Faculty Bibliography

Fanconi anemia (FA) is a rare genetic disease characterized by loss-of-function variants in any of the 22 previously identified genes (FANCA-FANCW) that encode proteins participating in the repair of DNA interstrand crosslinks (ICLs). Patient phenotypes are variable, but may include developmental abnormalities, early onset pancytopenia, and predisposition to hematologic and solid tumors. Here, we describe two unrelated families with multiple pregnancy losses and offspring presenting with severe developmental and hematologic abnormalities leading to death in utero or in early life. Homozygous loss-of-function variants in FAAP100 were identified in affected children of both families. The FAAP100 protein associates with FANCB and FANCL, the E3 ubiquitin ligase responsible for the monoubiquitination of FANCD2 and FANCI, which is necessary for FA pathway function. Patient-derived cells exhibited phenotypes consistent with FA. Expression of the wild-type FAAP100 cDNA, but not the patient-derived variants, rescued the observed cellular phenotypes. This establishes FAAP100 deficiency as a cause of Fanconi anemia, with FAAP100 gaining an alias as FANCX. The extensive developmental malformations of individuals with FAAP100 loss-of-function variants are among the most severe across previously described FA phenotypes, indicating that the FA pathway is essential for human development.

Mutations that accumulate in the human male germline with age are a major driver of genetic diversity and contribute to genetic diseases. However, aging-related male germline mutation rates have not been measured directly in germline cells (sperm) at the level of individuals. We developed a study design in which we recalled 23 sperm donors with prior banked samples to provide new sperm samples. The old and new sequential sperm samples were separated by long timespans, ranging from 10 to 33 years. We profiled these samples by high-fidelity duplex sequencing and demonstrate that direct high-fidelity sequencing of sperm yields cohort-wide mutation rates and patterns consistent with prior family-based (trio) studies. In every individual, we detected an increase in sperm mutation burden between the two sequential samples, yielding individual-specific measurements of germline mutation rate. Deep whole-genome sequencing of sequential sperm samples from two individuals followed by targeted validation measured remarkably stable mosaicism of clonal mutations that likely arose during embryonic and germline development, suggesting that age did not substantially impact the diversity of spermatogonial stem cell pools in these individuals. Our application of high-fidelity and deep whole-genome sequencing to sequential sperm samples provides insight into aging-related mutation processes in the male germline.

BACKGROUND:The diagnosis and treatment of tumors often depend on molecular-genetic data. However, rapid and iterative access to molecular data is not currently feasible during surgery, complicating intraoperative diagnosis and precluding measurement of tumor cell burdens at surgical margins to guide resections. METHODS:Here, we introduce Ultra-Rapid droplet digital PCR (UR-ddPCR), a technology that achieves the fastest measurement, to date, of mutation burdens in tissue samples, from tissue to result in 15 min. Our workflow substantially reduces the time from tissue biopsy to molecular diagnosis and provides a highly accurate means of quantifying residual tumor infiltration at surgical margins. FINDINGS/RESULTS: = 0.995). CONCLUSIONS:The technology and workflow developed here enable intraoperative molecular-genetic assays with unprecedented speed and sensitivity. We anticipate that our method will facilitate novel point-of-care diagnostics and molecularly guided surgeries that improve clinical outcomes. FUNDING/BACKGROUND:This study was funded by the National Institutes of Health and NYU Grossman School of Medicine institutional funds. Reagents and instruments were provided in kind by Bio-Rad.

Hirschsprung disease (HSCR) exhibits extensive genetic heterogeneity, with 72% of cases involving pathogenic variants in 10 genes forming a gene regulatory network (GRN) essential for enteric nervous system (ENS) development. The receptor tyrosine kinase gene RET is the most significant contributor, implicated in 12%-50% of individuals depending on the phenotype. RET plays a critical role in ENS precursor proliferation and migration, and defects in these processes lead to HSCR. However, the functional impact of RET pathogenic variants and their mechanisms of disease remain poorly understood. To address this, we investigated proliferative and migratory phenotypes in a RET-dependent neural crest-derived cell line harboring one of five missense (c.166C>A [p.Leu56Met]; c.532G>C [p.Glu178Gln]; c.2372A>T [p.Tyr791Phe]; c.2765C>A [p.Ser922Tyr]; or c.2994T>A [p.Phe998Leu]) or three nonsense (c.612C>A, c.2308C>T, or c.2943C>G) heterozygous pathogenic RET variants. Using cDNA- and CRISPR-based prime reverse insertion mechanism engineering (PRIME) editing coupled with quantitative proliferation and migration assays, we observed significant losses in proliferation and migration in three missense (c.612C>A [p.Tyr204^∗]; c.2308C>T [p.Arg770^∗]; and c.2943C>G [p.Tyr981^∗]) and all nonsense variants. Notably, the c.2372A>T (p.Tyr791Phe) missense variant, whose pathogenicity has been debated, appears benign. Importantly, the severity of migration loss did not consistently correlate with proliferation defects, and the phenotypic severity of nonsense variants was independent of their position within the RET protein. This study highlights the necessity of targeted functional assays to accurately assess the pathogenicity of HSCR-associated variants rather than relying solely on bioinformatics predictions, which could be refined by incorporating functional data.

Coding and enhancer variants of the RET receptor tyrosine kinase gene contribute to ~50% of Hirschsprung disease (HSCR) risk, a congenital disorder of disrupted enteric nervous system (ENS) development. The greatest contribution of this risk is from a common variant (rs2435357) in an ENS-active, SOX10-bound RET enhancer (MCS+9.7) that reduces RET gene expression in vivo and triggers expression changes in other ENS genes in the human fetal gut. To uncover the cellular basis of RET-mediated aganglionosis, we used CRISPR/Cas9 to delete (Δ) the homologous mouse enhancer (mcs+9.7). We used single cell RNA sequencing and high-resolution immunofluorescence to demonstrate four significant features of the developing E14.5 gut of Δmcs+9.7/Δmcs+9.7 embryos: (1) a small (5%) yet significant reduction in Ret gene expression in only two major cell types - early differentiating neurons and fate-restricted inhibitory motor neurons; (2) no significant cellular loss in the ENS; and, (3) loss of expression of 19 cell cycle regulator genes suggesting a proliferative defect. To identify the Ret functional threshold for normal ENS development, we also generated, in combination with the Ret CFP null allele, (4) Δmcs+9.7/CFP double heterozygote mice which reduced Ret gene expression in the ENS to 42% with severe loss of inhibitory motor neurons, an effect restricted to the hindgut and driven by proliferative loss. Thus, Ret gene expression drives proliferation of ENS progenitor cells and hindgut-specific inhibitory motor neuron development, and that HSCR aganglionosis arises from a cascade of cellular defects triggered by >50% loss of Ret function.

Despite the extensive genetic heterogeneity of Hirschsprung disease (HSCR; congenital colonic aganglionosis) 72% of patients harbor pathogenic variants in 10 genes that form a gene regulatory network (GRN) controlling the development of the enteric nervous system (ENS). Among these genes, the receptor tyrosine kinase gene RET is the most significant contributor, accounting for pathogenic variants in 12%-50% of patients depending on phenotype. RET plays a critical role in the proliferation and migration of ENS precursors, and defects in these processes lead to HSCR. However, despite the gene's importance in HSCR, the functional consequences of RET pathogenic variants and their mechanism of disease remain poorly understood. To address this, we investigated the proliferative and migratory phenotypes in a RET-dependent neural crest-derived cell line harboring one of five missense (L56M, E178Q, Y791F, S922Y, F998L) or three nonsense (Y204X, R770X, Y981X) pathogenic heterozygous variants. Using a combination of cDNA-based and CRISPR-based PRIME editing coupled with quantitative proliferation and migration assays, we detected significant losses in cell proliferation and migration in three missense (E178Q, S922Y, F998L) and all nonsense variants. Our data suggests that the Y791F variant, whose pathogenicity has been debated, is likely not pathogenic. Importantly, the severity of migration loss did not consistently correlate with proliferation defects, and the phenotypic severity of nonsense variants was independent of their position within the RET protein. This study highlights the necessity and feasibility of targeted functional assays to accurately assess the pathogenicity of HSCR-associated variants, rather than relying solely on machine learning predictions, which could themselves be refined by incorporating such functional data.

Microsatellites are highly mutable sequences that can serve as markers for relationships among individuals or cells within a population. The accuracy and resolution of reconstructing these relationships depends on the fidelity of microsatellite profiling and the number of microsatellites profiled. However, current methods for targeted profiling of microsatellites incur significant "stutter" artifacts that interfere with accurate genotyping, and sequencing costs preclude whole-genome microsatellite profiling of a large number of samples. We developed a novel method for accurate and cost-effective targeted profiling of a panel of more than 150,000 microsatellites per sample, along with a computational tool for designing large-scale microsatellite panels. Our method addresses the greatest challenge for microsatellite profiling-"stutter" artifacts-with a low-temperature hybridization capture that significantly reduces these artifacts. We also developed a computational tool for accurate genotyping of the resulting microsatellite sequencing data that uses an ensemble approach integrating three microsatellite genotyping tools, which we optimize by analysis of de novo microsatellite mutations in human trios. Altogether, our suite of experimental and computational tools enables high-fidelity, large-scale profiling of microsatellites, which may find utility in diverse applications such as lineage tracing, population genetics, ecology, and forensics.

BACKGROUND/UNASSIGNED:Inter-individual variation in blood pressure (BP) arises in part from sequence variants within enhancers modulating the expression of causal genes. We propose that these genes, active in tissues relevant to BP physiology, can be identified from tissue-level epigenomic data and genotypes of BP-phenotyped individuals. METHODS/UNASSIGNED:We used chromatin accessibility data from the heart, adrenal, kidney, and artery to identify cis-regulatory elements (CREs) in these tissues and estimate the impact of common human single-nucleotide variants within these CREs on gene expression, using machine learning methods. To identify causal genes, we performed a gene-wise association test. We conducted analyses in 2 separate large-scale cohorts: 77 822 individuals from the Genetic Epidemiology Research on Adult Health and Aging and 315 270 individuals from the UK Biobank. RESULTS/UNASSIGNED:<0.0001). These results enabled tissue expression prediction of these 988 to 2875 putative BP genes in individuals of both cohorts to construct an expression polygenic score. This score explained ≈27% of the reported single-nucleotide variant heritability, substantially higher than expected from prior studies. CONCLUSIONS/UNASSIGNED:Our work demonstrates the power of tissue-restricted comprehensive CRE analysis, followed by CRE-based expression prediction, for understanding BP regulation in relevant tissues and provides dual-modality supporting evidence, CRE and expression, for the causality genes.

BACKGROUND:We examined effects of single-nucleotide variants (SNVs) of IL1RN, the gene encoding the anti-inflammatory interleukin 1 receptor antagonist (IL-1Ra), on the cytokine release syndrome (CRS) and mortality in patients with acute severe respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. METHODS:IL1RN CTA haplotypes formed from 3 SNVs (rs419598, rs315952, rs9005) and the individual SNVs were assessed for association with laboratory markers of inflammation and mortality. We studied 2589 patients hospitalized with SARS-CoV-2 between March 2020 and March 2021. RESULTS:Mortality was 15.3% and lower in women than men (13.1% vs 17.3%, P = .0003). Carriers of the CTA-1/2 IL1RN haplotypes exhibited decreased inflammatory markers and increased plasma IL-1Ra. Evaluation of the individual SNVs of the IL1RN, carriers of the rs419598 C/C SNV exhibited significantly reduced inflammatory biomarker levels and numerically lower mortality compared to the C/T-T/T genotype (10.0% vs 17.8%, P = .052) in men, with the most pronounced association observed in male patients ≤74 years old, whose mortality was reduced by 80% (3.1% vs 14.0%, P = .030). CONCLUSIONS:The IL1RN haplotype CTA and C/C variant of rs419598 are associated with attenuation of the CRS and decreased mortality in men with acute SARS-CoV-2 infection. The data suggest that the IL1RN pathway modulates the severity of coronavirus disease 2019 (COVID-19) via endogenous anti-inflammatory mechanisms.

Mutations accumulate in the genome of every cell of the body throughout life, causing cancer and other diseases^1,2. Most mutations begin as nucleotide mismatches or damage in one of the two strands of the DNA before becoming double-strand mutations if unrepaired or misrepaired^3,4. However, current DNA-sequencing technologies cannot accurately resolve these initial single-strand events. Here we develop a single-molecule, long-read sequencing method (Hairpin Duplex Enhanced Fidelity sequencing (HiDEF-seq)) that achieves single-molecule fidelity for base substitutions when present in either one or both DNA strands. HiDEF-seq also detects cytosine deamination-a common type of DNA damage-with single-molecule fidelity. We profiled 134 samples from diverse tissues, including from individuals with cancer predisposition syndromes, and derive from them single-strand mismatch and damage signatures. We find correspondences between these single-strand signatures and known double-strand mutational signatures, which resolves the identity of the initiating lesions. Tumours deficient in both mismatch repair and replicative polymerase proofreading show distinct single-strand mismatch patterns compared to samples that are deficient in only polymerase proofreading. We also define a single-strand damage signature for APOBEC3A. In the mitochondrial genome, our findings support a mutagenic mechanism occurring primarily during replication. As double-strand DNA mutations are only the end point of the mutation process, our approach to detect the initiating single-strand events at single-molecule resolution will enable studies of how mutations arise in a variety of contexts, especially in cancer and ageing.