The Number of Overlapping AID Hotspots in Germline IGHV Genes Is Inversely Correlated with Mutation Frequency in Chronic Lymphocytic Leukemia

Abstract

The targeting of mutations by Activation-Induced Deaminase (AID) is a key step in generating antibody diversity at the Immunoglobulin (Ig) loci but is also implicated in B-cell malignancies such as chronic lymphocytic leukemia (CLL). AID has previously been shown to preferentially deaminate WRC (W = A/T, R = A/G) hotspots. WGCW sites, which contain an overlapping WRC hotspot on both DNA strands, mutate at much higher frequency than single hotspots. Human Ig heavy chain (IGHV) genes differ in terms of WGCW numbers, ranging from 4 for IGHV3-48*03 to as many as 12 in IGHV1-69*01. An absence of V-region mutations in CLL patients ("IGHV unmutated", or U-CLL) is associated with a poorer prognosis compared to "IGHV mutated" (M-CLL) patients. The reasons for this difference are still unclear, but it has been noted that particular IGHV genes associate with U-CLL vs M-CLL. For example, patients with IGHV1-69 clones tend to be U-CLL with a poor prognosis, whereas patients with IGHV3-30 tend to be M-CLL and have a better prognosis. Another distinctive feature of CLL is that ~30% of (mostly poor prognosis) patients can be classified into "stereotyped" subsets, each defined by HCDR3 similarity, suggesting selection, possibly for a self-antigen. We analyzed >1000 IGHV genes from CLL patients and found a highly significant statistical relationship between the number of WGCW hotspots in the germline V-region and the observed mutation frequency in patients. However, paradoxically, this correlation was inverse, with V-regions with more WGCW hotspots being less likely to be mutated, i.e., more likely to be U-CLL. The number of WGCW hotspots in particular, are more strongly correlated with mutation frequency than either non-overlapping (WRC) hotspots or more general models of mutability derived from somatic hypermutation data. Furthermore, this correlation is not observed in sequences from the B cell repertoires of normal individuals and those with autoimmune diseases.

Fig 1. Comparison of the number of WGCW hotspots to mean mutation frequency.

Number of WGCW hotspots in the germline IGHV gene sequence (horizontal axis) vs mean mutation frequency of each IGHV gene in CLL (vertical axis). Linear regression fit for all data points is shown by gray line; orange line shows fit with three outliers (encircled points) removed.

Fig 2

Profile of AID hotspots for (A) IGHV 3–21*01 and (B) IGHV3-48*03. Each colored dot in each panel represents an AID regular hot/cold/neutral spot as labeled at the edges. Double dots are overlapping hotspots (WGCW). CDR1 and CDR2 are indicated in gray shading. Although these genes have few WGCW hotspots, they both have a particularly dense region of regular GYW hotspots in CDR2, particularly so for IGHV 3–21*01. The blue boxes highlight dense clusters of non-overlapping WRC/GYW hotspots.

Fig 3. Comparison of the number of WGCW hotspots to mean mutation frequency aggregated by gene.

This plot is equivalent to Fig 1, except that each point here is aggregated by gene (e.g. IGHV3-23) by combining all patients with IGHV3-23 alleles (e.g. IGHV3-23*01, IGHV3-23*02, etc.) into single data points. The regression line (orange) was fit excluding the outliers (encircled points).