Advancing drug discovery using the power of the human genome.

Human genetics plays an increasingly important role in drug development and population health. Here we review the history of human genetics in the context of accelerating the discovery of therapies, present examples of how human genetics evidence supports successful drug targets, and discuss how polygenic risk scores could be beneficial in various clinical settings. We highlight the value of direct-to-consumer platforms in the era of fast-paced big data biotechnology, and how diverse genetic and health data can benefit society. © 2021 23andMe, Inc. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Correction to: Sequencing and curation strategies for identifying candidate glioblastoma treatments.

Following publication of the original article [1], it was reported that the given name of the fourteenth author was incorrectly published. The incorrect and the correct names are given below.

Sequencing and curation strategies for identifying candidate glioblastoma treatments.

BACKGROUND: Prompted by the revolution in high-throughput sequencing and its potential impact for treating cancer patients, we initiated a clinical research study to compare the ability of different sequencing assays and analysis methods to analyze glioblastoma tumors and generate real-time potential treatment options for physicians. METHODS: A consortium of seven institutions in New York City enrolled 30 patients with glioblastoma and performed tumor whole genome sequencing (WGS) and RNA sequencing (RNA-seq; collectively WGS/RNA-seq); 20 of these patients were also analyzed with independent targeted panel sequencing. We also compared results of expert manual annotations with those from an automated annotation system, Watson Genomic Analysis (WGA), to assess the reliability and time required to identify potentially relevant pharmacologic interventions. RESULTS: WGS/RNAseq identified more potentially actionable clinical results than targeted panels in 90% of cases, with an average of 16-fold more unique potentially actionable variants identified per individual; 84 clinically actionable calls were made using WGS/RNA-seq that were not identified by panels. Expert annotation and WGA had good agreement on identifying variants [mean sensitivity = 0.71, SD = 0.18 and positive predictive value (PPV) = 0.80, SD = 0.20] and drug targets when the same variants were called (mean sensitivity = 0.74, SD = 0.34 and PPV = 0.79, SD = 0.23) across patients. Clinicians used the information to modify their treatment plan 10% of the time. CONCLUSION: These results present the first comprehensive comparison of technical and machine augmented analysis of targeted panel and WGS/RNA-seq to identify potential cancer treatments.

Genome-wide analysis of insomnia in 1,331,010 individuals identifies new risk loci and functional pathways.

Insomnia is the second most prevalent mental disorder, with no sufficient treatment available. Despite substantial heritability, insight into the associated genes and neurobiological pathways remains limited. Here, we use a large genetic association sample (n = 1,331,010) to detect novel loci and gain insight into the pathways, tissue and cell types involved in insomnia complaints. We identify 202 loci implicating 956 genes through positional, expression quantitative trait loci, and chromatin mapping. The meta-analysis explained 2.6% of the variance. We show gene set enrichments for the axonal part of neurons, cortical and subcortical tissues, and specific cell types, including striatal, hypothalamic, and claustrum neurons. We found considerable genetic correlations with psychiatric traits and sleep duration, and modest correlations with other sleep-related traits. Mendelian randomization identified the causal effects of insomnia on depression, diabetes, and cardiovascular disease, and the protective effects of educational attainment and intracranial volume. Our findings highlight key brain areas and cell types implicated in insomnia, and provide new treatment targets.

Genome-wide somatic variant calling using localized colored de Bruijn graphs.

Reliable detection of somatic variations is of critical importance in cancer research. Here we present Lancet, an accurate and sensitive somatic variant caller, which detects SNVs and indels by jointly analyzing reads from tumor and matched normal samples using colored de Bruijn graphs. We demonstrate, through extensive experimental comparison on synthetic and real whole-genome sequencing datasets, that Lancet has better accuracy, especially for indel detection, than widely used somatic callers, such as MuTect, MuTect2, LoFreq, Strelka, and Strelka2. Lancet features a reliable variant scoring system, which is essential for variant prioritization, and detects low-frequency mutations without sacrificing the sensitivity to call longer insertions and deletions empowered by the local-assembly engine. In addition to genome-wide analysis, Lancet allows inspection of somatic variants in graph space, which augments the traditional read alignment visualization to help confirm a variant of interest. Lancet is available as an open-source program at https://github.com/nygenome/lancet.

Placenta and appetite genes GDF15 and IGFBP7 are associated with hyperemesis gravidarum.

Hyperemesis gravidarum (HG), severe nausea and vomiting of pregnancy, occurs in 0.3-2% of pregnancies and is associated with maternal and fetal morbidity. The cause of HG remains unknown, but familial aggregation and results of twin studies suggest that understanding the genetic contribution is essential for comprehending the disease etiology. Here, we conduct a genome-wide association study (GWAS) for binary (HG) and ordinal (severity of nausea and vomiting) phenotypes of pregnancy complications. Two loci, chr19p13.11 and chr4q12, are genome-wide significant (p < 5 × 10-8) in both association scans and are replicated in an independent cohort. The genes implicated at these two loci are GDF15 and IGFBP7 respectively, both known to be involved in placentation, appetite, and cachexia. While proving the casual roles of GDF15 and IGFBP7 in nausea and vomiting of pregnancy requires further study, this GWAS provides insights into the genetic risk factors contributing to the disease.

Comparing sequencing assays and human-machine analyses in actionable genomics for glioblastoma.

OBJECTIVE: To analyze a glioblastoma tumor specimen with 3 different platforms and compare potentially actionable calls from each. METHODS: Tumor DNA was analyzed by a commercial targeted panel. In addition, tumor-normal DNA was analyzed by whole-genome sequencing (WGS) and tumor RNA was analyzed by RNA sequencing (RNA-seq). The WGS and RNA-seq data were analyzed by a team of bioinformaticians and cancer oncologists, and separately by IBM Watson Genomic Analytics (WGA), an automated system for prioritizing somatic variants and identifying drugs. RESULTS: More variants were identified by WGS/RNA analysis than by targeted panels. WGA completed a comparable analysis in a fraction of the time required by the human analysts. CONCLUSIONS: The development of an effective human-machine interface in the analysis of deep cancer genomic datasets may provide potentially clinically actionable calls for individual patients in a more timely and efficient manner than currently possible. CLINICALTRIALSGOV IDENTIFIER: NCT02725684.

Indel variant analysis of short-read sequencing data with Scalpel.

As the second most common type of variation in the human genome, insertions and deletions (indels) have been linked to many diseases, but the discovery of indels of more than a few bases in size from short-read sequencing data remains challenging. Scalpel (http://scalpel.sourceforge.net) is an open-source software for reliable indel detection based on the microassembly technique. It has been successfully used to discover mutations in novel candidate genes for autism, and it is extensively used in other large-scale studies of human diseases. This protocol gives an overview of the algorithm and describes how to use Scalpel to perform highly accurate indel calling from whole-genome and whole-exome sequencing data. We provide detailed instructions for an exemplary family-based de novo study, but we also characterize the other two supported modes of operation: single-sample and somatic analysis. Indel normalization, visualization and annotation of the mutations are also illustrated. Using a standard server, indel discovery and characterization in the exonic regions of the example sequencing data can be completed in ∼5 h after read mapping.

Genetic Determinants of Cisplatin Resistance in Patients With Advanced Germ Cell Tumors.

Purpose Owing to its exquisite chemotherapy sensitivity, most patients with metastatic germ cell tumors (GCTs) are cured with cisplatin-based chemotherapy. However, up to 30% of patients with advanced GCT exhibit cisplatin resistance, which requires intensive salvage treatment, and have a 50% risk of cancer-related death. To identify a genetic basis for cisplatin resistance, we performed whole-exome and targeted sequencing of cisplatin-sensitive and cisplatin-resistant GCTs. Methods Men with GCT who received a cisplatin-containing chemotherapy regimen and had available tumor tissue were eligible to participate in this study. Whole-exome sequencing or targeted exon-capture-based sequencing was performed on 180 tumors. Patients were categorized as cisplatin sensitive or cisplatin resistant by using a combination of postchemotherapy parameters, including serum tumor marker levels, radiology, and pathology at surgical resection of residual disease. Results TP53 alterations were present exclusively in cisplatin-resistant tumors and were particularly prevalent among primary mediastinal nonseminomas (72%). TP53 pathway alterations including MDM2 amplifications were more common among patients with adverse clinical features, categorized as poor risk according to the International Germ Cell Cancer Collaborative Group (IGCCCG) model. Despite this association, TP53 and MDM2 alterations predicted adverse prognosis independent of the IGCCCG model. Actionable alterations, including novel RAC1 mutations, were detected in 55% of cisplatin-resistant GCTs. Conclusion In GCT, TP53 and MDM2 alterations were associated with cisplatin resistance and inferior outcomes, independent of the IGCCCG model. The finding of frequent TP53 alterations among mediastinal primary nonseminomas may explain the more frequent chemoresistance observed with this tumor subtype. A substantial portion of cisplatin-resistant GCTs harbor actionable alterations, which might respond to targeted therapies. Genomic profiling of patients with advanced GCT could improve current risk stratification and identify novel therapeutic approaches for patients with cisplatin-resistant disease.

Conpair: concordance and contamination estimator for matched tumor-normal pairs.

MOTIVATION: Sequencing of matched tumor and normal samples is the standard study design for reliable detection of somatic alterations. However, even very low levels of cross-sample contamination significantly impact calling of somatic mutations, because contaminant germline variants can be incorrectly interpreted as somatic. There are currently no sequence-only based methods that reliably estimate contamination levels in tumor samples, which frequently display copy number changes. As a solution, we developed Conpair, a tool for detection of sample swaps and cross-individual contamination in whole-genome and whole-exome tumor-normal sequencing experiments. RESULTS: On a ladder of in silico contaminated samples, we demonstrated that Conpair reliably measures contamination levels as low as 0.1%, even in presence of copy number changes. We also estimated contamination levels in glioblastoma WGS and WXS tumor-normal datasets from TCGA and showed that they strongly correlate with tumor-normal concordance, as well as with the number of germline variants called as somatic by several widely-used somatic callers. AVAILABILITY AND IMPLEMENTATION: The method is available at: https://github.com/nygenome/conpair CONTACT: egrabowska@gmail.com or mczody@nygenome.orgSupplementary information: Supplementary data are available at Bioinformatics online.

Differential burden of rare protein truncating variants in Alzheimer's disease patients compared to centenarians.

We compared coding region variants of 53 cognitively healthy centenarians and 45 patients with Alzheimer's disease (AD), all of Ashkenazi Jewish (AJ) ancestry. Despite the small sample size, the known AD risk variant APOE4 reached genome-wide significance, indicating the advantage of utilizing 'super-controls'. We restricted our subsequent analysis to rare variants observed at most once in the 1000 Genomes database and having a minor allele frequency below 2% in our AJ sample. We compared the burden of predicted protein altering variants between cases and controls as normalized by the level of rare synonymous variants. We observed an increased burden among AD subjects for predicted loss-of-function (LoFs) variants defined as stop-gain, frame shift, initiation codon (INIT) and splice site mutations (n = 930, OR = 1.3, P = 1.5×E-5). There was no enrichment across all rare protein altering variants defined as missense plus LoFs, in frame indels and stop-loss variants (n = 13 014, OR = 0.97, P = 0.47). Among LoFs, the strongest burden was observed for INIT (OR = 2.16, P = 0.0097) and premature stop variants predicted to cause non-sense-mediated decay in the majority of transcripts (NMD) (OR = 1.98, P = 0.02). Notably, this increased burden of NMD, INIT and splice variants was more pronounced in a set of 1397 innate immune genes (OR = 4.55, P = 0.0043). Further comparison to additional exomes indicates that the difference in LoF burden originated both from the AD and centenarian sample. In summary, we observed an overall increased burden of rare LoFs in AD subjects as compared to centenarians, and this enrichment is more pronounced for innate immune genes.

Integrative genetic analysis of mouse and human AML identifies cooperating disease alleles.

t(8;21) is one of the most frequent chromosomal abnormalities observed in acute myeloid leukemia (AML). However, expression of AML1-ETO is not sufficient to induce transformation in vivo. Consistent with this observation, patients with this translocation harbor additional genetic abnormalities, suggesting a requirement for cooperating mutations. To better define the genetic landscape in AML and distinguish driver from passenger mutations, we compared the mutational profiles of AML1-ETO-driven mouse models of leukemia with the mutational profiles of human AML patients. We identified TET2 and PTPN11 mutations in both mouse and human AML and then demonstrated the ability of Tet2 loss and PTPN11 D61Y to initiate leukemogenesis in concert with expression of AML1-ETO in vivo. This integrative genetic profiling approach allowed us to accurately predict cooperating events in t(8;21)(+) AML in a robust and unbiased manner, while also revealing functional convergence in mouse and human AML.

The Influence of Microdeletions and Microduplications of 16p11.2 on Global Transcription Profiles.

Copy number variants (CNVs) of a 600 kb region on 16p11.2 are associated with neurodevelopmental disorders and changes in brain volume. The authors hypothesize that abnormal brain development associated with this CNV can be attributed to changes in transcriptional regulation. The authors determined the effects of 16p11.2 dosage on gene expression by transcription profiling of lymphoblast cell lines derived from 6 microdeletion carriers, 15 microduplication carriers and 15 controls. Gene dosage had a significant influence on the transcript abundance of a majority (20/34) of genes within the CNV region. In addition, a limited number of genes were dysregulated in trans. Genes most strongly correlated with patient head circumference included SULT1A, KCTD13, and TMEM242. Given the modest effect of 16p11.2 copy number on global transcriptional regulation in lymphocytes, larger studies utilizing neuronal cell types may be needed in order to elucidate the signaling pathways that influence brain development in this genetic disorder.

Genome-wide association study of schizophrenia in Ashkenazi Jews.

Schizophrenia is a common, clinically heterogeneous disorder associated with lifelong morbidity and early mortality. Several genetic variants associated with schizophrenia have been identified, but the majority of the heritability remains unknown. In this study, we report on a case-control sample of Ashkenazi Jews (AJ), a founder population that may provide additional insights into genetic etiology of schizophrenia. We performed a genome-wide association analysis (GWAS) of 592 cases and 505 controls of AJ ancestry ascertained in the US. Subsequently, we performed a meta-analysis with an Israeli AJ sample of 913 cases and 1640 controls, followed by a meta-analysis and polygenic risk scoring using summary results from Psychiatric GWAS Consortium 2 schizophrenia study. The U.S. AJ sample showed strong evidence of polygenic inheritance (pseudo-R(2) ∼9.7%) and a SNP-heritability estimate of 0.39 (P = 0.00046). We found no genome-wide significant associations in the U.S. sample or in the combined US/Israeli AJ meta-analysis of 1505 cases and 2145 controls. The strongest AJ specific associations (P-values in 10(-6) -10(-7) range) were in the 22q 11.2 deletion region and included the genes TBX1, GLN1, and COMT. Supportive evidence (meta P < 1 × 10(-4) ) was also found for several previously identified genome-wide significant findings, including the HLA region, CNTN4, IMMP2L, and GRIN2A. The meta-analysis of the U.S. sample with the PGC2 results provided initial genome-wide significant evidence for six new loci. Among the novel potential susceptibility genes is PEPD, a gene involved in proline metabolism, which is associated with a Mendelian disorder characterized by developmental delay and cognitive deficits.

Whole-Exome Sequencing of Metastatic Cancer and Biomarkers of Treatment Response.

IMPORTANCE: Understanding molecular mechanisms of response and resistance to anticancer therapies requires prospective patient follow-up and clinical and functional validation of both common and low-frequency mutations. We describe a whole-exome sequencing (WES) precision medicine trial focused on patients with advanced cancer. OBJECTIVE: To understand how WES data affect therapeutic decision making in patients with advanced cancer and to identify novel biomarkers of response. DESIGN, SETTING, AND PATIENTS: Patients with metastatic and treatment-resistant cancer were prospectively enrolled at a single academic center for paired metastatic tumor and normal tissue WES during a 19-month period (February 2013 through September 2014). A comprehensive computational pipeline was used to detect point mutations, indels, and copy number alterations. Mutations were categorized as category 1, 2, or 3 on the basis of actionability; clinical reports were generated and discussed in precision tumor board. Patients were observed for 7 to 25 months for correlation of molecular information with clinical response. MAIN OUTCOMES AND MEASURES: Feasibility, use of WES for decision making, and identification of novel biomarkers. RESULTS: A total of 154 tumor-normal pairs from 97 patients with a range of metastatic cancers were sequenced, with a mean coverage of 95X and 16 somatic alterations detected per patient. In total, 16 mutations were category 1 (targeted therapy available), 98 were category 2 (biologically relevant), and 1474 were category 3 (unknown significance). Overall, WES provided informative results in 91 cases (94%), including alterations for which there is an approved drug, there are therapies in clinical or preclinical development, or they are considered drivers and potentially actionable (category 1-2); however, treatment was guided in only 5 patients (5%) on the basis of these recommendations because of access to clinical trials and/or off-label use of drugs. Among unexpected findings, a patient with prostate cancer with exceptional response to treatment was identified who harbored a somatic hemizygous deletion of the DNA repair gene FANCA and putative partial loss of function of the second allele through germline missense variant. Follow-up experiments established that loss of FANCA function was associated with platinum hypersensitivity both in vitro and in patient-derived xenografts, thus providing biologic rationale and functional evidence for his extreme clinical response. CONCLUSIONS AND RELEVANCE: The majority of advanced, treatment-resistant tumors across tumor types harbor biologically informative alterations. The establishment of a clinical trial for WES of metastatic tumors with prospective follow-up of patients can help identify candidate predictive biomarkers of response.

Disease variants in genomes of 44 centenarians.

To identify previously reported disease mutations that are compatible with extraordinary longevity, we screened the coding regions of the genomes of 44 Ashkenazi Jewish centenarians. Individual genome sequences were generated with 30× coverage on the Illumina HiSeq 2000 and single-nucleotide variants were called with the genome analysis toolkit (GATK). We identified 130 coding variants that were annotated as "pathogenic" or "likely pathogenic" based on the ClinVar database and that are infrequent in the general population. These variants were previously reported to cause a wide range of degenerative, neoplastic, and cardiac diseases with autosomal dominant, autosomal recessive, and X-linked inheritance. Several of these variants are located in genes that harbor actionable incidental findings, according to the recommendations of the American College of Medical Genetics. In addition, we found risk variants for late-onset neurodegenerative diseases, such as the APOE ε4 allele that was even present in a homozygous state in one centenarian who did not develop Alzheimer's disease. Our data demonstrate that the incidental finding of certain reported disease variants in an individual genome may not preclude an extraordinarily long life. When the observed variants are encountered in the context of clinical sequencing, it is thus important to exercise caution in justifying clinical decisions.

Comparative sequencing analysis reveals high genomic concordance between matched primary and metastatic colorectal cancer lesions.

BACKGROUND: Colorectal cancer is the second leading cause of cancer death in the United States, with over 50,000 deaths estimated in 2014. Molecular profiling for somatic mutations that predict absence of response to anti-EGFR therapy has become standard practice in the treatment of metastatic colorectal cancer; however, the quantity and type of tissue available for testing is frequently limited. Further, the degree to which the primary tumor is a faithful representation of metastatic disease has been questioned. As next-generation sequencing technology becomes more widely available for clinical use and additional molecularly targeted agents are considered as treatment options in colorectal cancer, it is important to characterize the extent of tumor heterogeneity between primary and metastatic tumors. RESULTS: We performed deep coverage, targeted next-generation sequencing of 230 key cancer-associated genes for 69 matched primary and metastatic tumors and normal tissue. Mutation profiles were 100% concordant for KRAS, NRAS, and BRAF, and were highly concordant for recurrent alterations in colorectal cancer. Additionally, whole genome sequencing of four patient trios did not reveal any additional site-specific targetable alterations. CONCLUSIONS: Colorectal cancer primary tumors and metastases exhibit high genomic concordance. As current clinical practices in colorectal cancer revolve around KRAS, NRAS, and BRAF mutation status, diagnostic sequencing of either primary or metastatic tissue as available is acceptable for most patients. Additionally, consistency between targeted sequencing and whole genome sequencing results suggests that targeted sequencing may be a suitable strategy for clinical diagnostic applications.

Genome-wide mapping of IBD segments in an Ashkenazi PD cohort identifies associated haplotypes.

The recent series of large genome-wide association studies in European and Japanese cohorts established that Parkinson disease (PD) has a substantial genetic component. To further investigate the genetic landscape of PD, we performed a genome-wide scan in the largest to date Ashkenazi Jewish cohort of 1130 Parkinson patients and 2611 pooled controls. Motivated by the reduced disease allele heterogeneity and a high degree of identical-by-descent (IBD) haplotype sharing in this founder population, we conducted a haplotype association study based on mapping of shared IBD segments. We observed significant haplotype association signals at three previously implicated Parkinson loci: LRRK2 (OR = 12.05, P = 1.23 × 10(-56)), MAPT (OR = 0.62, P = 1.78 × 10(-11)) and GBA (multiple distinct haplotypes, OR > 8.28, P = 1.13 × 10(-11) and OR = 2.50, P = 1.22 × 10(-9)). In addition, we identified a novel association signal on chr2q14.3 coming from a rare haplotype (OR = 22.58, P = 1.21 × 10(-10)) and replicated it in a secondary cohort of 306 Ashkenazi PD cases and 2583 controls. Our results highlight the power of our haplotype association method, particularly useful in studies of founder populations, and reaffirm the benefits of studying complex diseases in Ashkenazi Jewish cohorts.

Protein interaction network of alternatively spliced isoforms from brain links genetic risk factors for autism.

Increased risk for autism spectrum disorders (ASD) is attributed to hundreds of genetic loci. The convergence of ASD variants have been investigated using various approaches, including protein interactions extracted from the published literature. However, these datasets are frequently incomplete, carry biases and are limited to interactions of a single splicing isoform, which may not be expressed in the disease-relevant tissue. Here we introduce a new interactome mapping approach by experimentally identifying interactions between brain-expressed alternatively spliced variants of ASD risk factors. The Autism Spliceform Interaction Network reveals that almost half of the detected interactions and about 30% of the newly identified interacting partners represent contribution from splicing variants, emphasizing the importance of isoform networks. Isoform interactions greatly contribute to establishing direct physical connections between proteins from the de novo autism CNVs. Our findings demonstrate the critical role of spliceform networks for translating genetic knowledge into a better understanding of human diseases.