Nicholas K. Akers

Genome-wide association study of follicular lymphoma identifies a risk locus at 6p21.32

To identify susceptibility loci for non-Hodgkin lymphoma subtypes, we conducted a three-stage genome-wide association study. We identified two variants associated with follicular lymphoma at 6p21.32 (rs10484561, combined P = 1.12 × 10−29 and rs7755224, combined P = 2.00 × 10−19; r2 = 1.0), supporting the idea that major histocompatibility complex genetic variation influences follicular lymphoma susceptibility. We also found confirmatory evidence of a previously reported association between chronic lymphocytic leukemia/small lymphocytic lymphoma and rs735665 (combined P = 4.24 × 10−9).

Coding Variants at Hexa-allelic Amino Acid 13 of HLA-DRB1 Explain Independent SNP Associations with Follicular Lymphoma Risk

Non-Hodgkin lymphoma represents a diverse group of blood malignancies, of which follicular lymphoma (FL) is a common subtype. Previous genome-wide association studies (GWASs) have identified in the human leukocyte antigen (HLA) class II region multiple independent SNPs that are significantly associated with FL risk. To dissect these signals and determine whether coding variants in HLA genes are responsible for the associations, we conducted imputation, HLA typing, and sequencing in three independent populations for a total of 689 cases and 2,446 controls. We identified a hexa-allelic amino acid polymorphism at position 13 of the HLA-DR beta chain that showed the strongest association with FL within the major histocompatibility complex (MHC) region (multiallelic p = 2.3 × 10⁻¹⁵). Out of six possible amino acids that occurred at that position within the population, we classified two as high risk (Tyr and Phe), two as low risk (Ser and Arg), and two as moderate risk (His and Gly). There was a 4.2-fold difference in risk (95% confidence interval = 2.9-6.1) between subjects carrying two alleles encoding high-risk amino acids and those carrying two alleles encoding low-risk amino acids (p = 1.01 × 10⁻¹⁴). This coding variant might explain the complex SNP associations identified by GWASs and suggests a common HLA-DR antigen-driven mechanism for the pathogenesis of FL and rheumatoid arthritis.

Multi-locus HLA class I and II allele and haplotype associations with follicular lymphoma.

Follicular lymphoma (FL) is an indolent, sometimes, fatal disease characterized by recurrence at progressively shorter intervals and is frequently refractive to therapy. Genome-wide association studies have identified single nucleotide polymorphisms (SNPs) in the human leukocyte antigen (HLA) region on chromosome 6p21.32-33 that are statistically significantly associated with FL risk. Low to medium resolution typing of single or multiple HLA genes has provided an incomplete picture of the total genetic risk imparted by this highly variable region. To gain further insight into the role of HLA alleles in lymphomagenesis and to investigate the independence of validated SNPs and HLA alleles with FL risk, high-resolution HLA typing was conducted using next-generation sequencing in 222 non-Hispanic White FL cases and 220 matched controls from a larger San Francisco Bay Area population-based case-control study of lymphoma. A novel protective association was found between the DPB1*03:01 allele and FL risk [odds ratio (OR) = 0.39, 95% confidence interval (CI) = 0.21-0.68]. Extended haplotypes DRB1*01:01-DQA1*01:01-DQB1*05:01 (OR = 2.01, 95% CI = 1.22-3.38) and DRB1*15-DQA1*01-DQB1*06 (OR = 0.55, 95% CI = 0.36-0.82) also influenced FL risk. Moreover, DRB1*15-DQA1*01-DQB1*06 was highly correlated with an established FL risk locus, rs2647012. These results provide further insight into the critical roles of HLA alleles and SNPs in FL pathogenesis that involve multi-locus effects across the HLA region.

Association of HLA-DQB1 alleles with risk of follicular lymphoma

In a recent genome-wide association study of follicular lymphoma (FL), we identified novel risk alleles on chromosome 6p21.33 that appeared to be part of an extended haplotype including HLA-DRB1*0101, DQA1*0101, and DQB1*0501. To follow up on these findings, we obtained 2–4 digit HLA-DQB1 allelotypes on a subset of 265 cases of FL and 757 controls using a novel assay that applies multiplexed ligation-dependent probe amplification (MLPA). We confirmed a positive association between FL and the HLA-DQB1*05 allele group (OR = 1.70, 95% CI 1.28–2.27; adjusted p-value = 0.013) and also identified an allele group inversely associated with FL risk, HLA-DQB1*06 (OR = 0.51, 95% CI 0.38–0.69; adjusted p-value = 4.46 × 10−5). Although these findings require verification, the role of HLA class II proteins in B-cell survival and proliferation makes this a biologically plausible association.

Common variants within 6p21.31 locus are associated with chronic lymphocytic leukaemia and potentially other non-Hodgkin lymphoma subtypes

A recent meta‐analysis of three genome‐wide association studies of chronic lymphocytic leukaemia (CLL) identified two common variants at the 6p21.31 locus that are associated with CLL risk. To verify and further explore the association of these variants with other non‐Hodgkin lymphoma (NHL) subtypes, we genotyped 1196 CLL cases, 1699 NHL cases, and 2410 controls. We found significant associations between the 6p21.31 variants and CLL risk (rs210134: P = 0·01; rs210142: P = 6·8 × 10−3). These variants also showed a trend towards association with some of the other NHL subtypes. Our results validate the prior work and support specific genetic pathways for risk among NHL subtypes.

Associations between arsenic (+3 oxidation state) methyltransferase (AS3MT) and N‐6 adenine‐specific DNA methyltransferase 1 (N6AMT1) polymorphisms, arsenic metabolism, and cancer risk in a chilean population

Inter‐individual differences in arsenic metabolism have been linked to arsenic‐related disease risks. Arsenic (+3) methyltransferase (AS3MT) is the primary enzyme involved in arsenic metabolism, and we previously demonstrated in vitro that N‐6 adenine‐specific DNA methyltransferase 1 (N6AMT1) also methylates the toxic inorganic arsenic (iAs) metabolite, monomethylarsonous acid (MMA), to the less toxic dimethylarsonic acid (DMA). Here, we evaluated whether AS3MT and N6AMT1 gene polymorphisms alter arsenic methylation and impact iAs‐related cancer risks. We assessed AS3MT and N6AMT1 polymorphisms and urinary arsenic metabolites (%iAs, %MMA, %DMA) in 722 subjects from an arsenic‐cancer case‐control study in a uniquely exposed area in northern Chile. Polymorphisms were genotyped using a custom designed multiplex, ligation‐dependent probe amplification (MLPA) assay for 6 AS3MT SNPs and 14 tag SNPs in the N6AMT1 gene. We found several AS3MT polymorphisms associated with both urinary arsenic metabolite profiles and cancer risk. For example, compared to wildtypes, individuals carrying minor alleles in AS3MT rs3740393 had lower %MMA (mean difference = −1.9%, 95% CI: −3.3, −0.4), higher %DMA (mean difference = 4.0%, 95% CI: 1.5, 6.5), and lower odds ratios for bladder (OR = 0.3; 95% CI: 0.1–0.6) and lung cancer (OR = 0.6; 95% CI: 0.2–1.1). Evidence of interaction was also observed for both lung and bladder cancer between these polymorphisms and elevated historical arsenic exposures. Clear associations were not seen for N6AMT1. These results are the first to demonstrate a direct association between AS3MT polymorphisms and arsenic‐related internal cancer risk. This research could help identify subpopulations that are particularly vulnerable to arsenic‐related disease. Environ. Mol. Mutagen. 58:411–422, 2017.

Follicular lymphoma-protective HLA class II variants correlate with increased HLA-DQB1 protein expression.

Multiple follicular lymphoma (FL) susceptibility single-nucleotide polymorphisms in the human leukocyte antigen (HLA) class I and II regions have been identified, including rs6457327, rs3117222, rs2647012, rs10484561, rs9268853 and rs2621416. Here we validated previous expression quantitative trait loci results with real-time reverse transcription quantitative PCR and investigated protein expression in B-lymphoblastoid cell lines and primary dendritic cells using flow cytometry, cell-based enzyme-linked immunosorbent assay and western blotting. We confirmed that FL-protective rs2647012-linked variants, in high linkage disequilibrium with the extended haplotype DRB1*15:01-DQA1*01:02-DQB1*06:02, correlate with increased HLA-DQB1 expression. This association remained significant at the protein level and was reproducible across different cell types. We also found that differences in HLA-DQB1 expression were not related to changes in activation markers or class II, major histocompatibility complex, transactivator expression, suggesting the role of an alternative regulatory mechanism. However, functional analysis using RegulomeDB did not reveal any relevant regulatory candidates. Future studies should focus on the clinical relevance of increased HLA-DQB1 protein expression facilitating tumor cell removal through increased immune surveillance.

Multiplexed, ligation-dependent probe amplification for rapid and inexpensive HLA-DQB1 allelotyping

Many effective options exist to accurately type DNA for human leukocyte antigen (HLA) alleles. However, most of the existing methods are excessively costly in terms of overall monetary costs, DNA requirements, and proprietary software. We present a novel assay capable of resolving heterozygous HLA-DQB1 allelotypes at two digits, with even greater specificity for the HLA-DQB1*06 allele family, by using the multiplexed ligation-dependent probe amplification technology. This assay provides more specific allele data than genome-wide analysis and is more affordable than sequencing, making it a useful intermediate for researchers seeking to accurately allelotype human DNA samples.

Abstract 2764: High-resolution HLA typing by next-generation sequencing and risk of follicular lymphoma

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL The molecular basis of follicular lymphoma (FL) has been fairly well characterized, although its root causes remain less clear. Recently, in two genome-wide association studies of European-Americans, we identified novel genetic susceptibility loci for FL, narrowed down to two independent regions on chromosome 6p21.3 in the major histocompatibility complex (MHC) (Skibola et al., Nat Gen 41(8):873-5., 2009; Conde et al., Nat Gen 42(8):661-4, 2010). The first susceptibility locus, rs6457327 (combined allelic p-value=4.7 × 10-11 in 645 patients and 3,377 controls), is part of a 26-kb region of high linkage disequilibrium (LD) in the MHC Class 1 region at 6p21.33 in close proximity to the psoriasis susceptibility region 1 (PSORS1). A second locus at 6p21.32, in the MHC Class II region upstream of HLA-DQB1, two SNPs in high LD, rs10484561 and rs7755224, were associated with 2-fold increased risks for FL in 1,465 patients and 6,958 controls (combined p-values=1.12×10-29, 2.00×10-19). Using tag SNPs as surrogates for HLA allelotypes, the latter loci were found to be part of an extended haplotype that includes HLA-DRB1\*0101-HLA-DQA1\*0101-HLA-DQB1*0501 and that was associated with increased FL risk [odds ratio (OR) = 2.07, 95% confidence interval (CI) = 1.40-3.06]. These findings were further validated by direct typing of HLA-DQB1 alleles in a subset of 265 FL cases and 757 controls using multiplexed, ligation-dependent probe amplification. We verified the positive association between FL and the HLA-DQB1*05 allele group (OR=1.56, 95% CI 1.22-1.99; adjusted p-value=7.7×10-3), and found an inverse association between the HLA-DQB1*06 allele group and FL risk (OR=0.53, 95% CI 0.40-0.69; adjusted p-value=1.98×10-5). To gain further insight into the role of HLA alleles in FL susceptibility, high-resolution HLA typing by next-generation sequencing of the HLA class I (A, B, C) and class II (DRB1/3/4/5, DQA1, DQB1, DPB1) genes was conducted in 222 FL patients and 220 sex- and age-matched controls. HLA typing was performed by sequencing exon 2 for the class II genes and exons 2-4 for the class I genes, as described previously (Bentley, G et al. Tissue Antigens. 74:393-403, 2009), and data analysis is underway. To our knowledge, this study will present the highest resolution (up to 8-digit) HLA typing reported in a sample of European-Americans, and is an important next step to determine the role of MHC genetic variation in the pathogenesis of FL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2764. doi:10.1158/1538-7445.AM2011-2764

Abstract 4717: A search for overlapping HLA risk loci between NHL and autoimmune diseases

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Non-Hodgkin lymphoma (NHL) is a group of B- and T-cell neoplasms originating primarily from the lymph nodes, and represents the most common hematological malignancy in the United States. We recently conducted a genome-wide association study (GWAS) using DNA pools and identified a new risk locus for follicular lymphoma (FL) in the Class 1 human leukocyte antigen (HLA) region on chromosome 6. Currently, we are following up these results in a second GWAS using a subset of 757 NHL cases and 811 controls from a larger case-control study of NHL based in the San Francisco Bay Area using the Illumina HumanCNV370-Duo BeadChip. In the preliminary scan, we found additional evidence of Class 1 HLA loci that influence FL susceptibility, as well as loci in the Class 2 region, suggesting the importance of HLA genetic variation in FL risk. There is also clear evidence that HLA Class 1 and Class 2 loci confer genetic susceptibility to autoimmune diseases such as Crohns disease (CD), type 1 diabetes (T1D), rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). Thus, because a history of autoimmune disease may increase risk of NHL, we explored whether HLA-region risk loci are common between these autoimmune diseases and NHL. Using genetic data available from the Wellcome Trust Case-Control Consortium (WTCCC) for CD and the software package BEAGLE 3.0.3, we imputed SNPs to look for association signals in the HLA region. SNPs were imputed based on CD genotyped SNPs and haplotype information from unrelated CEU samples in the HapMap phase II data. A Cochran-Armitage test for an additive genetic effect implemented in PLINK v1.05 was subsequently performed to test the genotype-phenotype associations of genotyped and imputed SNPs. Our preliminary results suggest associations in the HLA Class 2 region near the HLA-DQA2 and HLA-DQB2 genes, providing evidence that overlapping risk loci may exist between FL and CD. We are currently expanding our analysis to include genetic data for other autoimmune diseases available from WTCCC such as T1D and RA. These analyses will contribute new data about genetic variation in plausible HLA region pathways and will help to identify potentially functional genetic factors that contribute to lymphoma susceptibility. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4717.