels for congenital erythrocytosis. As far equally nosotros know, this is the first time that this extended selection of both erythrocytosis and hereditary haemochromatosis‐associated genes was included in the molecular‐genetic analysis of erythrocytosis. We believe, this selection of genes represents a good initial step toward better diagnosis of patients with idiopathic erythrocytosis. For farther investigation of the aetiology of congenital erythrocytosis cases, we propose full screening of the non‐coding regions of erythrocytosis‐associated genes for all statistically significant variants that are potentially associated with increased expansion of RBCs, followed by whole‐exome sequencing or whole‐genome sequencing, if necessary.

CONFLICT OF Interest

RK and AV are employees of Kemomed Ltd., Kemomed Enquiry and Evolution. The other authors declare that they accept no disharmonize of involvement.

Supporting information

ACKNOWLEDGEMENTS

This study was supported by the Slovenian Inquiry Bureau, grant no. L3‐9279, grant no. P1‐0390 and Young Researcher funding to AK and the University Medical Centre, Ljubljana, grant no. 20170073.

Information AVAILABILITY Argument

The data that support the findings of this report are available from the respective author upon reasonable request.

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