Association Analysis Between JAK2^V617F And Flanking SNPs In The Western Population And Development Of A New Method For Detection Of JAK2^V617F

Ph- myeloproliferative neoplasms (MPNs) represent a heterogeneous group of chronic diseases characterized by increased expansion of hematopoietic cells of the myeloid lineage, including polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). MPNs mainly affect older people with an average age of onset of 55 years. JAK2^V617F, an activation mutation form of tyrosine kinase JAK2, is found in the majority of patients with MPNs. Studies have demonstrated that JAK2^V617F can cause MPNs, it was designated a major diagnosis criterion for Ph-MPN by WHO in 2008. But it remains unclear how this single abnormality gives rise to distinct clinical entities, there likely are additional genetic events that contribute to the pathogenesis of these disorders. Earlier studies have demonstrated that the 46/1 haplotype (or ?GGCC') on the JAK2 genes has a strong association with development of MPNs and the occurrence of JAK2^V617F. We genotyped JAK2 and 3 flanking SNPs by using a sensitive nested-AS-PCR strategy of 962 DNA samples from western population, and performed association study by using SNPStats. We find that the 5bp deletion allele of rs56241661, the G allele of rs10974944 and the C allele of rs12343867 are risky alleles, they are strongly associated with JAK2^V617F (each of analysis has a OR value of around 3, P<0.0001). Haplotype structure of the JAK2 locus shows JAK2^V617F site and these 3 SNPs locate in a LD block approximately 3kb apart. The haplotype consisting of 3 risky alleles is strong associated with JAK2^V617F positive MPNs (OR=2.16, P<0.0001). Our data suggest these 3 SNPs contribute to JAK2^V617F positive MPN predisposition, this is important for revealing the underlying cause of these neoplasms.The finding of JAK2^V617F is a major step forward in understanding the pathogenesis of MPNs, various methods have been developed to detect JAK2^V617F for diagnostic purposes. However, a highly sensitive method is still needed for the earliest possible detection and for disease prevention. In the present study, we developed a method dubbed restriction fragment nested allele-specific PCR (RFN-AS-PCR). The method consists of three steps: 1) initial amplification of DNA samples with PCR primers surrounding the JAK2^V617F mutation site, 2) digestion of the PCR products with restriction enzyme BsaXI which only cleaves the wild type allele. In this critical step, BsaXI removes from the wild type PCR product a 30 bp fragment containing the ^V617F mutation site, which effectively eliminates the chance that the full-length wild type product is re-generated in the subsequent PCR, and 3) detection of JAK2^V617F by allele-specific PCR with nested primers. We tested the sensitivity of the method by using purified plasmid DNAs and blood cell DNAs containing known proportions of JAK2^V617F. We were able to detect JAK2^V617F with a sensitivity of 0.001%. We further analyzed blood cell DNA samples from 105 healthy donors with normal blood cell counts and found 3 JAK2^V617F-positive cases, which would have remained undetected using a less sensitive method. In summary, we have developed a highly sensitive method that will allow for detection of JAK2^V617F at a very early stage. This method may have major implications in diagnosis and prevention of MPNs and related diseases.