Faculty Bibliography

Somatic mutations accumulate throughout life in every cell, and this process constitutes one of the hallmarks of aging-genomic instability. Caloric restriction (CR) has been shown to extend lifespan across diverse species. Using high-fidelity duplex DNA sequencing of bulk liver, bulk kidney, hepatocytes, and cerebellar neurons, we found that CR in mice reduces genome-wide somatic mutation burdens across multiple tissues and cell types. CR reduced both substitution and insertion/deletion burdens, with the magnitude of these effects varying across sample types. CR also decreased the activity of the enigmatic single-base substitution (SBS) mutational process SBS5 that gives rise to most mutations in mammals. Surprisingly, the mutation burden reduction from CR was greatest in transcriptionally inactive regions. This work illuminates links between diet, aging, and genomic integrity and establishes genomic integrity as a modifiable axis of aging.

Neurons accumulate somatic mutations with age, but how mutation processes vary among neuronal types remains unclear. Characterizing this variability may elucidate the role of genome integrity in brain function and disease and reveal determinants of mutation rates and patterns. Using high-fidelity duplex DNA sequencing, we profiled somatic mutations across the lifespan in human cerebellar Purkinje and granule neurons, which differ markedly in size and physiology. Surprisingly, they exhibited similar substitution rates, including rates of SBS5, the body's predominant mutational signature, whose mechanism is unknown. However, their substitution patterns and insertion/deletion rates and patterns differed, with transcription associated with these differences. In surviving granule neurons from five cerebellar ataxias, we detected only a small disease effect on mutation profiles. Our work indicates that neuronal types can differ in aging-related mutagenesis and that key features distinguishing Purkinje and granule neurons are unlikely, in these neurons, to be major determinants of SBS5 activity.

Episignatures are increasingly valuable for variant interpretation in rare neurodevelopmental disorders, especially when optimized to capture the impact of specific variant types and locations across a gene. Here, we generated a next-generation DNA methylation (DNAm) episignature for Kabuki syndrome type 1 (KS1) using the largest cohort studied to date, aiming to clarify the epigenomic and phenotypic effects of diverse KMT2D variant types and positions. Genome-wide DNAm profiles were obtained for 110 individuals with KMT2D variants and 854 controls using microarrays and long-read sequencing (LRS). Differentially methylated loci were enriched in genes involved in embryonic and nervous system development and were leveraged to construct a support-vector machine classifier for detecting pathogenic KMT2D variants. The classifier achieved 97% sensitivity and 100% specificity in validation cohorts and outperformed in silico tools, demonstrating stronger concordance with clinical presentation. Missense variants in the C-terminal region (exon 48) of KMT2D and the N-terminal plant homeodomain (PHD)-type zinc fingers were predominantly classified as pathogenic, highlighting regions enriched for pathogenic variants. Missense variants in the central region (exons 31-39) were more often predicted benign for KS1, consistent with potential association with a different syndrome, highlighting the classifier's specificity for KS1. Test performance was consistent across array and LRS platforms, and classifier scores reflected levels of mosaicism detected by LRS. The KS1 episignature also positively classified pathogenic KDM6A variants associated with KS2. These findings represent a significant advance in the evolution of episignature development, demonstrating diagnostic and interpretive value of a KS1 signature in resolving uncertain or complex cases.

INTRODUCTION/UNASSIGNED:have recently been identified as an ultra-rare cause of pediatric neurodevelopmental disorders (NDDs). However, the neurological and behavioral manifestations, genotype-phenotype correlations, and underlying disease mechanisms remain poorly understood due to the limited number of reported families. METHODS/UNASSIGNED:We describe a cohort of families presenting with microcephaly, global developmental delay, speech impairment, seizures and/or EEG abnormalities, movement disorders and severe behavioral disorders. Clinical assessments and brain imaging studies were conducted over a 10-year follow-up period. Genetic analysis was performed via whole-exome sequencing (WES), and structural modeling was used to investigate the functional impact of the identified variants. RESULTS/UNASSIGNED:-tubulin heterodimer formation, impairing binding stability at this critical interaction site. DISCUSSION/UNASSIGNED:-tubulin interactions.

From fertilization onwards, the cells of the human body acquire variations in their DNA sequence, known as somatic mutations. These postzygotic mutations arise from intrinsic errors in DNA replication and repair, as well as from exposure to mutagens. Somatic mutations have been implicated in some diseases, but a fundamental understanding of the frequency, type and patterns of mutations across healthy human tissues has been limited. This is primarily due to the small proportion of cells harbouring specific somatic variants within an individual, making them more challenging to detect than inherited variants. Here we describe the Somatic Mosaicism across Human Tissues Network, which aims to create a reference catalogue of somatic mutations and their clonal patterns across 19 different tissue sites from 150 non-diseased donors and develop new technologies and computational tools to detect somatic mutations and assess their phenotypic consequences, including clonal expansions. This strategy enables a comprehensive examination of the mutational landscape across the human body, and provides a comparison baseline for somatic mutation in diseases. This will lead to a deep understanding of somatic mutations and clonal expansions across the lifespan, as well as their roles in health, in ageing and, by comparison, in diseases.

Many chemotherapeutic agents impair cancer growth by inducing DNA damage. The impact of these agents on mutagenesis in normal cells, including sperm, is largely unknown. Here, we applied high-fidelity duplex sequencing to 94 samples from 36 individuals exposed to diverse chemotherapies and 32 controls. We found that many of the sperm samples from men exposed to chemotherapy, the mutation burden was elevated as compared to controls and the expected burden based on trio studies, with one subject having >10-fold increase over expected for age. Saliva from this same individual also had a markedly higher mutation burden. We then validated this finding using other tissues, also finding an increased mutation burden in the blood and liver of many subjects exposed to chemotherapy as compared to unexposed controls. Similarly, mice treated with three cycles of cisplatin had an increased mutation burden in sperm but also in the liver, and hematopoietic progenitor cells. These results suggest an association between cancer therapies and mutation burden, with implications for counseling cancer patients considering banking sperm prior to therapy and for cancer survivors considering the tradeoffs of using banked sperm as compared to conceiving naturally.

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.

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.