Functional interrogation of DNA damage response variants with base editing screens.

Mutations in DNA damage response (DDR) genes endanger genome integrity and predispose to cancer and genetic disorders. Here, using CRISPR-dependent cytosine base editing screens, we identify > 2,000 sgRNAs that generate nucleotide variants in 86 DDR genes, resulting in altered cellular fitness upon DNA damage. Among those variants, we discover loss- and gain-of-function mutants in the Tudor domain of the DDR regulator 53BP1 that define a non-canonical surface required for binding the deubiquitinase USP28. Moreover, we characterize variants of the TRAIP ubiquitin ligase that define a domain, whose loss renders cells resistant to topoisomerase I inhibition. Finally, we identify mutations in the ATM kinase with opposing genome stability phenotypes and loss-of-function mutations in the CHK2 kinase previously categorized as variants of uncertain significance for breast cancer. We anticipate that this resource will enable the discovery of additional DDR gene functions and expedite studies of DDR variants in human disease.

Tissue adaptation and clonal segregation of human memory T cells in barrier sites.

T lymphocytes migrate to barrier sites after exposure to pathogens, providing localized immunity and long-term protection. Here, we obtained blood and tissues from human organ donors to examine T cells across major barrier sites (skin, lung, jejunum), associated lymph nodes, lymphoid organs (spleen, bone marrow), and in circulation. By integrating single-cell protein and transcriptome profiling, we demonstrate that human barrier sites contain tissue-resident memory T (TRM) cells that exhibit site-adapted profiles for residency, homing and function distinct from circulating memory T cells. Incorporating T cell receptor and transcriptome analysis, we show that circulating memory T cells are highly expanded, display extensive overlap between sites and exhibit effector and cytolytic functional profiles, while TRM clones exhibit site-specific expansions and distinct functional capacities. Together, our findings indicate that circulating T cells are more disseminated and differentiated, while TRM cells exhibit tissue-specific adaptation and clonal segregation, suggesting that strategies to promote barrier immunity require tissue targeting.

Tissue Determinants of Human NK Cell Development, Function, and Residence.

Immune responses in diverse tissue sites are critical for protective immunity and homeostasis. Here, we investigate how tissue localization regulates the development and function of human natural killer (NK) cells, innate lymphocytes important for anti-viral and tumor immunity. Integrating high-dimensional analysis of NK cells from blood, lymphoid organs, and mucosal tissue sites from 60 individuals, we identify tissue-specific patterns of NK cell subset distribution, maturation, and function maintained across age and between individuals. Mature and terminally differentiated NK cells with enhanced effector function predominate in blood, bone marrow, spleen, and lungs and exhibit shared transcriptional programs across sites. By contrast, precursor and immature NK cells with reduced effector capacity populate lymph nodes and intestines and exhibit tissue-resident signatures and site-specific adaptations. Together, our results reveal anatomic control of NK cell development and maintenance as tissue-resident populations, whereas mature, terminally differentiated subsets mediate immunosurveillance through diverse peripheral sites. VIDEO ABSTRACT.

Site-specific development and progressive maturation of human tissue-resident memory T cells over infancy and childhood.

Infancy and childhood are critical life stages for generating immune memory to protect against pathogens; however, the timing, location, and pathways for memory development in humans remain elusive. Here, we investigated T cells in mucosal sites, lymphoid tissues, and blood from 96 pediatric donors aged 0-10 years using phenotypic, functional, and transcriptomic profiling. Our results revealed that memory T cells preferentially localized in the intestines and lungs during infancy and accumulated more rapidly in mucosal sites compared with blood and lymphoid organs, consistent with site-specific antigen exposure. Early life mucosal memory T cells exhibit distinct functional capacities and stem-like transcriptional profiles. In later childhood, they progressively adopt proinflammatory functions and tissue-resident signatures, coincident with increased T cell receptor (TCR) clonal expansion in mucosal and lymphoid sites. Together, our findings identify staged development of memory T cells targeted to tissues during the formative years, informing how we might promote and monitor immunity in children.

Lineage specification of human dendritic cells is marked by IRF8 expression in hematopoietic stem cells and multipotent progenitors.

The origin and specification of human dendritic cells (DCs) have not been investigated at the clonal level. Through the use of clonal assays, combined with statistical computation, to quantify the yield of granulocytes, monocytes, lymphocytes and three subsets of DCs from single human CD34+ progenitor cells, we found that specification to the DC lineage occurred in parallel with specification of hematopoietic stem cells (HSCs) to the myeloid and lymphoid lineages. This started as a lineage bias defined by specific transcriptional programs that correlated with the combinatorial 'dose' of the transcription factors IRF8 and PU.1, which was transmitted to most progeny cells and was reinforced by upregulation of IRF8 expression driven by the hematopoietic cytokine FLT3L during cell division. We propose a model in which specification to the DC lineage is driven by parallel and inheritable transcriptional programs in HSCs and is reinforced over cell division by recursive interactions between transcriptional programs and extrinsic signals.

Spatial map of human T cell compartmentalization and maintenance over decades of life.

Mechanisms for human memory T cell differentiation and maintenance have largely been inferred from studies of peripheral blood, though the majority of T cells are found in lymphoid and mucosal sites. We present here a multidimensional, quantitative analysis of human T cell compartmentalization and maintenance over six decades of life in blood, lymphoid, and mucosal tissues obtained from 56 individual organ donors. Our results reveal that the distribution and tissue residence of naive, central, and effector memory, and terminal effector subsets is contingent on both their differentiation state and tissue localization. Moreover, T cell homeostasis driven by cytokine or TCR-mediated signals is different in CD4+ or CD8+ T cell lineages, varies with their differentiation stage and tissue localization, and cannot be inferred from blood. Our data provide an unprecedented spatial and temporal map of human T cell compartmentalization and maintenance, supporting distinct pathways for human T cell fate determination and homeostasis.

Common variants increase risk for congenital diaphragmatic hernia within the context of de novo variants.

Congenital diaphragmatic hernia (CDH) is a severe congenital anomaly often accompanied by other structural anomalies and/or neurobehavioral manifestations. Rare de novo protein-coding variants and copy-number variations contribute to CDH in the population. However, most individuals with CDH remain genetically undiagnosed. Here, we perform integrated de novo and common-variant analyses using 1,469 CDH individuals, including 1,064 child-parent trios and 6,133 ancestry-matched, unaffected controls for the genome-wide association study. We identify candidate CDH variants in 15 genes, including eight novel genes, through deleterious de novo variants. We further identify two genomic loci contributing to CDH risk through common variants with similar effect sizes among Europeans and Latinx. Both loci are in putative transcriptional regulatory regions of developmental patterning genes. Estimated heritability in common variants is ∼19%. Strikingly, there is no significant difference in estimated polygenic risk scores between isolated and complex CDH or between individuals harboring deleterious de novo variants and individuals without these variants. The data support a polygenic model as part of the CDH genetic architecture.

Reference-informed prediction of alternative splicing and splicing-altering mutations from sequences.

Alternative splicing plays a crucial role in protein diversity and gene expression regulation in higher eukaryotes, and mutations causing dysregulated splicing underlie a range of genetic diseases. Computational prediction of alternative splicing from genomic sequences not only provides insight into gene-regulatory mechanisms but also helps identify disease-causing mutations and drug targets. However, the current methods for the quantitative prediction of splice site usage still have limited accuracy. Here, we present DeltaSplice, a deep neural network model optimized to learn the impact of mutations on quantitative changes in alternative splicing from the comparative analysis of homologous genes. The model architecture enables DeltaSplice to perform "reference-informed prediction" by incorporating the known splice site usage of a reference gene sequence to improve its prediction on splicing-altering mutations. We benchmarked DeltaSplice and several other state-of-the-art methods on various prediction tasks, including evolutionary sequence divergence on lineage-specific splicing and splicing-altering mutations in human populations and neurodevelopmental disorders, and demonstrated that DeltaSplice outperformed consistently. DeltaSplice predicted ∼15% of splicing quantitative trait loci (sQTLs) in the human brain as causal splicing-altering variants. It also predicted splicing-altering de novo mutations outside the splice sites in a subset of patients affected by autism and other neurodevelopmental disorders (NDDs), including 19 genes with recurrent splicing-altering mutations. Integration of splicing-altering mutations with other types of de novo mutation burdens allowed the prediction of eight novel NDD-risk genes. Our work expanded the capacity of in silico splicing models with potential applications in genetic diagnosis and the development of splicing-based precision medicine.

PLS3 missense variants affecting the actin-binding domains cause X-linked congenital diaphragmatic hernia and body-wall defects.

Congenital diaphragmatic hernia (CDH) is a relatively common and genetically heterogeneous structural birth defect associated with high mortality and morbidity. We describe eight unrelated families with an X-linked condition characterized by diaphragm defects, variable anterior body-wall anomalies, and/or facial dysmorphism. Using linkage analysis and exome or genome sequencing, we found that missense variants in plastin 3 (PLS3), a gene encoding an actin bundling protein, co-segregate with disease in all families. Loss-of-function variants in PLS3 have been previously associated with X-linked osteoporosis (MIM: 300910), so we used in silico protein modeling and a mouse model to address these seemingly disparate clinical phenotypes. The missense variants in individuals with CDH are located within the actin-binding domains of the protein but are not predicted to affect protein structure, whereas the variants in individuals with osteoporosis are predicted to result in loss of function. A mouse knockin model of a variant identified in one of the CDH-affected families, c.1497G>C (p.Trp499Cys), shows partial perinatal lethality and recapitulates the key findings of the human phenotype, including diaphragm and abdominal-wall defects. Both the mouse model and one adult human male with a CDH-associated PLS3 variant were observed to have increased rather than decreased bone mineral density. Together, these clinical and functional data in humans and mice reveal that specific missense variants affecting the actin-binding domains of PLS3 might have a gain-of-function effect and cause a Mendelian congenital disorder.

Thalidomide for Recurrent Bleeding Due to Small-Intestinal Angiodysplasia.

BACKGROUND: Recurrent bleeding from the small intestine accounts for 5 to 10% of cases of gastrointestinal bleeding and remains a therapeutic challenge. Thalidomide has been evaluated for the treatment of recurrent bleeding due to small-intestinal angiodysplasia (SIA), but confirmatory trials are lacking. METHODS: We conducted a multicenter, double-blind, randomized, placebo-controlled trial to investigate the efficacy and safety of thalidomide for the treatment of recurrent bleeding due to SIA. Eligible patients with recurrent bleeding (at least four episodes of bleeding during the previous year) due to SIA were randomly assigned to receive thalidomide at an oral daily dose of 100 mg or 50 mg or placebo for 4 months. Patients were followed for at least 1 year after the end of the 4-month treatment period. The primary end point was effective response, which was defined as a reduction of at least 50% in the number of bleeding episodes that occurred during the year after the end of thalidomide treatment as compared with the number that occurred during the year before treatment. Key secondary end points were cessation of bleeding without rebleeding, blood transfusion, hospitalization because of bleeding, duration of bleeding, and hemoglobin levels. RESULTS: Overall, 150 patients underwent randomization: 51 to the 100-mg thalidomide group, 49 to the 50-mg thalidomide group, and 50 to the placebo group. The percentages of patients with an effective response in the 100-mg thalidomide group, 50-mg thalidomide group, and placebo group were 68.6%, 51.0%, and 16.0%, respectively (P<0.001 for simultaneous comparison across the three groups). The results of the analyses of the secondary end points supported those of the primary end point. Adverse events were more common in the thalidomide groups than in the placebo group overall; specific events included constipation, somnolence, limb numbness, peripheral edema, dizziness, and elevated liver-enzyme levels. CONCLUSIONS: In this placebo-controlled trial, treatment with thalidomide resulted in a reduction in bleeding in patients with recurrent bleeding due to SIA. (Funded by the National Natural Science Foundation of China and the Shanghai Municipal Education Commission, Gaofeng Clinical Medicine; ClinicalTrials.gov number, NCT02707484.).

Neither cardiac mitochondrial DNA variation nor copy number contribute to congenital heart disease risk.

The well-established manifestation of mitochondrial mutations in functional cardiac disease (e.g., mitochondrial cardiomyopathy) prompted the hypothesis that mitochondrial DNA (mtDNA) sequence and/or copy number (mtDNAcn) variation contribute to cardiac defects in congenital heart disease (CHD). MtDNAcns were calculated and rare, non-synonymous mtDNA mutations were identified in 1,837 CHD-affected proband-parent trios, 116 CHD-affected singletons, and 114 paired cardiovascular tissue/blood samples. The variant allele fraction (VAF) of heteroplasmic variants in mitochondrial RNA from 257 CHD cardiovascular tissue samples was also calculated. On average, mtDNA from blood had 0.14 rare variants and 52.9 mtDNA copies per nuclear genome per proband. No variation with parental age at proband birth or CHD-affected proband age was seen. mtDNAcns in valve/vessel tissue (320 ± 70) were lower than in atrial tissue (1,080 ± 320, p = 6.8E-21), which were lower than in ventricle tissue (1,340 ± 280, p = 1.4E-4). The frequency of rare variants in CHD-affected individual DNA was indistinguishable from the frequency in an unaffected cohort, and proband mtDNAcns did not vary from those of CHD cohort parents. In both the CHD and the comparison cohorts, mtDNAcns were significantly correlated between mother-child, father-child, and mother-father. mtDNAcns among people with European (mean = 52.0), African (53.0), and Asian haplogroups (53.5) were calculated and were significantly different for European and Asian haplogroups (p = 2.6E-3). Variant heteroplasmic fraction (HF) in blood correlated well with paired cardiovascular tissue HF (r = 0.975) and RNA VAF (r = 0.953), which suggests blood HF is a reasonable proxy for HF in heart tissue. We conclude that mtDNA mutations and mtDNAcns are unlikely to contribute significantly to CHD risk.

The genetic architecture of pediatric cardiomyopathy.

To understand the genetic contribution to primary pediatric cardiomyopathy, we performed exome sequencing in a large cohort of 528 children with cardiomyopathy. Using clinical interpretation guidelines and targeting genes implicated in cardiomyopathy, we identified a genetic cause in 32% of affected individuals. Cardiomyopathy sub-phenotypes differed by ancestry, age at diagnosis, and family history. Infants < 1 year were less likely to have a molecular diagnosis (p < 0.001). Using a discovery set of 1,703 candidate genes and informatic tools, we identified rare and damaging variants in 56% of affected individuals. We see an excess burden of damaging variants in affected individuals as compared to two independent control sets, 1000 Genomes Project (p < 0.001) and SPARK parental controls (p < 1 × 10-16). Cardiomyopathy variant burden remained enriched when stratified by ancestry, variant type, and sub-phenotype, emphasizing the importance of understanding the contribution of these factors to genetic architecture. Enrichment in this discovery candidate gene set suggests multigenic mechanisms underlie sub-phenotype-specific causes and presentations of cardiomyopathy. These results identify important information about the genetic architecture of pediatric cardiomyopathy and support recommendations for clinical genetic testing in children while illustrating differences in genetic architecture by age, ancestry, and sub-phenotype and providing rationale for larger studies to investigate multigenic contributions.

SHINE: protein language model-based pathogenicity prediction for short inframe insertion and deletion variants.

Accurate variant pathogenicity predictions are important in genetic studies of human diseases. Inframe insertion and deletion variants (indels) alter protein sequence and length, but not as deleterious as frameshift indels. Inframe indel Interpretation is challenging due to limitations in the available number of known pathogenic variants for training. Existing prediction methods largely use manually encoded features including conservation, protein structure and function, and allele frequency to infer variant pathogenicity. Recent advances in deep learning modeling of protein sequences and structures provide an opportunity to improve the representation of salient features based on large numbers of protein sequences. We developed a new pathogenicity predictor for SHort Inframe iNsertion and dEletion (SHINE). SHINE uses pretrained protein language models to construct a latent representation of an indel and its protein context from protein sequences and multiple protein sequence alignments, and feeds the latent representation into supervised machine learning models for pathogenicity prediction. We curated training data from ClinVar and gnomAD, and created two test datasets from different sources. SHINE achieved better prediction performance than existing methods for both deletion and insertion variants in these two test datasets. Our work suggests that unsupervised protein language models can provide valuable information about proteins, and new methods based on these models can improve variant interpretation in genetic analyses.

Rare and de novo variants in 827 congenital diaphragmatic hernia probands implicate LONP1 as candidate risk gene.

Congenital diaphragmatic hernia (CDH) is a severe congenital anomaly that is often accompanied by other anomalies. Although the role of genetics in the pathogenesis of CDH has been established, only a small number of disease-associated genes have been identified. To further investigate the genetics of CDH, we analyzed de novo coding variants in 827 proband-parent trios and confirmed an overall significant enrichment of damaging de novo variants, especially in constrained genes. We identified LONP1 (lon peptidase 1, mitochondrial) and ALYREF (Aly/REF export factor) as candidate CDH-associated genes on the basis of de novo variants at a false discovery rate below 0.05. We also performed ultra-rare variant association analyses in 748 affected individuals and 11,220 ancestry-matched population control individuals and identified LONP1 as a risk gene contributing to CDH through both de novo and ultra-rare inherited largely heterozygous variants clustered in the core of the domains and segregating with CDH in affected familial individuals. Approximately 3% of our CDH cohort who are heterozygous with ultra-rare predicted damaging variants in LONP1 have a range of clinical phenotypes, including other anomalies in some individuals and higher mortality and requirement for extracorporeal membrane oxygenation. Mice with lung epithelium-specific deletion of Lonp1 die immediately after birth, most likely because of the observed severe reduction of lung growth, a known contributor to the high mortality in humans. Our findings of both de novo and inherited rare variants in the same gene may have implications in the design and analysis for other genetic studies of congenital anomalies.

Return of genetic research results in 21,532 individuals with autism.

PURPOSE: The aim of this study is to identify likely pathogenic (LP) and pathogenic (P) genetic results for autism that can be returned to participants in SPARK (SPARKforAutism.org): a large recontactable cohort of people with autism in the United States. We also describe the process to return these clinically confirmed genetic findings. METHODS: We present results from microarray genotyping and exome sequencing of 21,532 individuals with autism and 17,785 of their parents. We returned LP and P (American College of Medical Genetics criteria) copy-number variants, chromosomal aneuploidies, and variants in genes with strong evidence of association with autism and intellectual disability. RESULTS: We identified 1903 returnable LP/P variants in 1861 individuals with autism (8.6%). 89.5% of these variants were not known to participants. The diagnostic genetic result was returned to 589 participants (53% of those contacted). Features associated with a higher probability of having a returnable result include cognitive and medically complex features, being female, being White (versus non-White) and being diagnosed more than 20 years ago. We also find results among autistics across the spectrum, as well as in transmitting parents with neuropsychiatric features but no autism diagnosis. CONCLUSION: SPARK offers an opportunity to assess returnable results among autistic people who have not been ascertained clinically. SPARK also provides practical experience returning genetic results for a behavioral condition at a large scale.

Heterogeneity of comprehensive clinical phenotype and longitudinal adaptive function and correlation with computational predictions of severity of missense genotypes in KIF1A-associated neurological disorder.

PURPOSE: Pathogenic variants in kinesin family member 1A (KIF1A) are associated with KIF1A-associated neurological disorder. We report the clinical phenotypes and correlate genotypes of individuals with KIF1A-associated neurological disorder. METHODS: Medical history and adaptive function were assessed longitudinally. In-person evaluations included neurological, motor, ophthalmologic, and cognitive assessments. RESULTS: We collected online data on 177 individuals. Fifty-seven individuals were also assessed in-person. Most individuals had de novo heterozygous missense likely pathogenic/pathogenic KIF1A variants. The most common characteristics were hypotonia, spasticity, ataxia, seizures, optic nerve atrophy, cerebellar atrophy, and cognitive impairment. Mean Vineland adaptive behavior composite score (VABS-ABC) was low (M = 62.9, SD = 19.1). The mean change in VABS-ABC over time was -3.1 (SD = 7.3). The decline in VABS-ABC was associated with the age at first assessment and abnormal electroencephalogram/seizure. There was a positive correlation between evolutionary scale model (ESM) score for the variants and final VABS-ABC (P = .003). Abnormal electroencephalogram/seizure, neuroimaging result, and ESM explain 34% of the variance in final VABS-ABC (P < .001). CONCLUSION: In-person assessment confirmed caregiver report and identified additional visual deficits. Adaptive function declined over time consistent with both the neurodevelopmental and neurodegenerative nature of the condition. Using ESM score assists in predicting phenotype across a wide range of unique variants.

Homozygous missense variants in YKT6 result in loss of function and are associated with developmental delay, with or without severe infantile liver disease and risk for hepatocellular carcinoma.

PURPOSE: YKT6 plays important roles in multiple intracellular vesicle trafficking events but has not been associated with Mendelian diseases. METHODS: We report 3 unrelated individuals with rare homozygous missense variants in YKT6 who exhibited neurological disease with or without a progressive infantile liver disease. We modeled the variants in Drosophila. We generated wild-type and variant genomic rescue constructs of the fly ortholog dYkt6 and compared their ability in rescuing the loss-of-function phenotypes in mutant flies. We also generated a dYkt6KozakGAL4 allele to assess the expression pattern of dYkt6. RESULTS: Two individuals are homozygous for YKT6 [NM_006555.3:c.554A>G p.(Tyr185Cys)] and exhibited normal prenatal course followed by failure to thrive, developmental delay, and progressive liver disease. Haplotype analysis identified a shared homozygous region flanking the variant, suggesting a common ancestry. The third individual is homozygous for YKT6 [NM_006555.3:c.191A>G p.(Tyr64Cys)] and exhibited neurodevelopmental disorders and optic atrophy. Fly dYkt6 is essential and is expressed in the fat body (analogous to liver) and central nervous system. Wild-type genomic rescue constructs can rescue the lethality and autophagic flux defects, whereas the variants are less efficient in rescuing the phenotypes. CONCLUSION: The YKT6 variants are partial loss-of-function alleles, and the p.(Tyr185Cys) is more severe than p.(Tyr64Cys).

Clinical characteristics, longitudinal adaptive functioning, and association with electroencephalogram activity in PPP2R5D-related neurodevelopmental disorder.

Protein phosphatase 2 regulatory subunit B56δ related neurodevelopmental disorder (PPP2R5D-related NDD) is largely caused by de novo heterozygous missense PPP2R5D variants. We report medical characteristics, longitudinal adaptive functioning, and in-person neurological, motor, cognitive, and electroencephalogram (EEG) activity for PPP2R5D-related NDD. Forty-two individuals (median age 6 years, range = 0.8-25.3) with pathogenic/likely pathogenic PPP2R5D variants were assessed, and almost all variants were missense (97.6%) and de novo (85.7%). Common clinical symptoms were developmental delay, hypotonia, macrocephaly, seizures, autism, behavioral challenges, and sleep problems. The mean Gross motor functional measure-66 was 60.2 ± 17.3% and the mean Revised upper limb module score was 25.9 ± 8.8. The Vineland-3 adaptive behavior composite score (VABS-3 ABC) at baseline was low (M = 61.7 ± 16.8). VABS-3 growth scale value scores increased from baseline in all subdomains (range = 0.6-5.9) after a mean follow-up of 1.3 ± 0.3 years. EEG beta and gamma power were negatively correlated with VABS-3 score; p < 0.05. Individuals had a mean Quality-of-life inventory-disability score of 74.7 ± 11.4. Twenty caregivers (80%) had a risk of burnout based on the Caregiver burden inventory. Overall, the most common clinical manifestations of PPP2R5D-related NDD were impaired cognitive, adaptive function, and motor skills; and EEG activity was associated with adaptive functioning. This clinical characterization describes the natural history in preparation for clinical trials.

A Polymer-Based Polar Stationary Phase Grafted With Modified Lysine for Hydrophilic Interaction Chromatography.

The high hydrophobicity and chemical inertness of poly(styrene-divinylbenzene) (PS-DVB) microspheres make their surface hydrophilic modification difficult. Here we describe a facile way to convert PS-DVB microspheres to hydrophilic, then can be used as polar stationary phase for hydrophilic interaction chromatography. This approach utilizes the grafting of an acrylamide-terminated lysine zwitterionic monomer onto PS-DVB microspheres via free radical polymerization. The obtained stationary phase shows good hydrophilicity and a typical retention mechanism of hydrophilic interaction chromatography toward several model polar analytes. It also exhibits obvious zwitterionic properties and is capable of separating cationic and anionic analytes simultaneously. The column shows negligible bleeding level, much superior to silica-based ones.