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Primary familial and congenital polycythemia (PFCP) is characterized by erythrocytosis with normal arterial Po2, blood P50, and serum erythropoietin (EPO) levels. In two PFCP families EPO receptor (EPOR) polymorphisms cosegregated with PFCP. A heterozygous insertion of G at EPORnucleotide 5975 was identified in genomic DNA from polycythemic members of family no. 2. 5974insG shifts the reading frame at codon 430, predicting amino acid substitutions and truncation of the last 64 amino acids. Wild-type and mutant EPORtranscripts were detected in erythroid progenitors from affected individuals. Burst-forming units-erythroid from patients exhibited increased colony size and sensitivity to EPO. Transfected Ba/F3 cells expressing EPOR5974insG exhibited increased EPO sensitivity compared with cells expressing wild-type EPOR.The functional effect of this EPORmutation was directly compared with the other C-terminal mutations reported in unrelated PFCP families by expression in Ba/F3 cells. The transfected cells with another primary polycythemia associated EPORmutant construct (G6002A) also exhibited increased sensitivity to EPO.

A family in which two male siblings were affected with Wiskott-Aldrich syndrome (WAS) was studied using G-6-PD isoenzymes as an X-linked marker in order to investigate the nature of cellular abnormalities. Isolated peripheral blood cell types from the doubly heterozygous mother of the affected males seemingly failed to express the G-6-PD allele in cis position with the WAS allele while her cultured skin fibroblasts expressed both G-6-PD alleles. Additionally, a histogram analysis of platelet size revealed a single population of abnormally small platelets in the affected propositus, whereas the heterozygous mother had no appreciable small platelet subpopulation. In vitro culture of hemopoietic progenitor cells of the heterozygous mother showed that the majority of progenitor cells did not express the WAS allele. However, a small number of cells expressing the G-6-PD type linked with the WAS allele were detected. The proportion of the latter progenitors was significantly higher among more primitive progenitors (those giving rise to later appearing colonies). This observation suggests that selection against cells expressing the Wiskott-Aldrich defect takes place in the hemopoietic system of the heterozygous female and offers a possible means of carrier detection in some women. Linkage studies in this family revealed one example of probable recombination between the loci for WAS and G-6-PD among three informative subjects, suggesting that these two loci may not be closely linked on the X-chromosome.

Polycythemia vera (PV) is an acquired myeloproliferative clonal stem cell disorder characterized by cytokine hypersensitivity. The erythroid colony forming assay has been useful in PV diagnosis. However, studies aiming to elucidate molecular mechanism in PV pathogenesis often require large numbers of cells from a specific erythroid stage. For this purpose, we had adapted the mouse expansion assay described by Karur et al (Blood, 2006 in E-Pub) and differential display method described by Zhang et al ( Blood , 2003, 102; 3938) to develop a human erythroid expansion protocol. In this study, we used peripheral blood mononuclear cells (PB-MNCs) from PV patients and healthy donors, and expanded them along erythroid lineage in 21 day culture. Through the culture, we took samples at 8 timepoints (days 1, 7, 9, 11, 14, 16, 19, 21) to evaluate differentiation patterns. We had generated 5 different regions using CD71 (transferrin receptor) and CD235a (glycophorin A) and characterized these regions by standard morphology analysis. This protocol allowed differentiation of PB-MNCs to all erythroid stages ending with late normoblast, reticulocyte, and mature erythrocytes (which do not survive well in this culture environment).

Primary familial and congenital polycythemia (PFCP) is a rare inherited disorder presenting with elevated red blood cell mass, elevated hemoglobin concentration and low levels of erythropoietin. Ten mutations in the erythropoietin receptor (EPOR) gene to date have been associated with PFCP. All of these mutations result in deletion of 59 to 82 amino acids from the carboxy terminal of EpoR which has been shown to contain a negative regulatory domain. Here, we describe a 2-year old boy of French-Canadian descent presenting with polycythemia and splenomegaly. Sequencing of the EPORgene showed the proband to be heterozygous for a G to A transition in nucleotide 6002 (G6002A). The mutation generates a stop codon instead of tryptophan at amino acid 439, leading to a truncated EpoR. The association of the G6002A mutation in the EPORgene with PFCP has been previously described in a large Finnish family (dela Chapelle et al., Proc Natl Acad Sci USA 1993; 90: 4495) and in a 16-year old boy of English descent (Percy et al., Br J Hematol 1998; 100:407). The G6002A mutation in both cases was considered to have arisen independently based on differences in a microsatellite polymorphism in the 5′UT of EPORand the absence of the mutation in the immediate family of the English boy. We studied our proband's parents for the G6002A EPORmutation and did not find it. Their parentage was confirmed using 24 different microsatellite markers. This indicates that the G6002A mutation in the proband arose de novo. Since the mutation arose de novo, in vitromethycellulose cultures of erythroid progenitors isolated from peripheral blood of the proband were grown in the presence of increasing concentrations of Epo to rule out genetic mosaicism. The erythroid progenitors showed hypersensitivity to Epo as is characteristic of PFCP. However, we did not find evidence supportive of genetic mosaicism as all 70 BFU-E colonies analyzed were heterozygous for the G6002A mutation. Previously, two other polycythemia-associated EPORmutations, 5974insG (Sokol et al., Blood 1995; 86:15) and 5959G>T (Kralovics et al., Am J Hematol 2001; 68:115) were shown to have arisen de novo. This case is thus the fourth instance out of 13 reported cases of polycythemia-associated EPORmutations that has arisen de novo. Because of the rarity of polycythemia-associated EPORmutations, their frequent de novooccurrence suggests that these mutations do not have a selective advantage but are detrimental. Their possible association with increased risk of thromboembolic and atherosclerotic disease due to chronically augmented Epo signaling is being explored by ongoing clinical studies.