Faculty Bibliography

Numerous applications in molecular biology and genomics require characterization of mutant DNA molecules present at low levels within a larger sample of non-mutant DNA. This is often achieved either by selectively amplifying mutant DNA, or by sequencing all the DNA followed by computational identification of the mutant DNA. However, selective amplification is challenging for insertions and deletions (indels). Additionally, sequencing all the DNA in a sample may not be cost effective when only the presence of a mutation needs to be ascertained rather than its allelic fraction. The MutS protein evolved to detect DNA heteroduplexes in which the two DNA strands are mismatched. Prior methods have utilized MutS to enrich mutant DNA by hybridizing mutant to non-mutant DNA to create heteroduplexes. However, the purity of heteroduplex DNA these methods achieve is limited because they can only feasibly perform one or two enrichment cycles. We developed a MutS-magnetic bead system that enables rapid serial enrichment cycles. With six cycles, we achieve complete purification of heteroduplex indel DNA originally present at a 5% fraction and over 40-fold enrichment of heteroduplex DNA originally present at a 1% fraction. This system may enable novel approaches for enriching mutant DNA for targeted sequencing. This article is protected by copyright. All rights reserved.

Au-Kline syndrome (AKS) is a neurodevelopmental disorder associated with multiple malformations and a characteristic facial gestalt. The first individuals ascertained carried de novo loss-of-function (LoF) variants in HNRNPK. Here, we report 32 individuals with AKS (26 previously unpublished), including 13 with de novo missense variants. We propose new clinical diagnostic criteria for AKS that differentiate it from the clinically overlapping Kabuki syndrome and describe a significant phenotypic expansion to include individuals with missense variants who present with subtle facial features and few or no malformations. Many gene-specific DNA methylation (DNAm) signatures have been identified for neurodevelopmental syndromes. Because HNRNPK has roles in chromatin and epigenetic regulation, we hypothesized that pathogenic variants in HNRNPK may be associated with a specific DNAm signature. Here, we report a unique DNAm signature for AKS due to LoF HNRNPK variants, distinct from controls and Kabuki syndrome. This DNAm signature is also identified in some individuals with de novo HNRNPK missense variants, confirming their pathogenicity and the phenotypic expansion of AKS to include more subtle phenotypes. Furthermore, we report that some individuals with missense variants have an "intermediate" DNAm signature that parallels their milder clinical presentation, suggesting the presence of an epi-genotype phenotype correlation. In summary, the AKS DNAm signature may help elucidate the underlying pathophysiology of AKS. This DNAm signature also effectively supported clinical syndrome delineation and is a valuable aid for variant interpretation in individuals where a clinical diagnosis of AKS is unclear, particularly for mild presentations.

BACKGROUND:The availability of whole-genome sequencing data in large studies has enabled the assessment of coding and noncoding variants across the allele frequency spectrum for their associations with blood pressure. METHODS:We conducted a multiancestry whole-genome sequencing analysis of blood pressure among 51 456 Trans-Omics for Precision Medicine and Centers for Common Disease Genomics program participants (stage-1). Stage-2 analyses leveraged array data from UK Biobank (N=383 145), Million Veteran Program (N=318 891), and Reasons for Geographic and Racial Differences in Stroke (N=10 643) participants, along with whole-exome sequencing data from UK Biobank (N=199 631) participants. RESULTS:, respectively). DISCUSSION/CONCLUSIONS:We report one promising but unconfirmed rare variant for blood pressure and, more importantly, contribute insights for future blood pressure sequencing studies. Our findings suggest promise of aggregate analyses to complement single variant analysis strategies and the need for larger, diverse samples, and family studies to enable robust rare variant identification.

Type I interferon (IFN) is critical in our defense against viral infections. Increased type I IFN pathway activation is a genetic risk factor for systemic lupus erythematosus (SLE), and a number of common risk alleles contribute to the high IFN trait. We hypothesized that these common gain-of-function IFN pathway alleles may be associated with protection from mortality in acute COVID-19. We studied patients admitted with acute COVID-19 (756 European-American and 398 African-American ancestry). Ancestral backgrounds were analyzed separately, and mortality after acute COVID-19 was the primary outcome. In European-American ancestry, we found that a haplotype of interferon regulatory factor 5 (IRF5) and alleles of protein kinase cGMP-dependent 1 (PRKG1) were associated with mortality from COVID-19. Interestingly, these were much stronger risk factors in younger patients (OR=29.2 for PRKG1 in ages 45-54). Variants in the IRF7 and IRF8 genes were associated with mortality from COVID-19 in African-American subjects, and these genetic effects were more pronounced in older subjects. Combining genetic information with blood biomarker data such as C-reactive protein, troponin, and D-dimer resulted in significantly improved predictive capacity, and in both ancestral backgrounds the risk genotypes were most relevant in those with positive biomarkers (OR for death between 14 and 111 in high risk genetic/biomarker groups). This study confirms the critical role of the IFN pathway in defense against COVID-19 and viral infections, and supports the idea that some common SLE risk alleles exert protective effects in anti-viral immunity. BACKGROUND: We find that a number of IFN pathway lupus risk alleles significantly impact mortality following COVID-19 infection. These data support the idea that type I IFN pathway risk alleles for autoimmune disease may persist in high frequency in modern human populations due to a benefit in our defense against viral infections. TRANSLATIONAL SIGNIFICANCE: We develop multivariate prediction models which combine genetics and known biomarkers of severity to result in greatly improved prediction of mortality in acute COVID-19. The specific associated alleles provide some clues about key points in our defense against COVID-19.

Systolic and diastolic blood pressure (S/DBP) are highly correlated modifiable risk factors for cardiovascular disease (CVD). We report here a bidirectional Mendelian Randomization (MR) and horizontal pleiotropy analysis of S/DBP summary statistics from the UK Biobank (UKB)-International Consortium for Blood Pressure (ICBP) (UKB-ICBP) BP genome-wide association study and construct a composite genetic risk score (GRS) by including pleiotropic variants. The composite GRS captures greater (1.11-3.26 fold) heritability for BP traits and increases (1.09- and 2.01-fold) Nagelkerke's R² for hypertension and CVD. We replicated 118 novel BP horizontal pleiotropic variants including 18 novel BP loci using summary statistics from the Million Veteran Program (MVP) study. An additional 219 novel BP signals and 40 novel loci were identified after a meta-analysis of the UKB-ICBP and MVP summary statistics but without further independent replication. Our study provides further insight into BP regulation and provides a novel way to construct a GRS by including pleiotropic variants for other complex diseases.

BACKGROUND:While large genome-wide association studies have identified nearly one thousand loci associated with variation in blood pressure, rare variant identification is still a challenge. In family-based cohorts, genome-wide linkage scans have been successful in identifying rare genetic variants for blood pressure. This study aims to identify low frequency and rare genetic variants within previously reported linkage regions on chromosomes 1 and 19 in African American families from the Trans-Omics for Precision Medicine (TOPMed) program. Genetic association analyses weighted by linkage evidence were completed with whole genome sequencing data within and across TOPMed ancestral groups consisting of 60,388 individuals of European, African, East Asian, Hispanic, and Samoan ancestries. RESULTS:). CONCLUSIONS:Low frequency and rare variants in RCN3 contributes blood pressure variation. This study demonstrates that focusing association analyses in linkage regions greatly reduces multiple-testing burden and improves power to identify novel rare variants associated with blood pressure traits.

PURPOSE/OBJECTIVE:Hirschsprung disease (HSCR) is a developmental disorder of the enteric nervous system (ENS) characterized by congenital aganglionosis arising from coding variants in ENS genes causing partial or total loss-of-function. Low-penetrance, common, noncoding variants at RET, SEMA3 and NRG1 loci are also associated with HSCR, with small-to-moderate loss of gene expression mediated through sequence variants in cis-regulatory elements (CRE) as another causal mechanism. Since these latter variants are common, many individuals carry multiple risk variants. However, the extent and combinatorial effects of all putative CRE variants within and across these loci on HSCR is unknown. METHODS:Using 583 HSCR subjects, one of the largest samples of European ancestry studied, and genotyping 56 tag variants, we evaluated association of all common variants overlapping putative gut CREs and fine-mapped causal variants at RET, SEMA3 and NRG1. RESULTS:We demonstrate that 28 and 8 tag variants, several of which are genetically independent, overlap putative-enhancers at the RET and SEMA3 loci, respectively, as well as two fine-mapped tag variants at the NRG1 locus, are significantly associated with HSCR. Importantly, disease risk increases with increasing numbers of risk alleles from multiple variants within and across these loci, varying >25-fold across individuals. CONCLUSION/CONCLUSIONS:This increasing allele number-dependent risk, we hypothesize, arises from HSCR-relevant ENS cells sensing the reduced gene expression at multiple ENS genes since their developmental effects are integrated through gene regulatory networks.

The major genetic risk factors for Hirschsprung disease (HSCR) are three common polymorphisms within cis-regulatory elements (CREs) of the receptor tyrosine kinase gene RET, which reduce its expression during enteric nervous system (ENS) development. These risk variants attenuate binding of the transcription factors RARB, GATA2, and SOX10 to their cognate CREs, reduce RET gene expression, and dysregulate other ENS and HSCR genes in the RET-EDNRB gene regulatory network (GRN). Here, we use siRNA, ChIP, and CRISPR-Cas9 deletion analyses in the SK-N-SH cell line to ask how many additional HSCR-associated risk variants reside in RET CREs that affect its gene expression. We identify 22 HSCR-associated variants in candidate RET CREs, of which seven have differential allele-specific in vitro enhancer activity, and four of these seven affect RET gene expression; of these, two enhancers are bound by the transcription factor PAX3. We also show that deleting multiple variant-containing enhancers leads to synergistic effects on RET gene expression. These, coupled with our prior results, show that common sequence variants in at least 10 RET enhancers affect HSCR risk, seven with experimental evidence of affecting RET gene expression, extending the known RET-EDNRB GRN to reveal an extensive regulatory code modulating disease risk at a single gene.