Contributions
Abstract: PB1847
Type: Publication Only
Background
The mutational status of immunoglobulin heavy variable (IGHV) genes has been established as one of the most reliable prognostic markers in CLL. Unmutated CLL (U-CLL), is associated with an aggressive phenotype and shorter survival than patients with less than 98% identity to germ line (“mutated CLL”). The molecular mechanisms underlying these subtypes are incompletely understood. Human immunoglobulin (Ig) genes are diversified in mature B cells by distinct processes known as Ig heavy chain class switch recombination and Ig variable region exon somatic hypermutation. These DNA modification processes are initiated by activation-induced cytidine deaminase (AID), a DNA cytidine deaminase predominantly expressed in activated B cells. In malignant lymphoid tumors of germinal origin, activation-induced deaminase appears to be pathogenic. More recent work has suggested that WGCW sites, and in particular certain AGCT sites, may play a special role as AID “entry sites” that not only mutate at high frequency, but also facilitate further mutations close to the site of the original mutation and throughout the V region.
Aims
In this study, we investigated the frequency and mutation status of IGHV in a cohort of 489 CLL patients from Mediaterranean region of Spain. We also analyzed the relationship between the number of WGCW hotspots in the germline V-region and the observed mutation frequency in this cohort.
Methods
Initial FASTA files with IGH (BIOMED-2) sequences from 489 patients sequenced by Sanger were converted to FASTQ with simulated qualities to test our IGH NGS-designed analysis pipeline. Reads are aligned against IGHV, IGHD and IGHJ genes from IMGT References Database using BWA mem, and then mapping statistics are generated to select the 10 more represented alleles for each sample. IGHV and IGHJ genes are determined and compared with IMGT-VQuest results. Homology and mutational status in IGHV is achieved by performing local alignment (EMBOSS Water) against the candidate alleles, and those with a homology percent less than 85 are discarded (30 out of 489 samples). All the sequences were productive. IGHD allele is determined extracting CDR3 according to IMGT Unique numbering recommendations and then the subset is aligned against IGHD alleles using BLAST, choosing the hit with higher score. WGCW motif count was performed on each sequence using custom R scripts. Secondary AID hotspots (WRC, GYW, SYC, GRS) were quantified separately not involving possible overlappings with WGCW motif.
Results
The mean number of AID hotspots (WGCW) in the germline V region of U-CLL cases (11 motifs), is significantly higher than for M-CLL patients (6,8 motifs) whereas in the secondary hotspots we could not find any significative differences (Fig1.a). We compared the mutational status of each V gene, to the mean number of WGCW motifs (vertical axis) in the corresponding germline V region, and contradictory, V regions with more WGCW motifs are more likely to be U-CLL (Fig1.b). All of patients with IGHV1-69 rearrangement have a mean mutation frequency of 0.35%, and paradoxical, this germline sequence contains a mean of 13 WGCW sites; whereas cases expressing IGHV3-15 or IGHV3-72 clones, are commonly mutated, with a mean mutation frequency of 6% and 5.2%, respectively, and contains less hotspots (5-7 WGCW sites) (Fig1.c).
Conclusion
We confirm a geographical-dependent leukaemic repertoire and reproduced previous studies which observed both V-regions with more WGCW hotspots in the germline sequences are more likely to be unmutated and this relationship is restricted to WGCW hotspots.
Session topic: 5. Chronic lymphocytic leukemia and related disorders – Biology & Translational Research
Keyword(s): Chronic Lymphocytic Leukemia, Mutation status
Abstract: PB1847
Type: Publication Only
Background
The mutational status of immunoglobulin heavy variable (IGHV) genes has been established as one of the most reliable prognostic markers in CLL. Unmutated CLL (U-CLL), is associated with an aggressive phenotype and shorter survival than patients with less than 98% identity to germ line (“mutated CLL”). The molecular mechanisms underlying these subtypes are incompletely understood. Human immunoglobulin (Ig) genes are diversified in mature B cells by distinct processes known as Ig heavy chain class switch recombination and Ig variable region exon somatic hypermutation. These DNA modification processes are initiated by activation-induced cytidine deaminase (AID), a DNA cytidine deaminase predominantly expressed in activated B cells. In malignant lymphoid tumors of germinal origin, activation-induced deaminase appears to be pathogenic. More recent work has suggested that WGCW sites, and in particular certain AGCT sites, may play a special role as AID “entry sites” that not only mutate at high frequency, but also facilitate further mutations close to the site of the original mutation and throughout the V region.
Aims
In this study, we investigated the frequency and mutation status of IGHV in a cohort of 489 CLL patients from Mediaterranean region of Spain. We also analyzed the relationship between the number of WGCW hotspots in the germline V-region and the observed mutation frequency in this cohort.
Methods
Initial FASTA files with IGH (BIOMED-2) sequences from 489 patients sequenced by Sanger were converted to FASTQ with simulated qualities to test our IGH NGS-designed analysis pipeline. Reads are aligned against IGHV, IGHD and IGHJ genes from IMGT References Database using BWA mem, and then mapping statistics are generated to select the 10 more represented alleles for each sample. IGHV and IGHJ genes are determined and compared with IMGT-VQuest results. Homology and mutational status in IGHV is achieved by performing local alignment (EMBOSS Water) against the candidate alleles, and those with a homology percent less than 85 are discarded (30 out of 489 samples). All the sequences were productive. IGHD allele is determined extracting CDR3 according to IMGT Unique numbering recommendations and then the subset is aligned against IGHD alleles using BLAST, choosing the hit with higher score. WGCW motif count was performed on each sequence using custom R scripts. Secondary AID hotspots (WRC, GYW, SYC, GRS) were quantified separately not involving possible overlappings with WGCW motif.
Results
The mean number of AID hotspots (WGCW) in the germline V region of U-CLL cases (11 motifs), is significantly higher than for M-CLL patients (6,8 motifs) whereas in the secondary hotspots we could not find any significative differences (Fig1.a). We compared the mutational status of each V gene, to the mean number of WGCW motifs (vertical axis) in the corresponding germline V region, and contradictory, V regions with more WGCW motifs are more likely to be U-CLL (Fig1.b). All of patients with IGHV1-69 rearrangement have a mean mutation frequency of 0.35%, and paradoxical, this germline sequence contains a mean of 13 WGCW sites; whereas cases expressing IGHV3-15 or IGHV3-72 clones, are commonly mutated, with a mean mutation frequency of 6% and 5.2%, respectively, and contains less hotspots (5-7 WGCW sites) (Fig1.c).
Conclusion
We confirm a geographical-dependent leukaemic repertoire and reproduced previous studies which observed both V-regions with more WGCW hotspots in the germline sequences are more likely to be unmutated and this relationship is restricted to WGCW hotspots.
Session topic: 5. Chronic lymphocytic leukemia and related disorders – Biology & Translational Research
Keyword(s): Chronic Lymphocytic Leukemia, Mutation status