Organoid Modeling of the Tumor Immune Microenvironment.

In vitro cancer cultures, including three-dimensional organoids, typically contain exclusively neoplastic epithelium but require artificial reconstitution to recapitulate the tumor microenvironment (TME). The co-culture of primary tumor epithelia with endogenous, syngeneic tumor-infiltrating lymphocytes (TILs) as a cohesive unit has been particularly elusive. Here, an air-liquid interface (ALI) method propagated patient-derived organoids (PDOs) from >100 human biopsies or mouse tumors in syngeneic immunocompetent hosts as tumor epithelia with native embedded immune cells (T, B, NK, macrophages). Robust droplet-based, single-cell simultaneous determination of gene expression and immune repertoire indicated that PDO TILs accurately preserved the original tumor T cell receptor (TCR) spectrum. Crucially, human and murine PDOs successfully modeled immune checkpoint blockade (ICB) with anti-PD-1- and/or anti-PD-L1 expanding and activating tumor antigen-specific TILs and eliciting tumor cytotoxicity. Organoid-based propagation of primary tumor epithelium en bloc with endogenous immune stroma should enable immuno-oncology investigations within the TME and facilitate personalized immunotherapy testing.

DNA demethylase activity maintains intestinal cells in an undifferentiated state following loss of APC.

Although genome-wide hypomethylation is a hallmark of many cancers, roles for active DNA demethylation during tumorigenesis are unknown. Here, loss of the APC tumor suppressor gene causes upregulation of a DNA demethylase system and the concomitant hypomethylation of key intestinal cell fating genes. Notably, this hypomethylation maintained zebrafish intestinal cells in an undifferentiated state that was released upon knockdown of demethylase components. Mechanistically, the demethylase genes are directly activated by Pou5f1 and Cebpß and are indirectly repressed by retinoic acid, which antagonizes Pou5f1 and Cebpß. Apc mutants lack retinoic acid as a result of the transcriptional repression of retinol dehydrogenase l1 via a complex that includes Lef1, Groucho2, Ctbp1, Lsd1, and Corest. Our findings imply a model wherein APC controls intestinal cell fating through a switch in DNA methylation dynamics. Wild-type APC and retinoic acid downregulate demethylase components, thereby promoting DNA methylation of key genes and helping progenitors commit to differentiation.

Genetic variants associated with syncope implicate neural and autonomic processes.

AIMS: Syncope is a common and clinically challenging condition. In this study, the genetics of syncope were investigated to seek knowledge about its pathophysiology and prognostic implications. METHODS AND RESULTS: This genome-wide association meta-analysis included 56 071 syncope cases and 890 790 controls from deCODE genetics (Iceland), UK Biobank (United Kingdom), and Copenhagen Hospital Biobank Cardiovascular Study/Danish Blood Donor Study (Denmark), with a follow-up assessment of variants in 22 412 cases and 286 003 controls from Intermountain (Utah, USA) and FinnGen (Finland). The study yielded 18 independent syncope variants, 17 of which were novel. One of the variants, p.Ser140Thr in PTPRN2, affected syncope only when maternally inherited. Another variant associated with a vasovagal reaction during blood donation and five others with heart rate and/or blood pressure regulation, with variable directions of effects. None of the 18 associations could be attributed to cardiovascular or other disorders. Annotation with regard to regulatory elements indicated that the syncope variants were preferentially located in neural-specific regulatory regions. Mendelian randomization analysis supported a causal effect of coronary artery disease on syncope. A polygenic score (PGS) for syncope captured genetic correlation with cardiovascular disorders, diabetes, depression, and shortened lifespan. However, a score based solely on the 18 syncope variants performed similarly to the PGS in detecting syncope risk but did not associate with other disorders. CONCLUSION: The results demonstrate that syncope has a distinct genetic architecture that implicates neural regulatory processes and a complex relationship with heart rate and blood pressure regulation. A shared genetic background with poor cardiovascular health was observed, supporting the importance of a thorough assessment of individuals presenting with syncope.

Evaluating use of changing technologies for rapid next-generation sequencing in pediatrics.

BACKGROUND: Rapid next-generation sequencing (NGS) offers the potential to shorten the diagnostic process and improve the care of acutely ill children. The goal of this study was to report our findings, including benefits and limitations, of a targeted NGS panel and rapid genome sequencing (rGS) in neonatal and pediatric acute clinical care settings. METHODS: Retrospective analysis of patient characteristics, diagnostic yields, turnaround time, and changes in management for infants and children receiving either RapSeq, a targeted NGS panel for 4500+ genes, or rGS, at the University of Utah Hospital and Primary Children's Hospital, from 2015 to 2020. RESULTS: Over a 5-year period, 142 probands underwent rapid NGS: 66 received RapSeq and 76 rGS. Overall diagnostic yield was 39%. In the majority of diagnostic cases, there were one or more changes in clinical care management. Of note, 7% of diagnoses identified by rGS would not have been identified by RapSeq. CONCLUSIONS: Our results indicate that rapid NGS impacts acute pediatric care in real-life clinical settings. Although affected by patient selection criteria, diagnostic yields were similar to those from clinical trial settings. Future studies are needed to determine relative advantages, including cost, turnaround time, and benefits for patients, of each approach in specific clinical circumstances. IMPACT: The use of comprehensive Mendelian gene panels and genome sequencing in the clinical setting allows for early diagnosis of patients in neonatal, pediatric, and cardiac intensive care units and impactful change in management. Diagnoses led to significant changes in management for several patients in lower acuity inpatient units supporting further exploration of the utility of rapid sequencing in these settings. This study reviews the limitations of comparing sequencing platforms in the clinical setting and the variables that should be considered in evaluating diagnostic rates across studies.

Variant in the synaptonemal complex protein SYCE2 associates with pregnancy loss through effect on recombination.

Two-thirds of all human conceptions are lost, in most cases before clinical detection. The lack of detailed understanding of the causes of pregnancy losses constrains focused counseling for future pregnancies. We have previously shown that a missense variant in synaptonemal complex central element protein 2 (SYCE2), in a key residue for the assembly of the synaptonemal complex backbone, associates with recombination traits. Here we show that it also increases risk of pregnancy loss in a genome-wide association analysis on 114,761 women with reported pregnancy loss. We further show that the variant associates with more random placement of crossovers and lower recombination rate in longer chromosomes but higher in the shorter ones. These results support the hypothesis that some pregnancy losses are due to failures in recombination. They further demonstrate that variants with a substantial effect on the quality of recombination can be maintained in the population.

GWAS meta-analysis reveals key risk loci in essential tremor pathogenesis.

Essential tremor (ET) is a prevalent neurological disorder with a largely unknown underlying biology. In this genome-wide association study meta-analysis, comprising 16,480 ET cases and 1,936,173 controls from seven datasets, we identify 12 sequence variants at 11 loci. Evaluating mRNA expression, splicing, plasma protein levels, and coding effects, we highlight seven putative causal genes at these loci, including CA3 and CPLX1. CA3 encodes Carbonic Anhydrase III and carbonic anhydrase inhibitors have been shown to decrease tremors. CPLX1, encoding Complexin-1, regulates neurotransmitter release. Through gene-set enrichment analysis, we identify a significant association with specific cell types, including dopaminergic and GABAergic neurons, as well as biological processes like Rho GTPase signaling. Genetic correlation analyses reveals a positive association between ET and Parkinson's disease, depression, and anxiety-related phenotypes. This research uncovers risk loci, enhancing our knowledge of the complex genetics of this common but poorly understood disorder, and highlights CA3 and CPLX1 as potential therapeutic targets.

Variants at the Interleukin 1 Gene Locus and Pericarditis.

Importance: Recurrent pericarditis is a treatment challenge and often a debilitating condition. Drugs inhibiting interleukin 1 cytokines are a promising new treatment option, but their use is based on scarce biological evidence and clinical trials of modest sizes, and the contributions of innate and adaptive immune processes to the pathophysiology are incompletely understood. Objective: To use human genomics, transcriptomics, and proteomics to shed light on the pathogenesis of pericarditis. Design, Setting, and Participants: This was a meta-analysis of genome-wide association studies of pericarditis from 5 countries. Associations were examined between the pericarditis-associated variants and pericarditis subtypes (including recurrent pericarditis) and secondary phenotypes. To explore mechanisms, associations with messenger RNA expression (cis-eQTL), plasma protein levels (pQTL), and CpG methylation of DNA (ASM-QTL) were assessed. Data from Iceland (deCODE genetics, 1983-2020), Denmark (Copenhagen Hospital Biobank/Danish Blood Donor Study, 1977-2022), the UK (UK Biobank, 1953-2021), the US (Intermountain, 1996-2022), and Finland (FinnGen, 1970-2022) were included. Data were analyzed from September 2022 to August 2023. Exposure: Genotype. Main Outcomes and Measures: Pericarditis. Results: In this genome-wide association study of 4894 individuals with pericarditis (mean [SD] age at diagnosis, 51.4 [17.9] years, 2734 [67.6%] male, excluding the FinnGen cohort), associations were identified with 2 independent common intergenic variants at the interleukin 1 locus on chromosome 2q14. The lead variant was rs12992780 (T) (effect allele frequency [EAF], 31%-40%; odds ratio [OR], 0.83; 95% CI, 0.79-0.87; P = 6.67 × 10-16), downstream of IL1B and the secondary variant rs7575402 (A or T) (EAF, 45%-55%; adjusted OR, 0.89; 95% CI, 0.85-0.93; adjusted P = 9.6 × 10-8). The lead variant rs12992780 had a smaller odds ratio for recurrent pericarditis (0.76) than the acute form (0.86) (P for heterogeneity = .03) and rs7575402 was associated with CpG methylation overlapping binding sites of 4 transcription factors known to regulate interleukin 1 production: PU.1 (encoded by SPI1), STAT1, STAT3, and CCAAT/enhancer-binding protein ß (encoded by CEBPB). Conclusions and Relevance: This study found an association between pericarditis and 2 independent sequence variants at the interleukin 1 gene locus. This finding has the potential to contribute to development of more targeted and personalized therapy of pericarditis with interleukin 1-blocking drugs.

Rapid genome diagnosis of alveolar capillary dysplasia leading to treatment in a child with respiratory and cardiac failure.

Alveolar capillary dysplasia (ACD) is a fatal disorder that typically presents in the neonatal period with refractory hypoxemia and pulmonary hypertension. Lung biopsy is traditionally required to establish the diagnosis. We report a 22-mo-old male who presented with anemia, severe pulmonary hypertension, and right heart failure. He had a complicated hospital course resulting in cardiac arrest and requirement for extracorporeal membrane oxygenation. Computed tomography of the chest showed a heterogenous pattern of interlobular septal thickening and pulmonary edema. The etiology of his condition was unknown, lung biopsy was contraindicated because of his medical fragility, and discussions were held to move to palliative care. Rapid whole-genome sequencing (rWGS) was performed. In 2 d it resulted, revealing a novel FOXF1 gene pathogenic variant that led to the presumptive diagnosis of atypical ACD. Cases of atypical ACD have been reported with survival in patients using medical therapy or lung transplantation. Based on the rWGS diagnosis and more favorable potential of atypical ACD, aggressive medical treatment was pursued. The patient was discharged home after 67 d in the hospital; he is currently doing well more than 30 mo after his initial presentation with only one subsequent hospitalization and no requirement for lung transplantation. Our case reveals the potential for use of rWGS in a critically ill child in which the diagnosis is unknown. rWGS and other advanced genetic tests can guide clinical management and expand our understanding of atypical ACD and other conditions.

Obesity variants in the GIPR gene do not associate with risk of fracture or bone mineral density.

OBJECTIVE: The objective of this study was to investigate the risk of fracture and bone mineral density (BMD) of sequence variants in GIPR that reduce the activity of the GIPR receptor and have been associated with reduced body mass index (BMI). METHODS: We analysed the association of three missense variants in GIPR, a common variant, rs1800437 (p.Glu354Gln), and two rare variants, rs139215588 (p.Arg190Gln) and rs143430880 (p.Glu288Gly), as well as a burden of predicted loss of function (LoF) variants with risk of fracture and with BMD in a large meta-analysis of up to 1.2 million participants. We analysed associations with fractures at different skeletal sites in the general population; any fractures, hip fractures, vertebral fractures and forearm fractures, and specifically non-vertebral and osteoporotic fractures in postmenopausal women. We also evaluated associations with BMD at the lumbar spine, femoral neck, and total body measured with dual-energy X-ray absorptiometry (DXA), and with BMD estimated from heel ultrasound (eBMD). RESULTS: None of the three missense variants in GIPR associated significantly with increased risk of fractures or with lower BMD. Burden of LoF variants in GIPR were not associated with fractures or with BMD measured with clinically validated DXA, but associated with eBMD. CONCLUSION: Missense variants in GIPR, or burden of LoF variants in the gene, do not associate with risk of fractures or with lower BMD.

HerediGene Population Study IT infrastructure: A model to support genomic research recruitment and precision public health.

The HerediGene Population Study is a large research study focused on identifying new genetic biomarkers for disease prevention, diagnosis, prognosis, and development of new therapeutics. A substantial IT infrastructure evolved to reach enrollment targets and return results to participants. More than 170,000 participants have been enrolled in the study to date, with 5.87% of those whole genome sequenced and 0.46% of those genotyped harboring pathogenic variants. Among other purposes, this infrastructure supports: (1) identifying candidates from clinical criteria, (2) monitoring for qualifying clinical events (e.g., blood draw), (3) contacting candidates, (4) obtaining consent electronically, (5) initiating lab orders, (6) integrating consent and lab orders into clinical workflow, (7) de-identifying samples and clinical data, (8) shipping/transmitting samples and clinical data, (9) genotyping/sequencing samples, (10) and re-identifying and returning results for participants where applicable. This study may serve as a model for similar genomic research and precision public health initiatives.

Rare variants with large effects provide functional insights into the pathology of migraine subtypes, with and without aura.

Migraine is a complex neurovascular disease with a range of severity and symptoms, yet mostly studied as one phenotype in genome-wide association studies (GWAS). Here we combine large GWAS datasets from six European populations to study the main migraine subtypes, migraine with aura (MA) and migraine without aura (MO). We identified four new MA-associated variants (in PRRT2, PALMD, ABO and LRRK2) and classified 13 MO-associated variants. Rare variants with large effects highlight three genes. A rare frameshift variant in brain-expressed PRRT2 confers large risk of MA and epilepsy, but not MO. A burden test of rare loss-of-function variants in SCN11A, encoding a neuron-expressed sodium channel with a key role in pain sensation, shows strong protection against migraine. Finally, a rare variant with cis-regulatory effects on KCNK5 confers large protection against migraine and brain aneurysms. Our findings offer new insights with therapeutic potential into the complex biology of migraine and its subtypes.

Clinical Utility of Genomic Testing in Cancer Care.

PURPOSE: In the era of personalized medicine, physicians rely on their understanding of clinical utility to assess the value of rapidly evolving genetic and genomic tests. Current definitions of the clinical utility of genetic testing sufficiently capture a range of benefits and risks that derive from positive and negative results of tests that assess one gene or a few genes. However, these definitions of clinical utility are inadequate to recognize the wider scope of benefits that accrue from more comprehensive genomic tests, which can develop data sets that inform clinical decision making as well as population health and scientific advancement in novel ways. METHODS: An expert roundtable discussion with leaders from multiple sectors of the health care ecosystem was convened to develop a contemporary, fuller definition of the clinical utility of genomic testing in cancer care. RESULTS: We present an updated definition and offer recommendations for successful implementation. CONCLUSION: Applying this expanded definition will encourage evidence-based use of genomic testing in cancer care by helping physicians and other health care decision makers account for the broader range of benefits and risks of testing for individual patients, health systems, population health, and scientific advancement.

On the edge-A diagnostic odyssey.

The diagnostic odyssey of a child with epileptic encephalopathy was resolved by rapid whole genome sequencing. This identified a rare form of pyridoxine responsive epilepsy due to a pathogenic variant in PLPBP. Access to such potentially life-changing diagnostic technology needs to expand in a thoughtful and equitable manner.

Germline variants of ATG7 in familial cholangiocarcinoma alter autophagy and p62.

Autophagy is a housekeeping mechanism tasked with eliminating misfolded proteins and damaged organelles to maintain cellular homeostasis. Autophagy deficiency results in increased oxidative stress, DNA damage and chronic cellular injury. Among the core genes in the autophagy machinery, ATG7 is required for autophagy initiation and autophagosome formation. Based on the analysis of an extended pedigree of familial cholangiocarcinoma, we determined that all affected family members had a novel germline mutation (c.2000C>T p.Arg659* (p.R659*)) in ATG7. Somatic deletions of ATG7 were identified in the tumors of affected individuals. We applied linked-read sequencing to one tumor sample and demonstrated that the ATG7 somatic deletion and germline mutation were located on distinct alleles, resulting in two hits to ATG7. From a parallel population genetic study, we identified a germline polymorphism of ATG7 (c.1591C>G p.Asp522Glu (p.D522E)) associated with increased risk of cholangiocarcinoma. To characterize the impact of these germline ATG7 variants on autophagy activity, we developed an ATG7-null cell line derived from the human bile duct. The mutant p.R659* ATG7 protein lacked the ability to lipidate its LC3 substrate, leading to complete loss of autophagy and increased p62 levels. Our findings indicate that germline ATG7 variants have the potential to impact autophagy function with implications for cholangiocarcinoma development.

The Development of an Infrastructure to Facilitate the Use of Whole Genome Sequencing for Population Health.

The clinical use of genomic analysis has expanded rapidly resulting in an increased availability and utility of genomic information in clinical care. We have developed an infrastructure utilizing informatics tools and clinical processes to facilitate the use of whole genome sequencing data for population health management across the healthcare system. Our resulting framework scaled well to multiple clinical domains in both pediatric and adult care, although there were domain specific challenges that arose. Our infrastructure was complementary to existing clinical processes and well-received by care providers and patients. Informatics solutions were critical to the successful deployment and scaling of this program. Implementation of genomics at the scale of population health utilizes complicated technologies and processes that for many health systems are not supported by current information systems or in existing clinical workflows. To scale such a system requires a substantial clinical framework backed by informatics tools to facilitate the flow and management of data. Our work represents an early model that has been successful in scaling to 29 different genes with associated genetic conditions in four clinical domains. Work is ongoing to optimize informatics tools; and to identify best practices for translation to smaller healthcare systems.

Characterization of the consensus mucosal microbiome of colorectal cancer.

Dysbioisis is an imbalance of an organ's microbiome and plays a role in colorectal cancer pathogenesis. Characterizing the bacteria in the microenvironment of a cancer through genome sequencing has advantages compared to culture-based profiling. However, there are notable technical and analytical challenges in characterizing universal features of tumor microbiomes. Colorectal tumors demonstrate microbiome variation among different studies and across individual patients. To address these issues, we conducted a computational study to determine a consensus microbiome for colorectal cancer, analyzing 924 tumors from eight independent RNA-Seq data sets. A standardized meta-transcriptomic analysis pipeline was established with quality control metrics. Microbiome profiles across different cohorts were compared and recurrently altered microbial shifts specific to colorectal cancer were determined. We identified cancer-specific set of 114 microbial species associated with tumors that were found among all investigated studies. Firmicutes, Bacteroidetes, Proteobacteria and Actinobacteria were among the four most abundant phyla for the colorectal cancer microbiome. Member species of Clostridia were depleted and Fusobacterium nucleatum was one of the most enriched bacterial species in tumors. Associations between the consensus species and specific immune cell types were noted. Our results are available as a web data resource for other researchers to explore (https://crc-microbiome.stanford.edu).

The PATHFINDER Study: Assessment of the Implementation of an Investigational Multi-Cancer Early Detection Test into Clinical Practice.

To examine the extent of the evaluation required to achieve diagnostic resolution and the test performance characteristics of a targeted methylation cell-free DNA (cfDNA)-based multi-cancer early detection (MCED) test, ~6200 participants ≥50 years with (cohort A) or without (cohort B) ≥1 of 3 additional specific cancer risk factors will be enrolled in PATHFINDER (NCT04241796), a prospective, longitudinal, interventional, multi-center study. Plasma cfDNA from blood samples will be analyzed to detect abnormally methylated DNA associated with cancer (i.e., cancer "signal") and a cancer signal origin (i.e., tissue of origin). Participants with a "signal detected" will undergo further diagnostic evaluation per guiding physician discretion; those with a "signal not detected" will be advised to continue guideline-recommended screening. The primary objective will be to assess the number and types of subsequent diagnostic tests needed for diagnostic resolution. Based on microsimulations (using estimates of cancer incidence and dwell times) of the typical risk profiles of anticipated participants, the median (95% CI) number of participants with a "signal detected" result is expected to be 106 (87-128). Subsequent diagnostic evaluation is expected to detect 52 (39-67) cancers. The positive predictive value of the MCED test is expected to be 49% (39-58%). PATHFINDER will evaluate the integration of a cfDNA-based MCED test into existing clinical cancer diagnostic pathways. The study design of PATHFINDER is described here.

A genome-wide meta-analysis uncovers six sequence variants conferring risk of vertigo.

Vertigo is the leading symptom of vestibular disorders and a major risk factor for falls. In a genome-wide association study of vertigo (Ncases = 48,072, Ncontrols = 894,541), we uncovered an association with six common sequence variants in individuals of European ancestry, including missense variants in ZNF91, OTOG, OTOGL, and TECTA, and a cis-eQTL for ARMC9. The association of variants in ZNF91, OTOGL, and OTOP1 was driven by an association with benign paroxysmal positional vertigo. Using previous reports of sequence variants associating with age-related hearing impairment and motion sickness, we found eight additional variants that associate with vertigo. Although disorders of the auditory and the vestibular system may co-occur, none of the six genome-wide significant vertigo variants were associated with hearing loss and only one was associated with age-related hearing impairment. Our results uncovered sequence variants associating with vertigo in a genome-wide association study and implicated genes with known roles in inner ear development, maintenance, and disease.

An elevated neutrophil-to-lymphocyte ratio associates with weight loss and cachexia in cancer.

Systemic inflammation is present during and serves as a diagnostic tool for cancer-associated cachexia and is detrimental to serum 25-hydroxyvitamin D (25(OH)D) concentrations in non-cancer conditions. The neutrophil-to-lymphocyte ratio (NLR) is a desirable measure of systemic inflammation because it is easily calculated from a routine complete blood cell count with differentials. We sought to determine if an elevation in the NLR associates with greater weight loss, cachexia, and lower serum 25-hydroxyvitamin D (25(OH)D) concentrations in patients with advanced cancer. Advanced colon, lung, and prostate cancer patients (stages III/IV; n = 50) were retrospectively studied and separated into one of two groups: 1) Above (n = 25) or 2) Below (n = 25) the median NLR of 3.15 determined at diagnosis. Around the time of diagnosis, serum 25(OH)D and body weight were assessed, while body weight was assessed again at a later date. Weight loss and cachexia were significantly (both p < 0.05) greater and there was a trend (p < 0.10) for lower serum 25(OH)D concentrations in the Above group. We conclude that an elevation in the NLR associates with greater weight loss and cachexia, and potentially, a lower serum 25(OH)D concentration in patients with advanced colon, lung, or prostate cancer.

Discovery of TITIN Gene Truncating Variant Mutations and 5-Year Outcomes in Patients With Nonischemic Dilated Cardiomyopathy.

Genetic factors play an important role in nonischemic dilated cardiomyopathy (NIDC). However, prime opportunities remain for genetic discovery and prognostic understanding. TITIN gene truncating variant mutations (TTNtv) are of interest because of their frequent appearance in NIDC series. We sought to discover known and novel TTNtv mutations in a NIDC cohort and assess 5-year outcomes. Patients with NIDC entered into the INSPIRE Registry with ≥3 years of follow-up were studied. Whole exome sequencing (WES) was performed using an Illumina Novaseq platform. Genetic analysis used Sentieon software and the GRCh38 human reference genome. Variant calls were annotated with ClinVar. Five-year outcomes were determined by functional assessment and ejection fraction (EF) as recovered (EF ≥50%), persistent (EF 21% to 49%), or progressive (left ventricular assist device, transplant, heart failure [HF] or arrhythmic death, or EF ≤20%). The study comprised 229 NIDC patients (age = 50 ± 15 years, 58% men). TTNtv's were discovered in 27 patients with 22 unique mutations; (7 known, 15 novel). TTNtv+ patients more frequently presented with severe NIDC (EF ≤20%) (p = 0.032). By 5-year, outcomes were worse in TTNtv+ patients (p = 0.027), and patients less often recovered (11% vs. 30%). Prognosis was similar with known and novel mutations. Nongenetic (e.g., environmental) cocausal risk factors for HF were frequently present, and these factors frequently appeared to act in concert with genetic variants to precipitate clinical HF. In conclusion, our study expands the library of likely pathogenic TTN mutations and increases our understanding of their clinical impact in association with other HF risk factors.