The 46/1 Haplotype Frequency Is Not Increased in Patients of Essential Thrombocythemia with CALR Mutations
Authors: Gau Jyh-Pyng , Chen Chih-Cheng , Liu Chia-Jen , Yu Yuan-Bin , Hsiao Liang-Tsai , Liu Jin-Hwang , Chen Po-Min , Tzeng Cheng-Hwai 
Published: October 2019

Abstract
Purpose
The frequency of 46/1 haplotype in myeloproliferative neoplasms (MPN) patients with JAK2V617F mutation or CALR mutations were determined to explore their association.
Patients and Methods
MPN patients were diagnosed based on WHO criteria. Allele-specific PCR and PCR with direct sequencing were utilized to identify various mutations in MPNs and genotype of the 46/1 haplotype-associated SNP (C nucleotide at rs12343867).
Results
From Oct 2011 to Aug 2014, 187 cases of MPN with diagnosis of polycythemia vera (PV) and essential thrombocythemia (ET) were recruited for study after obtaining written informed consent. The 187 MPN patients included 126 cases with JAK2V617F mutation (PV 62, ET 64). The frequency of 46/1 haplotype-tagged SNP rs12343867 genotype was significantly higher in the 187 MPN patients (CC 15%, CT 51%, TT 34%; C allele frequency, 40%; p<0.001) in comparison to the 100 normal local control (CC 8%, CT 35%, TT 57%; C allele frequency 26%). In subgroup analysis, the C allele frequency is significantly higher in JAK2V617F-positive PV (51%, p <0.001) and ET (41%, p=0.005) compared to normal controls.
When the location of JAK2V617F mutation was determined in the 37 cases of PV and 32 cases of ET with heterozygous 46/1 haplotype, the mutation was located at the cis-46/1 haplotype in 86.4% (32/37) PV patients and 87.5% (28/32) ET patients, respectively. The proportion of the cis- location for JAK2V617F mutation in either PV or ET was significantly higher than the presumed incidence of 50% (p value < 0.001) based on random occurrence.
CALR gene mutations were analyzed and no CALR mutation was found in the 10 patients of PV without JAK2V617F mutation. Among the 51 patients of ET without JAK2V617F mutation, 38 (75%) patients harbored CALR mutations and 3 patients had MPL mutation. All 3 MPL-mutated ET patients had MPLW515L mutation, whereas no MPLW515K or MPLS505N mutation was identified. No mutation could be identified in the remaining 10 ET patients (triple negative). Consistent with the earlier reports, a majority of mutations in CALR gene were type 1 mutation (c.1092_143del, n = 20 [52.6%]) and type 2 (c.1154_1155insTTGTC; n = 10 [26.3%]). The frequency of 46/1 haplotype-tagged genotype in the 38 ET patients with CALR mutations were CC 2 (5%), CT 16 (42%) and TT 20 (53%) (Table 1). The C allele frequency was 27%, which is not significantly different from that of normal control (p value = 0.879).
Table 1 Summary of genotyping results for the 46/1 haplotype Category Total C/C C/T T/T 46/1 frequency P-value All MPNs 187 28(15%) 95(51%) 64(34%) 40% <0.001 All PV 72 14(19%) 43(60%) 15(21%) 49% <0.001 PV-JAK2V617F(+) 62 13(21%) 37(60%) 12(19%) 51% <0.001 PV-triple negative 10 1(10%) 6(60%) 3(30%) 40% 0.187 All ET 115 14(12%) 52(45%) 49(43%) 35% 0.046 ET-JAK2V617F(+) 64 10(16%) 32(50%) 22(34%) 41% 0.005 ET-CALR(+) 38 2( 5%) 16(42%) 20(53%) 27% 0.879 ET-MPL(+) 3 0( 0%) 1(33%) 2(67%) 17% 1.000 ET-triple negative 10 2(20%) 3(30%) 5(50%) 35% 0.424 Local control 100 8( 8%) 35(35%) 57(57%) 26% -
Comparison of 46/1 allele frequency between patient groups and normal control
Conclusions
The frequency of 46/1 haplotype in normal population of Taiwan is similar to that in Caucasians. The frequency of 46/1 haplotype is higher in both JAK2V617F- positive PV and ET patients, while similar to normal population in ET patients with CALR mutations. The effect of 46/1 haplotype could be functioning only in the context of JAK2V617F mutation.

Disclosures
No relevant conflicts of interest to declare.