The Polygenic and Monogenic Basis of Blood Traits and Diseases.

Blood cells play essential roles in human health, underpinning physiological processes such as immunity, oxygen transport, and clotting, which when perturbed cause a significant global health burden. Here we integrate data from UK Biobank and a large-scale international collaborative effort, including data for 563,085 European ancestry participants, and discover 5,106 new genetic variants independently associated with 29 blood cell phenotypes covering a range of variation impacting hematopoiesis. We holistically characterize the genetic architecture of hematopoiesis, assess the relevance of the omnigenic model to blood cell phenotypes, delineate relevant hematopoietic cell states influenced by regulatory genetic variants and gene networks, identify novel splice-altering variants mediating the associations, and assess the polygenic prediction potential for blood traits and clinical disorders at the interface of complex and Mendelian genetics. These results show the power of large-scale blood cell trait GWAS to interrogate clinically meaningful variants across a wide allelic spectrum of human variation.

Meta-analysis of up to 622,409 individuals identifies 40 novel smoking behaviour associated genetic loci.

Smoking is a major heritable and modifiable risk factor for many diseases, including cancer, common respiratory disorders and cardiovascular diseases. Fourteen genetic loci have previously been associated with smoking behaviour-related traits. We tested up to 235,116 single nucleotide variants (SNVs) on the exome-array for association with smoking initiation, cigarettes per day, pack-years, and smoking cessation in a fixed effects meta-analysis of up to 61 studies (up to 346,813 participants). In a subset of 112,811 participants, a further one million SNVs were also genotyped and tested for association with the four smoking behaviour traits. SNV-trait associations with P < 5 × 10-8 in either analysis were taken forward for replication in up to 275,596 independent participants from UK Biobank. Lastly, a meta-analysis of the discovery and replication studies was performed. Sixteen SNVs were associated with at least one of the smoking behaviour traits (P < 5 × 10-8) in the discovery samples. Ten novel SNVs, including rs12616219 near TMEM182, were followed-up and five of them (rs462779 in REV3L, rs12780116 in CNNM2, rs1190736 in GPR101, rs11539157 in PJA1, and rs12616219 near TMEM182) replicated at a Bonferroni significance threshold (P < 4.5 × 10-3) with consistent direction of effect. A further 35 SNVs were associated with smoking behaviour traits in the discovery plus replication meta-analysis (up to 622,409 participants) including a rare SNV, rs150493199, in CCDC141 and two low-frequency SNVs in CEP350 and HDGFRP2. Functional follow-up implied that decreased expression of REV3L may lower the probability of smoking initiation. The novel loci will facilitate understanding the genetic aetiology of smoking behaviour and may lead to the identification of potential drug targets for smoking prevention and/or cessation.

Exome Chip Meta-analysis Fine Maps Causal Variants and Elucidates the Genetic Architecture of Rare Coding Variants in Smoking and Alcohol Use.

BACKGROUND: Smoking and alcohol use have been associated with common genetic variants in multiple loci. Rare variants within these loci hold promise in the identification of biological mechanisms in substance use. Exome arrays and genotype imputation can now efficiently genotype rare nonsynonymous and loss of function variants. Such variants are expected to have deleterious functional consequences and to contribute to disease risk. METHODS: We analyzed ∼250,000 rare variants from 16 independent studies genotyped with exome arrays and augmented this dataset with imputed data from the UK Biobank. Associations were tested for five phenotypes: cigarettes per day, pack-years, smoking initiation, age of smoking initiation, and alcoholic drinks per week. We conducted stratified heritability analyses, single-variant tests, and gene-based burden tests of nonsynonymous/loss-of-function coding variants. We performed a novel fine-mapping analysis to winnow the number of putative causal variants within associated loci. RESULTS: Meta-analytic sample sizes ranged from 152,348 to 433,216, depending on the phenotype. Rare coding variation explained 1.1% to 2.2% of phenotypic variance, reflecting 11% to 18% of the total single nucleotide polymorphism heritability of these phenotypes. We identified 171 genome-wide associated loci across all phenotypes. Fine mapping identified putative causal variants with double base-pair resolution at 24 of these loci, and between three and 10 variants for 65 loci. Twenty loci contained rare coding variants in the 95% credible intervals. CONCLUSIONS: Rare coding variation significantly contributes to the heritability of smoking and alcohol use. Fine-mapping genome-wide association study loci identifies specific variants contributing to the biological etiology of substance use behavior.

Rare and low-frequency coding variants in CXCR2 and other genes are associated with hematological traits.

Hematological traits are important clinical parameters. To test the effects of rare and low-frequency coding variants on hematological traits, we analyzed hemoglobin concentration, hematocrit levels, white blood cell (WBC) counts and platelet counts in 31,340 individuals genotyped on an exome array. We identified several missense variants in CXCR2 associated with reduced WBC count (gene-based P = 2.6 × 10(-13)). In a separate family-based resequencing study, we identified a CXCR2 frameshift mutation in a pedigree with congenital neutropenia that abolished ligand-induced CXCR2 signal transduction and chemotaxis. We also identified missense or splice-site variants in key hematopoiesis regulators (EPO, TFR2, HBB, TUBB1 and SH2B3) associated with blood cell traits. Finally, we were able to detect associations between a rare somatic JAK2 mutation (encoding p.Val617Phe) and platelet count (P = 3.9 × 10(-22)) as well as hemoglobin concentration (P = 0.002), hematocrit levels (P = 9.5 × 10(-7)) and WBC count (P = 3.1 × 10(-5)). In conclusion, exome arrays complement genome-wide association studies in identifying new variants that contribute to complex human traits.

No evidence of interaction between known lipid-associated genetic variants and smoking in the multi-ethnic PAGE population.

Genome-wide association studies (GWAS) have identified many variants that influence high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, and/or triglycerides. However, environmental modifiers, such as smoking, of these known genotype-phenotype associations are just recently emerging in the literature. We have tested for interactions between smoking and 49 GWAS-identified variants in over 41,000 racially/ethnically diverse samples with lipid levels from the Population Architecture Using Genomics and Epidemiology (PAGE) study. Despite their biological plausibility, we were unable to detect significant SNP × smoking interactions.

TP53 deletion is not an adverse feature in multiple myeloma treated with total therapy 3.

Contrary to Total Therapy (TT) 2 for multiple myeloma patients, FGFR3- translocation bore no adverse effects on outcome in TT3 with added bortezomib. Del TP53, another poor-risk feature in TT2 and present in 10% of 441 patients treated, was examined for its prognostic consequences in TT3. Not affecting rate or duration of complete response, TP53 haplo-insufficiency also did not compromise, in the 83% with genomically defined low-risk myeloma, survival or event-free survival. FGFR3+ and FGFR3- molecular subgroups fared worse in the presence of del TP53 when applying TT2 but not TT3. Thus, the prognostic implications of del TP53 were protocol-, genome-defined risk- and molecular subgroup-dependent.

F18-fluorodeoxyglucose positron emission tomography in the context of other imaging techniques and prognostic factors in multiple myeloma.

F18-fluorodeoxyglucose positron emission tomography (FDG-PET) is a powerful tool to investigate the role of tumor metabolic activity and its suppression by therapy for cancer survival. As part of Total Therapy 3 for newly diagnosed multiple myeloma, metastatic bone survey, magnetic resonance imaging, and FDG-PET scanning were evaluated in 239 untreated patients. All 3 imaging techniques showed correlations with prognostically relevant baseline parameters: the number of focal lesions (FLs), especially when FDG-avid by PET-computed tomography, was positively linked to high levels of beta-2-microglobulin, C-reactive protein, and lactate dehydrogenase; among gene expression profiling parameters, high-risk and proliferation-related parameters were positively and low-bone-disease molecular subtype inversely correlated with FL. The presence of more than 3 FDG-avid FLs, related to fundamental features of myeloma biology and genomics, was the leading independent parameter associated with inferior overall and event-free survival. Complete FDG suppression in FL before first transplantation conferred significantly better outcomes and was only opposed by gene expression profiling-defined high-risk status, which together accounted for approximately 50% of survival variability (R(2) test). Our results provide a rationale for testing the hypothesis that myeloma survival can be improved by altering treatment in patients in whom FDG suppression cannot be achieved after induction therapy.

Cytogenetic abnormalities in multiple myeloma: poor prognosis linked to concomitant detection in random and focal lesion bone marrow samples and associated with high-risk gene expression profile.

The clinical significance of cytogenetic abnormalities (CA) present in randomly sampled (RS) or focal lesion (FL) bone marrow sites was examined in 419 untreated myeloma patients. Among 290 patients with gene expression profiling (GEP) data generated from RS sites, GEP-defined high-risk was present in 52% of the RS+/FL+ group but in only 9% of the remainder (P < 0.001). The RS+/FL+ constellation (18%) was an independent predictor of poor survival, also after adjusting for GEP-derived risk and TP53 status (Hazard ratio = 2.42, P = 0.004). The prevalence of high-risk myeloma in the RS+/FL+ group may reflect a dissemination-prone condition not shared by the other three groups.

Genetic associations with thalidomide mediated venous thrombotic events in myeloma identified using targeted genotyping.

A venous thromboembolism (VTE) with the subsequent risk of pulmonary embolism is a major concern in the treatment of patients with multiple myeloma with thalidomide. The susceptibility to developing a VTE in response to thalidomide therapy is likely to be influenced by both genetic and environmental factors. To test genetic variation associated with treatment related VTE in patient peripheral blood DNA, we used a custom-built molecular inversion probe (MIP)-based single nucleotide polymorphism (SNP) chip containing 3404 SNPs. SNPs on the chip were selected in "functional regions" within 964 genes spanning 67 molecular pathways thought to be involved in the pathogenesis, treatment response, and side effects associated with myeloma therapy. Patients and controls were taken from 3 large clinical trials: Medical Research Council (MRC) Myeloma IX, Hovon-50, and Eastern Cooperative Oncology Group (ECOG) EA100, which compared conventional treatments with thalidomide in patients with myeloma. Our analysis showed that the set of SNPs associated with thalidomide-related VTE were enriched in genes and pathways important in drug transport/metabolism, DNA repair, and cytokine balance. The effects of the SNPs associated with thalidomide-related VTE may be functional at the level of the tumor cell, the tumor-related microenvironment, and the endothelium. The clinical trials described in this paper have been registered as follows: MRC Myeloma IX: ISRCTN68454111; Hovon-50: NCT00028886; and ECOG EA100: NCT00033332.

Genomic variation in myeloma: design, content, and initial application of the Bank On A Cure SNP Panel to detect associations with progression-free survival.

BACKGROUND: We have engaged in an international program designated the Bank On A Cure, which has established DNA banks from multiple cooperative and institutional clinical trials, and a platform for examining the association of genetic variations with disease risk and outcomes in multiple myeloma. We describe the development and content of a novel custom SNP panel that contains 3404 SNPs in 983 genes, representing cellular functions and pathways that may influence disease severity at diagnosis, toxicity, progression or other treatment outcomes. A systematic search of national databases was used to identify non-synonymous coding SNPs and SNPs within transcriptional regulatory regions. To explore SNP associations with PFS we compared SNP profiles of short term (less than 1 year, n = 70) versus long term progression-free survivors (greater than 3 years, n = 73) in two phase III clinical trials. RESULTS: Quality controls were established, demonstrating an accurate and robust screening panel for genetic variations, and some initial racial comparisons of allelic variation were done. A variety of analytical approaches, including machine learning tools for data mining and recursive partitioning analyses, demonstrated predictive value of the SNP panel in survival. While the entire SNP panel showed genotype predictive association with PFS, some SNP subsets were identified within drug response, cellular signaling and cell cycle genes. CONCLUSION: A targeted gene approach was undertaken to develop an SNP panel that can test for associations with clinical outcomes in myeloma. The initial analysis provided some predictive power, demonstrating that genetic variations in the myeloma patient population may influence PFS.

Thalidomide arm of Total Therapy 2 improves complete remission duration and survival in myeloma patients with metaphase cytogenetic abnormalities.

Total Therapy 2 examined the clinical benefit of adding thalidomide up-front to a tandem transplant regimen for newly diagnosed patients with multiple myeloma. When initially reported with a median follow-up of 42 months, complete response rate and event-free survival were superior among the 323 patients randomized to thalidomide, whereas overall survival was indistinguishable from that of the 345 patients treated on the control arm. With further follow-up currently at a median of 72 months, survival plots segregated 5 years after initiation of therapy in favor of thalidomide (P = .09), reaching statistical significance for the one third of patients exhibiting cytogenetic abnormalities (CAs; P = .02), a well-recognized adverse prognostic feature. The duration of complete remission was also superior in the cohort presenting with CAs such that, at 7 years from onset of complete remission, 45% remained relapse-free as opposed to 20% on the control arm (P = .05). These observations were confirmed when examined by multivariate analysis demonstrating that thalidomide reduced the hazard of death by 41% among patients with CA-positive disease (P = .008). Because two thirds of patients without CAs have remained alive at 7 years, the presently emerging separation in favor of thalidomide may eventually reach statistical significance as well.

Completion of premaintenance phases in total therapies 2 and 3 improves clinical outcomes in multiple myeloma: an important variable to be considered in clinical trial designs.

BACKGROUND: Total Therapy (TT) programs are complex and their execution over the course of several years is fraught with patient attrition due to failure and toxicity of therapy and patient/physician acceptance. METHODS: The impact of completion versus noncompletion of intended treatment steps was examined in protocols TT2 (n=668) and TT3 (n=303) on overall survival (OS) and event-free survival (EFS). RESULTS: By using appropriate landmarks of 36 months with TT2 and 18 months with TT3, representing the maxima to completion of premaintenance phases, postconsolidation OS was superior for 211 patients completing versus 311 patients not completing premaintenance steps on TT2 (P=.001), which also pertained to the 161 patients completing versus 47 not completing intended treatment steps on TT3 (P=.01). On multivariate analysis that included all patients, completion of therapy independently favored longer OS and EFS in the context of both standard prognostic factors and gene expression profiling-defined risk; in addition, TT3 prolonged EFS over results obtained with TT2. CONCLUSIONS: 1) Completion of intended therapy was a significant independent variable conferring superior OS and EFS in TT programs; and 2) after adjusting for completion of therapy, EFS was still superior with TT3 versus TT2, supporting the beneficial role of bortezomib included in TT3. Collectively, these data point to the importance of designing clinical trials that balance the treatment requirements for disease control with host acceptance and tolerance.

Eight-year median survival in multiple myeloma after total therapy 2: roles of thalidomide and consolidation chemotherapy in the context of total therapy 1.

In comparison to total therapy 1 (TT1), the phase 3 trial total therapy 2 (TT2) evaluated the benefit of up-front administration of thalidomide (THAL); TT2 also introduced post-transplant consolidation chemotherapy. With median follow-up times of 5 and 12 years, respectively, outcome comparisons were made of 668 patient's enrolled on TT2 and 231 patients treated on TT1. Complete response (CR) rates were similar at 40% for TT1 and TT2 without THAL versus 60% on the THAL arm of TT2. CR durations were similar with either TT2 arm and both were superior to results of TT1. Event-free and overall survivals were extended from 2.6 to 5.7 years, respectively, with TT1 to 4.8 and 8.0 years with TT2. TT2's major advance vis-à-vis TT1 pertained to the subgroup without cytogenetic abnormalities (CA), supporting the role of post-transplant consolidation therapy, whereas the improved survival of the CA subgroup on the experimental versus control arm of TT2 attests to the role of THAL in this setting. Adjusting for prognostic variables in multivariate and pair-mate analyses, TT2 was superior to TT1 in terms of CR duration, event-free and overall survival. These results provide a basis for the prospective evaluation of the consolidation strategy in a randomized clinical trial design.

An analysis of the clinical and biologic significance of TP53 loss and the identification of potential novel transcriptional targets of TP53 in multiple myeloma.

TP53 is a tumor suppressor gene that functions as transcriptional regulator influencing cellular responses to DNA damage. Here we explored the clinical and transcriptional effects of TP53 expression in multiple myeloma (MM). We found that low expression of TP53, seen in approximately 10% of newly diagnosed patients, is highly correlated with TP53 deletion, an inferior clinical outcome, and represents an independent risk factor. Analysis of the expression of 122 known TP53 target genes in TP53-high vs -low MM cells from 351 newly diagnosed cases, revealed that only a few were highly correlated with TP53 expression. To elucidate TP53 regulatory networks in MM, we overexpressed TP53 in 4 MM cell lines. Gene expression profiling of these cell lines detected 85 significantly differentially expressed genes, with 50 up-regulated and 35 down-regulated. Unsupervised hierarchical clustering of myeloma samples from 351 newly diagnosed and 90 relapsed patients using the 85 putative TP53 target genes revealed 2 major subgroups showing a strong correlation with TP53 expression and survival. These data suggest that loss of TP53 expression in MM confers high risk and probably results in the deregulation of a novel set of MM-specific TP53-target genes. TP53 target gene specificity may be unique to different cell lineages.

Sustained complete remissions in multiple myeloma linked to bortezomib in total therapy 3: comparison with total therapy 2.

Total therapy 3 (TT3), incorporating bortezomib up-front into a tandem transplant regimen for newly diagnosed multiple myeloma (MM), effected 2-year complete response (CR) estimates >90%, which appeared superior to results reported for total therapy 2 (TT2). With median follow-up times of 2 years with TT3 and 5 years with TT2, the clinical outcomes of 303 patients in the former and 668 in the latter trial were compared, including the subset of 607 patients with gene expression profiling (GEP) data. With similar baseline prognostic factors, event-free survival (EFS) (P = 0.0002) and CR duration (P = 0.003) were superior with TT3 vs. TT2 with a strong trend noted also for improved overall survival (OS) (P = 0.16). In the GEP-defined FGFR3 subgroup, TT3 imparted significantly superior OS, EFS and CR duration vis-à-vis TT2. Matching 300 patients each by standard prognostic factors, TT3 yielded superior EFS and CR duration and borderline superior OS. The advantage of TT3 still pertained when the comparison was limited to patients who completed TT2 consolidation rapidly within 24 months. Our data strongly suggest that the addition of bortezomib in TT3 was accountable for its superior performance rather than greater compliance with protocol completion as a result of greater dose-density in TT3 vs. TT2.

Cytogenetically defined myelodysplasia after melphalan-based autotransplantation for multiple myeloma linked to poor hematopoietic stem-cell mobilization: the Arkansas experience in more than 3,000 patients treated since 1989.

Myelodysplastic syndrome (MDS) is a well-recognized complication of chemotherapy for multiple myeloma (MM). Serial bone marrow metaphase examinations were performed for MM restaging in 3,077 patients undergoing high-dose therapy (HDT). MDS-associated cytogenetic abnormalities (MDS-CAs) were observed in 105 of 2,418 patients in whom cytogenetic data were available after HDT. MDS-CAs occurred transiently in 72 patients and on 3 successive occasions (persistent MDS-CAs) in 33 patients, for 10-year estimates of 4% and 2%, respectively; only 21 patients developed overt clinical MDS and 5, acute myeloblastic leukemia (AML). MDS-CA development was linked to lower CD34 yield at collection, longer time interval from MM diagnosis to HDT, older age, and lower platelet recovery after HDT; persistent MDS-CAs were predicted by CD34 yield of less than 3 x 10(6)/kg and need for more than 2 apheresis procedures. Applying a tertile frequency distribution over time to all 105 patients with MDS-CAs, its detection early after HDT was associated with longer time interval from diagnosis and low pre-HDT platelet count (likely resulting from pre-HDT damage), whereas late-onset MDS-CAs were noted among patients treated with Total Therapy 2 and Total Therapy 3 that applied post-HDT consolidation chemotherapy (suggesting possible post-HDT damage). While the risk of MDS-CAs was low and clinical MDS occurred infrequently, monitoring after post-HDT consolidation chemotherapy appears warranted.