Susan L. Slager

Discovery and prioritization of somatic mutations in diffuse large B-cell lymphoma (DLBCL) by whole-exome sequencing

To gain insight into the genomic basis of diffuse large B-cell lymphoma (DLBCL), we performed massively parallel whole-exome sequencing of 55 primary tumor samples from patients with DLBCL and matched normal tissue. We identified recurrent mutations in genes that are well known to be functionally relevant in DLBCL, including MYD88, CARD11, EZH2, and CREBBP. We also identified somatic mutations in genes for which a functional role in DLBCL has not been previously suspected. These genes include MEF2B, MLL2, BTG1, GNA13, ACTB, P2RY8, PCLO, and TNFRSF14. Further, we show that BCL2 mutations commonly occur in patients with BCL2/IgH rearrangements as a result of somatic hypermutation normally occurring at the IgH locus. The BCL2 point mutations are primarily synonymous, and likely caused by activation-induced cytidine deaminase–mediated somatic hypermutation, as shown by comprehensive analysis of enrichment of mutations in WRCY target motifs. Those nonsynonymous mutations that are observed tend to be found outside of the functionally important BH domains of the protein, suggesting that strong negative selection against BCL2 loss-of-function mutations is at play. Last, by using an algorithm designed to identify likely functionally relevant but infrequent mutations, we identify KRAS, BRAF, and NOTCH1 as likely drivers of DLBCL pathogenesis in some patients. Our data provide an unbiased view of the landscape of mutations in DLBCL, and this in turn may point toward new therapeutic strategies for the disease.

An autosomal genomic screen for autism

Autism is a severe neurodevelopmental disorder defined by social and communication deficits and ritualistic-repetitive behaviors that are detectable in early childhood. The etiology of idiopathic autism is strongly genetic, and oligogenic transmission is likely. The first stage of a two-stage genomic screen for autism was carried out by the Collaborative Linkage Study of Autism on individuals affected with autism from 75 families ascertained through an affected sib-pair. The strongest multipoint results were for regions on chromosomes 13 and 7. The highest maximum multipoint heterogeneity LOD (MMLS/het) score is 3.0 at D13S800 (approximately 55 cM from the telomere) under the recessive model, with an estimated 35% of families linked to this locus. The next highest peak is an MMLS/het score of 2.3 at 19 cM, between D13S217 and D13S1229. Our third highest MMLS/het score of 2.2 is on chromosome 7 and is consistent with the International Molecular Genetic Study of Autism Consortium report of a possible susceptibility locus somewhere within 7q31-33. These regions and others will be followed up in the second stage of our study by typing additional markers in both the original and a second set of identically ascertained autism families, which are currently being collected. By comparing results across a number of studies, we expect to be able to narrow our search for autism susceptibility genes to a small number of genomic regions.

Mutations in CHEK2 Associated with Prostate Cancer Risk

The DNA-damage-signaling pathway has been implicated in all human cancers. However, the genetic defects and the mechanisms of this pathway in prostate carcinogenesis remain poorly understood. In this study, we analyzed CHEK2, the upstream regulator of p53 in the DNA-damage-signaling pathway, in several groups of patients with prostate cancer. A total of 28 (4.8%) germline CHEK2 mutations (16 of which were unique) were found among 578 patients. Additional screening for CHEK2 mutations in 149 families with familial prostate cancer revealed 11 mutations (5 unique) in nine families. These mutations included two frameshift and three missense mutations. Importantly, 16 of 18 unique CHEK2 mutations identified in both sporadic and familial cases were not detected among 423 unaffected men, suggesting a pathological effect of CHEK2 mutations in prostate cancer development. Analyses of the two frameshift mutations in Epstein Barr virus-transformed cell lines, using reverse-transcriptase polymerase chain reaction and western blot analysis, revealed abnormal splicing for one mutation and dramatic reduction of CHEK2 protein levels in both cases. Overall, our data suggest that mutations in CHEK2 may contribute to prostate cancer risk and that the DNA-damage-signaling pathway may play an important role in the development of prostate cancer.

Inherited Mutations in 17 Breast Cancer Susceptibility Genes Among a Large Triple-Negative Breast Cancer Cohort Unselected for Family History of Breast Cancer

PURPOSE Recent advances in DNA sequencing have led to the development of breast cancer susceptibility gene panels for germline genetic testing of patients. We assessed the frequency of mutations in 17 predisposition genes, including BRCA1 and BRCA2, in a large cohort of patients with triple-negative breast cancer (TNBC) unselected for family history of breast or ovarian cancer to determine the utility of germline genetic testing for those with TNBC. PATIENTS AND METHODS Patients with TNBC (N = 1,824) unselected for family history of breast or ovarian cancer were recruited through 12 studies, and germline DNA was sequenced to identify mutations. RESULTS Deleterious mutations were identified in 14.6% of all patients. Of these, 11.2% had mutations in the BRCA1 (8.5%) and BRCA2 (2.7%) genes. Deleterious mutations in 15 other predisposition genes were detected in 3.7% of patients, with the majority observed in genes involved in homologous recombination, including PALB2 (1.2%) and BARD1, RAD51D, RAD51C, and BRIP1 (0.3% to 0.5%). Patients with TNBC with mutations were diagnosed at an earlier age (P < .001) and had higher-grade tumors (P = .01) than those without mutations. CONCLUSION Deleterious mutations in predisposition genes are present at high frequency in patients with TNBC unselected for family history of cancer. Mutation prevalence estimates suggest that patients with TNBC, regardless of age at diagnosis or family history of cancer, should be considered for germline genetic testing of BRCA1 and BRCA2. Although mutations in other predisposition genes are observed among patients with TNBC, better cancer risk estimates are needed before these mutations are used for clinical risk assessment in relatives.

A genomewide association study of citalopram response in major depressive disorder.

BACKGROUND Antidepressant response is likely influenced by genetic constitution, but the actual genes involved have yet to be determined. We have carried out a genomewide association study to determine whether common DNA variation influences antidepressant response. METHODS Our sample is derived from Level 1 participants in the Sequenced Treatment Alternatives to Relieve Depression (STAR*D) study, all treated with citalopram. Association for the response phenotype included 883 responders and 608 nonresponders. For the remission phenotype, 743 subjects that achieved remission were compared with 608 nonresponders. We used a subset of single nucleotide polymorphisms (SNPs; n = 430,198) from the Affymetrix 500K and 5.0 Human SNP Arrays, and association analysis was carried out after correcting for population stratification. RESULTS We identified three SNPs associated with response with p values less than 1 x 10(-5) near the UBE3C gene (rs6966038, p = 4.65 x 10(-7)), another 100 kb away from BMP7 (rs6127921, p = 3.45 x 10(-6)), and a third that is intronic in the RORA gene (rs809736, p = 8.19 x 10(-6)). These same SNPs were also associated with remission. Thirty-nine additional SNPs are of interest with p values < or = .0001 for the response and remission phenotypes. CONCLUSIONS Although the findings reported here do not meet a genomewide threshold for significance, the regions identified from this study provide targets for independent replication and novel pathways to investigate mechanisms of antidepressant response. This study was not placebo controlled, making it possible that we are also observing associations to nonspecific aspects of drug treatment of depression.

Investigation of serotonin-related genes in antidepressant response

In this study, we sought out to test the hypothesis that genetic factors may influence antidepressant response to fluoxetine. The investigation focused on seven candidate genes in the serotonergic pathway involved in the synthesis, transport, recognition, and degradation of serotonin. Our clinical sample consisted of 96 subjects with unipolar major depression treated with fluoxetine with response variables assessed after a 12-week trial. Patient data were also collected to investigate the pattern of drug response. Using a high-throughput single-nucleotide polymorphism (SNP) genotyping platform and capillary electrophoresis, we genotyped patients at 110 SNPs and four repeat polymorphisms located in seven candidate genes (HTR1A, HTR2A, HTR2C, MAOA, SLC6A4, TPH1, and TPH2). Statistical tests performed included single-locus and haplotype association tests, and linkage disequilibrium (LD) estimation. Little evidence of population stratification was observed in the sample with 20 random SNPs using a genomic control procedure. Our most intriguing result involved three SNPs in the TPH1 gene and one SNP in the SLC6A4 gene, which show significant single-locus association when response to fluoxetine is compared to nonresponse (P=0.02–0.04). All odds ratios indicated an increased risk of not responding to fluoxetine. In the specific response vs nonspecific and nonresponse comparison, three SNPs in the TPH2 gene (P=0.02–0.04) were positively associated and one SNP in the HTR2A gene (P=0.02) was negatively associated. When comparing specific response to nonspecific response, we found significant negative associations in three SNPs in the HTR2A gene (P=0.001–0.03) and two SNPs in the MAOA gene (P=0.03–0.05). We observed variable, although strong LD, in each gene and unexpectedly low numbers of estimated haplotypes, formed from tagged SNPs. Significant haplotype associations were found in all but the HTR1A and HTR2C genes. Although these data should be interpreted cautiously due to the small sample size, these results implicate TPH1 and SLC6A4 in general response, and HTR2A, TPH2, and MAOA in the specificity of response to fluoxetine. Intriguingly, we observe that a number of the less frequent alleles of many of the SNP markers were associated with the nonresponse and nonspecific phenotypes.

Sequence Analysis of the Serotonin Transporter and Associations with Antidepressant Response

BACKGROUND The serotonin transporter is the molecular target of many antidepressants, and the gene (SLC6A4) encoding this protein has been associated with response to selective serotonin reuptake inhibitors (SSRIs). We sought to test further the hypothesis that SLC6A4 is associated with SSRI response by resequencing this gene in subjects with major depression. METHODS The sequence of all exons, parts of all introns, and the promoter region containing a polymorphic repeat polymorphism (HTTLPR) previously associated with SSRI response was determined for 96 subjects, and variants were tested for association to treatment response with fluoxetine. RESULTS We screened a total of 712 kilobases of sequence and found 27 SLC6A4 variants, 21 of which were previously undescribed. Seventeen were seen on one chromosome each, including three of the five exonic variants. One polymorphism (rs25531), just upstream of the HTTLPR, showed evidence of an association with treatment response, and biochemical experiments showed this polymorphism altered binding of nuclear extracts to a consensus sequence for the activator protein 2 transcription factor, which is believed to be a critical factor in regulating neural gene expression in mammals. CONCLUSIONS These results support an association between response to SSRIs and deoxyribonucleic acid variation at the serotonin transporter locus. We have also identified a potentially important functional variant that contributes to this association and a possible biologic mechanism that could mediate its effect.

Cigarette Smoking and Colorectal Cancer Risk by Molecularly Defined Subtypes

BACKGROUND Cigarette smoking is an established risk factor for colorectal cancer. Because colorectal carcinogenesis is a heterogeneous process, we investigated whether cigarette smoking is differentially associated with molecularly defined subtypes of colorectal cancer. METHODS We evaluated associations between smoking and incident colorectal cancer, overall and by microsatellite instability (MSI) phenotype (MSI-high vs MSI-low or microsatellite stable), CpG island methylator phenotype (CIMP positive or CIMP negative), and BRAF mutation status (BRAF mutation positive or BRAF mutation negative), among 37 399 participants in a population-based cohort study (the Iowa Womens Health Study). Cigarette smoking (and other exposures) was assessed by self-report at baseline in 1986, including smoking status (never and ever [former or current]), age at initiation, total duration, average number of cigarettes smoked per day, cumulative pack-years, and induction period. Vital status and state of residence were determined by mailed follow-up questionnaires in 1987, 1989, 1992, and 1997 and by linkage to Iowa death certificate records. Nonrespondents were checked via the National Death Index to identify descendants. Participants with newly diagnosed (ie, incident) colorectal cancer were identified through annual linkage with the Iowa Cancer Registry. Archived paraffin-embedded tumor tissue specimens were obtained for 555 patients with colorectal cancer who were diagnosed from January 1, 1986, through December 31, 2002, and MSI status, CIMP status, and BRAF status were determined. Multivariable Cox regression models were fit to estimate relative risks (RRs) and 95% confidence intervals (CIs). RESULTS Ever-smokers were at moderately increased risk for incident colorectal cancer (RR = 1.19, 95% CI = 1.05 to 1.35) compared with never-smokers. Higher risk estimates were observed for current smokers with MSI-high tumors (RR = 1.99, 95% CI = 1.26 to 3.14), CIMP-positive tumors (RR = 1.88, 95% CI = 1.22 to 2.90), and BRAF mutation-positive tumors (RR = 1.92, 95% CI = 1.22 to 3.02). Other smoking-related variables (ie, age at initiation, total duration, average number of cigarettes smoked per day, cumulative pack-years, and induction period) were also associated with MSI-high, CIMP-positive, and BRAF mutation-positive tumor subtypes. Conversely, cigarette smoking status (ever vs never) was not associated with the MSI-low or microsatellite stable (RR = 1.00, 95% CI = 0.79 to 1.25), CIMP-negative (RR = 1.02, 95% CI = 0.81 to 1.30), or BRAF mutation-negative subtypes (RR = 1.00, 95% CI = 0.65 to 1.27). CONCLUSIONS In this prospective study of older women, cigarette smoking was associated with the MSI-high, CIMP-positive, and BRAF mutation-positive colorectal cancer subtypes, which indicates that epigenetic modification may be functionally involved in smoking-related colorectal carcinogenesis.

Vitamin D Insufficiency and Prognosis in Non-Hodgkin's Lymphoma

PURPOSE Vitamin D insufficiency is common in the United States, with low levels linked in some studies to higher cancer incidence, including non-Hodgkins lymphoma (NHL). Recent data also suggest that vitamin D insufficiency is related to inferior prognosis in some cancers, although there are no data for NHL. PATIENTS AND METHODS We tested the hypothesis that circulating 25-hydroxyvitamin D [25(OH)D] levels are predictive of event-free survival (EFS) and overall survival (OS) in a prospective cohort of 983 newly diagnosed patients with NHL. 25(OH)D and 1,25-dihydroxyvitamin D [1,25(OH)(2)D] levels were measured by liquid chromatography-tandem mass spectrometry. RESULTS Mean age at diagnosis was 62 years (range, 19 to 94 years); 44% of patients had insufficient 25(OH)D levels (< 25 ng/mL) within 120 days of diagnosis. Median follow-up was 34.8 months; 404 events and 193 deaths (168 from lymphoma) occurred. After adjusting for known prognostic factors and treatment, 25(OH)D insufficient patients with diffuse large B-cell lymphoma (DLBCL) had inferior EFS (hazard ratio [HR], 1.41; 95% CI, 0.98 to 2.04) and OS (HR, 1.99; 95% CI, 1.27 to 3.13); 25(OH)D insufficient patients with T-cell lymphoma also had inferior EFS (HR, 1.94; 95% CI, 1.04 to 3.61) and OS (HR, 2.38; 95% CI, 1.04 to 5.41). There were no associations with EFS for the other NHL subtypes. Among patients with DLBCL and T-cell lymphoma, higher 1,25(OH)(2)D levels were associated with better EFS and OS, suggesting that any putative tumor 1-α-hydroxylase activity did not explain the 25(OH)D associations. CONCLUSION 25(OH)D insufficiency was associated with inferior EFS and OS in DLBCL and T-cell lymphoma. Whether normalizing vitamin D levels in these patients improves outcomes will require testing in future trials.

Case-Control Studies of Genetic Markers: Power and Sample Size Approximations for Armitage’s Test for Trend

The association of a candidate gene with disease can be efficiently evaluated by a case-control study in which allele frequencies are compared for diseased cases and unaffected controls. However, when the distribution of genotypes in the population deviates from Hardy-Weinberg proportions, the frequency of genotypes – rather than alleles – should be compared by the Armitage test for trend. We present formulas for power and sample size for studies that use Armitage’s trend test. The formulas make no assumptions about Hardy-Weinberg equilibrium, but do assume random ascertainment of cases and controls, all of whom are independent of one another. We demonstrate the accuracy of the formulas by simulations.