Faculty Bibliography

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.

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.

PURPOSE/OBJECTIVE(S): Olfactory neuroblastoma (ONB) and sinonasal undifferentiated carcinoma (SNUC) are rare sinonasal cancers with distinct clinical courses. Methylomic differences between these entities have not been previously explored. The aims of this study were to compare genome-wide methylomes of ONB and SNUC, and to explore candidate tissue of origin of SNUC using a comparative methylomics approach. MATERIALS/METHODS: We included a total of 87 sinonasal malignancy samples. Genome-wide methylome data were obtained with an SNP-based human microarray for 71 ONB-like samples (44 "definitive" ONB samples, 27 ONB-resembling samples) and 10 IDH2-mutatnt SNUC samples. RNA sequencing data for a further 3 ONB and 3 SNUC samples were included. Methylomes were compared between ONB and SNUC samples and to publicly available methylation data for 195 tissue samples representing an array of tumors and normal tissues using R. Strict quality control was performed.
RESULT(S): A total of 41 ONB and 9 SNUC samples passed QC and were included in the final analysis. A total of 89,016 differentially methylated probes had adjusted P-value < 0.01, the majority (90.4%) of which were hypermethylated in SNUC and located predominantly in regulatory regions, likely reflecting IDH2 mutation. The remaining 9.6% were hypomethylated in SNUC samples and were commonly located in intergenic regions. Top differentially methylated regions were GATA3, IGF2, LSP1, NKX6-2 and UNCX. NKX6-2 and UNCX were not expressed in SNUC RNAseq data. Gene set enrichment analysis (GSEA) of differentially hypermethylated CpG-island associated probes with at least 2.5-fold-decrease in SNUC RNA-seq data showed enrichment of EZH2 targets, H3K27 bound genes consistent with previous GSEA in IDH2-mutat sarcomas, in addition to WNT pathway, olfactory transduction, lipid & carbohydrate metabolism and genes pertaining to the nervous system (P < 0.0001). On Pearson's correlation-based analysis, ONB clustered with neuronal samples while SNUC clustered with medulloblastoma (MB) and malignant peripheral nerve sheath tumor (MPNST). On separate correlation, SNUC clustered with T-cell lymphoma, MPNST and MB. On examination of the top 300 differentially methylated probes, SNUC again clustered with MPNST and MB and with a neural crest cluster. On deconvolution analysis, both ONB and SNUC samples showed a predominant T-cell presence, which was significantly higher in SNUC (P < 0.001).
CONCLUSION(S): ONB and SNUC possess distinct methylomes. PRC2 complex dysregulation may represent a key driver of a genome-wide repressive phenotype in IDH2-mutant SNUC samples leading to a dedifferentiated phenotype and may be a potential avenue for targeted therapies in the future. The presence of T-cell infiltration in both tumors represents a potential avenue for exploration of therapeutic checkpoint inhibition. Genome-wide methylomics suggests SNUC may be derived from neuronal tissue and we are currently involved in further transomic approaches to independently validate this hypothesis.
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In medical genetics, the vast majority of patients with a currently known genetic basis harbor a rare deleterious allele explaining its Mendelian inheritance. Increasingly, for these phenotypes, we recognize exceptions to Mendelian expectations from non-penetrance of clinical disease to significant inter-individual variation in clinical manifestations, likely reflecting the actions of additional modifier genes. Despite recent progress, we still remain ignorant about the molecular basis for many rare disorders presumed to be Mendelian. The molecular evidence increasingly suggests a role for multiple genes in some of these cases, but for how many? In this article, I discuss why equating a phenotype as Mendelian or complex may be short-sighted or even erroneous. As we learn more about the functions of the human genome with its genes in networks, we should view the phenotype of an individual patient as arising from his or her total genomic deleterious burden in a set of functionally inter-related genes affecting that phenotype. This can sometimes arise from deleterious allele(s) at a single gene (Mendelian inheritance) creating a specific biochemical deficiency (or excess) but could just as frequently arise from the cumulative effects of multiple disease alleles (complex inheritance) leading to the same biochemical deficiency (or excess).

Systolic and diastolic blood pressure (S/

Massively parallel reporter assays (MPRAs) are a high-throughput method for evaluating in vitro activities of thousands of candidate cis-regulatory elements (CREs). In these assays, candidate sequences are cloned upstream or downstream from a reporter gene tagged by unique DNA sequences. However, tag sequences may themselves affect reporter gene expression and lead to major potential biases in the measured cis-regulatory activity. Here, we present a sequence-based method for correcting tag-sequence-specific effects and show that our method can significantly reduce this source of variation and improve the identification of functional regulatory variants by MPRAs. We also show that our model captures sequence features associated with post-transcriptional regulation of mRNA. Thus, this new method helps not only to improve detection of regulatory signals in MPRA experiments but also to design better MPRA protocols.

Background/Purpose: 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 alleles contribute to the genetic high IFN trait. In this study, we examine whether these common gain-of-function alleles in the type I IFN pathway are associated with protection from mortality in acute COVID-19.
Method(s): We studied IFN pathway SLE risk genes in patients with acute COVID-19 admitted to NYU Langone hospitals (751 European-American and 398 African-American ancestry). The samples were genotyped using low depth sequencing and imputation, and we analyzed data from the following SNPs: IRF5 (rs2004640, rs3807306, rs10488631, rs2280714), IRF7/PHRF (rs1131665, rs4963128), IRF8 (rs17445836, rs12444486), and PRKG1 (rs7897633). Ancestral backgrounds were analyzed separately, and mortality after acute COVID-19 was the primary outcome.
Result(s): We observed specific IRF5 haplotypes that are protective against SLE risk were associated with increased risk of mortality in acute COVID-19 patients in European-American ancestry (OR=3.74, p=0.015). Alleles of PRKG1 were also associated with mortality from COVID-19 in the European-American ancestry cohort (OR=1.80, p=0.0057), and this risk factor was particularly strong in younger patients (OR=29.2, p=0.01 in ages 45-54). IRF8 genotype at rs1244486 was associated with protection from mortality in COVID-19 in African-American subjects aged 65 and older (OR=0.34, p=0.04).
Conclusion(s): We find that a number of type I IFN pathway genes associated with risk of SLE also modulate risk of death during acute COVID-19. Similar to their associations with SLE, these alleles are variably associated with COVID-19 mortality across ancestral backgrounds, suggesting ancestral differences in the genetic regulation of the IFN pathway. These data confirm the critical role of the IFN pathway in our defense against viral infections, and support the idea that some common SLE risk alleles exert protective effects in anti-viral immunity

Over the past decade, genomic analyses of single cells-the fundamental units of life-have become possible. Single-cell DNA sequencing has shed light on biological questions that were previously inaccessible across diverse fields of research, including somatic mutagenesis, organismal development, genome function, and microbiology. Single-cell DNA sequencing also promises significant future biomedical and clinical impact, spanning oncology, fertility, and beyond. While single-cell approaches that profile RNA and protein have greatly expanded our understanding of cellular diversity, many fundamental questions in biology and important biomedical applications require analysis of the DNA of single cells. Here, we review the applications and biological questions for which single-cell DNA sequencing is uniquely suited or required. We include a discussion of the fields that will be impacted by single-cell DNA sequencing as the technology continues to advance. Expected final online publication date for the Annual Review of Genomics and Human Genetics Volume 22 is August 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

PURPOSE/OBJECTIVE:The endoplasmic reticulum membrane complex (EMC) is a highly conserved, multifunctional 10-protein complex related to membrane protein biology. In seven families, we identified 13 individuals with highly overlapping phenotypes who harbor a single identical homozygous frameshift variant in EMC10. METHODS:Using exome, genome, and Sanger sequencing, a recurrent frameshift EMC10 variant was identified in affected individuals in an international cohort of consanguineous families. Multiple families were independently identified and connected via Matchmaker Exchange and internal databases. We assessed the effect of the frameshift variant on EMC10 RNA and protein expression and evaluated EMC10 expression in normal human brain tissue using immunohistochemistry. RESULTS:A homozygous variant EMC10 c.287delG (Refseq NM_206538.3, p.Gly96Alafs*9) segregated with affected individuals in each family, who exhibited a phenotypic spectrum of intellectual disability (ID) and global developmental delay (GDD), variable seizures and variable dysmorphic features (elongated face, curly hair, cubitus valgus, and arachnodactyly). The variant arose on two founder haplotypes and results in significantly reduced EMC10 RNA expression and an unstable truncated EMC10 protein. CONCLUSION/CONCLUSIONS:We propose that a homozygous loss-of-function variant in EMC10 causes a novel syndromic neurodevelopmental phenotype. Remarkably, the recurrent variant is likely the result of a hypermutable site and arose on distinct founder haplotypes.