A Screening Approach for Inherited Erythrocytosis due to the VHL:c.598C > T Mutation (Chuvash Polycythemia)

Abstract

Genetic work-up of unexplained erythrocytosis that is suspected to be inherited in nature currently requires either laborious exon-by-exon gene panel testing by Sanger sequencing or expensive next-generation sequencing. A high prevalence of Chuvash polycythemia (61%) has been previously reported among north Indian erythrocytosis patients. We assessed PCR-RFLP for VHL c.598C > T mutation as a first-line test in 99 persons with JAK2 V617F-negative, unexplained erythrocytosis. We enrolled two groups: Group A (n = 38) had erythrocytosis patients (n = 33) or their first-degree relatives (n = 5), and, Group B with 61 healthy blood donation volunteers who were deferred after the discovery of unexplained high hemoglobin levels. Detailed history and clinical examination, hemogram, erythropoietin levels and PCR–RFLP for the VHL:c.598C > T;p.R200W mutation were done. In Group A, three (8%) persons aged 9, 13 and 30-years were homozygous for VHL:c.598C > T. Two were heterozygous (parents of a known case of Chuvash polycythemia). None of the Group B subjects had the Chuvash mutation. Erythropoietin levels in group A were low in 5/26 cases (19%) and normal in 18/26 (69%). In Group B, seven (11%) donors had normal values while the remaining 54 (89%) had high erythropoietin levels. Despite a lower frequency (8%) compared to literature, our results suggest that the relatively simpler PCR-RFLP for VHL:c.598C > T mutation may be considered for the initial genetic screening of unexplained, suspected congenital erythrocytosis in regions where Chuvash polycythemia comprises a large proportion of inherited erythrocytosis, after polycythemia vera and common acquired secondary causes are excluded.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12288-023-01668-9.

Introduction

Erythrocytosis, after excluding spurious causes, can be classified as secondary to increased Epo levels, or primary, i.e., erythropoietin (Epo)-independent disease [1–3]. Either type may be inherited or acquired [2]. Acquired primary (i.e., polycythemia vera) and secondary erythrocytosis are commoner and well-studied while inherited/congenital erythrocytosis are rarer. Mutations in the oxygen-sensing pathways’ regulatory protein-coding genes (VHL, EPAS1 and EGLN1) lead to secondary congenital erythrocytosis [4, 5].

The VHL tumor-suppressor gene has been linked to renal cell carcinomas, pheochromocytomas and other neoplasms [6]. Its protein is a negative regulator of the hypoxia-inducible transcription factors (HIFs). Loss-of-function mutations at the 3’-end of the VHL coding region are associated with polycythemia but do not lead to the VHL cancer syndromes [5, 7]. Homozygosity for a hypomorphic VHL: c.598C > T; p.R200W mutation leads to the rare autosomal-recessive Chuvash polycythemia. Initially described in the Chuvash Republic of Russia and in Italy, this disorder (with features of secondary as well as primary erythrocytosis) has been reported from India, Afghanistan, Turkey, Bangladesh and Pakistan [1, 8–11].

Genetic diagnosis of erythrocytosis requires an algorithmic testing approach that may be too resource-intensive for developing countries [12]. Next-generation testing (NGS) has, in recent years, enabled one-shot testing of all implicated genes, however, the work-up remains technically-complex and expensive [1, 13]. A prior study revealed a very high prevalence (61%) of Chuvash polycythemia among north Indian patients [10]. We therefore assessed the diagnostic value of first-line screening of unexplained erythrocytosis for the Chuvash mutation (VHL:c.598C > T) using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis.

Materials and Methods

This prospective clinical and laboratory-based study was cleared by the Institutional Ethics Committee. All subjects and/or their legal guardians provided written informed consent. We enrolled two subject groups: GROUP A had 38 patients with Hb > 16.5 g/dL in males and > 16.0 g/dL in females, persistent for at least 2 months [14]. Pediatric patients had Hb persistently higher than the age and sex-specific reference range. All available first-degree relatives of the patients were also included. GROUP B comprised of 61 self-reported healthy males who had volunteered to donate blood but were incidentally found to have erythrocytosis (Hb > 16.5 g/dL). Chronic and/or heavy cigarette smokers, JAK2 V617F-positive persons and those with chronic lung disease, right-to-left cardiac shunts, Epo-secreting tumors, obstructive sleep apnea or morbid obesity (BMI ≥ 40 kg/m² or ≥ 35 kg/m² with obesity-related health conditions, e.g., hypertension or diabetes) were excluded.

Subjects underwent detailed history and clinical examination, complete hemogram (LH780/DxH800 analyzers, Beckman Coulter Inc., FL, USA) and erythropoietin levels by ELISA (Biomerica, MDSS, GmbH, Hannovar, Germany). PCR–RFLP was done for the VHL:c.598C > T;p.R200W) mutation described in detail in the Supplementary Methods file. High-performance liquid chromatography (HPLC) was performed in Group A subjects (Variant II, Bio-Rad Laboratories, USA).

Results

Demographic, clinical and hemogram data are summarized in Table 1. Group A included 33 index cases with erythrocytosis and their five family members (two sets of two brothers each, two father-son duos and the parents of an already diagnosed case of Chuvash polycythemia i.e., homozygous VHL:c.598C > T;p.R200W mutation) [15]. PCR-RFLP revealed homozygosity for VHL:c.598C > T;p.R200W in 3 (8%) persons and heterozygosity in two individuals (parents of the known case of Chuvash polycythemia) in Group A (Supplementary Figs. 1 and 2). In Group B, none of the 61 prospective blood donors revealed the VHL:c.598C > T mutation.

Table 1.

Demographic, clinical and laboratory results across the two subject groups

Parameter	Group A (n = 38)	Group B (n = 61)
Demographic and clinical data
Age in years (median, range)	35, 6–70#	32, 18–64
Sex ratio (males:females)	37:1	61:0
Past phlebotomies (n,%)	15, 39% (therapeutic phlebotomies)	55, 90% (prior blood donations)
Clinical presentations	6 (16%): headache and/or a generalized feeling of unwellness, 2 (5%): stroke, 1 (3%) each: central retinal vein thrombosis and intermittent claudication. Remaining: High Hb detected following testing done for a plethoric appearance, family screening, or incidentally	Asymptomatic, detected on Hb estimation when volunteering for blood donation. Retrospectively, 5 (8%) recalled mild headache and/or irritability
Family history of erythrocytosis, n(%)	7 (18%)	–
Laboratory data
Hb (g/dL)	18.1 ± 2.3, 13.0–23.1	17.5 ± 1.0, 16.5–20.9
Hematocrit (%)	54.3 ± 7.8, 39.8–74.0	54.5 ± 3.4, 47.9–65.4
RBC count (million/μL)	6.16 ± 1.38, 4.35–10.55	5.75 ± 0.40, 4.81–7.04
MCV (fL)	89.5 ± 8.5, 65.0–112.9	90.4 ± 6.8, 80.0–109.2
MCH (pg)	30.0 ± 3.3, 20.5–37.8	30.4 ± 1.73, 27.3–36.1
MCHC (g/dL)	33.6 ± 1.9, 30.0–39.51	33.9 ± 2.3, 28.5–38.7
RDW-CV (%)	15.3 ± 2.7, 12.1–25.2	13.9 ± 1.7, 11.6–18.9
TLC (× 10^9/L)	8.1 ± 1.8, 4.2–13.1	7.2 ± 3.4, 3.6–14.1
Platelet count (× 10^9/L)	226 ± 72.4, 57–449	196 ± 65.8, 146–350
EPO levels (mIU/L)	15.7, 1.0–115.6	97.1, 3.4–643.6

Data are presented as mean ± standard deviation (range), except for EPO which is given as median (range)

EPO Erythropoietin; Hb Hemoglobin; MCV Mean corpuscular volume; MCH Mean corpuscular hemoglobin; MCHC Mean corpuscular hemoglobin concentration; RBC Red blood corpuscule; RDW-CV Red cell distribution width-Coefficient of variation; TLC Total leucocyte count

^#Four (11%) were children aged below 14 years, 11 (32%) were aged 21–30 years and nine (24%) were aged ≥ 60 years. Values in Group A include those from subjects on phlebotomies

All three Chuvash polycythemia patients (from Group A) had Hb greater than 21 gm%. Five persons in Group A had Hb < 16.5 g/dL due to a recent phlebotomy (n = 3) or were relatives of index cases (n = 2). In Group B, nine potential donors (15%) had Hb ≥ 18.5 g/dL. Average MCV and MCH in both groups were within normal range, two brothers in Group A had low MCV < 80 fL. They were subsequently found to be compound heterozygous for β-thalassemia trait and the high oxygen-affinity Hb Regina [16]. Three cases had macrocytosis (MCV > 104 fL). RDW-CV was elevated in 12 (32%) Group A subjects, of whom, 8 (67%) were undergoing phlebotomies for their erythrocytosis and thus possibly had iatrogenic iron deficiency.

Epo levels were estimated in 26 (68%) Group A and 61 (100%) Group B subjects (Table 1). In Group A, 5/26 cases (19%) had reduced Epo, 18/26 (69%) had normal values and 3/26 (12%) had high Epo levels. In Group B, 7 (11%) donors had normal values while the remaining 54 (88%) had high Epo levels. Epo was significantly higher in group B vis-à-vis group A (p < 0.001, students T-test).

Hb HPLC was done in 34 (89%) Group A cases as part of erythrocytosis work-up. Of them, 30 (88%) showed normal patterns. One person had HbD-Punjab trait. One 6-year-old boy with Hb 22.4 g/dL and high Epo levels (56 mIU/mL) had high HbF (35.8%) with normal range HbA2 (2.3%). His parents had normal Hb values (father’s Hb-15.1 g/dL and mother’s Hb 12.0 g/dL). Molecular genetic testing by Gap-PCR revealed an incidental heterozygous hereditary persistence of fetal hemoglobin-3 (HPFH-3), the common Indian ^Gγ^Aγ(δβ)⁰ form of HPFH in both the father and the index case, but did not explain his erythrocytosis. Two brothers were compound heterozygous for Hb Regina and β-thalassemia [16]. The dominant-acting Hb Regina explained their erythrocytosis.

Discussion

Genetic diagnosis of erythrocytosis after excluding acquired causes typically requires techniques like automated DNA sequencing or NGS [2, 13, 17]. This study’s shorter VHL PCR-RFLP “single test first” approach was modestly successful, diagnosing 3/38 group A cases (8%) with Chuvash polycythemia. Although low, this 8% diagnostic yield is not insignificant, since even high-coverage molecular genetic techniques yield diagnoses in only 4–29% (targeted NGS) and 13–73% (for extended Sanger sequencing) in unexplained erythrocytosis. [10, 13, 17–20] Hence, the simpler PCR-RFLP can be recommended in regions like northern India, the southern former Russian republics and eastern Europe for initial screening of unexplained, suspected congenital erythrocytosis. A comparison of targeted NGS, direct DNA sequencing by Sanger technique and PCR-RFLP for the diagnosis of inherited or congenital erythrocytosis is provided in the Supplementary Table 1.

There are only two prior Indian reports in indexed literature that describe patients with DNA-testing confirmed Chuvash polycythemia [10, 15]. The first study found this VHL substitution as the commonest homozygous mutation detected in unexplained erythrocytosis (11/18 cases) on Sanger sequencing [10]. Their higher frequency was likely due to more stringent patient selection criteria since they recruited patients before the WHO reduced the Hb cut-off in 2016 [14]. The other publication was a detailed case report of one of these 11 patients who had an amalgamated hemolytic + polycythemic phenotype due to the co-inheritance of Chuvash polycythemia with G6PD-deficiency [15]. Our study adds three more cases of Chuvash polycythemia to the 12 previously reported from India [10, 15, 21]. Out of these 15 cases, 14 were males.

We also studied 61 potential blood donors incidentally detected to have erythrocytosis. High Hb accounts for only a small fraction of the reasons for donor deferral [22]. The method of Hb screening used for blood donor selection, may have role to play in this observation, since screening may be done using qualitative methods such as copper sulfate, whereas a high Hb can only be picked up by quantitative methods like photometers or hematology analyzers. Moreover, the upper limit of acceptable Hb level for blood donation is not clearly defined in several donor selection criteria [23]. WHO recommends accepting donations from individuals with secondary erythrocytosis, provided that PV has been excluded [24]. Keeping in view the downward revision of the WHO cut-off for erythrocytosis in 2016 [14], the blood centre’s approach in potential donors with Hb between 16.5 and 18.5 g/dL also needs to be re-examined. Acceptance or deferral of these donors and the requirement of referral of such donors to the internal medicine/clinical hematology should be on the pattern of referral of donors found to be sero-reactive for transfusion transmissible infections.

We did not follow-up the deferred polycythemic donors due to logistic constraints. However, Hultcranz et al. [25] studied 15,38,019 Swedish and Danish blood donors between 1987 and 2012. In this cohort, 190 persons were subsequently found to develop a myeloproliferative neoplasm including PV. Hb > 17.5 g/dL in men and > 16.0 g/dL in women portended increased the risk of myocardial infarction (hazard ratios of 3.52 and 3.22 respectively). Ischemic stroke risk in the above groups was also increased (hazard ratios of 2.36 and 2.35 in polycythemic men and women respectively). Venous thromboembolism risk was however, not consistently elevated in individuals with high Hb [25].

This study has some limitations. Epo was unavailable in 12 group A patients and JAK2 exon 12 mutations were not excluded. Testing remained incomplete due to the stepwise nature of the work-up being interrupted by the COVID-19 pandemic. Follow-up blood counts would have been useful in group B to assess if polycythemia was a transient or a secondary phenomenon in them. Hb HPLC could have been done in group B (potential donors) had a follow-up been available. Erythrocytosis remained unexplained in all but five cases since further evaluation was not a part of this research plan. Subsequent steps would include confirmation of persistence of erythrocytosis in group B, and NGS in group A to reveal any genetic mutations.

In conclusion, our findings suggest that the PCR-RFLP for VHL:c.598C > T mutation may be considered for initial genetic screening of unexplained, suspected congenital erythrocytosis after excluding PV and common acquired secondary causes in regions where Chuvash polycythemia forms a significant proportion of inherited erythrocytosis.

Supplementary Information

Below is the link to the electronic supplementary material.

Funding

This work was supported by the Special Research Grant from the Medical Education and Research Cell, PGIMER, Chandigarh, India (to ND).

Data and Material Availability

All data mentioned in this paper are available with the corresponding author and will be provided upon request.

Code Availability

Not applicable.

Declarations

Conflict of interest

Nil for all the authors.

Consent to Participate

All subjects and/or their legal guardians gave written informed consent.

Consent for Publication

Consent for publication was obtained from the subjects.

Ethical Approval

This study was cleared by the Institutional Ethics Committee of the PGIMER, Chandigarh vide letter no. INT/IEC/2019/2568.