Background: Non-alcoholic fatty liver disease (NAFLD) is a common chronic liver illness with a genetically heterogeneous background that can be accompanied by considerable morbidity and attendant health care costs. The pathogenesis and progression of NAFLD is complex with many unanswered questions. We conducted genome-wide association studies (GWASs) using both adult and pediatric participants from the Electronic Medical Records and Genomics (eMERGE) Network to identify novel genetic contributors to this condition. Methods: First, a natural language processing (NLP) algorithm was developed, tested, and deployed at each site to identify 1106 NAFLD cases and 8571 controls and histological data from liver tissue in 235 available participants. These include 1242 pediatric participants (396 cases, 846 controls). The algorithm included billing codes, text queries, laboratory values, and medication records. Next, GWASs were performed on NAFLD cases and controls and case-only analyses using histologic scores and liver function tests adjusting for age, sex, site, ancestry, PC, and body mass index (BMI). Results: Consistent with previous results, a robust association was detected for the PNPLA3 gene cluster in participants with European ancestry. At the PNPLA3-SAMM50 region, three SNPs, rs738409, rs738408, and rs3747207, showed strongest association (best SNP rs738409 p = 1.70 × 10 − 20). This effect was consistent in both pediatric (p = 9.92 × 10 − 6) and adult (p = 9.73 × 10 − 15) cohorts. Additionally, this variant was also associated with disease severity and NAFLD Activity Score (NAS) (p = 3.94 × 10 − 8 , beta = 0.85). PheWAS analysis link this locus to a spectrum of liver diseases beyond NAFLD with a novel negative correlation with gout (p = 1.09 × 10 − 4). We also identified novel loci for NAFLD disease severity, including one novel locus for NAS score near IL17RA (rs5748926, p = 3.80 × 10 − 8), and another near ZFP90-CDH1 for fibrosis (rs698718, p = 2.74 × 10 − 11). Post-GWAS and gene-based analyses identified more than 300 genes that were used for functional and pathway enrichment analyses.
Genome-wide association studies of Systemic Lupus Erythematosus (SLE) nominate 3073 genetic variants at 91 risk loci. To systematically screen these variants for allelic transcriptional enhancer activity, we construct a massively parallel reporter assay (MPRA) library comprising 12,396 DNA oligonucleotides containing the genomic context around every allele of each SLE variant. Transfection into the Epstein-Barr virus-transformed B cell line GM12878 reveals 482 variants with enhancer activity, with 51 variants showing genotype-dependent (allelic) enhancer activity at 27 risk loci. Comparison of MPRA results in GM12878 and Jurkat T cell lines highlights shared and unique allelic transcriptional regulatory mechanisms at SLE risk loci. In-depth analysis of allelic transcription factor (TF) binding at and around allelic variants identifies one class of TFs whose DNA-binding motif tends to be directly altered by the risk variant and a second class of TFs that bind allelically without direct alteration of their motif by the variant. Collectively, our approach provides a blueprint for the discovery of allelic gene regulation at risk loci for any disease and offers insight into the transcriptional regulatory mechanisms underlying SLE.
Preterm birth (PTB), or birth that occurs earlier than 37 weeks of gestational age, is a major contributor to infant mortality and neonatal hospitalization.Mutations in the mitochondrial genome (mtDNA) have been linked to various rare mitochondrial disorders and may be a contributing factor in PTB given that maternal genetic factors have been strongly linked to PTB. However, to date, no study has found a conclusive connection between a particular mtDNA variant and PTB. Given the high mtDNA copy number per cell, an automated pipeline was developed for detecting mtDNA variants using low-coverage wholegenome sequencing (lcWGS) data. The pipeline was first validated against samples of known heteroplasmy, and then applied to 929 samples from a PTB cohort from diverse ethnic backgrounds with an average gestational age of 27.18 weeks (range: 21-30). Our new pipeline successfully identified haplogroups and a large number of mtDNA variants in this large PTB cohort, including 8 samples carrying known pathogenic variants and 47 samples carrying rare mtDNA variants. These results confirm that lcWGS can be utilized to reliably identify mtDNA variants. These mtDNA variants may make a contribution toward preterm birth in a small proportion of live births.
Early diagnosis of Turner syndrome enhances care, but in routine practice, even within larger referral centers, diagnosis is delayed. Our study examines the utility of an electronic health record (EHR) algorithm in identifying patients at high risk for TS. Six percent of those identified had missed diagnoses of TS.Turner syndrome results from a complete or partial loss of the X chromosome and affects 25-50 per 100,000 females (1). TS is relatively common in females with unexplained short stature, but the heterogenous phenotype may result in a delayed diagnosis if classic features (e.g. lymphedema and webbed neck) are not present at birth. Only 20-40% of affected individuals are diagnosed in the newborn period with the remaining cases caught during late childhood with abnormal growth or in adolescence with lack of pubertal development (2-4). Savendhahl et al evaluated the age of diagnosis in 81 girls with TS, of which about half were diagnosed in infancy. If not diagnosed in infancy, the mean age of diagnosis was 8.8 ± 5 years (delay in diagnosis averaged 7.7 ± 5.4 years) at a mean height of −2.9 standard deviations (SD) (5). Studies have shown that combined auxological measures (an absolute height SD < −2 and a distance to target height of > 2 SD) provide the best predictive value for pathological short stature, and in patients >3 years old the distance to target height is the most important criterion (4, 6, 7). Careful growth monitoring with attention to such measures is paramount to a timely diagnosis of TS, especially in cases of mosaicism where short stature may be the only obvious sign. Furthermore, with early detection and initiation
Genome-wide association studies of Systemic Lupus Erythematosus (SLE) nominate 3,073 genetic variants at 91 risk loci. To systematically screen these variants for allelic transcriptional enhancer activity, we constructed a massively parallel reporter assay (MPRA) library comprising 12,396 DNA oligonucleotides containing the genomic context around every allele of each SLE variant. Transfection into EBV-infected B cells revealed 482 variants with enhancer activity, with 51 variants showing genotype-dependent (allelic) enhancer activity at 27 risk loci. In-depth analysis of allelic transcription factor (TF) binding at and around these 51 variants identified one class of TFs whose DNA-binding motif tends to be directly altered by the risk variant and a second, larger class of TFs that also bind allelically but do not have their motifs directly altered by the variant. Collectively, our approach provides a blueprint for the discovery of allelic gene regulation at risk loci for any disease and offers insight into the transcriptional regulatory mechanisms underlying SLE.
ALL, the most common pediatric cancer, originates from progenitor B-cells in ~85% of cases. IKAROS Family Zinc Finger 1 (IKZF1) is an important locus for ALL. Our goal was to identify the functional variants, thereby nominating a potential causal mechanism for ALL. We evaluated all 27 genetic polymorphisms in disequilibrium at r2>0.8 with the tag, the most highly associated risk variant at IKZF1 locus. They map to intron 7, 3′ UTR and 3′ flanking sequence of IKZF1. Fidgetin-like protein 1 (FIGNL1) is 45,524 bases from the tag variant and is nominated by eQTL and DNA looping data along with IKZF1 as possible targets. Epstein-Barr virus (EBV) transformed GM12878 cells were transduced with lentiviral vectors for stable expression of fusion proteins dead (d) Cas9-VP64 and activator MS2-p65-HSF1 (synergistic activation mediator (SAM) system). We designed 27 single guide (sg) RNAs, one for each of the 27 variants and evaluated expression perturbation in cells transfected with pools and individual sgRNA. Expression of FIGNL1, but not IKZF1, was convincingly altered in this system. The reduction of FIGNL1 expression by ~50% was isolated to two neighboring variants separated by 1 kb. The risk allele at these variants increases FIGNL1 expression in wild type cells, however in EBV transformed B-cells FIGNL1 expression is decreased. Other data show that the EBV encoded DNA binding co-factor EBNA3C binds at or very near this locus in EBV transformed B cells. Our results nominate two closely located SNPs in the last intron of IKZF1 as potentially causal variants, controlling the expression of FIGNL1, which ordinarily participates in homologous recombination during DNA double-strand break repair, maintenance of genomic stability and prevention of cancer.
BACKGROUND: Turner syndrome (TS) results from a complete or partial loss of the second X chromosome and affects 25-50 per 100,000 females. TS is common in females with unexplained short stature, but the diagnosis is often not made until late childhood (8-9 years) if classic features are not present or recognized in infancy. This results in delayed medical intervention and screening for comorbid conditions. The aim of our study was to determine if an electronic health record (EHR) notification model improves the timing and rate of detection of TS. METHODS/RESULTS: A search of the EHR was performed to identify a cohort of females with idiopathic short stature (ISS) who were seen in the endocrine clinic from 2012-2017. Selection criteria included a height ≤ -2 SD, BMI > 5%-ile, absence of chronic illness, and presence of mid-parental height (MPH) data, which yielded 216 patients. Given that a height deflection from MPH %-ile is one of the better predictors of TS 1 , we focused on those ≥ 1 SD below MPH %-ile. Of these 189 patients, 72 (38%) hadn’t received prior genetic testing. This group formed our study population and microarray analysis was performed on available samples to assess for undiagnosed TS or other chromosomal abnormalities. A total of 39 patient samples were prospectively recruited or obtained from an IRB approved biobank (including 5 TS controls). Microarray data was generated for 37 samples, as 2 had an insufficient quantity of DNA, and data was manually analyzed by a cytogeneticist. We identified two cases of undiagnosed TS (6%) and one with a different chromosomal abnormality (3%). One of these patients returned to endocrine clinic prior to microarray analysis and had a karyotype of 45,X/46,XY. The two other chromosomal abnormalities were picked up by microarray. One had mosaic monosomy X (45,X/46,XX). The other had a 2.7 Mb deletion from chromosome 1 (1q25.3->1q31.1) and a 2.1 Mb duplication from 22q11.21, which has been associated with short stature. CONCLUSIONS: The EHR algorithm was effective at identifying patients at risk for TS that merit genetic testing. Of the ISS females ≥ 1 SD below their MPH %-ile, 38% never had a karyotype done and on further investigation two new cases of TS (6%) and one other chromosomal abnormality (3%) were found. Although not all patients had DNA available for microarray analysis, if we assume that all other cases were negative, the rate of undiagnosed TS would still approach 3%. Extrapolating this data to a larger scale suggests that the diagnosis of TS may be delayed for many females with ISS, even after evaluation in endocrine clinics. We recommend the implementation of additional tools in the clinic workflow, such as EHR alerts based on specific growth parameters, to increase clinical suspicion and testing for TS. REFERENCES: 1. Grote, FK, et al (2008). Developing evidence-based guidelines for referral for short stature.&nbs...
OBJECTIVES/GOALS: Juvenile idiopathic arthritis (JIA) is the most common childhood rheumatologic disease childhood and a cause of pain and potential disability. JIA has a strong genetic component and no known cure. The goal of this study is to evaluate allele-dependent effects of a novel JIA risk variant at 1q24.3. METHODS/STUDY POPULATION: JIA patients meeting criteria for the two most common disease subtypes (oligoarticular and RF neg polyarthritis) were genotyped using the Immunochip, an Illumina array with dense coverage of the HLA region and 186 other loci previously reported in autoimmune diseases. Phase I association findings (Hinks, 2013) and Phase II analysis (unpublished) of an expanded cohort (4,271 JIA and 14,390 controls) identified new risk loci, including rs78037977 at 1q24.3. We prioritized rs78037977 and predicted possible impacted mechanisms based on Bayesian predictions of attributable risk, the surrounding chromatin landscape, and transcription factor binding data. A luciferase reporter assay was used to assess allele-dependent enhancer activity. RESULTS/ANTICIPATED RESULTS: rs78037977 is located between FASLG and TNFSF18 at chromosome 1q24.3 is associated with JIA (p = 6.3x10−09), and explains 94% of the posterior probability at this locus; no other SNPs in linkage disequilibrium (r2>0.6). The chromatin landscape around rs78037977 contains H3K4Me1 and H3K27Ac marks, which are indicative of enhancer activity. Further, >160 transcription factors have chromatin immunoprecipitation followed by sequencing (ChIP-seq) peaks overlapping rs78037977 in various cellular contexts. In luciferase reporter assays, the region around rs78037977 containing the reference A allele had ~2-fold increased enhancer activity compared to the non-reference allele. DISCUSSION/SIGNIFICANCE OF IMPACT: This work provides in vitro evidence to support allele-dependent enhancer activity of a novel JIA-risk variant at 1q24.3. Our ongoing work investigates the effect of the DNA-containing region of rs78037977 on gene expression and differential transcription factor binding at rs78037977.
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