Tlr4 is monoallelically expressed in a tissue specific manner in mice (Peireira JP, Girard R, Chaby R, et al. Nat Immunol 4(5): 464–70, 2003). We characterized Tlr4 allelic expression in human BFU-E, CFU-E, and reticulocytes. The alleles were distinguished by a single nucleotide polymorphism (SNP) that resulted in creation of a new Bcc1 restriction enzyme cutting site. Individuals were genotyped for the SNP and only individuals heterozygous for the SNP were used in experiments. Polycythemia vera patients were diagnosed based on clinical presentation, the presence of the JAK2 V617F mutation, and clonality by X chromosome inactivation methods when available. Individual human BFU-E and CFU-E were grown from whole blood mononuclear cell fractions on methylcellulose media. Allelic expression of Tlr4 was assayed after reverse transcription of mRNA, nested PCR amplification of cDNA, and Bcc1 digestion of PCR product.

Normal human BFU-E and CFU-E colonies demonstrated monoallelic expression of both alleles as well as biallelic expression (n=1). Polycythemia vera patient BFU-E and CFU-E colonies demonstrated a similar pattern (n=2). Tlr4 allelic expression in reticulocyte RNA fractions was assayed as outlined above and by quantitative reverse transcription PCR methods. Normal human reticulocyte RNA fractions isolated from whole blood demonstrated biallelic expression (n=4). 3 of 5 polycythemia vera patient reticulocyte RNA fractions isolated from whole blood demonstrated skewed biallelic expression, and 2 of 5 polycythemia vera patients demonstrated monoallelic expression of either allele.

Tlr4 is located on chromosome 9 in humans. The finding of monoallelic expression in the reticulocyte RNA of 2 of 5 PV patients suggests that

  1. Tlr4 may be used as a phenotypic clonality marker in males and females, and

  2. a subset of polycythemia vera patients can be identified within the diagnostic population defined by clinical history and the JAK2 V617F mutation.

If the assumption that clonal proliferation follows somatic mutation is true, then at least 2 mutations are implied in the pathogenesis of polycythemia vera: one by the original demonstration of clonality by X-chromosome inactivation methods, and the other by the demonstration of acquired monoallelic Tlr4 expression in the BFU-E/CFU-E to reticulocyte transition. These data are now followed by a prospective study of Tlr4 expression in a large number of female and male patients to be correlated with quantitative JAK2 V617F measurements.

Topics:
clonality (genetic analysis), erythroid progenitor cells, hematological diseases, mutation, somatic, toll-like receptor 4, polycythemia vera, rna, polymerase chain reaction, dna restriction enzymes, dna, complementary

Disclosure: No relevant conflicts of interest to declare.

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2006, The American Society of Hematology
2006
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