Lan Jiang

Quality Control Procedures for Genome‐Wide Association Studies

Genome‐wide association studies (GWAS) are being conducted at an unprecedented rate in population‐based cohorts and have increased our understanding of the pathophysiology of complex disease. Regardless of context, the practical utility of this information will ultimately depend upon the quality of the original data. Quality control (QC) procedures for GWAS are computationally intensive, operationally challenging, and constantly evolving. Here we enumerate some of the challenges in QC of GWAS data and describe the approaches that the electronic MEdical Records and Genomics (eMERGE) network is using for quality assurance in GWAS data, thereby minimizing potential bias and error in GWAS results. We discuss common issues associated with QC of GWAS data, including data file formats, software packages for data manipulation and analysis, sex chromosome anomalies, sample identity, sample relatedness, population substructure, batch effects, and marker quality. We propose best practices and discuss areas of ongoing and future research. Curr. Protoc. Hum. Genet. 68:1.19.1‐1.19.18

Variation in ITGB3 is associated with whole-blood serotonin level and autism susceptibility

Autism is a pervasive developmental disorder affecting more males than females. Heritability estimates for autism can rise above 90%, and genes influencing the serotonin system are strong candidates for autism susceptibility genes, as drugs selectively acting on the serotonin system are some of the most effective treatments for maladaptive behaviors seen in autism. ITGB3 was recently identified as a male quantitative trait locus (QTL) for whole-blood serotonin levels in the Hutterites (P=0.0003). Here, we demonstrate associations between variation in ITGB3 and serotonin levels in two outbred samples (P=0.010 and 0.015). Lastly, we show that a coding variant of ITGB3 is associated with autism susceptibility in a large multiplex sample (P=0.00082), and that this variation has different effects in males and females (P=0.0018).

Common genetic polymorphism at 4q25 locus predicts atrial fibrillation recurrence after successful cardioversion

BACKGROUND Genome-wide association studies have identified numerous common polymorphisms associated with atrial fibrillation (AF). The 3 loci most strongly associated with AF occur at chromosome 4q25 (near PITX2), 16q22 (in ZFHX3), and 1q21 (in KCNN3). OBJECTIVE To evaluate whether timing of AF recurrence after direct current cardioversion (DCCV) is modulated by common AF susceptibility alleles. METHODS A total of 208 patients (age 65 ± 11 years; 77% men) with persistent AF underwent successful DCCV and were prospectively evaluated at 3, 6, and 12 months for AF recurrence. Four single nucleotide polymorphisms--rs2200733 and rs10033464 at 4q25, rs7193343 in ZFHX3, and rs13376333 in KCNN3--were genotyped. RESULTS The final study cohort consisted of 184 patients. In 162 (88%) patients, sinus rhythm was restored with DCCV, of which 108 (67%) had AF recurrence at a median of 60 (interquartile range 29-176) days. In multivariable analysis, the presence of any common single nucleotide polymorphism (rs2200733, rs10033464) at the 4q25 locus was an independent predictor of AF recurrence (hazard ratio 2.1; 95% confidence interval 1.21-3.30; P = .008). Furthermore, rs2200733 exhibited a graded allelic dose response for early AF recurrence (homozygous variants: 7 [interquartile range 4-56] days; heterozygous variants: 54 [28-135] days; and wild type: 64 [29-180] days; P = .03). CONCLUSIONS To our knowledge, this is the first study to evaluate whether genomic markers can predict timing of AF recurrence in patients undergoing elective DCCV. Our findings show that a common polymorphism on chromosome 4q25 (rs2200733) is an independent predictor of AF recurrence after DCCV and point to a potential role of stratification by genotype.

Racial disparity in amniotic fluid concentrations of tumor necrosis factor (TNF)-α and soluble TNF receptors in spontaneous preterm birth

OBJECTIVE Preterm birth rate in the United States is higher in blacks than whites. It has been hypothesized that a differential inflammatory response may explain this disparity. The objective of this study is to examine the inflammatory cytokine, tumor necrosis factor (TNF)-alpha and soluble TNF receptor concentrations (sTNFR1 and sTNFR2) in the amniotic fluid of black and white women at delivery. STUDY DESIGN Amniotic fluid samples were collected during active labor from 158 cases (preterm births, gestational age 22(0/7) weeks to 36(0/7) weeks, 52 black and 106 white) and 175 controls (term births, gestational age 37(0/7) weeks to 42(0/7) weeks, 87 black and 88 white) at Centennial Womens Hospital, Nashville, TN. Amniotic fluid TNF-alpha, sTNFR1, and sTNFR2 concentrations and the molar ratios of TNF-alpha to its receptors were compared between cases and controls within each racial group. RESULTS Median TNF-alpha concentration was associated with preterm birth when whites and blacks were analyzed together, with cases having higher values (191.5 pg/mL) than controls (68.9 pg/mL; P < .001). There were no significant associations with sTNFR1 or sTNFR2 concentrations between cases (2409.4 and 2934.3 pg/mL, respectively) and controls (2759.9 and 3084.1 pg/mL, respectively) when the racial groups were analyzed together (P = .08, P = .4, respectively). Black cases associated with higher TNF-alpha concentrations (1287.0 pg/mL in cases and 67.3 pg/mL in controls; P < .001). In whites there was no association between TNF-alpha and preterm birth (P = .3). The molar ratio of TNF-alpha/total sTNFR (R1 plus R2) associated with higher TNF-alpha in black cases, compared with black controls (P < .001). There was no significant association between white cases and controls for ligand receptor ratios (P = .3). CONCLUSION The TNF-alpha/sTNFR profile in pregnancy differs between racial groups, suggesting a difference in bioavailability of TNF-alpha. The larger molar ratio of TNF-alpha/sTNFR in black cases may be indicative of a TNF-alpha mediated pathological process of preterm birth in blacks but not in whites.

Ethnic differences in interleukin 6 (IL-6) and IL6 receptor genes in spontaneous preterm birth and effects on amniotic fluid protein levels

Preterm birth (PTB) is a significant neonatal health problem that is more common in African‐Americans (AA) than in European‐Americans (EA). Part of this disparity is likely to result from the differing genetic architectures of EA and AA. To begin assessing the role of these differences, patterns of genetic variation in two previously proposed candidate genes, encoding interleukin 6 (IL6) and its receptor (IL6R), were analyzed in mothers and fetuses from 496 EA birth‐events (149 cases and 347 controls) and 397 birth‐events in AA (76 cases and 321 controls). IL‐6 levels in amniotic fluid (AF) samples were determined in a subset of these pregnancies. Case‐control comparisons revealed a single SNP in IL6R associated with PTB (p=0.04 for allelic and p=0.05 for genotype association). In addition, all of the SNPs studied showed significant frequency differences between AA and EA in at least one comparison, significantly in excess of that expected from general population databases. Higher IL‐6 concentrations were associated with the IL6 SNP ‐661 in EA preterm samples (p=0.0056), and this result seems to be driven by microbial invasion of the amniotic cavity, indicating a gene by infection interaction. These findings indicate that, as a function of IL6 genotype, EA and AA women respond differently to infection with respect to their expression of IL‐6. Our data support differential genetic control of levels of IL‐6 in amniotic fluid between EA and AA.

Patterns of Genetic Variation in the Hypertension Candidate Gene GRK4: Ethnic Variation and Haplotype Structure

Association studies using single nucleotide polymorphisms (SNPs) have the potential to help unravel the genetic basis of hypertension. Nevertheless, to date, association studies of hypertension have yielded ambiguous results. It is becoming clear that such association studies must be interpreted within the context of the genetic structure of the populations being studied, and patterns of variation within specific genomic regions. With this in mind we analyzed genetic variation in the G protein‐coupled receptor kinase 4 (GRK4) gene, a gene whose product has recently been shown to inhibit the dopamine receptor D1 (DRD1) from increasing sodium excretion. We genotyped three previously identified GRK4 SNPs, as well as ten additional SNPs, over 71.6 kb of the GRK4 locus in four populations: African Americans, Asians, Hispanics and Caucasians. Haplotype structure varied among populations, with Hispanics and Caucasians having the most linkage disequilibrium (LD) among SNPs. African Americans had three shorter haplotype blocks, while patterns of markers in the Asian populations demonstrated less LD among markers, a pattern inconsistent with block structure. We observed limited haplotype diversity in each of the four populations, with differing haplotype frequencies among the ethnic groups. We also found substantial evidence for population differentiation, with the largest differences between the African‐American and Asian samples with FST values in the upper 90th percentile when compared to a genome‐wide distribution. However, for all population comparisons, FST values decreased sharply in the 3′ region of the gene. This pattern of differentiation among populations is consistent with selection in this part of the gene maintaining similar patterns of variation among otherwise divergent populations. Our results document not only different allele frequencies between populations, but differences in haplotype structure that may be important in evaluating association studies between hypertension and GRK4.

Pitfalls of Merging GWAS Data: Lessons Learned in the eMERGE Network and Quality Control Procedures to Maintain High Data Quality

Genome‐wide association studies (GWAS) are a useful approach in the study of the genetic components of complex phenotypes. Aside from large cohorts, GWAS have generally been limited to the study of one or a few diseases or traits. The emergence of biobanks linked to electronic medical records (EMRs) allows the efficient reuse of genetic data to yield meaningful genotype–phenotype associations for multiple phenotypes or traits. Phase I of the electronic MEdical Records and GEnomics (eMERGE‐I) Network is a National Human Genome Research Institute‐supported consortium composed of five sites to perform various genetic association studies using DNA repositories and EMR systems. Each eMERGE site has developed EMR‐based algorithms to comprise a core set of 14 phenotypes for extraction of study samples from each sites DNA repository. Each eMERGE site selected samples for a specific phenotype, and these samples were genotyped at either the Broad Institute or at the Center for Inherited Disease Research using the Illumina Infinium BeadChip technology. In all, approximately 17,000 samples from across the five sites were genotyped. A unified quality control (QC) pipeline was developed by the eMERGE Genomics Working Group and used to ensure thorough cleaning of the data. This process includes examination of sample and marker quality and various batch effects. Upon completion of the genotyping and QC analyses for each sites primary study, eMERGE Coordinating Center merged the datasets from all five sites. This larger merged dataset reentered the established eMERGE QC pipeline. Based on lessons learned during the process, additional analyses and QC checkpoints were added to the pipeline to ensure proper merging. Here, we explore the challenges associated with combining datasets from different genotyping centers and describe the expansion to eMERGE QC pipeline for merged datasets. These additional steps will be useful as the eMERGE project expands to include additional sites in eMERGE‐II, and also serve as a starting point for investigators merging multiple genotype datasets accessible through the National Center for Biotechnology Information in the database of Genotypes and Phenotypes. Our experience demonstrates that merging multiple datasets after additional QC can be an efficient use of genotype data despite new challenges that appear in the process. Genet. Epidemiol. 35:887–898, 2011.

A genome-wide linkage analysis of dementia in the Amish

Susceptibility genes for Alzheimers disease are proving to be highly challenging to detect and verify. Population heterogeneity may be a significant confounding factor contributing to this difficulty. To increase the power for disease susceptibility gene detection, we conducted a genome‐wide genetic linkage screen using individuals from the relatively isolated, genetically homogeneous, Amish population. Our genome linkage analysis used a 407‐microsatellite‐marker map (average density 7 cM) to search for autosomal genes linked to dementia in five Amish families from four Midwestern U.S. counties. Our highest two‐point lod score (3.01) was observed at marker D4S1548 on chromosome 4q31. Five other regions (10q22, 3q28, 11p13, 4q28, 19p13) also demonstrated suggestive linkage with markers having two‐point lod scores >2.0. While two of these regions are novel (4q31 and 11p13), the other regions lie close to regions identified in previous genome scans in other populations. Our results identify regions of the genome that may harbor genes involved in a subset of dementia patients, in particular the North American Amish community.

Variations in the α2A-adrenergic receptor gene and their functional effects

The α2A‐adrenergic receptor (ADRA2A) plays a central role in the regulation of systemic sympathetic activity and hence cardiovascular responses such as heart rate and blood pressure. The objectives of this study were to systematically search for variants in the ADRA2A gene, to define the genes haplotype structure, and to examine potential functional effects of these variants.

Variation in the alpha2B-adrenergic receptor gene (ADRA2B) and its relationship to vascular response in vivo.

The &agr;2B-adrenergic receptor (ADRA2B) plays an important role in vasoconstriction and blood pressure regulation. One common variant in the ADRA2B gene (del301–303) has been identified, and results in markedly decreased receptor desensitization in vitro but does not alter vascular sensitivity in vivo. Therefore, we fully characterized genetic variations in ADRA2B and related them to phenotype in vivo. We examined 5812 bp of contiguous sequence of ADRA2B (promoter, exonic, and 3′-untranslated region; 3′-UTR) using the polymerase chain reaction to amplify the genomic target followed by bidirectional sequencing (n=68). Haplotypes were inferred using an expectation maximization algorithm. Vasoconstriction in response to increasing doses of the highly selective &agr;2-adrenergic receptor agonist, dexmedetomidine (0.01–1000 ng/min) was measured in the dorsal hand vein using a linear variable differential transformer. The dose that produced 50% (ED50) of maximum venoconstriction (Emax) was determined for each subject from the individual dose–response curves. ED50 and Emax were compared in subjects with and without variant alleles and haplotypes of interest. We identified 24 variable sites, 12 in the promoter region, five in the coding region (including two previously described as non-synonymous variants) and seven in the 3′-UTR region. Four haplotypes were inferred, representing approximately 95% of the cohort. One haplotype, characterized by two single nucleotide polymorphisms in the promoter region, and one in the 3′-UTR, occurred in seven of 38 African-Americans, and was associated with a lower Emax, 61.3% [95% confidence interval (CI) 39.5–83.0, n=7] compared to 78.1% (CI 73.8–82.5) in wild-types (n=61) (P=0.02). There was no association between the nine common variants and dexmedetomidine ED50. We have described novel variants and haplotypes of the ADRA2B gene. These do not alter sensitivity to a selective &agr;2-adrenergic receptor agonist but some may decrease maximal venoconstriction in vivo.