Introduction

Familial erythrocytosis type 2 or Chuvash polycythemia (MIM #263400), an autosomal recessive condition, presents high hemoglobin levels and normal or high erythropoietin levels characteristics of primary and secondary erythrocytosis. As mutations in VHL gene are the cause of this disease, homozygosity or compound heterozygosity variants could be found in affected individuals¹-⁴.

Von Hippel-Lindau disease (VHL), an autosomal dominant inherited cancer syndrome, is characterized by hemangioblastoma, pheochromocytoma, clear cell renal carcinoma, pancreatic neuroendocrine tumors, and other tumors such as epididymal cysts in heterozygous patients, who carry only one germline mutation in the VHL gene⁵,⁶. Polycythemia is a rare manifestation of VHL (5-20% of cases), in which elevated hemoglobin levels are secondary to tumor-produced erythropoietin⁷. In patients with familial erythrocytosis type 2, no increased risk for the formation of VHL-associated tumors has been reported⁶,⁸-¹⁰.

We report a pediatric patient with both familial erythrocytosis type 2 and von Hippel-Lindau disease.

Clinical case

We describe a 9-year-and-5-month old male patient incidentally diagnosed with polyglobulia when performing routine laboratory studies after an accident at 8 years of age (hemoglobin, 21.4 mg/dl; hematocrit, 67%; mean corpuscular hemoglobin, 23.2; platelets, 229,000; leukocytes, 5,990; neutrophils, 3260; lymphocytes, 1,790; reticulocytes, 1.2%). The mother reported that the patient presented fatigue and peripheral cyanosis during exercise.

Several studies were conducted, including venous blood gas, lung VQ scan, echocardiogram, cardiac catheterization, bone marrow aspirate, bone biopsy, respiratory function analysis, and ocular fundus study, all of which were reported as normal.

The physical examination showed a tumor in the right testicle. A cyst in the right epididymis was identified by testicular ultrasound.

Due to von Hippel-Lindau disease's family history (Figure 1), a molecular genetic test was performed on his mother, in whom a deletion of exon 3 of the VHL gene (heterozygote) was identified. The mother of the patient died due to complications of a hemangioblastoma in the central nervous system.

Figure 1 Family pedigree of four generations with von Hippel-Lindau disease. 

The patient's DNA was obtained from a peripheral blood sample, identifying the deletion of exon 3 of the VHL gene by multiplex ligation-dependent probe amplification (Figure 2A).

Figure 2 A: multiplex ligation-dependent probe amplification (MLPA) in which the deletion of exon 3 was identified in both mother and patient compared with a wild type allele. B and C: partial electropherograms of exon 2 of the VHL gene in the mother and the child, respectively. The heterozygous change 416 C>G (p.Ser139Cys) is observed (red arrow). D: conservation of S139 in VHL in different mammal species. The protein sequences of VHL orthologs at positions 124-154 are aligned. The arrow indicates the position of S139. 

Due to the antecedent of polyglobulia, where affected individuals could be homozygous or compound heterozygotes, complete sequencing of the VHL gene was performed in the patient's sample. A new missense mutation was found: the change of cytosine by guanine at nucleotide 416 results in the change from a serine to a cysteine (p.Ser139Cys) (Figures 2B, 2C and 2D).

The patient was a compound heterozygote. According to the clinical picture and molecular biology studies, the diagnosis of familial erythrocytosis type 2 and von Hippel-Lindau disease was established.

Discussion

Familial erythrocytosis type 2 or Chuvash polycythemia was first described by Polyakova (1974), who reported 103 cases in 81 families, where all patients showed elevated hemoglobin and hematocrit levels¹. Additionally to the increase in hemoglobin and hematocrit, Sergeyeva et al. (1997) found elevation of erythropoietin, for which they suggested that the cause of this polycythemia could be related to genes different from those coding for erythropoietin and its receptor².

In 2002, Ang et al. reported that patients with familial erythrocytosis type 2 were homozygous for the c.598C>T mutation in the VHL gene. This mutation has been reported in Chuvashian, Italian, British, Bengalis, German, Turkish, African American, and Caucasian patients³,⁴,¹¹-¹⁴.

The pathophysiology of familial erythrocytosis type 2 is due to an alteration in oxygen homeostasis. The VHL tumor growth suppressor gene codes the VHL protein (pVHL), which plays a critical role in cell adaptation in response to hypoxia through downregulation of hypoxia-inducible factors (HIF)⁷.

The principal transcriptional activator of gene expression in hypoxic cells is HIF-1. The c.598C>T mutation in VHL causes a decrease in the affinity of pVHL for HIF-1a, resulting in lower ubiquitination under normal oxygen levels, leading to an increased expression of genes such as EPO (erythropoietin) and VEGF (vascular endothelial growth factor), explaining the relatively high levels of erythropoietin in these patients⁷,¹⁵,¹⁶.

It was thought that all patients with familial erythrocytosis type 2 should be homozygous for the c.598C>T mutation. However, Pastore et al. reported two Croatian homozygous patients for the c.571C>G mutation⁸,¹⁷. Both patients showed significantly higher levels of erythropoietin compared to patients bearing the c.598C>T mutation and no data of a primary polycythemia⁸,¹⁷.

To the present day, four mutations in the homozygous state that cause familial erythrocytosis type 2 have been described: c.598C>T, c.571C>G, c.413C>T, and c.586A>G⁸,⁹,¹³,¹⁷. Also, compound heterozygous variants have been reported⁹,¹⁷,¹⁸.

Patients with familial erythrocytosis type 2 do not appear to be at increased risk for tumor formation, and less than 20% of patients with VHL have polycythemia due to erythropoietin tumor production⁷. In 2012, Capodimonti et al. reported a patient with familial erythrocytosis type 2 and von Hippel-Lindau disease. The patient was diagnosed with polyglobulia and pheochromocytoma and was a compound heterozygote with the c.598C>T and c.388G>C mutations. The c.388G>C mutation has been previously reported in patients with von Hippel-Lindau disease¹⁰. The pheochromocytoma study performed in the patient only reported the c.388G>C mutation, indicating that the allele with the c.598C>T mutation was lost, explaining the tumor development according to the Knudson hypothesis. Familial erythrocytosis type 2 was clinically expressed due to the absence of a wild-type VHL allele allowing the expression of the c.598C>T mutation allele responsible for most cases of familial erythrocytosis type 2⁶,¹⁰.

The present case is a compound heterozygote. One of the mutations found in our patient, deletion of exon 3 in the VHL gene, has already been reported in patients with von Hippel-Lindau type 1 disease. In this case, we found a cyst in the patient's right epididymis, which has been associated with VHL disease. The second mutation found in the patient, c.416C>G in exon 2 of the VHL gene, has not been previously reported.

The nucleotide substitution changes the conserved amino acid serine to cysteine at the codon 139 (NP_000542.1) in the N-terminal domain (B-domain) of the VHL protein (Figure 2D). To test the potential impact of the amino acid substitution of this variant on the structure and function of the VHL protein, we used the web tools PolyPhen2, Mutationassesor, Provean, and SIFT. The prediction analysis for the novel variant S139C suggests it is deleterious (Condel score 0.59)¹⁹. This result explains the presence of polycythemia in the patient since individuals with familial erythrocytosis type 2 must be necessarily homozygous or compound heterozygotes. We know that the deletion of exon 3 was inherited from his mother. However, no information was available on the patient's father, nor were his current whereabouts to verify whether he is the carrier of the c.416C>G mutation found in the patient or whether it was a de novo mutation.

As part of our patient's follow-up, it is essential to monitor the possible complications associated with familial erythrocytosis type 2 since they are at high risk for peripheral thrombosis. Additionally, increased prevalence of vertebral hemangiomas, varicose veins, cardiovascular alterations, hypotension, and pulmonary hypertension have been reported¹⁶,²⁰-²².

According to the patient's age, VHL disease surveillance consists of an annual pediatric assessment (complete physical examination and neurological examination), ophthalmologic assessment, determination of plasma or 24-hour urine sample metanephrines, and abdominal ultrasound. In the case of biochemical alterations, an abdominal magnetic resonance image (MRI) should be obtained every 2-3 years. Also, an audiology assessment is recommended, which should be annual in case of hearing loss, tinnitus, or vertigo. Head and spinal cord MRI should be performed annually²³-²⁵.

This case is the second one reported worldwide where von Hippel-Lindau disease and familial erythrocytosis type 2 coexist in an individual. Despite the low frequency of familial type 2 erythrocytosis in VHL, the possibility of this diagnosis should be considered in a patient with a family or personal history of VHL in whom polyglobulia is present. On the one hand, it is important to avoid unnecessary invasive studies to try to explain the presence of polyglobulia, and on the other hand, it is necessary to ensure adequate follow-up and surveillance of both diseases.