Contributions
Abstract: PB1851
Type: Publication Only
Background
NOTCH1 mutations are associated with poor prognosis for chronic lymphocytic leukemia (CLL) patients. They are represented mostly by c.7541_754delCT deletion and, in the minority of cases, point mutations in the 3′UTR region of NOTCH1. Both types of mutations resulted in the removal of C-terminal PEST domain and deregulation of NOTCH1-dependent signaling pathways.
Aims
To evaluate the frequencies of mutations in the 3′UTR region of the NOTCH1 in CLL patients in Ukraine and the distribution of rs3124591 genotypes located in the amplified area.
Methods
Detection of mutations in the 3′UTR region of the NOTCH1 was performed by direct sequencing in 109 CLL patients in whom typical c.7544_7545delCT mutation was not found. IGHV rearrangement were analyzed by IMGT/V-QUEST (Brochet et al., 2008). Analysis of rs3124591 genotypes was performed using SNPstats tool (http://bioinfo.iconcologia.net/snpstats/start.htm).
Results
Mutations in the 3′UTR region of the NOTCH1 were revealed in 3 of 109 CLL patient (2.75%), all three among 92 cases with in-frame unmutated IGHV genes (5.4%). Non-coding mutations were represented by 139390152A>G (two cases) and 139390145A>G (one case). Two cases with mutations were related to subset #1, and one case belonged to stereotyped subset #28a. In addition to previously revealed c.7544_7545delCT mutations in 34 CLL patients, four of eight cases of subset #1 and both cases belonging to subset #28a in our CLL cohort had NOTCH1 mutations. The distribution of rs3124591 genotypes was as follows: CC genotype - 22 cases (20.2%), CT genotype - 47 cases (43.1%), and TT genotype - 40 cases (36.7%). The spectrum of used IGHV genes was significantly larger in TT homozygotes than in carriers of CT and CC genotypes (; p = 0.046). When in-frame unmutated IGHV rearrangements were analyzed, we found a reduced IGHV1-69 gene usage in carriers of TT genotype compared to carriers of CT and CC genotypes (14.7%, 38.5%, and 47.4%; p = 0.023) and, on the contrary, more frequent IGHJ4 gene usage (50.0%, 25.6%, and 21.1%; p = 0.046). In addition, the frequency of “major” stereotyped subsets (according to Agathangelidis et al., 2012) was higher in TT carriers compared to carriers of CT and CC genotypes (26.5%, 10.2%, and 5.5%; p = 0.021). The number of N nucleotides inserted in the VHD junctions in unmutated IGHV rearrangements was significantly less in carriers of TT genotype than in carriers of CT and CC genotypes (5.28 ± 0.74, 9.21 ± 0.94, and 9.21 ± 1.03, correspondingly; p = 0.003). The HCDR3 length in carriers of TT genotype was significantly shorter than in carriers of others genotypes (18.88 ± 0.71 a.a. vs 21.15 ± 0.68 a.a. in CT genotype, and 21.36 ± 1.13 in CC genotype; p=0.05). The comparison of CLL sequences with non-CLL sequences available from public databases showed that most cases that had HCDR3 homology with antibacterial or antiviral Ig clones were present in TT homozygotes (18.7% vs 6.5% in carriers of CT genotype and 0% in carriers of CC genotype; p=0.042). All CLL sequences homologous with autoreactive clones were revealed in carriers of TT (10.6%) and CT (8.7%) genotypes.
Conclusion
Our data confirmed current data on the association between the structure of the B-cell receptor and appearance of NOTCH1 mutations. Some features of HCDR3 structure were identified in carriers of TT and CC genotypes. Taking into account data of Cao et al. (2014) on the functional significance of this polymorphism, it can be suggested that rs3124591 can influence on the selection of B-cell clones during early stages of CLL development.
Session topic: 5. Chronic lymphocytic leukemia and related disorders – Biology & Translational Research
Keyword(s): Chronic Lymphocytic Leukemia, Mutation, Notch, SNP
Abstract: PB1851
Type: Publication Only
Background
NOTCH1 mutations are associated with poor prognosis for chronic lymphocytic leukemia (CLL) patients. They are represented mostly by c.7541_754delCT deletion and, in the minority of cases, point mutations in the 3′UTR region of NOTCH1. Both types of mutations resulted in the removal of C-terminal PEST domain and deregulation of NOTCH1-dependent signaling pathways.
Aims
To evaluate the frequencies of mutations in the 3′UTR region of the NOTCH1 in CLL patients in Ukraine and the distribution of rs3124591 genotypes located in the amplified area.
Methods
Detection of mutations in the 3′UTR region of the NOTCH1 was performed by direct sequencing in 109 CLL patients in whom typical c.7544_7545delCT mutation was not found. IGHV rearrangement were analyzed by IMGT/V-QUEST (Brochet et al., 2008). Analysis of rs3124591 genotypes was performed using SNPstats tool (http://bioinfo.iconcologia.net/snpstats/start.htm).
Results
Mutations in the 3′UTR region of the NOTCH1 were revealed in 3 of 109 CLL patient (2.75%), all three among 92 cases with in-frame unmutated IGHV genes (5.4%). Non-coding mutations were represented by 139390152A>G (two cases) and 139390145A>G (one case). Two cases with mutations were related to subset #1, and one case belonged to stereotyped subset #28a. In addition to previously revealed c.7544_7545delCT mutations in 34 CLL patients, four of eight cases of subset #1 and both cases belonging to subset #28a in our CLL cohort had NOTCH1 mutations. The distribution of rs3124591 genotypes was as follows: CC genotype - 22 cases (20.2%), CT genotype - 47 cases (43.1%), and TT genotype - 40 cases (36.7%). The spectrum of used IGHV genes was significantly larger in TT homozygotes than in carriers of CT and CC genotypes (; p = 0.046). When in-frame unmutated IGHV rearrangements were analyzed, we found a reduced IGHV1-69 gene usage in carriers of TT genotype compared to carriers of CT and CC genotypes (14.7%, 38.5%, and 47.4%; p = 0.023) and, on the contrary, more frequent IGHJ4 gene usage (50.0%, 25.6%, and 21.1%; p = 0.046). In addition, the frequency of “major” stereotyped subsets (according to Agathangelidis et al., 2012) was higher in TT carriers compared to carriers of CT and CC genotypes (26.5%, 10.2%, and 5.5%; p = 0.021). The number of N nucleotides inserted in the VHD junctions in unmutated IGHV rearrangements was significantly less in carriers of TT genotype than in carriers of CT and CC genotypes (5.28 ± 0.74, 9.21 ± 0.94, and 9.21 ± 1.03, correspondingly; p = 0.003). The HCDR3 length in carriers of TT genotype was significantly shorter than in carriers of others genotypes (18.88 ± 0.71 a.a. vs 21.15 ± 0.68 a.a. in CT genotype, and 21.36 ± 1.13 in CC genotype; p=0.05). The comparison of CLL sequences with non-CLL sequences available from public databases showed that most cases that had HCDR3 homology with antibacterial or antiviral Ig clones were present in TT homozygotes (18.7% vs 6.5% in carriers of CT genotype and 0% in carriers of CC genotype; p=0.042). All CLL sequences homologous with autoreactive clones were revealed in carriers of TT (10.6%) and CT (8.7%) genotypes.
Conclusion
Our data confirmed current data on the association between the structure of the B-cell receptor and appearance of NOTCH1 mutations. Some features of HCDR3 structure were identified in carriers of TT and CC genotypes. Taking into account data of Cao et al. (2014) on the functional significance of this polymorphism, it can be suggested that rs3124591 can influence on the selection of B-cell clones during early stages of CLL development.
Session topic: 5. Chronic lymphocytic leukemia and related disorders – Biology & Translational Research
Keyword(s): Chronic Lymphocytic Leukemia, Mutation, Notch, SNP