Primary Outcome Measures:
- Identify the molecular defect of a polycythemic disorder [ Time Frame: Weekly ] [ Designated as safety issue: No ]
Secondary Outcome Measures:
- Define disease causing lesions of the erythropoietin (EPO) and EPO receptor (EPOR) pathway, as well as test the hypothesis that the EPOR mutations can cause cardiovascular disease. [ Time Frame: Monthly ] [ Designated as safety issue: No ]
The proposed research is designed to characterize the clinical picture and genetic pattern of Primary and Familial Polycythemia (PFCP). The overall objective of this proposal is to examine the mechanisms of control of erythropoiesis concentrating on those that, when disturbed, result in polycythemia. We have described the disease entity of primary familial and congenital polycythemia (PFCP), which provides a convenient model for studies of alterations of the control of erythropoiesis. We will continue to define disease causing lesions of the erythropoietin (EPO) and EPO receptor (EPOR) pathway and will test the hypothesis that the EPOR mutations can cause cardiovascular disease. We will also test the hypothesis that the aberrations and/or dysregulation of factors other than EPO and EPOR can have an effect on control of erythrocyte production by uncovering the molecular defects leading to primary familial and congenital polycythemia (PFCP) and other congenital polycythemias.
Our hypothesis is that other genes than the EPOR mutations are causative of the PFCP. These will be sought for by genetic and cell biology means. The purpose of the study is to identify the molecular defect of a polycythemic disorder.
5-7 teaspoons of peripheral blood will be drawn on all study subjects. The subject will be encouraged to speak with family members and ask if they too would like to participate. Blood will be drawn from all affected, consenting individuals.
After DNA is obtained, linkage analysis and/or mutation analysis will be performed. Linkage analysis requires patient DNA and markers from known regions of the human genome. The markers used can be in the form of DNA probes called oligonucleotide primers used for polymerase chain reaction (PCR) which identifies benign changes in DNA known as polymorphisms. Once the polymorphisms are analyzed, a computer program for linkage analysis is utilized to decipher the statistical likelihood that a gene responsible for the disease under study resides in the region of the marker used in the analyses. The resultant logarithm of the odds (LOD score) provides a mathematical prediction (in log 10) of the likelihood of the gene location. Once the LOD score approximates a score of > 3.0 (i.e., 10 to the third or 1000:1 odds of the gene being in this region), the gene location is assumed to be "close by". This then, suggests that the DNA probe is close to the actual gene of interest and allows for genetic evaluation of other families with similar clinical disease since Mendel's law predicts co-segregation of disease with this DNA marker.
Once linkage analysis is performed and localization of the disease-causing gene to a region of the genome is established, the region may be narrowed and the gene cloned. Cloning of the gene then enables the analysis to proceed to mutation analysis using direct sequencing, denaturing high performance liquid chromatography (DHPLC) or single strand conformation polymorphisms (SSCP), thus discovering the actual abnormality within the gene that causes the disease. Depending on the number of different mutations, the spectrum of disease severity and physiologic mechanisms causing the disease can then be evaluated and discovered. Once the genes and responsible mutations are found, affected patients and relatives will be analyzed for diagnosis.
Purpose
