JAK2 Negative Erythrocytosis Associated with JAK2 GGCC_46/1 Haplotype, Calr rs1049481_G, and Normal Erythropoietin Level: Is This a New Entity?

Introduction. The JAK2 haplotype known as "GGCC or 46/1 haplotype" consists of a germline combination of single nucleotide polymorphisms (SNPs) that are inherited together and are frequently associated with the onset of myeloproliferative neoplasms (MPNs) positive for both JAK2 V617 and exon 12 mutations. It has been reported a significant association between JAK2 negative erythrocytosis and the simultaneous occurrence of JAK2 haplotype GGCC_46/1 and CALR rs1049481_G allele, mapping in the 3' UTR of the gene. In the present study, we investigated the presence of JAK2 haplotype GGCC_46/1 and CALR rs1049481_G allele in a more extensive series of erythrocytosis patients and evaluated a possible correlation with serum erythropoietin (EPO) level.

Methods. Fifty-nine erythrocytosis patients, diagnosed at our Center from 2009 to 2020, negative for canonical JAK2 mutations, and secondary causes, were included in this study. Forty-nine (83%) out of 59 cases met only two major WHO 2016 diagnostic criteria for polycythemia vera (PV); the remaining 10 cases were "JAK2 negative PV", as they also showed the minor criterion with subnormal serum EPO. Genomic DNA from all patients was extracted from peripheral blood granulocytes and analyzed for the presence of both SNPs. The occurrence of the JAK2 haplotype was investigated by analyzing rs10974944 tagging SNP, using distal JAK2 intron 12 primers (forward: 5'-CTGTGCAGTCCAAACCCATG-3' and reverse: 5'-TTCTCTGCTTGCTAGTGGGT-3'). This amplification generated a PCR product of 272 bp, revealing a C/G substitution by Sanger sequencing. PCR investigated the occurrence of the G/T allele in the SNP rs1049481 in the CALR gene with primers CALR-UTR-F 5'-GACAAGGAGGATGATGAGGACAAA-3' and CALR-UTR-R 5'- AAAAATGAAAGTTCTCGAGTCTCACAGA-3' generating a 205 bp product. In silico data from 2504 healthy individuals of the 1000G Project (1000G) were used as a control group. Fisher's exact test was used to compare the differences of both SNPs and the frequency of their alleles between cases and controls, respectively; the c-square test was employed for evaluating the distribution of JAK2 GGCC haplotype in erythrocytosis patients and controls.

Results. Thirty-eight (64.4%) and 21 (35.6%) cases resulted in being positive and negative for the JAK2 GGCC haplotype, respectively. The JAK2 GGCC haplotype occurred to be associated with erythrocytosis as a statistically significant difference in frequency was detected as respect to 2504 healthy individuals from the 1000G Project (p=0.0013). This finding was also confirmed considering the Genome Aggregation Database (gnomAD v2.1.1, https://gnomad.broadinstitute.org/)(p=0.0024). The association was also demonstrated in terms of allelic frequency (p=0.0131) and genotype distribution (p=0.0030). Regarding CALR rs1049481 SNP, a significant difference in the CALR rs1049481_G allelic rate was confirmed in our cohort compared to 1000G controls (p=0.0360). Overall, the association between JAK2 GGCC haplotype and CALR rs1049481_G was observed in 28/59 (47.5%) erythrocytosis cases compared to healthy individuals (628/2504, 25%), and this difference resulted in being statistically significant (p=0.0002). Based on the EPO level, erythrocytosis patients were divided into two groups: normal (49 cases) or subnormal (10 cases). Interestingly, the simultaneous presence of JAK2 GGCC haplotype and CALR rs1049481_G was statistically significantly associated with the erythrocytosis group showing normal EPO (p=0.0133).

Conclusions. This study suggests that the JAK2 GGCC haplotype and the presence of the CALR rs1049481_G allele were significantly associated with erythrocytosis cases, negative for canonical JAK2 mutations. Our findings are in line with recent literature evidence showing that germline predisposition factors such as SNPs and haplotypes may play a crucial role in MPN pathogenesis. Moreover, this study shows that patients showing two major WHO 2016 diagnostic criteria (erythrocytosis and panmyelosis) without JAK2 mutations and with normal EPO levels can benefit from the search for germline polymorphisms combination in JAK2 and CALR driver genes for a better diagnostic classification. Therefore, the presence of these polymorphisms could represent a novel minor criterion for the diagnosis of "JAK2 negative PV".