Multivariate Association Analysis Research Articles

Why No Signals? Cerebral Anatomy Predicts Success of Intraoperative Neuromonitoring During Correction of Scoliosis Secondary to Cerebral Palsy.

Intraoperative neuromonitoring (IONM) is widely used to reduce postoperative neurological complications during scoliosis correction. IONM allows intraoperative detection of neurological insults to the spinal cord and enables surgeons to react in real time. IONM failure rates can reach 61% in patients with cerebral palsy (CP). Factors decreasing the quality of IONM signals or making IONM impossible in CP patients undergoing scoliosis correction have not been well described. We categorized IONM data from 206 children with CP who underwent surgical scoliosis correction at a single institution from 2002 through 2013 into 3 groups: (1) "no signals," if neither somatosensory-evoked potentials (SSEP) nor transcranial motor-evoked potentials (TcMEP) could be obtained; (2) "no sensory," if no interpretable SSEP were obtained regardless of interpretable TcMEP; and (3) "no motor," if no interpretable TcMEP were obtained regardless of interpretable SSEP. We analyzed preexisting neuroimaging, available for 93 patients, and neurological status of the full cohort against these categories. Statistical analysis of univariate and multivariate associations was performed using logistic regression. Odds ratios (ORs) were calculated with significance set at P<0.05. Multivariate analysis showed significant associations of periventricular leukomalacia (PVL), hydrocephalus, and encephalomalacia with lack of meaningful and interpretable signals. Focal PVL (Fig. 1) was associated with no motor (OR=39.95; P=0.04). Moderate hydrocephalus was associated with no signals (OR=32.35; P<0.01), no motor (OR=10.14; P=0.04), and no sensory (OR=8.44; P=0.03). Marked hydrocephalus (Fig. 2) was associated with no motor (OR=20.46; P<0.01) and no signals (OR=8.83; P=0.01). Finally, encephalomalacia (Fig. 3) was associated with no motor (OR=6.99; P=0.01) and no signals (OR=4.26; P=0.03). Neuroanatomic findings of PVL, hydrocephalus, and encephalomalacia are significant predictors of limited IONM signals, especially TcMEP. Level IV.

Clinicopathological analysis of 241 thymic epithelial tumors-experience in the Shanghai Chest Hospital from 1997-2004.

To assess the correlation of WHO histological classification of thymomas and thymic carcinomas (TCs) with prognosis in recently treated patient cohort compared to a historical one from a single institution. Retrospective review of clinical charts and histological sections of 241 patients treated during 1997-2004. Univariate and multivariate analysis of associations between risk factors including gender, age, tumor size, myasthenia gravis, WHO histological subtype, Masaoka stage, resection status, (neo-)adjuvant therapies, and survival. The 5-year overall survival (OS) of A, AB, B1, B2, B3 thymomas and TCs patients was 100%, 100%, 94%, 80%, 94% and 45%. Five-year progression-free survival (PFS) was 100%, 96%, 78%, 80%, 78% and 39%, respectively. The 5-year OS of patients with Masaoka stage I, II, III and IV thymomas and TCs was 96%, 89%, 59% and 50%. (Neo-)adjuvant therapies were administered more often than in the historical cohort. Tumor-related death mainly occurred in patients with stage III, IV and B2, B3 thymomas and TCs. By univariate analysis, gender, tumor size, myasthenia gravis (MG) status, histotype, Masaoka stage, resection status and treatment were associated with OS. By multivariate analysis, histological subtype, Masaoka stage, and (neo-)adjuvant therapy were revealed as independent prognostic indicators. WHO histological subtype, Masaoka stage and (neo-)adjuvant treatment have remained independent determinants of OS in patients with thymomas and TCs. Compared with the historical cohort during 1969-1996, prognosis of patients with B2, B3 thymomas has improved, which may be partly due to the increased use of adjuvant therapies. Prognosis of patients with TCs remained unsatisfactory, suggesting that neoadjuvant treatment should be tested to improve survival.

Evidence for contribution of common genetic variants within chromosome 8p21.2-8p21.1 to restricted and repetitive behaviors in autism spectrum disorders.

BackgroundRestricted and Repetitive Behaviors (RRB), one of the core symptom categories for Autism Spectrum Disorders (ASD), comprises heterogeneous groups of behaviors. Previous research indicates that there are two or more factors (subcategories) within the RRB domain. In an effort to identify common variants associated with RRB, we have carried out a genome-wide association study (GWAS) using the Autism Genetic Resource Exchange (AGRE) dataset (n = 1,335, all ASD probands of European ancestry) for each identified RRB subcategory, while allowing for comparisons of associated single nucleotide polymorphisms (SNPs) with associated SNPs in the same set of probands analyzed using all the RRB subcategories as phenotypes in a multivariate linear mixed model. The top ranked SNPs were then explored in an independent dataset.ResultsUsing principal component analysis of item scores obtained from Autism Diagnostic Interview-Revised (ADI-R), two distinct subcategories within Restricted and Repetitive Behaviors were identified: Repetitive Sensory Motor (RSM) and Insistence on Sameness (IS). Quantitative RSM and IS scores were subsequently used as phenotypes in a GWAS using the AGRE ASD cohort. Although no associated SNPs with genome-wide significance (P < 5.0E-08) were detected when RSM or IS were analyzed independently, three SNPs approached genome-wide significance when RSM and IS were considered together using multivariate association analysis. These included the top IS-associated SNP, rs62503729 (P-value = 6.48E-08), which is located within chromosome 8p21.2-8p21.1, a locus previously linked to schizophrenia. Notably, all of the most significantly associated SNPs are located in close proximity to STMN4 and PTK2B, genes previously shown to function in neuron development. In addition, several of the top-ranked SNPs showed correlations with STMN4 mRNA expression in adult CEU (Caucasian and European descent) human prefrontal cortex. However, the association signals within chromosome 8p21.2-8p21.1 failed to replicate in an independent sample of 2,588 ASD probands; the insufficient sample size and between-study heterogeneity are possible explanations for the non-replication.ConclusionsOur analysis indicates that RRB in ASD can be represented by two distinct subcategories: RSM and IS. Subsequent univariate and multivariate genome-wide association studies of these RRB subcategories enabled the detection of associated SNPs at 8p21.2-8p21.1. Although these results did not replicate in an independent ASD dataset, genomic features of this region and pathway analysis suggest that common variants in 8p21.2-8p21.1 may contribute to RRB, particularly IS. Together, these observations warrant future studies to elucidate the possible contributions of common variants in 8p21.2-8p21.1 to the etiology of RSM and IS in ASD.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2475-y) contains supplementary material, which is available to authorized users.

Blood group antigen loci demonstrate multivariate genetic associations with circulating cellular adhesion protein levels in the Multi-Ethnic Study of Atherosclerosis.

The cellular adhesion pathway is critical in the pathophysiology of atherosclerosis, and genetic factors contributing to regulation of circulating levels of related proteins may be relevant to risk prediction of cardiovascular disease. In contrast to conducting separate genome-wide protein quantitative trait loci (pQTL) mapping analyses of each individual protein, joint genetic association analyses of multiple quantitative traits can leverage cross-trait co-variation and identify simultaneous regulatory effects on protein levels across the pathway. We conducted a multi-pQTL (mpQTL) analysis of 15 proteins related to cellular adhesion assayed on 2313 participants from the Multi-Ethnic Study of Atherosclerosis (MESA). We applied the MQFAM multivariate association analysis method in PLINK on normalized protein level residuals derived from univariate linear regression, adjusting for age, sex, and principal components of ancestry. Race/ethnicity-stratified analyses identified nine genome-wide significant (P < 5e-08) loci associated with co-variation of protein levels. Although the majority of these SNPs were in proximity to structural genes of the assayed proteins, we discovered multiple loci demonstrating co-association with the circulation of at least two proteins. Of these, two significant loci specific to non-Hispanic white participants, rs17074898 at ALOX5AP (P = 1.78E-08) and rs7521237 at KIAA1614 (P = 2.2E-08), would not have met statistical significance using univariate analyses. Moreover, common patterns of multi-protein associations were discovered at the ABO locus across race/ethnicity. These results indicate the biological relevance of blood group antigens on regulation of circulating cellular adhesion pathway proteins while also demonstrating race/ethnicity-specific co-regulatory effects.

Outcome of octogenarian versus (vs) young patients (pts) with metastatic renal cell carcinoma (mRCC), treated with sunitinib (su).

538 Background: Su is a standard treatment (tx) for mRCC. Octogenarian pts (aged ≥ 80) are often considered to be unfit for su tx, and recommendations for their tx is limited by the paucity of clinical trials data in this population. We aimed to study baseline characteristics and outcome of octogenarian vs young (aged ≤45) pts with mRCC treated with su. Methods: We performed an international multicenter retrospective study of pts with mRCC, who were treated with su in 8 centers across 2 different countries. We compared baseline characteristics and outcome of octogenarian versus young pts. The effect of very old age on response rate (RR), progression free survival (PFS) and overall survival (OS), was tested with adjustment of other known confounding risk factors using a chi-square test and partial likelihood test from cox model. Furthermore, univariate and multivariate analyses of association between clinicopathologic factors and age, and outcome were performed using the entire pt cohort. Results: Between 2004-2013, 36 octogenarian (group 1; median age 83) and 37 young (group 2; median age 42) mRCC were treated with su. The groups were balanced regarding the following baseline clinicopathologic characteristics: gender, HENG risk, past nephrectomy, mRCC histology, ≥ 2 metastatic sites, lung/liver/bone metastasis, prior targeted tx, smoking status, use of angiotensin system inhibitors (ASIs), pre-tx neutrophil to lymphocyte ratio (NLR) &gt;3, and sunitinib induced hypertension (HTN). In group 1 vs 2, 53% vs 27% (p=0.006) had dose reduction/treatment interruption d/t side effects. Clinical benefit (partial response + stable disease) in group 1 vs 2 was 76% vs 84%, while 24% vs 16% had disease progression within the first 3 months of tx (p=0.09). Median PFS was 11 vs 8 months (p=0.1). Median OS was 22 vs 20 months (p=0.7). In multivariate analyses of the entire pt cohort (n=73), age was not significantly associated with PFS or OS. Conclusions: Su is active in octogenarian mRCC pts. Vs young pts, a significantly higher proportion of octogenarian pts had dose reduction/treatment interruption d/t side effects.

Comparison of sunitinib (su) versus temsirolimus (tem) in patients (pts) with poor-risk metastatic renal cell carcinoma (prmRCC).

539 Background: Based on a single phase 3 study, the mTOR inhibitor tem was approved as 1st line therapy for prmRCC. However, in daily practice, prmRCC pts are often treated with the VEGFR and PDGFR inhibitor su. We aimed to compare the clinical effectiveness of su vs tem in prmRCC pts. Methods: We performed an international multicenter retrospective study of pts with prmRCC (HENG criteria), who were treated in 8 centers across 2 different countries. 31 pts were treated with 1st line tem. Each tem treated pt was individually matched with a 1st line su treated pt, by clinicopathologic factors. The effect of tx type (tem vs su) on clinical benefit, progression free survival (PFS) and overall survival (OS), was tested using a chi-square test and partial likelihood test from cox model. Furthermore, univariate and multivariate analyses of association between clinicopathologic factors and tx type (tem vs su), and outcome were performed using the entire pt cohort (n=62). Results: The groups were matched by age (median 65), gender (male 68%), prior nephrectomy (58%), renal cell carcinoma histology (clear cell 81%), smoking status (active in 35%), use of angiotensin system inhibitors (42%), and pre-tx neutrophil to lymphocyte ratio &gt;3 (58%). In tem vs su treated pts, clinical benefit (partial response + stable disease) was 61% (n=19) (partial response 6%, n=2) vs 71% (n=22) (partial response 29%, n=9) (p=0.62). Median PFS was 5 vs 8 mos (p=0.08), and median OS 9 vs 17 mos (p=0.03). In multivariate analyses of the entire pt cohort (n=62), su tx was independently associated with OS (HR 0.6, p=0.001). Conclusions: In prmRCC patient, the VEGFR inhibitor su may be associated with an improved outcome vs the mTOR inhibitor tem.

Pancreatic hyperechogenicity associated with hypoadiponectinemia and insulin resistance: A Japanese population study.

AIMTo examine the relationship between pancreatic hyperechogenicity and risk factors for metabolic syndrome.METHODSA general population-based survey of lifestyle-related diseases was conducted from 2005 to 2006 in Japan. The study involved 551 participants older than 40 year of age. Data for 472 non-diabetic adults were included in the analysis. The measures included the demographic factors, blood parameters, results of a 75 g oral glucose tolerance test, and abdominal ultrasonography. The echogenicity of the pancreas and liver was compared, and then the subjects were separated into two groups: cases with pancreatic hyperechogenicity (n = 208) and cases without (controls, n = 264). The differences between both groups were compared using an unpaired t-test or Fisher’s exact test. Multiple logistic regression analysis was used to determine the relationship between the pancreatic hyperechogenicity and clinical and biochemical parameters.RESULTSSubjects with pancreatic hyperechogenicity had decreased serum adiponectin concentration compared to control subjects [8.9 (6.5, 12.8) vs 11.1 (7.8, 15.9), P < 0.001] and more frequently exhibited features of metabolic syndrome. Logistic regression analysis showed that the following variables were significantly and independently associated with pancreatic hyperechogenicity: Presence of hypoadiponectinemia, increased body mass index (BMI), higher homeostasis model assessment of insulin resistance (HOMA-IR) score, and presence of fatty liver. Similar associations were also observed in subjects with pancreatic hyperechogenicity without fatty liver. Multivariate association analysis of data from participants without fatty liver showed that hypoadiponectinemia was significantly associated with pancreatic hyperechogenicity (OR = 0.93, 95%CI: 0.90 - 0.97, P < 0.001). This association was independent of other confounding variables. Additionally, an increased BMI and higher HOMA-IR score were significantly associated with pancreatic hyperechogenicity.CONCLUSIONPancreatic hyperechogenicity is independently associated with increased BMI, insulin resistance, and hypoadiponectinemia in the general population.

Patterns of Failure After Radical Cystectomy for pT3-4 Bladder Cancer: Implications for Adjuvant Radiation Therapy

In patients with muscle-invasive bladder cancer, local-regional failure (LF) has been reported to occur in up to 20% of patients following radical cystectomy. The goals of this study were to describe patterns of LF, as well as assess factors associated with LF in a cohort of patients with pT3-4 bladder cancer. This information may have implications towards the use of adjuvant radiation therapy. Patients with pathologic T3-4 N0-1 bladder cancer were examined from an institutional radical cystectomy database. Preoperative demographics and pathologic characteristics were examined. Outcomes included overall survival and LF. Local-regional failures were defined using follow-up imaging reports and scans, and the locations of LF were characterized. Variables were tested by univariate and multivariate analysis for association with LF and overall survival. A total of 334 patients had pT3-4 and N0-1 disease after radical cystectomy and bilateral pelvic lymph node dissection. Of these, 46% received perioperative chemotherapy. The median age was 71 years old, and median follow-up was 11 months. On univariate analysis, margin status, pT stage, and pN stage, were all associated with LF (P<.05), however, on multivariate analysis, only pT and pN stages were significantly associated with LF (P<.05). Three strata of risk were defined, including low-risk patients with pT3N0 disease, intermediate-risk patients with pT3N1 or pT4N0 disease, and high-risk patients with pT4N1 disease, who had a 2-year incidence of LF of 12%, 33%, and 72%, respectively. The most common sites of pelvic relapse included the external and internal iliac lymph nodes (LNs) and obturator LN regions. Notably, 34% of patients with LF had local-regional only disease at the time of recurrence. Patients with pT4 or N1 disease have a 2-year risk of LF that exceeds 30%. These patients may be the most likely to benefit from local adjuvant therapies.

Genome-Wide Association Analysis of Adaptation Using Environmentally Predicted Traits.

Current methods for studying the genetic basis of adaptation evaluate genetic associations with ecologically relevant traits or single environmental variables, under the implicit assumption that natural selection imposes correlations between phenotypes, environments and genotypes. In practice, observed trait and environmental data are manifestations of unknown selective forces and are only indirectly associated with adaptive genetic variation. In theory, improved estimation of these forces could enable more powerful detection of loci under selection. Here we present an approach in which we approximate adaptive variation by modeling phenotypes as a function of the environment and using the predicted trait in multivariate and univariate genome-wide association analysis (GWAS). Based on computer simulations and published flowering time data from the model plant Arabidopsis thaliana, we find that environmentally predicted traits lead to higher recovery of functional loci in multivariate GWAS and are more strongly correlated to allele frequencies at adaptive loci than individual environmental variables. Our results provide an example of the use of environmental data to obtain independent and meaningful information on adaptive genetic variation.

Effects of mitochondrial haplogroup N9a on type 2 diabetes mellitus and its associated complications.

A case-control study was conducted with the aim of identifying the predominant haplogroups associated with type 2 diabetes mellitus (T2DM) and its complications. In addition, the role of N9a in T2DM risk and complications was analyzed. Sequencing of the entire mitochondrial DNA was conducted in 235 patients and 244 controls in cohort 1, and six haplogroups (F, B4, D4, D5, M8a and N9a) associated with T2DM were classified. The frequency of N9a was further determined in cohort 2 (440 patients and 244 controls) and examined in two combined cohorts, including 675 patients with T2DM and 649 non-diabetic controls. Multivariate logistic regression analysis and association analysis were performed to investigate the association between genotypes, T2DM and diabetic nephropathy. M8a [P=0.011; odds ratio (OR), 3.49; 95% confidence interval (CI), 1.26-9.69] and haplogroup N9a (P=0.023; OR, 2.60; 95% CI, 1.11-6.05) were associated with an increased risk of T2DM. The frequency of N9a was higher in T2DM patients compared with that in the controls (6.2% vs. 4.3%) and associated with a mild risk (P=0.10; OR, 1.51; 95% CI, 0.92-2.49). N9a was significantly associated with an increased risk of diabetic nephropathy (P=0.024; OR, 2.15; 95% CI, 1.11-4.19). Previous findings of N9a being protective against T2DM were not replicated in the present study, although this haplogroup was associated with an increased risk of diabetic nephropathy.

Evaluating the role of admixture in cancer therapy via in vitro drug response and multivariate genome-wide associations.

We investigate the role of ethnicity and admixture in drug response across a broad group of chemotherapeutic drugs. Also, we generate hypotheses on the genetic variants driving differential drug response through multivariate genome-wide association studies. Immortalized lymphoblastoid cell lines from 589 individuals (Hispanic or non-Hispanic/Caucasian) were used to investigate dose-response for 28 chemotherapeutic compounds. Univariate and multivariate statistical models were used to elucidate associations between genetic variants and differential drug response as well as the role of ethnicity in drug potency and efficacy. For many drugs, the variability in drug response appears to correlate with self-reported race and estimates of genetic ancestry. Additionally, multivariate genome-wide association analyses offered interesting hypotheses governing these differential responses.

Association mapping in Salix viminalis L. (Salicaceae) – identification of candidate genes associated with growth and phenology

Willow species (Salix) are important as short‐rotation biomass crops for bioenergy, which creates a demand for faster genetic improvement and breeding through deployment of molecular marker‐assisted selection (MAS). To find markers associated with important adaptive traits, such as growth and phenology, for use in MAS, we genetically dissected the trait variation of a Salix viminalis (L.) population of 323 accessions. The accessions were sampled throughout northern Europe and were established at two field sites in Pustnäs, Sweden, and at Woburn, UK, offering the opportunity to assess the impact of genotype‐by‐environment interactions (G × E) on trait–marker associations. Field measurements were recorded for growth and phenology traits. The accessions were genotyped using 1536 SNP markers developed from phenology candidate genes and from genes previously observed to be differentially expressed in contrasting environments. Association mapping between 1233 of these SNPs and the measured traits was performed taking into account population structure and threshold selection bias. At a false discovery rate (FDR) of 0.2, 29 SNPs were associated with bud burst, leaf senescence, number of shoots or shoot diameter. The percentage of accession variation (Radj2) explained by these associations ranged from 0.3% to 4.4%, suggesting that the studied traits are controlled by many loci of limited individual impact. Despite this, a SNP in the EARLY FLOWERING 3 gene was repeatedly associated (FDR < 0.2) with bud burst. The rare homozygous genotype exhibited 0.4–1.0 lower bud burst scores than the other genotype classes on a five‐grade scale. Consequently, this marker could be promising for use in MAS and the gene deserves further study. Otherwise, associations were less consistent across sites, likely due to their small Radj2 estimates and to considerable G × E interactions indicated by multivariate association analyses and modest trait accession correlations across sites (0.32–0.61).

Long-Range Modulation of PAG1 Expression by 8q21 Allergy Risk Variants

The gene(s) whose expression is regulated by allergy risk variants is unknown for many loci identified through genome-wide association studies. Addressing this knowledge gap might point to new therapeutic targets for allergic disease. The aim of this study was to identify the target gene(s) and the functional variant(s) underlying the association between rs7009110 on chromosome 8q21 and allergies. Eight genes are located within 1 Mb of rs7009110. Multivariate association analysis of publicly available exon expression levels from lymphoblastoid cell lines (LCLs) identified a significant association between rs7009110 and the expression of a single gene, PAG1 (p = 0.0017), 732 kb away. Analysis of histone modifications and DNase I hypersensitive sites in LCLs identified four putative regulatory elements (PREs) in the region. Chromosome conformation capture confirmed that two PREs interacted with the PAG1 promoter, one in allele-specific fashion. To determine whether these PREs were functional, LCLs were transfected with PAG1 promoter-driven luciferase reporter constructs. PRE3 acted as a transcriptional enhancer for PAG1 exclusively when it carried the rs2370615:C allergy predisposing allele, a variant in complete linkage disequilibrium with rs7009110. As such, rs2370615, which overlaps RelA transcription factor (TF) binding in LCLs and was found to disrupt Foxo3a binding to PRE3, represents the putative functional variant in this locus. Our studies suggest that the risk-associated allele of rs2370615 predisposes to allergic disease by increasing PAG1 expression, which might promote B cell activation and have a pro-inflammatory effect. Inhibition of PAG1 expression or function might have therapeutic potential for allergic diseases.

Approximate score-based testing with application to multivariate trait association analysis.

For genome-wide association studies and DNA sequencing studies, several powerful score-based tests, such as kernel machine regression and sum of powered score tests, have been proposed in the last few years. However, extensions of these score-based tests to more complex models, such as mixed-effects models for analysis of multiple and correlated traits, have been hindered by the unavailability of the score vector, due to either no output from statistical software or no closed-form solution at all. We propose a simple and general method to asymptotically approximate the score vector based on an asymptotically normal and consistent estimate of a parameter vector to be tested and its (consistent) covariance matrix. The proposed method is applicable to both maximum-likelihood estimation and estimating function-based approaches. We use the derived approximate score vector to extend several score-based tests to mixed-effects models. We demonstrate the feasibility and possible power gains of these tests in association analysis of multiple and correlated quantitative or binary traits with both real and simulated data. The proposed method is easy to implement with a wide applicability.

A multivariate genome-wide association analysis of 10 LDL subfractions, and their response to statin treatment, in 1868 Caucasians.

We conducted a genome-wide association analysis of 7 subfractions of low density lipoproteins (LDLs) and 3 subfractions of intermediate density lipoproteins (IDLs) measured by gradient gel electrophoresis, and their response to statin treatment, in 1868 individuals of European ancestry from the Pharmacogenomics and Risk of Cardiovascular Disease study. Our analyses identified four previously-implicated loci (SORT1, APOE, LPA, and CETP) as containing variants that are very strongly associated with lipoprotein subfractions (log10Bayes Factor > 15). Subsequent conditional analyses suggest that three of these (APOE, LPA and CETP) likely harbor multiple independently associated SNPs. Further, while different variants typically showed different characteristic patterns of association with combinations of subfractions, the two SNPs in CETP show strikingly similar patterns - both in our original data and in a replication cohort - consistent with a common underlying molecular mechanism. Notably, the CETP variants are very strongly associated with LDL subfractions, despite showing no association with total LDLs in our study, illustrating the potential value of the more detailed phenotypic measurements. In contrast with these strong subfraction associations, genetic association analysis of subfraction response to statins showed much weaker signals (none exceeding log10Bayes Factor of 6). However, two SNPs (in APOE and LPA) previously-reported to be associated with LDL statin response do show some modest evidence for association in our data, and the subfraction response proles at the LPA SNP are consistent with the LPA association, with response likely being due primarily to resistance of Lp(a) particles to statin therapy. An additional important feature of our analysis is that, unlike most previous analyses of multiple related phenotypes, we analyzed the subfractions jointly, rather than one at a time. Comparisons of our multivariate analyses with standard univariate analyses demonstrate that multivariate analyses can substantially increase power to detect associations. Software implementing our multivariate analysis methods is available at http://stephenslab.uchicago.edu/software.html.

AB013. Long-range modulation of PAG1 expression by 8q21 allergy risk variants

The gene(s) whose expression is regulated by allergy risk variants is unknown for many loci identified through genome-wide association studies. Addressing this knowledge gap might point to new therapeutic targets for allergic disease. The aim of this study was to identify the target gene(s) and the functional variant(s) underlying the association between rs7009110 on chromosome 8q21 and allergies. Eight genes are located within 1 Mb of rs7009110. Multivariate association analysis of publicly available exon expression levels from lymphoblastoid cell lines (LCLs) identified a significant association between rs7009110 and the expression of a single gene: PAG1 (P=0.0017), 732 kb away. Analysis of histone modifications and DNase I hypersensitive sites in LCLs identified four putative regulatory elements (PREs) in the region. Chromosome conformation capture confirmed that two PREs interacted with the PAG1 promoter, one in allele-specific fashion. To determine if these PREs were functional, LCLs were transfected with PAG1 promoter-driven luciferase reporter constructs. PRE3 acted as a transcriptional enhancer for PAG1 exclusively when it carried the rs2370615:C allergy predisposing allele, a variant in complete linkage disequilibrium with rs7009110. As such, rs2370615, which overlaps RelA transcription factor (TF) binding in LCLs and was found to disrupt Foxo3a binding to PRE3, represents the putative functional variant in this locus. Our studies suggest that the risk-associated allele of rs2370615 predisposes to allergic disease by increasing PAG1 expression which may promote B-cell activation and have a pro-inflammatory effect. Inhibition of PAG1 expression or function may have therapeutic potential for allergic diseases.

Patients with metastatic chromophobe renal cell carcinoma treated with sunitinib therapy: Analysis of an international database regarding outcome and comparison to clear cell histology (mccRCC).

429 Background: Sunitinib (Su) is a standard therapy (tx) for mccRCC. Data on its activity in the rare variant of metastatic chromophobe renal cell carcinoma (mchRCC) are limited by very small or heterogeneous (mixed histology with papillary type, or mixed targeted therapies) studies. We analyzed the activity of Su in a relatively large and homogenous international cohort of mchRCC pts, in terms of outcome and comparison to mccRCC. Methods: Records from mchRCC pts treated with first-line Su in 9 centers across 4 countries were retrospectively reviewed. Univariate and multivariate analyses of association between clinicopathologic factors and outcome were performed. Subsequently, mchRCC pts were individually matched to mccRCC pts. We compared the response rate (RR), progression free survival (PFS), and overall survival (OS) between the groups. Results: Between 2004-2014, 33 pts (median age 64, 45% male) with mchRCC were treated with Su as first-line tx. 76% had a prior nephrectomy. HENG risk was good 27%, intermediate 55%, and poor 18%. 33% were active smokers, and 30% users of angiotensin system inhibitors (ASIs). 55%, 27%, and 33% had lung, liver, and bone metastases, respectively. 48% had a pre-tx neutrophil to lymphocyte ratio (NLR) &gt;3. 42% had dose reduction/tx interruption (DR/TI). Su-induced hypertension (HTN) occurred in 48%. 75% achieved a clinical benefit (partial response + stable disease), while 25% had disease progression within the first 3 months of tx. Median PFS and OS were 10 and 26 months, respectively. Factors associated with PFS were the HENG risk (HR 3.8, p=0.025) and pre-tx NLR &gt;3 (HR 0.6, p=0.012). Factors associated with OS were the HENG risk (HR 4.27, p=0.027), liver metastases (HR 4.6, p=0.029), and pre-treatment NLR &lt;3 (HR 0.5, p=0.04). Tx outcome was not significantly different between mchRCC pts and mccRCC pts, who were individually matched by HENG risk, nephrectomy/smoking status, pre-tx NLR, use of ASIs, DR/TI, and Su induced HTN. In mccRCC pts (p value versus mchRCC), 70% achieved a clinical benefit (p=0.58), and median PFS and OS were 9 (p=0.7) and 24 (p=0.6) months, respectively. Conclusions: In mchRCC pts, Su tx may have similar outcome to mccRCC pts.

Metformin use and outcome of sunitinib treatment in diabetic patients with metastatic renal cell carcinoma.

440 Background: Sunitinib (Su) is a standard treatment (tx) for metastatic renal cell carcinoma (mRCC). Pre-clinical and clinical studies in several cancer types suggest that the antidiabetic agent metformin (Met) has antitumor activity. Met may negatively regulate mTOR activity. Its effect on the outcome of targeted therapies in mRCC is poorly defined. We analyzed the effect of Met use on the outcome sunitinib tx in diabetic patients (pts) with mRCC. Methods: We performed a retrospective study of an unselected cohort of diabetic pts with mRCC, who were treated with Su in 7 centers across 2 countries. Pts were divided into 2 groups: (1) Met users and (2) Met naive. The effect of Met use on response rate (RR), progression free survival (PFS) and overall survival (OS), was tested with adjustment of other known confounding risk factors using a chisquare test and partial likelihood test from Cox model. Furthermore, univariate and multivariate analyses of association between clinicopathologic factors and Met use, and outcome were performed using the entire pt cohort. Results: Between 2004-2014, 108 diabetic pts with mRCC were treated with sunitinib. There were 52 Met users (group 1) and 56 nonusers (group 2). The groups were balanced regarding the following clinicopathologic factors: age, gender, HENG risk, past nephrectomy, mRCC histology, ≥2 metastatic sites, lung/liver/bone metastasis, prior targeted tx, smoking status, use of angiotensin system inhibitors (ASIs), pre-tx neutrophil to lymphocyte ratio (NLR) &gt;3, Su-induced hypertension (HTN), and Su dose reduction/tx interruption. Clinical benefit (partial response + stable disease) in group 1 vs. group 2 was 96% vs. 84%, while 4% vs. 16% had disease progression within the first 3 months of tx (p=0.054). Median PFS was 15 vs. 11.5 months (p=0.1). Median OS was 32 vs. 21 months (p=0.001). In multivariate analyses of the entire pt cohort (n=108), factors associated with PFS were active smoking (HR=2.7, p&lt;0.0001) and pre-tx NLR &gt;3 (HR 1.8, p=0.012). Factors associated with OS were Met use (HR 0.2, p&lt;0.0001), HENG risk (HR 3.3, p=0.008), active smoking (HR=2.9, p&lt;0.0001), liver metastases (HR 1.8, p=0.004), and pre-tx NLR &gt;3 (HR 3.3, p&lt;0.0001). Conclusions: Met use may improve the OS of diabetic pts with mRCC that are treated with Su.

Family-based association analysis: a fast and efficient method of multivariate association analysis with multiple variants.

BackgroundMany disease phenotypes are outcomes of the complicated interplay between multiple genes, and multiple phenotypes are affected by a single or multiple genotypes. Therefore, joint analysis of multiple phenotypes and multiple markers has been considered as an efficient strategy for genome-wide association analysis, and in this work we propose an omnibus family-based association test for the joint analysis of multiple genotypes and multiple phenotypes.ResultsThe proposed test can be applied for both quantitative and dichotomous phenotypes, and it is robust under the presence of population substructure, as long as large-scale genomic data is available. Using simulated data, we showed that our method is statistically more efficient than the existing methods, and the practical relevance is illustrated by application of the approach to obesity-related phenotypes.ConclusionsThe proposed method may be more statistically efficient than the existing methods. The application was developed in C++ and is available at the following URL: http://healthstat.snu.ac.kr/software/mfqls/.

Association of novelty-related behaviors and intravenous cocaine self-administration in Diversity Outbred mice

The preference for and reaction to novelty are strongly associated with addiction to cocaine and other drugs. However, the genetic variants and molecular mechanisms underlying these phenomena remain largely unknown. Although the relationship between novelty- and addiction-related traits has been observed in rats, studies in mice have failed to demonstrate this association. New, genetically diverse, high-precision mouse populations including Diversity Outbred (DO) mice provide an opportunity to assess an expanded range of behavioral variation enabling detection of associations of novelty- and addiction-related traits in mice. To examine the relationship between novelty- and addiction-related traits, male (n = 51) and female (n = 47) DO mice were tested on open field exploration, hole board exploration, and novelty preference followed by intravenous cocaine self-administration (IVSA; ten 2-h sessions of fixed ratio 1 and one 6-h session of progressive ratio). We observed high variation of cocaine IVSA in DO mice with 43 % reaching and 57 % not reaching conventional acquisition criteria. As a group, mice that did not reach these criteria still demonstrated significant lever discrimination. Mice experiencing catheter occlusion or other technical issues (n = 17) were excluded from the analysis. Novelty-related behaviors were positively associated with cocaine IVSA. Multivariate analysis of associations among novelty- and addiction-related traits revealed a large degree of shared variance (45 %). Covariation among cocaine IVSA and novelty-related phenotypes in DO mice indicates that this relationship is amenable to genetic dissection. The high genetic precision and phenotypic diversity in the DO may facilitate discovery of previously undetectable mechanisms underlying predisposition to develop addiction disorders.