Jonathan D. Mosley

Systematic comparison of phenome-wide association study of electronic medical record data and genome-wide association study data

Candidate gene and genome-wide association studies (GWAS) have identified genetic variants that modulate risk for human disease; many of these associations require further study to replicate the results. Here we report the first large-scale application of the phenome-wide association study (PheWAS) paradigm within electronic medical records (EMRs), an unbiased approach to replication and discovery that interrogates relationships between targeted genotypes and multiple phenotypes. We scanned for associations between 3,144 single-nucleotide polymorphisms (previously implicated by GWAS as mediators of human traits) and 1,358 EMR-derived phenotypes in 13,835 individuals of European ancestry. This PheWAS replicated 66% (51/77) of sufficiently powered prior GWAS associations and revealed 63 potentially pleiotropic associations with P < 4.6 × 10−6 (false discovery rate < 0.1); the strongest of these novel associations were replicated in an independent cohort (n = 7,406). These findings validate PheWAS as a tool to allow unbiased interrogation across multiple phenotypes in EMR-based cohorts and to enhance analysis of the genomic basis of human disease.

Genomewide linkage analysis of quantitative spirometric phenotypes in severe early-onset chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is a common, complex disease associated with substantial morbidity and mortality. COPD is defined by irreversible airflow obstruction; airflow obstruction is typically determined by reductions in quantitative spirometric indices, including forced expiratory volume at 1 s (FEV(1)) and the ratio of FEV(1) to forced vital capacity (FVC). To identify genetic determinants of quantitative spirometric phenotypes, an autosomal 10-cM genomewide scan of short tandem repeat (STR) polymorphic markers was performed in 72 pedigrees (585 individuals) ascertained through probands with severe early-onset COPD. Multipoint variance-component linkage analysis (using SOLAR) was performed for quantitative phenotypes, including FEV(1), FVC, and FEV(1)/FVC. In the initial genomewide scan, significant evidence for linkage to FEV(1)/FVC was demonstrated on chromosome 2q (LOD score 4.12 at 222 cM). Suggestive evidence was found for linkage to FEV(1)/FVC on chromosomes 1 (LOD score 1.92 at 120 cM) and 17 (LOD score 2.03 at 67 cM) and to FVC on chromosome 1 (LOD score 2.05 at 13 cM). The highest LOD score for FEV(1) in the initial genomewide scan was 1.53, on chromosome 12, at 36 cM. After inclusion of 12 additional STR markers on chromosome 12p, which had been previously genotyped in this population, suggestive evidence for linkage of FEV(1) (LOD score 2.43 at 37 cM) to this region was demonstrated. These observations provide both significant evidence for an early-onset COPD-susceptibility locus on chromosome 2 and suggestive evidence for linkage of spirometry-related phenotypes to several other genomic regions. The significant linkage of FEV(1)/FVC to chromosome 2q could reflect one or more genes influencing the development of airflow obstruction or dysanapsis.

FOXA1 is an essential determinant of ERα expression and mammary ductal morphogenesis

FOXA1, estrogen receptor α (ERα) and GATA3 independently predict favorable outcome in breast cancer patients, and their expression correlates with a differentiated, luminal tumor subtype. As transcription factors, each functions in the morphogenesis of various organs, with ERα and GATA3 being established regulators of mammary gland development. Interdependency between these three factors in breast cancer and normal mammary development has been suggested, but the specific role for FOXA1 is not known. Herein, we report that Foxa1 deficiency causes a defect in hormone-induced mammary ductal invasion associated with a loss of terminal end bud formation and ERα expression. By contrast, Foxa1 null glands maintain GATA3 expression. Unlike ERα and GATA3 deficiency, Foxa1 null glands form milk-producing alveoli, indicating that the defect is restricted to expansion of the ductal epithelium, further emphasizing the novel role for FOXA1 in mammary morphogenesis. Using breast cancer cell lines, we also demonstrate that FOXA1 regulates ERα expression, but not GATA3. These data reveal that FOXA1 is necessary for hormonal responsiveness in the developing mammary gland and ERα-positive breast cancers, at least in part, through its control of ERα expression.

Genome- and Phenome-Wide Analyses of Cardiac Conduction Identifies Markers of Arrhythmia Risk

Background— ECG QRS duration, a measure of cardiac intraventricular conduction, varies ≈2-fold in individuals without cardiac disease. Slow conduction may promote re-entrant arrhythmias. Methods and Results— We performed a genome-wide association study to identify genomic markers of QRS duration in 5272 individuals without cardiac disease selected from electronic medical record algorithms at 5 sites in the Electronic Medical Records and Genomics (eMERGE) network. The most significant loci were evaluated within the Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE) consortium QRS genome-wide association study meta-analysis. Twenty-three single-nucleotide polymorphisms in 5 loci, previously described by CHARGE, were replicated in the eMERGE samples; 18 single-nucleotide polymorphisms were in the chromosome 3 SCN5A and SCN10A loci, where the most significant single-nucleotide polymorphisms were rs1805126 in SCN5A with P=1.2×10−8 (eMERGE) and P=2.5×10−20 (CHARGE) and rs6795970 in SCN10A with P=6×10−6 (eMERGE) and P=5×10−27 (CHARGE). The other loci were in NFIA, near CDKN1A, and near C6orf204. We then performed phenome-wide association studies on variants in these 5 loci in 13859 European Americans to search for diagnoses associated with these markers. Phenome-wide association study identified atrial fibrillation and cardiac arrhythmias as the most common associated diagnoses with SCN10A and SCN5A variants. SCN10A variants were also associated with subsequent development of atrial fibrillation and arrhythmia in the original 5272 “heart-healthy” study population. Conclusions— We conclude that DNA biobanks coupled to electronic medical records not only provide a platform for genome-wide association study but also may allow broad interrogation of the longitudinal incidence of disease associated with genetic variants. The phenome-wide association study approach implicated sodium channel variants modulating QRS duration in subjects without cardiac disease as predictors of subsequent arrhythmias.

Screening for Acute IKr Block Is Insufficient to Detect Torsades de Pointes Liability Role of Late Sodium Current

Background— New drugs are routinely screened for IKr blocking properties thought to predict QT prolonging and arrhythmogenic liability. However, recent data suggest that chronic (hours) drug exposure to phosphoinositide 3-kinase inhibitors used in cancer can prolong QT by inhibiting potassium currents and increasing late sodium current (INa-L) in cardiomyocytes. We tested the extent to which IKr blockers with known QT liability generate arrhythmias through this pathway. Methods and Results— Acute exposure to dofetilide, an IKr blocker without other recognized electropharmacologic actions, produced no change in ion currents or action potentials in adult mouse cardiomyocytes, which lack IKr. By contrast, 2 to 48 hours of exposure to the drug generated arrhythmogenic afterdepolarizations and ≥15-fold increases in INa-L. Including phosphatidylinositol 3,4,5-trisphosphate, a downstream effector for the phosphoinositide 3-kinase pathway, in the pipette inhibited these effects. INa-L was also increased, and inhibitable by phosphatidylinositol 3,4,5-trisphosphate, with hours of dofetilide exposure in human-induced pluripotent stem cell–derived cardiomyocytes and in Chinese hamster ovary cells transfected with SCN5A, encoding sodium current. Cardiomyocytes from dofetilide-treated mice similarly demonstrated increased INa-L and afterdepolarizations. Other agents with variable IKr-blocking potencies and arrhythmia liability produced a range of effects on INa-L, from marked increases (E-4031, D-sotalol, thioridazine, and erythromycin) to little or no effect (haloperidol, moxifloxacin, and verapamil). Conclusions— Some but not all drugs designated as arrhythmogenic IKr blockers can generate arrhythmias by augmenting INa-L through the phosphoinositide 3-kinase pathway. These data identify a potential mechanism for individual susceptibility to proarrhythmia and highlight the need for a new paradigm to screen drugs for QT prolonging and arrhythmogenic liability.

Biobanks and Electronic Medical Records: Enabling Cost-Effective Research

Linking of data from electronic medical records to biological specimens enables cost-effective and rapid genomic analyses. The use of electronic medical record data linked to biological specimens in health care settings is expected to enable cost-effective and rapid genomic analyses. Here, we present a model that highlights potential advantages for genomic discovery and describe the operational infrastructure that facilitated multiple simultaneous discovery efforts.

Rapamycin inhibits multiple stages of c-Neu/ErbB2-induced tumor progression in a transgenic mouse model of HER2-positive breast cancer

Amplification of the HER2 (ErbB2, c-Neu) proto-oncogene in breast cancer is associated with poor prognosis and high relapse rates. HER2/ErbB2, in conjunction with ErbB3, signals through the Akt/phosphatidylinositol 3-kinase pathway and leads to the activation of mammalian target of rapamycin (mTOR), a critical mRNA translation regulator that controls cell growth. Gene expression analysis of mammary tumors collected from mouse mammary tumor virus-c-Neu transgenic mice revealed that mRNA levels of several mTOR pathway members were either up-regulated (p85/phosphatidylinositol 3-kinase and p70S6 kinase) or down-regulated (eIF-4E-BP1) in a manner expected to enhance signaling through this pathway. Treatment of these mice with the mTOR inhibitor rapamycin caused growth arrest and regression of primary tumors with no evidence of weight loss or generalized toxicity. The treatment effects were due to decreased proliferation, associated with reduced cyclin D1 expression, and increased cell death in primary tumors. Whereas many of the dead epithelial cells had the histopathologic characteristics of ischemic necrosis, rapamycin treatment was not associated with changes in microvascular density or apoptosis. Rapamycin also inhibited cellular proliferation in lung metastases. In summary, data from this preclinical model of ErbB2/Neu-induced breast cancer show that inhibition of the mTOR pathway with rapamycin blocks multiple stages of ErbB2/Neu-induced tumorigenic progression. [Mol Cancer Ther 2007;6(8):2188–97]

Exome sequencing implicates an increased burden of rare potassium channel variants in the risk of drug-induced long QT interval syndrome.

OBJECTIVES The aim of this study was to test the hypothesis that rare variants are associated with drug-induced long QT interval syndrome (diLQTS) and torsades de pointes. BACKGROUND diLQTS is associated with the potentially fatal arrhythmia torsades de pointes. The contribution of rare genetic variants to the underlying genetic framework predisposing to diLQTS has not been systematically examined. METHODS We performed whole-exome sequencing on 65 diLQTS patients and 148 drug-exposed control subjects of European descent. We used rare variant analyses (variable threshold and sequence kernel association test) and gene-set analyses to identify genes enriched with rare amino acid coding (AAC) variants associated with diLQTS. Significant associations were reanalyzed by comparing diLQTS patients with 515 ethnically matched control subjects from the National Heart, Lung, and Blood Grand Opportunity Exome Sequencing Project. RESULTS Rare variants in 7 genes were enriched in the diLQTS patients according to the sequence kernel association test or variable threshold compared with drug-exposed controls (p < 0.001). Of these, we replicated the diLQTS associations for KCNE1 and ACN9 using 515 Exome Sequencing Project control subjects (p < 0.05). A total of 37% of the diLQTS patients also had 1 or more rare AAC variants compared with 21% of control subjects (p = 0.009), in a pre-defined set of 7 congenital long QT interval syndrome (cLQTS) genes encoding potassium channels or channel modulators (KCNE1, KCNE2, KCNH2, KCNJ2, KCNJ5, KCNQ1, AKAP9). CONCLUSIONS By combining whole-exome sequencing with aggregated rare variant analyses, we implicate rare variants in KCNE1 and ACN9 as risk factors for diLQTS. Moreover, diLQTS patients were more burdened by rare AAC variants in cLQTS genes encoding potassium channel modulators, supporting the idea that multiple rare variants, notably across cLQTS genes, predispose to diLQTS.

Genetic variants in transcription factors are associated with the pharmacokinetics and pharmacodynamics of metformin.

One‐third of type 2 diabetes patients do not respond to metformin. Genetic variants in metformin transporters have been extensively studied as a likely contributor to this high failure rate. Here, we investigate, for the first time, the effect of genetic variants in transcription factors on metformin pharmacokinetics (PK) and response. Overall, 546 patients and healthy volunteers contributed their genome‐wide, pharmacokinetic (235 subjects), and HbA1c data (440 patients) for this analysis. Five variants in specificity protein 1 (SP1), a transcription factor that modulates the expression of metformin transporters, were associated with changes in treatment HbA1c (P < 0.01) and metformin secretory clearance (P < 0.05). Population pharmacokinetic modeling further confirmed a 24% reduction in apparent clearance in homozygous carriers of one such variant, rs784888. Genetic variants in other transcription factors, peroxisome proliferator–activated receptor‐α and hepatocyte nuclear factor 4‐α, were significantly associated with HbA1c change only. Overall, our study highlights the importance of genetic variants in transcription factors as modulators of metformin PK and response.

Lack of association between a polymorphism in the interleukin‐13 gene and total serum immunoglobulin E level among nuclear families in Costa Rica

Background IL‐13 has been implicated in the pathogenesis of asthma and in the regulation of IgE synthesis in humans. Single nucleotide polymorphisms (SNPs) in the IL‐13 gene have been associated with asthma and total serum IgE level in Caucasian populations.