Etiology of Polycythaemia

The terms polycythemia and erythrocytosis though used interchangeably describe different entities. Polycythemia (Gk, polys + kytos, cell, haima, blood) is an absolute increase in the mass of red cells. Erythrocytosis refers to only increase in the concentration of erythrocytes. Erythrocytosis may be due to increase in red cells (polycythemia, absolute erythrocytosis) or decrease in plasma volume (relative erythrocytosis).

Erythropoiesis and It’s Control

Erythrocyte are produced from haemopoietic stem cells (see Morphology of Erythroid Processors). Erythropoietin (EPO) prevents apoptosis of erythroid precurssors and increases erythrocyte production. Hypoxia stimulates erythropoietin production.

Erythropoietin is produced in response to hypoxia. Hypoxia sensing involves

• von-Hipple Lindau oncogene (VHL) protein
• Hypoxia inducible factor (HIF) of there are three forms HIF1, HIF2 and HIF3. HIF2 is involved in erythropoietin production in response to hypoxia.
• Prolyl-4-hydroxylase

Oxygen is critical for cell survival and rapid response to hypoxia is critical. Synthesizing proteins as effectors of response is slow process that may not be appropriate for a stress like hypoxia. Instead of synthesizing HIF in response to hypoxia the cells control the levels HIF by controlling it’s destruction. Cells on one hand synthesize HIF at a steady rate and on the other carry out a proteosomal degradation HIF by oxygen dependent mechanism.

Prolyl hydroxylase (PDH) is a constutively expressed enzyme that is involved in the process of marking HIF for proteosome mediated destruction. Proteosomal destruction of HIF also requires the von Hipple-Lindau oncogene protein. PHD is oxygen sensitive. Hypoxia reduces the PHD activity which in turn increases HIF levels by reducing its degradation. HIF induces the transcription of many genes involved in response to hypoxia including the erythropoietin gene. Increased HIF causes erythrocytosis. Mutations of the PDH and VHL gene have an effect similar to hypoxia. They increase erythrpoietin secretion and cause increased erythrocyte production.

Erythropoietin action is mediated by the erythropoietin receptor (ER). Binding of EPO to ER results in conformational change and the receptor is switched on. Receptors mediate their actions by a cascade of enzymatic actions. One of the most common mechanism of receptors action is activation of receptor tyrosine kinase activity. This allows the receptor to phosphorylate proteins and start a cascade of changes, usually phosphorylations, that lead to target actions. Not all receptors have tyrosine kinase activity. Those without tyrosine kinase activity rely of non-receptor tyrosine kinase for action. The erythropoietin receptor lacks tyrosine kinase activity essential for signal transduction. It associated with the non-receptor tyrosine kinase Janus kinase 2 (JAK2). JAK2 mediates the action of erythropoietin receptor (see ErythropoietinReceptor Signalling for details of erythropoietin production and action).

Table 1 – Causes of Polycythaemia

Primary Polycythaemia (serum erythropoietin low)
Inherited/Congenital	Acquired
Familial Erythrocytosis Type I	Polycythaemia Vera
Secondary Polycythaemia (serum erythropoietin normal/high)
Inherited/Congenital	Acquired
Disorders of Oxygen Sensing Familial Erythrocytosis Type II (VHL Mutations) Familial Erythrocytosis Type III (PHD2 mutations) Familial Erythrocytosis Type II (HIF2A Mutations) High Affinity Haemoglobins Inherited Methaemoglobinaemia Congenital heart disease with right to left shunt	Hypoxia Chronic lung disease Conginital heart disease with reversal of left to right shunt Smokers High altitude Ectopic erythropoetin seceretion Paraneoplastic Erythropoietin seceretion

Causes of Polycythemia (See Table 1)

Erythrocytosis may be primary or secondary, inherited or acquired. Primary erythrocytosis is erythropoietin independent. These patinets have low EPO levels. Patients with secondary erythropoiesis have have normal or high erythropoietin. The genetic defects associated with polycythaemia are listed in table 2.

1. Primary Polycythaemia (Polycythaemia with low serum erythropoietin)
   1. Inherited
      1. Familial erythrocytosis type I: Inherited erythrocytosis is an autosomal dominant disorder resulting from truncating mutation in the erythropoietin receptor that result in deletion of between 57 to 127 amino acids from the cytoplasmic domain of the receptor (see table 2 below). The cytoplasmic domain appears to be necessary for inhibition of receptor action. Truncation results in the loss of the SHP-1 binding site. SHP-1 dephosphorylates JAK2 and attenuates erythropoietin rceptor signaling. Deletion causing familial erythrocytosis type I result in a loss of cytoplasmic portion of the erythropoietin receptor resulting in a receptor that can not interact with SHP-1 and be the turned off. Pathways stimulated by the erythropoietin receptor are continuously activated and polycythaemia is seen. The erythropoietin levels are low.
   2. Accquired
      1. Polycythaemia Vera: Polycythaemia vera is an accquired polycythaemic myeloproliferative disease. Majority (95%) of the patients have the JAK2V617F mutation. Patients not having JAK2V617F mutation have mutations in exon 12. JKAK2 is the mediator of effects of erythropoietin receptor. These mutations result in increased erythropoietin sensitiviy and polycythaemia (see table 2 below). The erythropoietin levels are low.
2. Secondary Polycythaemia (Polycythaemia with normal or high serum erythropoietin
   1. Inherited
      1. Familial Erythrocytosis type 2 (Chuvash and other Polycythemias): Patients with familial erythrocytosis from the Chuvash republic of the former Soviet Union was found to have an Arg to Trp substitution at the amino acid position 200 (ARG200TRP) in the VHL gene. Other VHL mutations associated with erythrocytosis including VAL130LEU, ASP126TYR, PRO192SER and HIS191SER (see table 2 below). These patients have an abnormality in hypoxia sensing and result in an inappropriately high production for the degree of hypoxia. VHL muttions result in von Hipple-Lindau syndrome that predisposes the individula to renal cell carcinoma. No such predisposition is found in patients with type 2 familial erythrocytosis. Patients with Chuvash polycythaemia develop vertibral angiomas, are predisposed to venous thrombosis and respond abnormally to hypoxia
      2. Familial Erythrocytosis Type 3: Familial erythrocytosis type 3 results from mutations of the PDH2 gene resulting in a defect in oxygen sensing (see table 2 below). These patients have inappropriately normal erythropoietin levels. Identified mutations are listed in table above.
      3. Familial Erythrocytosis Type 4: Familial erythrocytosis type 4 is an autosomal dominant disorder resulting from gain of function mutations in the HIF2A gene that codes for the α subunit of hypixia inducible factor 2 (see table 2 below). The mutations cause a defect in oxygen sensing resulting in an inappropriately high production of erythropoietin for the oxygenation.
      4. Haemoglobin Anomalies Causing Polycythaemia: Polycythaemia is caused by high affinity haemoglobins and congenital methaemoglobinaemia (see table 2 below). Mutations resulting in high affinity haemoglobins result in impaired oxygen release. this is sensed as hypoxia, erythropoietin production increased and polycythaemia results. Congenital methaemoglobinca results in suboptimal oxygen delivery erythropoietin secretion and polycythaemia.
   2. Acquired polycythaemia
      1. Accquired secondary polycythaemia: Accquired secondary polycythemia is observed in the following conditions
         1. Conditions associated with hypoxia:
            • Chronic Obstructive pulmonary disease
            • Left to right shunt with eisenmengerization
            • Chronic Smokers
            • High altitude polycythaemia
         2. Conditions associated with ectopic erythropoietin secretion:
            • Solitary renal cysts
            • Polycystic kidney disease
            • Hydronephrosis
         3. Paraneoplastic erythropoietin secretion:
            • Renal cell carcinoma – most common
            • Hepatoma – Less common
            • Occasional associations: Adrenal tumours,  cerebellar hemangioblastoma, hemangiomas, pheochromocytomas, sarcomas, uterine fibroids and Wilms tumor.

Table 2 – Genetic Defects Causing Polycythaemia

Syndrome	Gene	Mutations
Inherited disorders
Familial Erythrocytosis Type I (Autosomal Dominant)	Erythropoietin Receptor	TRP439TER 1-BP INS, 5975G ASN487SER 7-BP DEL, NT5980 1-BP INS, 5967T TYR426TER 5968_5975DUP
Familial Erythrocytosis Type II (Autosomal reccssive)	VHL Gene	ARG200TRP (Chuvash Polycythaemia) VAL130LEU ASP126TYR PRO192SER HIS191ASP
Familail Erythrocytosis Type III (Autosomal Dominant, all patinets have been heterozygous)	PDH2 gene	PRO317ARG ARG371HIS HIS374ARG;
Familial Erythrocytosis Type IV (Autosomal DOminant)	HIF2A gene	GLY537TRP GLY537TRP MET535VAL
High Affinity Haemoglobin (Autosomal Dominant)	HBA, HBB	93 varients listed at http://globin.bx.psu.edu/hbvar/menu.html accessesd on 11th July 2013
Congenital Methemoglobinaemia	Cytochrome b5, Cytochrom b5 Reductase, Hemoglobin M	The specific mutations are described in the links to the genes in the adjacent columns
2,3 Bisphosphoglucerate Mutase deficiency	2,3-Bisphoglyceromutase	ARG89CYS; 1-BP DEL, 205C
Acquired
Polycythaemia Vera	JAK2	VAL617ILE (V617F), Exon 12 mutations