A genome-wide spectrum of tandem repeat expansions in 338,963 humans.

The Genome Aggregation Database (gnomAD), widely recognized as the gold-standard reference map of human genetic variation, has largely overlooked tandem repeat (TR) expansions, despite the fact that TRs constitute ∼6% of our genome and are linked to over 50 human diseases. Here, we introduce the TR-gnomAD (https://wlcb.oit.uci.edu/TRgnomAD), a biobank-scale reference of 0.86 million TRs derived from 338,963 whole-genome sequencing (WGS) samples of diverse ancestries (39.5% non-European samples). TR-gnomAD offers critical insights into ancestry-specific disease prevalence using disparities in TR unit number frequencies among ancestries. Moreover, TR-gnomAD is able to differentiate between common, presumably benign TR expansions, which are prevalent in TR-gnomAD, from those potentially pathogenic TR expansions, which are found more frequently in disease groups than within TR-gnomAD. Together, TR-gnomAD is an invaluable resource for researchers and physicians to interpret TR expansions in individuals with genetic diseases.

Genomic architecture of autism from comprehensive whole-genome sequence annotation.

Fully understanding autism spectrum disorder (ASD) genetics requires whole-genome sequencing (WGS). We present the latest release of the Autism Speaks MSSNG resource, which includes WGS data from 5,100 individuals with ASD and 6,212 non-ASD parents and siblings (total n = 11,312). Examining a wide variety of genetic variants in MSSNG and the Simons Simplex Collection (SSC; n = 9,205), we identified ASD-associated rare variants in 718/5,100 individuals with ASD from MSSNG (14.1%) and 350/2,419 from SSC (14.5%). Considering genomic architecture, 52% were nuclear sequence-level variants, 46% were nuclear structural variants (including copy-number variants, inversions, large insertions, uniparental isodisomies, and tandem repeat expansions), and 2% were mitochondrial variants. Our study provides a guidebook for exploring genotype-phenotype correlations in families who carry ASD-associated rare variants and serves as an entry point to the expanded studies required to dissect the etiology in the ∼85% of the ASD population that remain idiopathic.

The Parkinson's disease protein alpha-synuclein is a modulator of processing bodies and mRNA stability.

Alpha-synuclein (αS) is a conformationally plastic protein that reversibly binds to cellular membranes. It aggregates and is genetically linked to Parkinson's disease (PD). Here, we show that αS directly modulates processing bodies (P-bodies), membraneless organelles that function in mRNA turnover and storage. The N terminus of αS, but not other synucleins, dictates mutually exclusive binding either to cellular membranes or to P-bodies in the cytosol. αS associates with multiple decapping proteins in close proximity on the Edc4 scaffold. As αS pathologically accumulates, aberrant interaction with Edc4 occurs at the expense of physiologic decapping-module interactions. mRNA decay kinetics within PD-relevant pathways are correspondingly disrupted in PD patient neurons and brain. Genetic modulation of P-body components alters αS toxicity, and human genetic analysis lends support to the disease-relevance of these interactions. Beyond revealing an unexpected aspect of αS function and pathology, our data highlight the versatility of conformationally plastic proteins with high intrinsic disorder.

TCR+/BCR+ dual-expressing cells and their associated public BCR clonotype are not enriched in type 1 diabetes.

Ahmed and colleagues recently described a novel hybrid lymphocyte expressing both a B and T cell receptor, termed double expresser (DE) cells. DE cells in blood of type 1 diabetes (T1D) subjects were present at increased numbers and enriched for a public B cell clonotype. Here, we attempted to reproduce these findings. While we could identify DE cells by flow cytometry, we found no association between DE cell frequency and T1D status. We were unable to identify the reported public B cell clone, or any similar clone, in bulk B cells or sorted DE cells from T1D subjects or controls. We also did not observe increased usage of the public clone VH or DH genes in B cells or in sorted DE cells. Taken together, our findings suggest that DE cells and their alleged public clonotype are not enriched in T1D. This Matters Arising paper is in response to Ahmed et al. (2019), published in Cell. See also the response by Ahmed et al. (2021), published in this issue.

The Polygenic and Monogenic Basis of Blood Traits and Diseases.

Blood cells play essential roles in human health, underpinning physiological processes such as immunity, oxygen transport, and clotting, which when perturbed cause a significant global health burden. Here we integrate data from UK Biobank and a large-scale international collaborative effort, including data for 563,085 European ancestry participants, and discover 5,106 new genetic variants independently associated with 29 blood cell phenotypes covering a range of variation impacting hematopoiesis. We holistically characterize the genetic architecture of hematopoiesis, assess the relevance of the omnigenic model to blood cell phenotypes, delineate relevant hematopoietic cell states influenced by regulatory genetic variants and gene networks, identify novel splice-altering variants mediating the associations, and assess the polygenic prediction potential for blood traits and clinical disorders at the interface of complex and Mendelian genetics. These results show the power of large-scale blood cell trait GWAS to interrogate clinically meaningful variants across a wide allelic spectrum of human variation.

Comprehensive In Vivo Interrogation Reveals Phenotypic Impact of Human Enhancer Variants.

Establishing causal links between non-coding variants and human phenotypes is an increasing challenge. Here, we introduce a high-throughput mouse reporter assay for assessing the pathogenic potential of human enhancer variants in vivo and examine nearly a thousand variants in an enhancer repeatedly linked to polydactyly. We show that 71% of all rare non-coding variants previously proposed as causal lead to reporter gene expression in a pattern consistent with their pathogenic role. Variants observed to alter enhancer activity were further confirmed to cause polydactyly in knockin mice. We also used combinatorial and single-nucleotide mutagenesis to evaluate the in vivo impact of mutations affecting all positions of the enhancer and identified additional functional substitutions, including potentially pathogenic variants hitherto not observed in humans. Our results uncover the functional consequences of hundreds of mutations in a phenotype-associated enhancer and establish a widely applicable strategy for systematic in vivo evaluation of human enhancer variants.

Coatopathies: Genetic Disorders of Protein Coats.

Protein coats are supramolecular complexes that assemble on the cytosolic face of membranes to promote cargo sorting and transport carrier formation in the endomembrane system of eukaryotic cells. Several types of protein coats have been described, including COPI, COPII, AP-1, AP-2, AP-3, AP-4, AP-5, and retromer, which operate at different stages of the endomembrane system. Defects in these coats impair specific transport pathways, compromising the function and viability of the cells. In humans, mutations in subunits of these coats cause various congenital diseases that are collectively referred to as coatopathies. In this article, we review the fundamental properties of protein coats and the diseases that result from mutation of their constituent subunits.

Finding Needles in the Haystack: Strategies for Uncovering Noncoding Regulatory Variants.

Despite accumulating evidence implicating noncoding variants in human diseases, unraveling their functionality remains a significant challenge. Systematic annotations of the regulatory landscape and the growth of sequence variant data sets have fueled the development of tools and methods to identify causal noncoding variants and evaluate their regulatory effects. Here, we review the latest advances in the field and discuss potential future research avenues to gain a more in-depth understanding of noncoding regulatory variants.

Structural and functional properties of mSWI/SNF chromatin remodeling complexes revealed through single-cell perturbation screens.

The mammalian SWI/SNF (mSWI/SNF or BAF) family of chromatin remodeling complexes play critical roles in regulating DNA accessibility and gene expression. The three final-form subcomplexes-cBAF, PBAF, and ncBAF-are distinct in biochemical componentry, chromatin targeting, and roles in disease; however, the contributions of their constituent subunits to gene expression remain incompletely defined. Here, we performed Perturb-seq-based CRISPR-Cas9 knockout screens targeting mSWI/SNF subunits individually and in select combinations, followed by single-cell RNA-seq and SHARE-seq. We uncovered complex-, module-, and subunit-specific contributions to distinct regulatory networks and defined paralog subunit relationships and shifted subcomplex functions upon perturbations. Synergistic, intra-complex genetic interactions between subunits reveal functional redundancy and modularity. Importantly, single-cell subunit perturbation signatures mapped across bulk primary human tumor expression profiles both mirror and predict cBAF loss-of-function status in cancer. Our findings highlight the utility of Perturb-seq to dissect disease-relevant gene regulatory impacts of heterogeneous, multi-component master regulatory complexes.

Germline genetic contribution to the immune landscape of cancer.

Understanding the contribution of the host's genetic background to cancer immunity may lead to improved stratification for immunotherapy and to the identification of novel therapeutic targets. We investigated the effect of common and rare germline variants on 139 well-defined immune traits in ∼9000 cancer patients enrolled in TCGA. High heritability was observed for estimates of NK cell and T cell subset infiltration and for interferon signaling. Common variants of IFIH1, TMEM173 (STING1), and TMEM108 were associated with differential interferon signaling and variants mapping to RBL1 correlated with T cell subset abundance. Pathogenic or likely pathogenic variants in BRCA1 and in genes involved in telomere stabilization and Wnt-ß-catenin also acted as immune modulators. Our findings provide evidence for the impact of germline genetics on the composition and functional orientation of the tumor immune microenvironment. The curated datasets, variants, and genes identified provide a resource toward further understanding of tumor-immune interactions.

The contemporary management of cancers of the sinonasal tract in adults.

Sinonasal malignancies make up <5% of all head and neck neoplasms, with an incidence of 0.5-1.0 per 100,000. The outcome of these rare malignancies has been poor, whereas significant progress has been made in the management of other cancers. The objective of the current review was to describe the incidence, causes, presentation, diagnosis, treatment, and recent developments of malignancies of the sinonasal tract. The diagnoses covered in this review included sinonasal undifferentiated carcinoma, sinonasal adenocarcinoma, sinonasal squamous cell carcinoma, and esthesioneuroblastoma, which are exclusive to the sinonasal tract. In addition, the authors covered malignances that are likely to be encountered in the sinonasal tract-primary mucosal melanoma, NUT (nuclear protein of the testis) carcinoma, and extranodal natural killer cell/T-cell lymphoma. For the purpose of keeping this review as concise and focused as possible, sarcomas and malignancies that can be classified as salivary gland neoplasms were excluded.

Mime-seq 2.0: a method to sequence microRNAs from specific mouse cell types.

Many microRNAs (miRNAs) are expressed with high spatiotemporal specificity during organismal development, with some being limited to rare cell types, often embedded in complex tissues. Yet, most miRNA profiling efforts remain at the tissue and organ levels. To overcome challenges in accessing the microRNomes from tissue-embedded cells, we had previously developed mime-seq (miRNome by methylation-dependent sequencing), a technique in which cell-specific miRNA methylation in C. elegans and Drosophila enabled chemo-selective sequencing without the need for cell sorting or biochemical purification. Here, we present mime-seq 2.0 for profiling miRNAs from specific mouse cell types. We engineered a chimeric RNA methyltransferase that is tethered to Argonaute protein and efficiently methylates miRNAs at their 3'-terminal 2'-OH in mouse and human cell lines. We also generated a transgenic mouse for conditional expression of this methyltransferase, which can be used to direct methylation of miRNAs in a cell type of choice. We validated the use of this mouse model by profiling miRNAs from B cells and bone marrow plasma cells.

BK Channelopathies and KCNMA1-Linked Disease Models.

Novel KCNMA1 variants, encoding the BK K+ channel, are associated with a debilitating dyskinesia and epilepsy syndrome. Neurodevelopmental delay, cognitive disability, and brain and structural malformations are also diagnosed at lower incidence. More than half of affected individuals present with a rare negative episodic motor disorder, paroxysmal nonkinesigenic dyskinesia (PNKD3). The mechanistic relationship of PNKD3 to epilepsy and the broader spectrum of KCNMA1-associated symptomology is unknown. This review summarizes patient-associated KCNMA1 variants within the BK channel structure, functional classifications, genotype-phenotype associations, disease models, and treatment. Patient and transgenic animal data suggest delineation of gain-of-function (GOF) and loss-of-function KCNMA1 neurogenetic disease, validating two heterozygous alleles encoding GOF BK channels (D434G and N999S) as causing seizure and PNKD3. This discovery led to a variant-defined therapeutic approach for PNKD3, providing initial insight into the neurological basis. A comprehensive clinical definition of monogenic KCNMA1-linked disease and the neuronal mechanisms currently remain priorities for continued investigation.

PRIM1 deficiency causes a distinctive primordial dwarfism syndrome.

DNA replication is fundamental for cell proliferation in all organisms. Nonetheless, components of the replisome have been implicated in human disease, and here we report PRIM1 encoding the catalytic subunit of DNA primase as a novel disease gene. Using a variant classification agnostic approach, biallelic mutations in PRIM1 were identified in five individuals. PRIM1 protein levels were markedly reduced in patient cells, accompanied by replication fork asymmetry, increased interorigin distances, replication stress, and prolonged S-phase duration. Consequently, cell proliferation was markedly impaired, explaining the patients' extreme growth failure. Notably, phenotypic features distinct from those previously reported with DNA polymerase genes were evident, highlighting differing developmental requirements for this core replisome component that warrant future investigation.

Pharmacogenomics Beyond Single Common Genetic Variants: The Way Forward.

Interindividual variability in genes encoding drug-metabolizing enzymes, transporters, receptors, and human leukocyte antigens has a major impact on a patient's response to drugs with regard to efficacy and safety. Enabled by both technological and conceptual advances, the field of pharmacogenomics is developing rapidly. Major progress in omics profiling methods has enabled novel genotypic and phenotypic characterization of patients and biobanks. These developments are paralleled by advances in machine learning, which have allowed us to parse the immense wealth of data and establish novel genetic markers and polygenic models for drug selection and dosing. Pharmacogenomics has recently become more widespread in clinical practice to personalize treatment and to develop new drugs tailored to specific patient populations. In this review, we provide an overview of the latest developments in the field and discuss the way forward, including how to address the missing heritability, develop novel polygenic models, and further improve the clinical implementation of pharmacogenomics.

Assessing autosomal aneuploidy in ancient genomes.

Using genetic methods, aneuploidies can be detected in ancient human remains, which is so far the only way to reliably prove their existence in the past. As highlighted in recent studies by Rohrlach et al. and by Anastasiadou et al., this initial step enables a deeper exploration of the history of rare diseases, encompassing the social and historical contexts of the afflicted individuals.

RNA-based medicine: from molecular mechanisms to therapy.

RNA-based therapeutics have the potential to revolutionize the treatment and prevention of human diseases. While early research faced setbacks, it established the basis for breakthroughs in RNA-based drug design that culminated in the extraordinarily fast development of mRNA vaccines to combat the COVID-19 pandemic. We have now reached a pivotal moment where RNA medicines are poised to make a broad impact in the clinic. In this review, we present an overview of different RNA-based strategies to generate novel therapeutics, including antisense and RNAi-based mechanisms, mRNA-based approaches, and CRISPR-Cas-mediated genome editing. Using three rare genetic diseases as examples, we highlight the opportunities, but also the challenges to wide-ranging applications of this class of drugs.

Impact of genome build on RNA-seq interpretation and diagnostics.

Transcriptomics is a powerful tool for unraveling the molecular effects of genetic variants and disease diagnosis. Prior studies have demonstrated that choice of genome build impacts variant interpretation and diagnostic yield for genomic analyses. To identify the extent genome build also impacts transcriptomics analyses, we studied the effect of the hg19, hg38, and CHM13 genome builds on expression quantification and outlier detection in 386 rare disease and familial control samples from both the Undiagnosed Diseases Network and Genomics Research to Elucidate the Genetics of Rare Disease Consortium. Across six routinely collected biospecimens, 61% of quantified genes were not influenced by genome build. However, we identified 1,492 genes with build-dependent quantification, 3,377 genes with build-exclusive expression, and 9,077 genes with annotation-specific expression across six routinely collected biospecimens, including 566 clinically relevant and 512 known OMIM genes. Further, we demonstrate that between builds for a given gene, a larger difference in quantification is well correlated with a larger change in expression outlier calling. Combined, we provide a database of genes impacted by build choice and recommend that transcriptomics-guided analyses and diagnoses are cross referenced with these data for robustness.

De novo variants in FRYL are associated with developmental delay, intellectual disability, and dysmorphic features.

FRY-like transcription coactivator (FRYL) belongs to a Furry protein family that is evolutionarily conserved from yeast to humans. The functions of FRYL in mammals are largely unknown, and variants in FRYL have not previously been associated with a Mendelian disease. Here, we report fourteen individuals with heterozygous variants in FRYL who present with developmental delay, intellectual disability, dysmorphic features, and other congenital anomalies in multiple systems. The variants are confirmed de novo in all individuals except one. Human genetic data suggest that FRYL is intolerant to loss of function (LoF). We find that the fly FRYL ortholog, furry (fry), is expressed in multiple tissues, including the central nervous system where it is present in neurons but not in glia. Homozygous fry LoF mutation is lethal at various developmental stages, and loss of fry in mutant clones causes defects in wings and compound eyes. We next modeled four out of the five missense variants found in affected individuals using fry knockin alleles. One variant behaves as a severe LoF variant, whereas two others behave as partial LoF variants. One variant does not cause any observable defect in flies, and the corresponding human variant is not confirmed to be de novo, suggesting that this is a variant of uncertain significance. In summary, our findings support that fry is required for proper development in flies and that the LoF variants in FRYL cause a dominant disorder with developmental and neurological symptoms due to haploinsufficiency.

The impact of clinical genome sequencing in a global population with suspected rare genetic disease.

There is mounting evidence of the value of clinical genome sequencing (cGS) in individuals with suspected rare genetic disease (RGD), but cGS performance and impact on clinical care in a diverse population drawn from both high-income countries (HICs) and low- and middle-income countries (LMICs) has not been investigated. The iHope program, a philanthropic cGS initiative, established a network of 24 clinical sites in eight countries through which it provided cGS to individuals with signs or symptoms of an RGD and constrained access to molecular testing. A total of 1,004 individuals (median age, 6.5 years; 53.5% male) with diverse ancestral backgrounds (51.8% non-majority European) were assessed from June 2016 to September 2021. The diagnostic yield of cGS was 41.4% (416/1,004), with individuals from LMIC sites 1.7 times more likely to receive a positive test result compared to HIC sites (LMIC 56.5% [195/345] vs. HIC 33.5% [221/659], OR 2.6, 95% CI 1.9-3.4, p < 0.0001). A change in diagnostic evaluation occurred in 76.9% (514/668) of individuals. Change of management, inclusive of specialty referrals, imaging and testing, therapeutic interventions, and palliative care, was reported in 41.4% (285/694) of individuals, which increased to 69.2% (480/694) when genetic counseling and avoidance of additional testing were also included. Individuals from LMIC sites were as likely as their HIC counterparts to experience a change in diagnostic evaluation (OR 6.1, 95% CI 1.1-∞, p = 0.05) and change of management (OR 0.9, 95% CI 0.5-1.3, p = 0.49). Increased access to genomic testing may support diagnostic equity and the reduction of global health care disparities.