A whole-genome reference panel of 14,393 individuals for East Asian populations accelerates discovery of rare functional variants.

Underrepresentation of non-European (EUR) populations hinders growth of global precision medicine. Resources such as imputation reference panels that match the study population are necessary to find low-frequency variants with substantial effects. We created a reference panel consisting of 14,393 whole-genome sequences including more than 11,000 Asian individuals. Genome-wide association studies were conducted using the reference panel and a population-specific genotype array of 72,298 subjects for eight phenotypes. This panel yields improved imputation accuracy of rare and low-frequency variants within East Asian populations compared with the largest reference panel. Thirty-nine previously unidentified associations were found, and more than half of the variants were East Asian specific. We discovered genes with rare protein-altering variants, including LTBP1 for height and GPR75 for body mass index, as well as putative regulatory mechanisms for rare noncoding variants with cell type-specific effects. We suggest that this dataset will add to the potential value of Asian precision medicine.

Chromatin alternates between A and B compartments at kilobase scale for subgenic organization.

Nuclear compartments are prominent features of 3D chromatin organization, but sequencing depth limitations have impeded investigation at ultra fine-scale. CTCF loops are generally studied at a finer scale, but the impact of looping on proximal interactions remains enigmatic. Here, we critically examine nuclear compartments and CTCF loop-proximal interactions using a combination of in situ Hi-C at unparalleled depth, algorithm development, and biophysical modeling. Producing a large Hi-C map with 33 billion contacts in conjunction with an algorithm for performing principal component analysis on sparse, super massive matrices (POSSUMM), we resolve compartments to 500 bp. Our results demonstrate that essentially all active promoters and distal enhancers localize in the A compartment, even when flanking sequences do not. Furthermore, we find that the TSS and TTS of paused genes are often segregated into separate compartments. We then identify diffuse interactions that radiate from CTCF loop anchors, which correlate with strong enhancer-promoter interactions and proximal transcription. We also find that these diffuse interactions depend on CTCF's RNA binding domains. In this work, we demonstrate features of fine-scale chromatin organization consistent with a revised model in which compartments are more precise than commonly thought while CTCF loops are more protracted.

Comprehensive characterization of maternal, fetal, and neonatal microbiomes supports prenatal colonization of the gastrointestinal tract.

In this study, we aimed to comprehensively characterize the microbiomes of various samples from pregnant women and their neonates, and to explore the similarities and associations between mother-neonate pairs, sample collection sites, and obstetrical factors. We collected samples from vaginal discharge and amniotic fluid in pregnant women and umbilical cord blood, gastric liquid, and meconium from neonates. We identified 19,597,239 bacterial sequences from 641 samples of 141 pregnant women and 178 neonates. By applying rigorous filtering criteria to remove contaminants, we found evidence of microbial colonization in traditionally considered sterile intrauterine environments and the fetal gastrointestinal track. The microbiome distribution was strongly grouped by sample collection site, rather than the mother-neonate pairs. The distinct bacterial composition in meconium, the first stool passed by newborns, supports that microbial colonization occurs during normal pregnancy. The microbiome in neonatal gastric liquid was similar, but not identical, to that in maternal amnionic fluid, as expected since fetuses swallow amnionic fluid in utero and their urine returns to the fluid under normal physiological conditions. Establishing a microbiome library from various samples formed only during pregnancy is crucial for understanding human development and identifying microbiome modifications in obstetrical complications.

Whole-Genome Sequencing and Genomic Variant Analysis of Kazakh Individuals.

Kazakhstan, the ninth-largest country in the world, is located along the Great Silk Road and connects Europe with Asia. Historically, its territory has been inhabited by nomadic tribes, and modern-day Kazakhstan is a multiethnic country with a dominant Kazakh population. We sequenced and analyzed the genomes of five ethnic Kazakhs at high coverage using the Illumina HiSeq2000 next-generation sequencing platform. The five Kazakhs yielded a total number of base pairs ranging from 87,308,581,400 to 107,526,741,301. On average, 99.06% were properly mapped. Based on the Het/Hom and Ti/Tv ratios, the quality of the genomic data ranged from 1.35 to 1.49 and from 2.07 to 2.08, respectively. Genetic variants were identified and annotated. Functional analysis of the genetic variants identified several variants that were associated with higher risks of metabolic and neurogenerative diseases. The present study showed high levels of genetic admixture of Kazakhs that were comparable to those of other Central Asians. These whole-genome sequence data of healthy Kazakhs could contribute significantly to biomedical studies of common diseases as their findings could allow better insight into the genotype-phenotype relations at the population level.

ClinPharmSeq: A targeted sequencing panel for clinical pharmacogenetics implementation.

The accurate identification of genetic variants contributing to therapeutic drug response or adverse effects is the first step in implementation of precision drug therapy. Targeted sequencing has recently become a common methodology for large-scale studies of genetic variation thanks to its favorable balance between low cost, high throughput, and deep coverage. Here, we present ClinPharmSeq, a targeted sequencing panel of 59 genes with associations to pharmacogenetic (PGx) phenotypes, as a platform to explore the relationship between drug response and genetic variation, both common and rare. For validation, we sequenced DNA from 64 ethnically diverse Coriell samples with ClinPharmSeq to call star alleles (haplotype patterns) in 27 genes using the bioinformatics tool PyPGx. These reference samples were extensively characterized by multiple laboratories using PGx testing assays and, more recently, whole genome sequencing. We found that ClinPharmSeq can consistently generate deep-coverage data (mean = 274x) with high uniformity (30x or above = 94.8%). Our genotype analysis identified a total of 185 unique star alleles from sequencing data, and showed that diplotype calls from ClinPharmSeq are highly concordant with that from previous publications (97.6%) and whole genome sequencing (97.9%). Notably, all 19 star alleles with complex structural variation including gene deletions, duplications, and hybrids were recalled with 100% accuracy. Altogether, these results demonstrate that the ClinPharmSeq platform offers a feasible path for broad implementation of PGx testing and optimization of individual drug treatments.

Indigenous Ancestry and Admixture in the Uruguayan Population.

The Amerindian group known as the Charrúas inhabited Uruguay at the timing of European colonial contact. Even though they were extinguished as an ethnic group as a result of a genocide, Charrúan heritage is part of the Uruguayan identity both culturally and genetically. While mitochondrial DNA studies have shown evidence of Amerindian ancestry in living Uruguayans, here we undertake whole-genome sequencing of 10 Uruguayan individuals with self-declared Charruan heritage. We detect chromosomal segments of Amerindian ancestry supporting the presence of indigenous genetic ancestry in living descendants. Specific haplotypes were found to be enriched in "Charrúas" and rare in the rest of the Amerindian groups studied. Some of these we interpret as the result of positive selection, as we identified selection signatures and they were located mostly within genes related to the infectivity of specific viruses. Historical records describe contacts of the Charrúas with other Amerindians, such as Guaraní, and patterns of genomic similarity observed here concur with genomic similarity between these groups. Less expected, we found a high genomic similarity of the Charrúas to Diaguita from Argentinian and Chile, which could be explained by geographically proximity. Finally, by fitting admixture models of Amerindian and European ancestry for the Uruguayan population, we were able to estimate the timing of the first pulse of admixture between European and Uruguayan indigenous peoples in approximately 1658 and the second migration pulse in 1683. Both dates roughly concurring with the Franciscan missions in 1662 and the foundation of the city of Colonia in 1680 by the Spanish.

Genomic and transcriptomic analyses reveal a tandem amplification unit of 11 genes and mutations in mismatch repair genes in methotrexate-resistant HT-29 cells.

DHFR gene amplification is commonly present in methotrexate (MTX)-resistant colon cancer cells and acute lymphoblastic leukemia. In this study, we proposed an integrative framework to characterize the amplified region by using a combination of single-molecule real-time sequencing, next-generation optical mapping, and chromosome conformation capture (Hi-C). We identified an amplification unit spanning 11 genes, from the DHFR gene to the ATP6AP1L gene position, with high adjusted interaction frequencies on chromosome 5 (~2.2 Mbp) and a twenty-fold tandemly amplified region, and novel inversions at the start and end positions of the amplified region as well as frameshift insertions in most of the MSH and MLH genes were detected. These mutations might stimulate chromosomal breakage and cause the dysregulation of mismatch repair. Characterizing the tandem gene-amplified unit may be critical for identifying the mechanisms that trigger genomic rearrangements. These findings may provide new insight into the mechanisms underlying the amplification process and the evolution of drug resistance.

Classification of High-Grade Serous Ovarian Carcinoma by Epithelial-to-Mesenchymal Transition Signature and Homologous Recombination Repair Genes.

High-grade serous ovarian cancer (HGSOC) is one of the deadliest cancers that can occur in women. This study aimed to investigate the molecular characteristics of HGSOC through integrative analysis of multi-omics data. We used fresh-frozen, chemotherapy-naïve primary ovarian cancer tissues and matched blood samples of HGSOC patients and conducted next-generation whole-exome sequencing (WES) and RNA sequencing (RNA-seq). Genomic and transcriptomic profiles were comprehensively compared between patients with germline BRCA1/2 mutations and others with wild-type BRCA1/2. HGSOC samples initially divided into two groups by the presence of germline BRCA1/2 mutations showed mutually exclusive somatic mutation patterns, yet the implementation of high-dimensional analysis of RNA-seq and application of epithelial-to-mesenchymal (EMT) index onto the HGSOC samples revealed that they can be divided into two subtypes; homologous recombination repair (HRR)-activated type and mesenchymal type. Patients with mesenchymal HGSOC, characterized by the activation of the EMT transcriptional program, low genomic alteration and diverse cell-type compositions, exhibited significantly worse overall survival than did those with HRR-activated HGSOC (p = 0.002). In validation with The Cancer Genome Atlas (TCGA) HGSOC data, patients with a high EMT index (≥the median) showed significantly worse overall survival than did those with a low EMT index (

Evaluation of low-pass genome sequencing in polygenic risk score calculation for Parkinson's disease.

BACKGROUND: Low-pass sequencing (LPS) has been extensively investigated for applicability to various genetic studies due to its advantages over genotype array data including cost-effectiveness. Predicting the risk of complex diseases such as Parkinson's disease (PD) using polygenic risk score (PRS) based on the genetic variations has shown decent prediction accuracy. Although ultra-LPS has been shown to be effective in PRS calculation, array data has been favored to the majority of PRS analysis, especially for PD. RESULTS: Using eight high-coverage WGS, we assessed imputation approaches for downsampled LPS data ranging from 0.5 × to 7.0 × . We demonstrated that uncertain genotype calls of LPS diminished imputation accuracy, and an imputation approach using genotype likelihoods was plausible for LPS. Additionally, comparing imputation accuracies between LPS and simulated array illustrated that LPS had higher accuracies particularly at rare frequencies. To evaluate ultra-low coverage data in PRS calculation for PD, we prepared low-coverage WGS and genotype array of 87 PD cases and 101 controls. Genotype imputation of array and downsampled LPS were conducted using a population-specific reference panel, and we calculated risk scores based on the PD-associated SNPs from an East Asian meta-GWAS. The PRS models discriminated cases and controls as previously reported when both LPS and genotype array were used. Also strong correlations in PRS models for PD between LPS and genotype array were discovered. CONCLUSIONS: Overall, this study highlights the potentials of LPS under 1.0 × followed by genotype imputation in PRS calculation and suggests LPS as attractive alternatives to genotype array in the area of precision medicine for PD.

Whole genome sequencing reveals a frameshift mutation and a large deletion in YY1AP1 in a girl with a panvascular artery disease.

BACKGROUND: Rare diseases are pathologies that affect less than 1 in 2000 people. They are difficult to diagnose due to their low frequency and their often highly heterogeneous symptoms. Rare diseases have in general a high impact on the quality of life and life expectancy of patients, which are in general children or young people. The advent of high-throughput sequencing techniques has improved diagnosis in several different areas, from pediatrics, achieving a diagnostic rate of 41% with whole genome sequencing (WGS) and 36% with whole exome sequencing, to neurology, achieving a diagnostic rate between 47 and 48.5% with WGS. This evidence has encouraged our group to pursue a molecular diagnosis using WGS for this and several other patients with rare diseases. RESULTS: We used whole genome sequencing to achieve a molecular diagnosis of a 7-year-old girl with a severe panvascular artery disease that remained for several years undiagnosed. We found a frameshift variant in one copy and a large deletion involving two exons in the other copy of a gene called YY1AP1. This gene is related to Grange syndrome, a recessive rare disease, whose symptoms include stenosis or occlusion of multiple arteries, congenital heart defects, brachydactyly, syndactyly, bone fragility, and learning disabilities. Bioinformatic analyses propose these mutations as the most likely cause of the disease, according to its frequency, in silico predictors, conservation analyses, and effect on the protein product. Additionally, we confirmed one mutation in each parent, supporting a compound heterozygous status in the child. CONCLUSIONS: In general, we think that this finding can contribute to the use of whole genome sequencing as a diagnosis tool of rare diseases, and in particular, it can enhance the set of known mutations associated with different diseases.

Direct conversion of adult human fibroblasts into functional endothelial cells using defined factors.

We aimed to directly convert adult human dermal fibroblasts (aHDFs) into functional endothelial cells (ECs). Lentiviral vectors encoding endothelial transcription factors (TFs) were constructed. We examined whether five TFs (FOXO1, ER71, KLF2, TAL1, and LMO2) used for the generation of mouse induced ECs (iECs) could convert the aHDFs into human iECs. Twenty-eight days after transduction with lentiviral constructs, 32.1 ± 5.1% cells expressed vascular endothelial (VE)-cadherin. Factor screening revealed that only three factors (3F: ER71, KLF2, and TAL1) were necessary to induce VE-cadherin (+) cells (49.4 ± 3.5%). However, whole transcriptome sequencing showed that VE-cadherin (+) cells were not completely reprogrammed. Mature iECs double-positive for VE-cadherin/Pecam1 (DP cells) with a cobblestone appearance were obtained at a frequency of only 5.1 ± 0.6%. Using whole transcriptome analysis, the potential factors that could block the conversion were screened. Among candidates TWIST1-knockdown enhanced efficiency of conversion. Rosiglitazone, an inhibitor of epithelial-mesenchymal transition (EMT), also improved the conversion efficiency. Moreover, a 2nd second-stage conversion process, in which VE-cadherin (+) cells were incubated for additional two weeks, further enhanced the efficiency. The final protocol for 6 weeks yielded a conversion rate of 19.6 ± 3.0% iECs, defined by DP cells depicting the nature of mature ECs in various analyses. Further analyses revealed that the genetic and epigenetic profiles of iECs resembled those of functional ECs. Collectively, aHDFs can be converted into functional ECs through the transduction of ER71, KLF2, and TAL1, combined with two EMT inhibitors (siTWIST1 and rosiglitazone), followed by 2nd stage conversion.

Integrative Genomic and Transcriptomic Analyses of Tumor Suppressor Genes and Their Role on Tumor Microenvironment and Immunity in Lung Squamous Cell Carcinoma.

Non-small-cell lung cancers (NSCLCs) are largely classified into lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC), which have different therapeutic options according to its molecular profiles and immune checkpoint expression, especially PD-L1, which is a suppressive factor in the tumor microenvironment. The tumor microenvironment can be altered by the genomic mutations on specific innate immune genes as well as tumor suppressor genes, so it is essential to comprehend the association between tumor microenvironment and tumor suppressor genes to discover the promising immunotherapeutic strategy to overcome the resistance of immune check point blockade. In this study, we aimed to analyze how the somatic mutations in tumor suppressor genes affect the tumor immune microenvironment through a comprehensive analysis of mutational profiling on the representative tumor suppressor genes (TP53, CDKN2A, PTEN, RB1, BRCA1, BRCA2) and immune gene expression in The Cancer Genome Atlas (TCGA) 155 lung squamous cell carcinoma (LUSC) and 196 lung adenocarcinoma (LUAD) samples. Several microenvironmental factors, such as the infiltrating immune and stromal cells, were suppressed by the mutated tumor suppressor genes in LUSC, unlike in the LUAD samples. In particular, infiltrating immune cells such as macrophage, neutrophil, and dendritic cells were significantly reduced in tumors with mutated tumor suppressor genes' group. In addition, the gene expressions for interleukin production and lymphocyte differentiation and PGC, C7, HGF, PLA2G2A, IL1RL1, CCR2, ALOX15B, CXCL11, FCN3 were significantly down-regulated, which were key immune genes for the cross-talk between LUSC microenvironment and tumor suppressors. Therefore, we generated evidence that TSG mutations in LUSC have an impact on tumor immune microenvironment, which suggests that TSG non-mutated patients will have the more inflamed tumors and are more likely to respond to immune checkpoint blockade therapy.

A High Quality Asian Genome Assembly Identifies Features of Common Missing Regions.

The current human reference genome (GRCh38), with its superior quality, has contributed significantly to genome analysis. However, GRCh38 may still underrepresent the ethnic genome, specifically for Asians, though exactly what we are missing is still elusive. Here, we juxtaposed GRCh38 with a high-contiguity genome assembly of one Korean (AK1) to show that a part of AK1 genome is missing in GRCh38 and that the missing regions harbored ~1390 putative coding elements. Furthermore, we found that multiple populations shared some certain parts in the missing genome when we analyzed the "unmapped" (to GRCh38) reads of fourteen individuals (five East-Asians, four Europeans, and five Africans), amounting to ~5.3 Mb (~0.2% of AK1) of the total genomic regions. The recovered AK1 regions from the "unmapped reads", which were the estimated missing regions that did not exist in GRCh38, harbored candidate coding elements. We verified that most of the common (shared by ≥7 individuals) missing regions exist in human and chimpanzee DNA. Moreover, we further identified the occurrence mechanism and ethnic heterogeneity as well as the presence of the common missing regions. This study illuminates a potential advantage of using a pangenome reference and brings up the need for further investigations on the various features of regions globally missed in GRCh38.

Severe reactive astrocytes precipitate pathological hallmarks of Alzheimer's disease via H2O2- production.

Although the pathological contributions of reactive astrocytes have been implicated in Alzheimer's disease (AD), their in vivo functions remain elusive due to the lack of appropriate experimental models and precise molecular mechanisms. Here, we show the importance of astrocytic reactivity on the pathogenesis of AD using GiD, a newly developed animal model of reactive astrocytes, where the reactivity of astrocytes can be manipulated as mild (GiDm) or severe (GiDs). Mechanistically, excessive hydrogen peroxide (H2O2) originated from monoamine oxidase B in severe reactive astrocytes causes glial activation, tauopathy, neuronal death, brain atrophy, cognitive impairment and eventual death, which are significantly prevented by AAD-2004, a potent H2O2 scavenger. These H2O2--induced pathological features of AD in GiDs are consistently recapitulated in a three-dimensional culture AD model, virus-infected APP/PS1 mice and the brains of patients with AD. Our study identifies H2O2 from severe but not mild reactive astrocytes as a key determinant of neurodegeneration in AD.

Population-specific and trans-ancestry genome-wide analyses identify distinct and shared genetic risk loci for coronary artery disease.

To elucidate the genetics of coronary artery disease (CAD) in the Japanese population, we conducted a large-scale genome-wide association study of 168,228 individuals of Japanese ancestry (25,892 cases and 142,336 controls) with genotype imputation using a newly developed reference panel of Japanese haplotypes including 1,781 CAD cases and 2,636 controls. We detected eight new susceptibility loci and Japanese-specific rare variants contributing to disease severity and increased cardiovascular mortality. We then conducted a trans-ancestry meta-analysis and discovered 35 additional new loci. Using the meta-analysis results, we derived a polygenic risk score (PRS) for CAD, which outperformed those derived from either Japanese or European genome-wide association studies. The PRS prioritized risk factors among various clinical parameters and segregated individuals with increased risk of long-term cardiovascular mortality. Our data improve the clinical characterization of CAD genetics and suggest the utility of trans-ancestry meta-analysis for PRS derivation in non-European populations.

Alterations of transcriptome signatures in head trauma-related neurodegenerative disorders.

Chronic traumatic encephalopathy (CTE) is a neurodegenerative disease that is associated with repetitive traumatic brain injury (TBI). CTE is known to share similar neuropathological features with Alzheimer's disease (AD), but little is known about the molecular properties in CTE. To better understand the neuropathological mechanism of TBI-related disorders, we conducted transcriptome sequencing analysis of CTE including AD and CTE with AD (CTE/AD) post-mortem human brain samples. Through weighted gene co-expression network analysis (WGCNA) and principal component analysis (PCA), we characterized common and unique transcriptome signatures among CTE, CTE/AD, and AD. Interestingly, synapse signaling-associated gene signatures (such as synaptotagmins) were commonly down-regulated in CTE, CTE/AD, and AD. Quantitative real-time PCR (qPCR) and Western blot analyses confirmed that the levels of synaptotagmin 1 (SYT1) were markedly decreased in CTE and AD compared to normal. In addition, calcium/calmodulin-dependent protein kinase II (CaMKII), protein kinase A (PKA), protein kinase C (PKC), and AMPA receptor genes that play a pivotal role in memory function, were down-regulated in head trauma-related disorders. On the other hand, up-regulation of cell adhesion molecules (CAMs) associated genes was only found in CTE. Our results indicate that dysregulation of synaptic transmission- and memory function-related genes are closely linked to the pathology of head injury-related disorder and AD. Alteration of CAMs-related genes may be specific pathological markers for the CTE pathology.

The tumor immune microenvironmental analysis of 2,033 transcriptomes across 7 cancer types.

Understanding the tumor microenvironment is important to efficiently identify appropriate patients for immunotherapies in a variety of cancers. Here, we presented the tumor microenvironmental analysis of 2,033 cancer samples across 7 cancer types: colon adenocarcinoma, skin cutaneous melanoma, kidney renal papillary cell carcinoma, sarcoma, pancreatic adenocarcinoma, glioblastoma multiforme, and pheochromocytoma / paraganglioma from The Cancer Genome Atlas cohort. Unsupervised hierarchical clustering based on the gene expression profiles separated the cancer samples into two distinct clusters, and characterized those into immune-competent and immune-deficient subtypes using the estimated abundances of infiltrated immune and stromal cells. We demonstrated differential tumor microenvironmental characteristics of immune-competent subtypes across 7 cancer types, particularly immunosuppressive tumor microenvironment features in kidney renal papillary cell carcinoma with significant poorer survival rates and immune-supportive features in sarcoma and skin cutaneous melanoma. Additionally, differential genomic instability patterns between the subtypes were found across the cancer types, and discovered that immune-competent subtypes in most of cancer types had significantly higher immune checkpoint gene expressions. Overall, this study suggests that our subtyping approach based on transcriptomic data could contribute to precise prediction of immune checkpoint inhibitor responses in a wide range of cancer types.

Genomic and Transcriptomic Characteristics According to Size of Papillary Thyroid Microcarcinoma.

It is controversial as to whether papillary thyroid microcarcinoma (PTMC) has some genomic and transcriptomic characteristics that differentiate between an early-stage lesion that would eventually evolve into the larger papillary thyroid cancer (PTC), and an occult indolent cancer in itself. To investigate this, we comprehensively elucidated the genomic and transcriptomic landscapes of PTMCs of different sizes, using a large-scaled database. This study included 3435 PTCs, 1985 of which were PTMCs. We performed targeted next-generation sequencing for 221 PTCs and integrated these data with the data including The Cancer Genome Atlas (TCGA) project. The frequency of v-raf murine sarcoma viral oncogene homolog B (BRAF)V600E mutation was higher in PTMCs >0.5 cm than that in very small PTMCs (≤0.5 cm) and decreased again in PTCs >2 cm. Among PTMCs, the prevalence of mutations in rat sarcoma (RAS) and telomerase reverse transcriptase (TERT) promoter was not significantly different according to their size, but lower than in large PTCs. There was no change in the tumor mutational burden, the number of driver mutations, and transcriptomic profiles with tumor size, among PTMCs and all PTCs. Although a few genes with differential expression and TERT promoter mutations were found in a few PTMCs, our findings showed that there were no useful genomic or transcriptomic characteristics for the prediction of the future progression of PTMC.

Recent Improvements in Genomic and Transcriptomic Understanding of Anaplastic and Poorly Differentiated Thyroid Cancers.

Anaplastic thyroid cancer (ATC) is a lethal human cancer with a 5-year survival rate of less than 10%. Recently, its genomic and transcriptomic characteristics have been extensively elucidated over 5 years owing to advance in high throughput sequencing. These efforts have extended molecular understandings into the progression mechanisms and therapeutic vulnerabilities of aggressive thyroid cancers. In this review, we provide an overview of genomic and transcriptomic alterations in ATC and poorly-differentiated thyroid cancer, which are distinguished from differentiated thyroid cancers. Clinically relevant genomic alterations and deregulated signaling pathways will be able to shed light on more effective prevention and stratified therapeutic interventions for affected patients.