High-throughput RNA isoform sequencing using programmed cDNA concatenation.

Full-length RNA-sequencing methods using long-read technologies can capture complete transcript isoforms, but their throughput is limited. We introduce multiplexed arrays isoform sequencing (MAS-ISO-seq), a technique for programmably concatenating complementary DNAs (cDNAs) into molecules optimal for long-read sequencing, increasing the throughput >15-fold to nearly 40 million cDNA reads per run on the Sequel IIe sequencer. When applied to single-cell RNA sequencing of tumor-infiltrating T cells, MAS-ISO-seq demonstrated a 12- to 32-fold increase in the discovery of differentially spliced genes.

Multi-ancestry genome-wide association analyses improve resolution of genes and pathways influencing lung function and chronic obstructive pulmonary disease risk.

Lung-function impairment underlies chronic obstructive pulmonary disease (COPD) and predicts mortality. In the largest multi-ancestry genome-wide association meta-analysis of lung function to date, comprising 580,869 participants, we identified 1,020 independent association signals implicating 559 genes supported by ≥2 criteria from a systematic variant-to-gene mapping framework. These genes were enriched in 29 pathways. Individual variants showed heterogeneity across ancestries, age and smoking groups, and collectively as a genetic risk score showed strong association with COPD across ancestry groups. We undertook phenome-wide association studies for selected associated variants as well as trait and pathway-specific genetic risk scores to infer possible consequences of intervening in pathways underlying lung function. We highlight new putative causal variants, genes, proteins and pathways, including those targeted by existing drugs. These findings bring us closer to understanding the mechanisms underlying lung function and COPD, and should inform functional genomics experiments and potentially future COPD therapies.

Mechanisms of response and resistance to combined decitabine and ipilimumab for advanced myeloid disease.

The challenge of eradicating leukemia in patients with acute myelogenous leukemia (AML) after initial cytoreduction has motivated modern efforts to combine synergistic active modalities including immunotherapy. Recently, the ETCTN/CTEP 10026 study tested the combination of the DNA methyltransferase inhibitor decitabine together with the immune checkpoint inhibitor ipilimumab for AML/myelodysplastic syndrome (MDS) either after allogeneic hematopoietic stem cell transplantation (HSCT) or in the HSCT-naïve setting. Integrative transcriptome-based analysis of 304 961 individual marrow-infiltrating cells for 18 of 48 subjects treated on study revealed the strong association of response with a high baseline ratio of T to AML cells. Clinical responses were predominantly driven by decitabine-induced cytoreduction. Evidence of immune activation was only apparent after ipilimumab exposure, which altered CD4+ T-cell gene expression, in line with ongoing T-cell differentiation and increased frequency of marrow-infiltrating regulatory T cells. For post-HSCT samples, relapse could be attributed to insufficient clearing of malignant clones in progenitor cell populations. In contrast to AML/MDS bone marrow, the transcriptomes of leukemia cutis samples from patients with durable remission after ipilimumab monotherapy showed evidence of increased infiltration with antigen-experienced resident memory T cells and higher expression of CTLA-4 and FOXP3. Altogether, activity of combined decitabine and ipilimumab is impacted by cellular expression states within the microenvironmental niche of leukemic cells. The inadequate elimination of leukemic progenitors mandates urgent development of novel approaches for targeting these cell populations to generate long-lasting responses. This trial was registered at www.clinicaltrials.gov as #NCT02890329.

Large-scale sequencing identifies multiple genes and rare variants associated with Crohn's disease susceptibility.

Genome-wide association studies (GWASs) have identified hundreds of loci associated with Crohn's disease (CD). However, as with all complex diseases, robust identification of the genes dysregulated by noncoding variants typically driving GWAS discoveries has been challenging. Here, to complement GWASs and better define actionable biological targets, we analyzed sequence data from more than 30,000 patients with CD and 80,000 population controls. We directly implicate ten genes in general onset CD for the first time to our knowledge via association to coding variation, four of which lie within established CD GWAS loci. In nine instances, a single coding variant is significantly associated, and in the tenth, ATG4C, we see additionally a significantly increased burden of very rare coding variants in CD cases. In addition to reiterating the central role of innate and adaptive immune cells as well as autophagy in CD pathogenesis, these newly associated genes highlight the emerging role of mesenchymal cells in the development and maintenance of intestinal inflammation.

Accounting for population structure in genetic studies of cystic fibrosis.

CFTR F508del (c.1521_1523delCTT, p.Phe508delPhe) is the most common pathogenic allele underlying cystic fibrosis (CF), and its frequency varies in a geographic cline across Europe. We hypothesized that genetic variation associated with this cline is overrepresented in a large cohort (N > 5,000) of persons with CF who underwent whole-genome sequencing and that this pattern could result in spurious associations between variants correlated with both the F508del genotype and CF-related outcomes. Using principal-component (PC) analyses, we showed that variation in the CFTR region disproportionately contributes to a PC explaining a relatively high proportion of genetic variance. Variation near CFTR was correlated with population structure among persons with CF, and this correlation was driven by a subset of the sample inferred to have European ancestry. We performed genome-wide association studies comparing persons with CF with one versus two copies of the F508del allele; this allowed us to identify genetic variation associated with the F508del allele and to determine that standard PC-adjustment strategies eliminated the significant association signals. Our results suggest that PC adjustment can adequately prevent spurious associations between genetic variants and CF-related traits and are therefore effective tools to control for population structure even when population structure is confounded with disease severity and a common pathogenic variant.

Combined tumor and immune signals from genomes or transcriptomes predict outcomes of checkpoint inhibition in melanoma.

Immune checkpoint blockade (CPB) improves melanoma outcomes, but many patients still do not respond. Tumor mutational burden (TMB) and tumor-infiltrating T cells are associated with response, and integrative models improve survival prediction. However, integrating immune/tumor-intrinsic features using data from a single assay (DNA/RNA) remains underexplored. Here, we analyze whole-exome and bulk RNA sequencing of tumors from new and published cohorts of 189 and 178 patients with melanoma receiving CPB, respectively. Using DNA, we calculate T cell and B cell burdens (TCB/BCB) from rearranged TCR/Ig sequences and find that patients with TMBhigh and TCBhigh or BCBhigh have improved outcomes compared to other patients. By combining pairs of immune- and tumor-expressed genes, we identify three gene pairs associated with response and survival, which validate in independent cohorts. The top model includes lymphocyte-expressed MAP4K1 and tumor-expressed TBX3. Overall, RNA or DNA-based models combining immune and tumor measures improve predictions of melanoma CPB outcomes.

Diet Quality Scores Are Positively Associated with Whole Blood-Derived Mitochondrial DNA Copy Number in the Framingham Heart Study.

BACKGROUND: The association between diet quality and mitochondrial DNA copy number (mtDNA-CN) remains to be examined. OBJECTIVES: We aimed to study the relation between diet quality and mtDNA-CN. METHODS: We analyzed data from 2931 Framingham Heart Study (FHS) participants (mean age of 57 y, 55% females). Whole-genome sequencing was used to calculate mtDNA-CN from whole-blood samples. We examined the cross-sectional associations between 3 diet quality scores, the Dietary Approaches to Stop Hypertension (DASH) score, the Alternative Healthy Eating Index (AHEI), and the Mediterranean diet score (MDS), and mtDNA-CN. Linear mixed models were used to account for maternal lineage. RESULTS: We observed that a higher DASH score was positively associated with mtDNA-CN after adjusting for sex, age, energy intake, smoking status, alcohol intake, and physical activity level. A 1-SD increase in the DASH score was associated with a 0.042-SD greater mtDNA-CN (95% CI: 0.007, 0.077; P = 0.02). Similarly, for each SD increase in AHEI and MDS, the mtDNA-CN SD increased by 0.056 (95% CI: 0.019, 0.092; P = 0.003) and 0.047 (95% CI: 0.01, 0.083; P = 0.01), respectively. Diet quality scores were associated with neutrophil and lymphocyte counts but not platelet counts, e.g., for a 1-SD increase in the DASH, neutrophils decreased by 0.8% (95% CI: 0.5%, 1.1%; P = 4.1 × 10-6), lymphocytes increased by 0.7% (95% CI: 0.4%, 1%, P = 1.2 × 10-5), and there was no significant change in platelet number (0.1 × 1000/µL; 95% CI: -1.6, 1.9; P = 0.89). Further adjustment for neutrophil, lymphocyte, and platelet counts and the associations between diet quality scores and mtDNA-CN were completely attenuated to nonsignificant (P = 0.95, 0.54, and 0.91, respectively). CONCLUSIONS: We observed that higher diet quality is associated with a greater whole-blood derived mtDNA-CN in middle-aged to older adult FHS participants, and that blood cell composition, particularly neutrophil counts, attenuated the association between diet quality and mtDNA-CN.

Molecular and cellular features of CTLA-4 blockade for relapsed myeloid malignancies after transplantation.

Relapsed myeloid disease after allogeneic stem cell transplantation (HSCT) remains largely incurable. We previously demonstrated the potent activity of immune checkpoint blockade in this clinical setting with ipilimumab or nivolumab. To define the molecular and cellular pathways by which CTLA-4 blockade with ipilimumab can reinvigorate an effective graft-versus-leukemia (GVL) response, we integrated transcriptomic analysis of leukemic biopsies with immunophenotypic profiling of matched peripheral blood samples collected from patients treated with ipilimumab following HSCT on the Experimental Therapeutics Clinical Trials Network 9204 trial. Response to ipilimumab was associated with transcriptomic evidence of increased local CD8+ T-cell infiltration and activation. Systemically, ipilimumab decreased naïve and increased memory T-cell populations and increased expression of markers of T-cell activation and costimulation such as PD-1, HLA-DR, and ICOS, irrespective of response. However, responding patients were characterized by higher turnover of T-cell receptor sequences in peripheral blood and showed increased expression of proinflammatory chemokines in plasma that was further amplified by ipilimumab. Altogether, these data highlight the compositional T-cell shifts and inflammatory pathways induced by ipilimumab both locally and systemically that associate with successful GVL outcomes. This trial was registered at www.clinicaltrials.gov as #NCT01822509.

Performance and Implementation Evaluation of the Abbott BinaxNOW Rapid Antigen Test in a High-Throughput Drive-Through Community Testing Site in Massachusetts.

Rapid diagnostic tests (RDTs) for SARS-CoV-2 antigens (Ag) that can be performed at point of care (POC) can supplement molecular testing and help mitigate the COVID-19 pandemic. Deployment of an Ag RDT requires an understanding of its operational and performance characteristics under real-world conditions and in relevant subpopulations. We evaluated the Abbott BinaxNOW COVID-19 Ag card in a high-throughput, drive-through, free community testing site in Massachusetts using anterior nasal (AN) swab reverse transcriptase PCR (RT-PCR) for clinical testing. Individuals presenting for molecular testing in two of seven lanes were offered the opportunity to also receive BinaxNOW testing. Dual AN swabs were collected from symptomatic and asymptomatic children (≤18 years of age) and adults. BinaxNOW testing was performed in a testing pod with temperature/humidity monitoring. One individual performed testing and official result reporting for each test, but most tests had a second independent reading to assess interoperator agreement. Positive BinaxNOW results were scored as faint, medium, or strong. Positive BinaxNOW results were reported to patients by phone, and they were instructed to isolate pending RT-PCR results. The paired RT-PCR result was the reference for sensitivity and specificity calculations. Of 2,482 participants, 1,380 adults and 928 children had paired RT-PCR/BinaxNOW results and complete symptom data. In this study, 974/1,380 (71%) adults and 829/928 (89%) children were asymptomatic. BinaxNOW had 96.5% (95% confidence interval [CI], 90.0 to 99.3) sensitivity and 100% (95% CI, 98.6 to 100.0) specificity in adults within 7 days of symptoms and 84.6% (95% CI, 65.1 to 95.6) sensitivity and 100% (95% CI, 94.5 to 100.0) specificity in children within 7 days of symptoms. Sensitivity and specificity in asymptomatic adults were 70.2% (95% CI, 56.6 to 81.6) and 99.6% (95% CI, 98.9 to 99.9), respectively, and in asymptomatic children, they were 65.4% (95% CI, 55.6 to 74.4) and 99.0% (95% CI, 98.0 to 99.6), respectively. By cycle threshold (CT ) value cutoff, sensitivity in all subgroups combined (n = 292 RT-PCR-positive individuals) was 99.3% with CT values of ≤25, 95.8% with CT values of ≤30, and 81.2% with CT values of ≤35. Twelve false-positive BinaxNOW results (out of 2,308 tests) were observed; in all 12, the test bands were faint but otherwise normal and were noted by both readers. One invalid BinaxNOW result was identified. Interoperator agreement (positive versus negative BinaxNOW result) was 100% (n = 2,230/2,230 double reads). Each operator was able to process 20 RDTs per hour. In a separate set of 30 specimens (from individuals with symptoms ≤7 days) run at temperatures below the manufacturer's recommended range (46 to 58.5°F), sensitivity was 66.7% and specificity 95.2%. BinaxNOW had very high specificity in both adults and children and very high sensitivity in newly symptomatic adults. Overall, 95.8% sensitivity was observed with CT values of ≤30. These data support public health recommendations for use of the BinaxNOW test in adults with symptoms for ≤7 days without RT-PCR confirmation. Excellent interoperator agreement indicates that an individual can perform and read the BinaxNOW test alone. A skilled laboratorian can perform and read 20 tests per hour. Careful attention to temperature is critical.

Cross-Site Concordance Evaluation of Tumor DNA and RNA Sequencing Platforms for the CIMAC-CIDC Network.

PURPOSE: Whole-exome (WES) and RNA sequencing (RNA-seq) are key components of cancer immunogenomic analyses. To evaluate the consistency of tumor WES and RNA-seq profiling platforms across different centers, the Cancer Immune Monitoring and Analysis Centers (CIMAC) and the Cancer Immunologic Data Commons (CIDC) conducted a systematic harmonization study. EXPERIMENTAL DESIGN: DNA and RNA were centrally extracted from fresh frozen and formalin-fixed paraffin-embedded non-small cell lung carcinoma tumors and distributed to three centers for WES and RNA-seq profiling. In addition, two 10-plex HapMap cell line pools with known mutations were used to evaluate the accuracy of the WES platforms. RESULTS: The WES platforms achieved high precision (> 0.98) and recall (> 0.87) on the HapMap pools when evaluated on loci using > 50× common coverage. Nonsynonymous mutations clustered by tumor sample, achieving an index of specific agreement above 0.67 among replicates, centers, and sample processing. A DV200 > 24% for RNA, as a putative presequencing RNA quality control (QC) metric, was found to be a reliable threshold for generating consistent expression readouts in RNA-seq and NanoString data. MedTIN > 30 was likewise assessed as a reliable RNA-seq QC metric, above which samples from the same tumor across replicates, centers, and sample processing runs could be robustly clustered and HLA typing, immune infiltration, and immune repertoire inference could be performed. CONCLUSIONS: The CIMAC collaborating laboratory platforms effectively generated consistent WES and RNA-seq data and enable robust cross-trial comparisons and meta-analyses of highly complex immuno-oncology biomarker data across the NCI CIMAC-CIDC Network.

Premature Menopause, Clonal Hematopoiesis, and Coronary Artery Disease in Postmenopausal Women.

BACKGROUND: Premature menopause is an independent risk factor for cardiovascular disease in women, but mechanisms underlying this association remain unclear. Clonal hematopoiesis of indeterminate potential (CHIP), the age-related expansion of hematopoietic cells with leukemogenic mutations without detectable malignancy, is associated with accelerated atherosclerosis. Whether premature menopause is associated with CHIP is unknown. METHODS: We included postmenopausal women from the UK Biobank (n=11 495) aged 40 to 70 years with whole exome sequences and from the Women's Health Initiative (n=8111) aged 50 to 79 years with whole genome sequences. Premature menopause was defined as natural or surgical menopause occurring before age 40 years. Co-primary outcomes were the presence of any CHIP and CHIP with variant allele frequency >0.1. Logistic regression tested the association of premature menopause with CHIP, adjusted for age, race, the first 10 principal components of ancestry, smoking, diabetes, and hormone therapy use. Secondary analyses considered natural versus surgical premature menopause and gene-specific CHIP subtypes. Multivariable-adjusted Cox models tested the association between CHIP and incident coronary artery disease. RESULTS: The sample included 19 606 women, including 418 (2.1%) with natural premature menopause and 887 (4.5%) with surgical premature menopause. Across cohorts, CHIP prevalence in postmenopausal women with versus without a history of premature menopause was 8.8% versus 5.5% (P<0.001), respectively. After multivariable adjustment, premature menopause was independently associated with CHIP (all CHIP: odds ratio, 1.36 [95% 1.10-1.68]; P=0.004; CHIP with variant allele frequency >0.1: odds ratio, 1.40 [95% CI, 1.10-1.79]; P=0.007). Associations were larger for natural premature menopause (all CHIP: odds ratio, 1.73 [95% CI, 1.23-2.44]; P=0.001; CHIP with variant allele frequency >0.1: odds ratio, 1.91 [95% CI, 1.30-2.80]; P<0.001) but smaller and nonsignificant for surgical premature menopause. In gene-specific analyses, only DNMT3A CHIP was significantly associated with premature menopause. Among postmenopausal middle-aged women, CHIP was independently associated with incident coronary artery disease (hazard ratio associated with all CHIP: 1.36 [95% CI, 1.07-1.73]; P=0.012; hazard ratio associated with CHIP with variant allele frequency >0.1: 1.48 [95% CI, 1.13-1.94]; P=0.005). CONCLUSIONS: Premature menopause, especially natural premature menopause, is independently associated with CHIP among postmenopausal women. Natural premature menopause may serve as a risk signal for predilection to develop CHIP and CHIP-associated cardiovascular disease.

Infantile Myelofibrosis and Myeloproliferation with CDC42 Dysfunction.

Studies of genetic blood disorders have advanced our understanding of the intrinsic regulation of hematopoiesis. However, such genetic studies have only yielded limited insights into how interactions between hematopoietic cells and their microenvironment are regulated. Here, we describe two affected siblings with infantile myelofibrosis and myeloproliferation that share a common de novo mutation in the Rho GTPase CDC42 (Chr1:22417990:C>T, p.R186C) due to paternal germline mosaicism. Functional studies using human cells and flies demonstrate that this CDC42 mutant has altered activity and thereby disrupts interactions between hematopoietic progenitors and key tissue microenvironmental factors. These findings suggest that further investigation of this and other related disorders may provide insights into how hematopoietic cell-microenvironment interactions play a role in human health and can be disrupted in disease. In addition, we suggest that deregulation of CDC42 may underlie more common blood disorders, such as primary myelofibrosis.

Genetic Interleukin 6 Signaling Deficiency Attenuates Cardiovascular Risk in Clonal Hematopoiesis.

BACKGROUND: Clonal hematopoiesis of indeterminate potential (CHIP) refers to clonal expansion of hematopoietic stem cells attributable to acquired leukemic mutations in genes such as DNMT3A or TET2. In humans, CHIP associates with prevalent myocardial infarction. In mice, CHIP accelerates atherosclerosis and increases IL-6/IL-1ß expression, raising the hypothesis that IL-6 pathway antagonism in CHIP carriers would decrease cardiovascular disease (CVD) risk. METHODS: We analyzed exome sequences from 35 416 individuals in the UK Biobank without prevalent CVD, to identify participants with DNMT3A or TET2 CHIP. We used the IL6R p.Asp358Ala coding mutation as a genetic proxy for IL-6 inhibition. We tested the association of CHIP status with incident CVD events (myocardial infarction, coronary revascularization, stroke, or death), and whether it was modified by IL6R p.Asp358Ala. RESULTS: We identified 1079 (3.0%) individuals with CHIP, including 432 (1.2%) with large clones (allele fraction >10%). During 6.9-year median follow-up, CHIP associated with increased incident CVD event risk (hazard ratio, 1.27 [95% CI, 1.04-1.56], P=0.019), with greater risk from large CHIP clones (hazard ratio, 1.59 [95% CI, 1.21-2.09], P<0.001). IL6R p.Asp358Ala attenuated CVD event risk among participants with large CHIP clones (hazard ratio, 0.46 [95% CI, 0.29-0.73], P<0.001) but not in individuals without CHIP (hazard ratio, 0.95 [95% CI, 0.89-1.01], P=0.08; Pinteraction=0.003). In 9951 independent participants, the association of CHIP status with myocardial infarction similarly varied by IL6R p.Asp358Ala (Pinteraction=0.036). CONCLUSIONS: CHIP is associated with increased risk of incident CVD. Among carriers of large CHIP clones, genetically reduced IL-6 signaling abrogated this risk.

Impaired human hematopoiesis due to a cryptic intronic GATA1 splicing mutation.

Studies of allelic variation underlying genetic blood disorders have provided important insights into human hematopoiesis. Most often, the identified pathogenic mutations result in loss-of-function or missense changes. However, assessing the pathogenicity of noncoding variants can be challenging. Here, we characterize two unrelated patients with a distinct presentation of dyserythropoietic anemia and other impairments in hematopoiesis associated with an intronic mutation in GATA1 that is 24 nucleotides upstream of the canonical splice acceptor site. Functional studies demonstrate that this single-nucleotide alteration leads to reduced canonical splicing and increased use of an alternative splice acceptor site that causes a partial intron retention event. The resultant altered GATA1 contains a five-amino acid insertion at the C-terminus of the C-terminal zinc finger and has no observable activity. Collectively, our results demonstrate how altered splicing of GATA1, which reduces levels of the normal form of this master transcription factor, can result in distinct changes in human hematopoiesis.

Triggered Palliative Care for Late-Stage Dementia: A Pilot Randomized Trial.

CONTEXT: Persons with late-stage dementia have limited access to palliative care. OBJECTIVE: The objective of this study was to test dementia-specific specialty palliative care triggered by hospitalization. METHODS: This pilot randomized controlled trial enrolled 62 dyads of persons with late-stage dementia and family decision-makers on admission to hospital. Intervention dyads received dementia-specific specialty palliative care consultation plus postacute transitional care. Control dyads received usual care and educational information. The primary outcome was 60-day hospital or emergency department visits. Secondary patient- and family-centered outcomes were patient comfort, family distress, palliative care domains addressed in the treatment plan, and access to hospice or community-based palliative care. Secondary decision-making outcomes were discussion of prognosis, goals of care, completion of Medical Orders for Scope of Treatment (MOST), and treatment decisions. RESULTS: Of 137 eligible dyads, 62 (45%) were enrolled. The intervention proved feasible, with protocol completion ranging from 77% (family two-week call) to 93% (initial consultation). Hospital and emergency department visits did not differ (intervention vs. control, 0.68 vs. 0.53 transfers per 60 days, P = 0.415). Intervention patients had more palliative care domains addressed and were more likely to receive hospice (25% vs. 3%, P < 0.019). Intervention families were more likely to discuss prognosis (90% vs. 3%, P < 0.001) and goals of care (90% vs. 25%, P < 0.001) and to have a MOST at 60-day follow-up (79% vs. 30%, P < 0.001). More intervention families made decisions to avoid rehospitalization (13% vs. 0%, P = 0.033). CONCLUSION: Specialty palliative care consultation for hospitalized patients with late-stage dementia is feasible and promising to improve decision-making and some treatment outcomes.

Recall by genotype and cascade screening for familial hypercholesterolemia in a population-based biobank from Estonia.

PURPOSE: Large-scale, population-based biobanks integrating health records and genomic profiles may provide a platform to identify individuals with disease-predisposing genetic variants. Here, we recall probands carrying familial hypercholesterolemia (FH)-associated variants, perform cascade screening of family members, and describe health outcomes affected by such a strategy. METHODS: The Estonian Biobank of Estonian Genome Center, University of Tartu, comprises 52,274 individuals. Among 4776 participants with exome or genome sequences, we identified 27 individuals who carried FH-associated variants in the LDLR, APOB, or PCSK9 genes. Cascade screening of 64 family members identified an additional 20 carriers of FH-associated variants. RESULTS: Via genetic counseling and clinical management of carriers, we were able to reclassify 51% of the study participants from having previously established nonspecific hypercholesterolemia to having FH and identify 32% who were completely unaware of harboring a high-risk disease-associated genetic variant. Imaging-based risk stratification targeted 86% of the variant carriers for statin treatment recommendations. CONCLUSION: Genotype-guided recall of probands and subsequent cascade screening for familial hypercholesterolemia is feasible within a population-based biobank and may facilitate more appropriate clinical management.

Neoantigen vaccine generates intratumoral T cell responses in phase Ib glioblastoma trial.

Neoantigens, which are derived from tumour-specific protein-coding mutations, are exempt from central tolerance, can generate robust immune responses1,2 and can function as bona fide antigens that facilitate tumour rejection3. Here we demonstrate that a strategy that uses multi-epitope, personalized neoantigen vaccination, which has previously been tested in patients with high-risk melanoma4-6, is feasible for tumours such as glioblastoma, which typically have a relatively low mutation load1,7 and an immunologically 'cold' tumour microenvironment8. We used personalized neoantigen-targeting vaccines to immunize patients newly diagnosed with glioblastoma following surgical resection and conventional radiotherapy in a phase I/Ib study. Patients who did not receive dexamethasone-a highly potent corticosteroid that is frequently prescribed to treat cerebral oedema in patients with glioblastoma-generated circulating polyfunctional neoantigen-specific CD4+ and CD8+ T cell responses that were enriched in a memory phenotype and showed an increase in the number of tumour-infiltrating T cells. Using single-cell T cell receptor analysis, we provide evidence that neoantigen-specific T cells from the peripheral blood can migrate into an intracranial glioblastoma tumour. Neoantigen-targeting vaccines thus have the potential to favourably alter the immune milieu of glioblastoma.

Whole-Genome Sequencing to Characterize Monogenic and Polygenic Contributions in Patients Hospitalized With Early-Onset Myocardial Infarction.

BACKGROUND: The relative prevalence and clinical importance of monogenic mutations related to familial hypercholesterolemia and of high polygenic score (cumulative impact of many common variants) pathways for early-onset myocardial infarction remain uncertain. Whole-genome sequencing enables simultaneous ascertainment of both monogenic mutations and polygenic score for each individual. METHODS: We performed deep-coverage whole-genome sequencing of 2081 patients from 4 racial subgroups hospitalized in the United States with early-onset myocardial infarction (age ≤55 years) recruited with a 2:1 female-to-male enrollment design. We compared these genomes with those of 3761 population-based control subjects. We first identified individuals with a rare, monogenic mutation related to familial hypercholesterolemia. Second, we calculated a recently developed polygenic score of 6.6 million common DNA variants to quantify the cumulative susceptibility conferred by common variants. We defined high polygenic score as the top 5% of the control distribution because this cutoff has previously been shown to confer similar risk to that of familial hypercholesterolemia mutations. RESULTS: The mean age of the 2081 patients presenting with early-onset myocardial infarction was 48 years, and 66% were female. A familial hypercholesterolemia mutation was present in 36 of these patients (1.7%) and was associated with a 3.8-fold (95% CI, 2.1-6.8; P<0.001) increased odds of myocardial infarction. Of the patients with early-onset myocardial infarction, 359 (17.3%) carried a high polygenic score, associated with a 3.7-fold (95% CI, 3.1-4.6; P<0.001) increased odds. Mean estimated untreated low-density lipoprotein cholesterol was 206 mg/dL in those with a familial hypercholesterolemia mutation, 132 mg/dL in those with high polygenic score, and 122 mg/dL in those in the remainder of the population. Although associated with increased risk in all racial groups, high polygenic score demonstrated the strongest association in white participants ( P for heterogeneity=0.008). CONCLUSIONS: Both familial hypercholesterolemia mutations and high polygenic score are associated with a >3-fold increased odds of early-onset myocardial infarction. However, high polygenic score has a 10-fold higher prevalence among patients presents with early-onset myocardial infarction. CLINICAL TRIAL REGISTRATION: URL: https://www.clinicaltrials.gov . Unique identifier: NCT00597922.

Loss of Protocadherin-12 Leads to Diencephalic-Mesencephalic Junction Dysplasia Syndrome.

OBJECTIVE: To identify causes of the autosomal-recessive malformation, diencephalic-mesencephalic junction dysplasia (DMJD) syndrome. METHODS: Eight families with DMJD were studied by whole-exome or targeted sequencing, with detailed clinical and radiological characterization. Patient-derived induced pluripotent stem cells were derived into neural precursor and endothelial cells to study gene expression. RESULTS: All patients showed biallelic mutations in the nonclustered protocadherin-12 (PCDH12) gene. The characteristic clinical presentation included progressive microcephaly, craniofacial dysmorphism, psychomotor disability, epilepsy, and axial hypotonia with variable appendicular spasticity. Brain imaging showed brainstem malformations and with frequent thinned corpus callosum with punctate brain calcifications, reflecting expression of PCDH12 in neural and endothelial cells. These cells showed lack of PCDH12 expression and impaired neurite outgrowth. INTERPRETATION: DMJD patients have biallelic mutations in PCDH12 and lack of protein expression. These patients present with characteristic microcephaly and abnormalities of white matter tracts. Such pathogenic variants predict a poor outcome as a result of brainstem malformation and evidence of white matter tract defects, and should be added to the phenotypic spectrum associated with PCDH12-related conditions. Ann Neurol 2018;84:646-655.