Ago2 and a miRNA reduce Topoisomerase 1 for enhancing DNA cleavage in antibody diversification by activation-induced cytidine deaminase.

Activation-induced cytidine deaminase (AID) is the essential enzyme for imprinting immunological memory through class switch recombination (CSR) and somatic hypermutation (SHM) of the immunoglobulin (Ig) gene. AID-dependent reduction of Topoisomerase 1 (Top1) promotes DNA cleavage that occurs upon Ig gene diversification, whereas the mechanism behind AID-induced Top1 reduction remains unclear. Here, we clarified the contribution of the microRNA-Ago2 complex in AID-dependent Top1 decrease. Ago2 binds to Top1 3'UTR with two regions of AID-dependent Ago2-binding sites (5'- and 3'dABs). Top1 3'UTR knockout (3'UTRKO) in B lymphoma cells leads to decreases in DNA break efficiency in the IgH gene accompanied by a reduction in CSR and SHM frequencies. Furthermore, AID-dependent Top1 protein reduction and Ago2-binding to Top1 mRNA are down-regulated in 3'UTRKO cells. Top1 mRNA in the highly translated fractions of the sucrose gradient is decreased in an AID-dependent and Top1 3'UTR-mediated manner, resulting in a decrease in Top1 protein synthesis. Both AID and Ago2 localize in the mRNA-binding protein fractions and they interact with each other. Furthermore, we found some candidate miRNAs which possibly bind to 5'- and 3'dAB in Top1 mRNA. Among them, miR-92a-3p knockdown induces the phenotypes of 3'UTRKO cells to wild-type cells whereas it does not impact on 3'UTRKO cells. Taken together, the Ago2-miR-92a-3p complex will be recruited to Top1 3'UTR in an AID-dependent manner and posttranscriptionally reduces Top1 protein synthesis. These consequences cause the increase in a non-B-DNA structure, enhance DNA cleavage by Top1 in the Ig gene and contribute to immunological memory formation.

OLIG2 is an in vivo bookmarking transcription factor in the developing neural tube in mouse.

Cells possess intrinsic features that are inheritable via epigenetic regulation, such as DNA methylation and histone modification. These inheritable features maintain a unique gene expression pattern, underlying cellular memory. Because of the degradation or displacement of mitotic chromosomes, most transcription factors do not contribute to cellular memory. However, accumulating in vitro evidence indicates that some transcription factors can be retained in mitotic chromosomes called as bookmarking. Such transcription factors may contribute to a novel third mechanism of cellular memory. Since most findings of transcription factor bookmarking have been reported in vitro, little is currently known in vivo. In the neural tube of mouse embryos, we discovered that OLIG2, a basic helix loop helix (bHLH) transcription factor that regulates proliferation of neural progenitors and the cell fate of motoneurons and oligodendrocytes, binds to chromatin through every cell cycle including M-phase. OLIG2 chromosomal localization coincides with mitotic cell features such as the phosphorylation of histone H3, KI67, and nuclear membrane breakdown. Chromosomal localization of OLIG2 is regulated by an N-terminus triple serine motif. Photobleaching analysis revealed slow OLIG2 mobility, suggesting a high affinity of OLIG2 to DNA. In Olig2 N-terminal deletion mutant mice, motoneurons and oligodendrocyte progenitor numbers are reduced in the neural tube, suggesting that the bookmarking regulatory domain is important for OLIG2 function. We conclude that OLIG2 is a de novo in vivo bookmarking transcription factor. Our results demonstrate the presence of in vivo bookmarking in a living organism and illustrate a novel function of transcription factors.

Changes in Multiple microRNA Levels with Antidepressant Treatment Are Associated with Remission and Interact with Key Pathways: A Comprehensive microRNA Analysis.

Individual treatment outcomes to antidepressants varies widely, yet the determinants to this difference remain elusive. MicroRNA (miRNA) gene expression regulation in major depressive disorder (MDD) has attracted interest as a biomarker. This 4-week randomized controlled trial examined changes in the plasma miRNAs that correlated with the treatment outcomes of mirtazapine (MIR) and selective serotonin reuptake inhibitor (SSRI) monotherapy. Pre- and post- treatment, we comprehensively analyzed the miRNA levels in MDD patients, and identified the gene pathways linked to these miRNAs in 46 patients. Overall, 141 miRNA levels significantly demonstrated correlations with treatment remission after 4 weeks of MIR, with miR-1237-5p showing the most robust and significant correlation after Bonferroni correction. These 141 miRNAs displayed a negative correlation with remission, indicating a decreasing trend. These miRNAs were associated with 15 pathways, including TGF-ß and MAPK. Through database searches, the genes targeted by these miRNAs with the identified pathways were compared, and it was found that MAPK1, IGF1, IGF1R, and BRAF matched. Alterations in specific miRNAs levels before and after MIR treatment correlated with remission. The miRNAs mentioned in this study have not been previously reported. No other studies have investigated treatment with MIR. The identified miRNAs also correlated with depression-related genes and pathways.

Raman Multi-Omic Snapshot and Statistical Validation of Structural Differences between Herpes Simplex Type I and Epstein-Barr Viruses.

Raman spectroscopy was applied to study the structural differences between herpes simplex virus Type I (HSV-1) and Epstein-Barr virus (EBV). Raman spectra were first collected with statistical validity on clusters of the respective virions and analyzed according to principal component analysis (PCA). Then, average spectra were computed and a machine-learning approach applied to deconvolute them into sub-band components in order to perform comparative analyses. The Raman results revealed marked structural differences between the two viral strains, which could mainly be traced back to the massive presence of carbohydrates in the glycoproteins of EBV virions. Clear differences could also be recorded for selected tyrosine and tryptophan Raman bands sensitive to pH at the virion/environment interface. According to the observed spectral differences, Raman signatures of known biomolecules were interpreted to link structural differences with the viral functions of the two strains. The present study confirms the unique ability of Raman spectroscopy for answering structural questions at the molecular level in virology and, despite the structural complexity of viral structures, its capacity to readily and reliably differentiate between different virus types and strains.

Development and validation of a vascularity-based architectural classification for clear cell renal cell carcinoma: correlation with conventional pathological prognostic factors, gene expression patterns, and clinical outcomes.

The prognostic significance of an architectural grading system for clear cell renal cell carcinoma (ccRCC) has recently been demonstrated. The present study aimed to establish a vascularity-based architectural classification using the cohort of 436 patients with localized ccRCC who underwent extirpative surgery and correlated the findings with conventional pathologic factors, gene expression, and prognosis. First, we assessed architectural patterns in the highest-grade area on hematoxylin and eosin-stained slides, then separately evaluated our surrogate score for vascularity. We grouped nine architectural patterns into three categories based on the vascular network score. "Vascularity-based architectural classification" was defined: category 1: characterized by enrichment of the vascular network, including compact/small nested, macrocyst/microcystic, and tubular/acinar patterns; category 2: characterized by a widely spaced-out vascular network, including alveolar/large nested, thick trabecular/insular, papillary/pseudopapillary patterns; category 3: characterized by scattered vascularity without a vascular network, including solid sheets, rhabdoid and sarcomatoid patterns. Adverse pathological prognostic factors such as TNM stage, WHO/ISUP grade, and necrosis were significantly associated with category 3, followed by category 2 (all p < 0.001). We successfully validated the classification using The Cancer Genome Atlas (TCGA) cohort (n = 162), and RNA-sequencing data available from TCGA showed that the angiogenesis gene signature was significantly enriched in category 1 compared to categories 2 and 3, whereas the immune gene signature was significantly enriched in category 3 compared to categories 1 and 2. In univariate analysis, vascularity-based architectural classification showed the best accuracy in pathological prognostic factors for predicting recurrence-free survival (c-index = 0.786). The predictive accuracy of our model which integrated WHO/ISUP grade, necrosis, TNM stage, and vascularity-based architectural classification was greater than conventional risk models (c-index = 0.871 vs. 0.755-0.843). Our findings suggest that the vascularity-based architectural classification is prognostically useful and may help stratify patients appropriately for management based on their likelihood of post-surgical recurrence.

Accelerated Angiogenesis of Human Umbilical Vein Endothelial Cells Under Negative Pressure Was Associated With the Regulation of Gene Expression Involved in the Proliferation and Migration.

ABSTRACT: Negative pressure has been used as a preferred therapy for wound healing; however, the mechanisms by which negative pressure promotes tissue restoration remain unclear. In the present study, RNA-sequencing analysis was performed to identify differentially expressed genes in human umbilical vein endothelial cells (HUVECs) exposed to negative pressure. Cell viability and DNA synthesis were examined using the cell counting kit-8 assay and bromodeoxyuridine incorporation, respectively. Cell migration was assessed using tube formation, Transwell, and wound healing assays. Activity of the serine/threonine kinase (AKT) signaling pathway was also examined by measuring the levels of phospho-paxicillin, phospho-focal adhesion kinase (p-FAK), and p-AKT1. The exposure of HUVECs to negative pressure enhanced cell proliferation and DNA synthesis. Negative pressure enhanced the migration and invasion of HUVECs, which was accompanied by upregulation of genes involved in angiogenesis, extracellular matrix organization, and cytoskeletal organization. The mRNA levels of growth factors, including placental growth factor and platelet-derived growth factor B, also increased. In addition, phosphorylation of paxicillin, focal adhesion kinase, and AKT increased under negative pressure. Collectively, the findings of this study demonstrated that negative pressure stimulates the angiogenic activity of HUVECs by increasing their proliferation and migration via activation of the AKT signaling pathway.

Multiple Pre-Treatment miRNAs Levels in Untreated Major Depressive Disorder Patients Predict Early Response to Antidepressants and Interact with Key Pathways.

Major depressive disorder (MDD) is a life-impairing disorder, and early successful treatment is important for a favorable prognosis. However, early response to antidepressants differs widely among individuals, and is difficult to predict pre-treatment. As miRNAs have been reported to play important roles in depression, identification of miRNAs associated with antidepressant treatment responses and their interacting genes and pathways will be beneficial in understanding the predictors and molecular mechanisms of depression treatment. This randomized control trial examined miRNAs correlated with the early therapeutic effect of selective serotonin reuptake inhibitors (SSRIs; paroxetine or sertraline) and mirtazapine monotherapy. Before medication, we comprehensively analyzed the miRNA expression of 92 depressed participants and identified genes and pathways interacting with miRNAs. A total of 228 miRNAs were significantly correlated with depressive symptoms improvements after 2 weeks of SSRIs treatment, with miR-483.5p showing the most robust correlation. These miRNAs are involved in 21 pathways, including TGF-ß, glutamatergic synapse, long-term depression, and the mitogen-activated protein kinase (MAPK) signaling pathways. Using these miRNAs enabled us to predict SSRI response at week 2 with a 57% difference. This study shows that pre-treatment levels of miRNAs could be used to predict early responses to antidepressant administration, a knowledge of genes, and an identification of genes and pathways associated with the antidepressant response.

Legacy Data Confound Genomics Studies.

Recent reports have identified differences in the mutational spectra across human populations. Although some of these reports have been replicated in other cohorts, most have been reported only in the 1000 Genomes Project (1kGP) data. While investigating an intriguing putative population stratification within the Japanese population, we identified a previously unreported batch effect leading to spurious mutation calls in the 1kGP data and to the apparent population stratification. Because the 1kGP data are used extensively, we find that the batch effects also lead to incorrect imputation by leading imputation servers and a small number of suspicious GWAS associations. Lower quality data from the early phases of the 1kGP thus continue to contaminate modern studies in hidden ways. It may be time to retire or upgrade such legacy sequencing data.

PDK2 leads to cisplatin resistance through suppression of mitochondrial function in ovarian clear cell carcinoma.

Ovarian clear cell carcinoma (CCC) exhibits an association with endometriosis, resistance to oxidative stress, and poor prognosis owing to its resistance to conventional platinum-based chemotherapy. A greater understanding of the molecular characteristics and pathogenesis of ovarian cancer subtypes may facilitate the development of targeted therapeutic strategies, although the mechanism of drug resistance in ovarian CCC has yet to be determined. In this study, we assessed exome sequencing data to identify new therapeutic targets of mitochondrial function in ovarian CCC because of the central role of mitochondria in redox homeostasis. Copy number analyses revealed that chromosome 17q21-24 (chr.17q21-24) amplification was associated with recurrence in ovarian CCC. Cell viability assays identified an association between cisplatin resistance and chr.17q21-24 amplification, and mitochondrion-related genes were enriched in patients with chr.17q21-24 amplification. Patients with high expression of pyruvate dehydrogenase kinase 2 (PDK2) had a worse prognosis than those with low PDK2 expression. Furthermore, inhibition of PDK2 synergistically enhanced cisplatin sensitivity by activating the electron transport chain and by increasing the production of mitochondrial reactive oxygen species. Mouse xenograft models showed that inhibition of PDK2 with cisplatin inhibited tumor growth. This evidence suggests that targeting mitochondrial metabolism and redox homeostasis is an attractive therapeutic strategy for improving drug sensitivity in ovarian CCC.

Genome-wide association study of individual differences of human lymphocyte profiles using large-scale cytometry data.

Human immune systems are very complex, and the basis for individual differences in immune phenotypes is largely unclear. One reason is that the phenotype of the immune system is so complex that it is very difficult to describe its features and quantify differences between samples. To identify the genetic factors that cause individual differences in whole lymphocyte profiles and their changes after vaccination without having to rely on biological assumptions, we performed a genome-wide association study (GWAS), using cytometry data. Here, we applied computational analysis to the cytometry data of 301 people before receiving an influenza vaccine, and 1, 7, and 90 days after the vaccination to extract the feature statistics of the lymphocyte profiles in a nonparametric and data-driven manner. We analyzed two types of cytometry data: measurements of six markers for B cell classification and seven markers for T cell classification. The coordinate values calculated by this method can be treated as feature statistics of the lymphocyte profile. Next, we examined the genetic basis of individual differences in human immune phenotypes with a GWAS for the feature statistics, and we newly identified seven significant and 36 suggestive single-nucleotide polymorphisms associated with the individual differences in lymphocyte profiles and their change after vaccination. This study provides a new workflow for performing combined analyses of cytometry data and other types of genomics data.

Positive renal familial history in IgA nephropathy is associated with worse renal outcomes: a single-center longitudinal study.

BACKGROUND: IgA nephropathy (IgAN) is the most common primary glomerulonephritis worldwide. Although most IgAN cases are sporadic, few show a familial aggregation. However, the prevalence and prognosis of IgAN individuals with positive familial history (FH) of renal disorders remains uncertain. To address these issues, we conducted a longitudinal observational study on a single-institution cohort of patients with biopsy-proven IgAN. METHODS: A total of 467 IgAN patients who underwent renal biopsy during 1994 to 2019 were ascertained to have positive- or negative-FH by history taking and were followed for an average of 8.9 years. We compared the clinical and pathological features of the two subgroups. The primary outcome, a composite of a hard endpoint (end-stage renal disease [ESRD]) and surrogate endpoint (a 50% or more reduction in the estimated glomerular filtration rate [eGFR] from baseline), was evaluated. To estimate the risk for progression to ESRD, a Cox proportional hazards analysis was performed for a subset of patients who underwent follow-up for > 2 years and had an eGFR > 30 mL/min/1.73 m2 at baseline (n = 389; observation, 8.7 years). RESULTS: Positive-FH subtype accounted for 11.6% (n = 54) of all IgAN patients. At baseline, there were no significant differences between the positive- and negative-FH subgroups regarding age, sex, comorbid disease, MEST-C score, observation period, and therapeutic interventions. However, the eGFR value at baselines was significantly lower in the positive-FH subgroup than in the negative-FH subgroup (P < 0.01). On multivariate analysis, positive-FH emerged an independent determinant of poorer renal outcomes (odds ratio, 2.31; 95% confidence interval, 1.10-4.85; P = 0.03), after adjusting for confounding factors. eGFR at follow-up was significantly lower in the positive-FH subgroup than in the negative-FH subgroup after adjustment for age and observation period. CONCLUSIONS: Positive-FH was found in 11.6% of all IgAN patients, consistent with the incidence seen in previous literature. A significantly lower eGFR at baseline and last follow-up and unfavorable renal outcomes in the positive-FH subgroup suggest that certain genetic risk factors predisposing to renal failure may exist in a fraction of our IgAN cohort. (331 words).

Association of ALPL variants with serum alkaline phosphatase and bone traits in the general Japanese population: The Nagahama Study.

Although alkaline phosphatase (ALP) activity is relatively low in carriers of recessive type hypophosphatasia (HPP), most are asymptomatic and therefore do not undergo medical evaluations. We analyzed the association of ALP-encoding ALPL variants with serum ALP and bone traits in the general Japanese population. Study participants (n = 9671) were from the Nagahama Study, which was a longitudinal cohort study of an apparently healthy general Japanese population. ALPL variants were analyzed by whole-genome sequencing or TaqMan probe assays using DNA extracted from peripheral blood samples. The speed of sound in calcaneal bone was assessed by quantitative ultrasound (QUS) and used as surrogate measures of bone mineral density. We identified 13 ALPL variants. Minor allele frequencies of three variants were higher than expected. Variant c.529G > A has been reported as a possible pathogenic variant for adult type HPP. Variants c.979C > T and c.1559delT are reported as pathogenic variants for perinatal severe HPP or infantile HPP. The allele frequencies of c.529G > A, c.979C > T, and c.1559delT were 0.0107, 0.0040, and 0.0014, respectively. Serum ALP activity was significantly lower and differed among the three variants (P < 0.001), as well as between individuals with and without any of the three variants (P < 0.001). Serum ALP activity was inversely associated with QUS values, although no direct association was observed between the ALPL variants and QUS values. An association between serum ALP activity and QUS was confirmed; however, we failed to detect an association between ALPL variants and bone traits in the general Japanese population.

Contribution of a Non-classical HLA Gene, HLA-DOA, to the Risk of Rheumatoid Arthritis.

Despite the progress in human leukocyte antigen (HLA) causal variant mapping, independent localization of major histocompatibility complex (MHC) risk from classical HLA genes is challenging. Here, we conducted a large-scale MHC fine-mapping analysis of rheumatoid arthritis (RA) in a Japanese population (6,244 RA cases and 23,731 controls) population by using HLA imputation, followed by a multi-ethnic validation study including east Asian and European populations (n = 7,097 and 23,149, respectively). Our study identified an independent risk of a synonymous mutation at HLA-DOA, a non-classical HLA gene, on anti-citrullinated protein autoantibody (ACPA)-positive RA risk (p = 1.4 × 10(-9)), which demonstrated a cis-expression quantitative trait loci (cis-eQTL) effect on HLA-DOA expression. Trans-ethnic comparison revealed different linkage disequilibrium (LD) patterns in HLA-DOA and HLA-DRB1, explaining the observed HLA-DOA variant risk heterogeneity among ethnicities, which was most evident in the Japanese population. Although previous HLA fine-mapping studies have identified amino acid polymorphisms of the classical HLA genes as driving genetic susceptibility to disease, our study additionally identifies the dosage contribution of a non-classical HLA gene to disease etiology. Our study contributes to the understanding of HLA immunology in human diseases and suggests the value of incorporating additional ancestry in MHC fine-mapping.

A Comprehensive Genomic Analysis Reveals the Genetic Landscape of Mitochondrial Respiratory Chain Complex Deficiencies.

Mitochondrial disorders have the highest incidence among congenital metabolic disorders characterized by biochemical respiratory chain complex deficiencies. It occurs at a rate of 1 in 5,000 births, and has phenotypic and genetic heterogeneity. Mutations in about 1,500 nuclear encoded mitochondrial proteins may cause mitochondrial dysfunction of energy production and mitochondrial disorders. More than 250 genes that cause mitochondrial disorders have been reported to date. However exact genetic diagnosis for patients still remained largely unknown. To reveal this heterogeneity, we performed comprehensive genomic analyses for 142 patients with childhood-onset mitochondrial respiratory chain complex deficiencies. The approach includes whole mtDNA and exome analyses using high-throughput sequencing, and chromosomal aberration analyses using high-density oligonucleotide arrays. We identified 37 novel mutations in known mitochondrial disease genes and 3 mitochondria-related genes (MRPS23, QRSL1, and PNPLA4) as novel causative genes. We also identified 2 genes known to cause monogenic diseases (MECP2 and TNNI3) and 3 chromosomal aberrations (6q24.3-q25.1, 17p12, and 22q11.21) as causes in this cohort. Our approaches enhance the ability to identify pathogenic gene mutations in patients with biochemically defined mitochondrial respiratory chain complex deficiencies in clinical settings. They also underscore clinical and genetic heterogeneity and will improve patient care of this complex disorder.

Regional genetic differences among Japanese populations and performance of genotype imputation using whole-genome reference panel of the Tohoku Medical Megabank Project.

BACKGROUND: Genotype imputation from single-nucleotide polymorphism (SNP) genotype data using a haplotype reference panel consisting of thousands of unrelated individuals from populations of interest can help to identify strongly associated variants in genome-wide association studies. The Tohoku Medical Megabank (TMM) project was established to support the development of precision medicine, together with the whole-genome sequencing of 1070 human genomes from individuals in the Miyagi region (Northeast Japan) and the construction of the 1070 Japanese genome reference panel (1KJPN). Here, we investigated the performance of 1KJPN for genotype imputation of Japanese samples not included in the TMM project and compared it with other population reference panels. RESULTS: We found that the 1KJPN population was more similar to other Japanese populations, Nagahama (south-central Japan) and Aki (Shikoku Island), than to East Asian populations in the 1000 Genomes Project other than JPT, suggesting that the large-scale collection (more than 1000) of Japanese genomes from the Miyagi region covered many of the genetic variations of Japanese in mainland Japan. Moreover, 1KJPN outperformed the phase 3 reference panel of the 1000 Genomes Project (1KGPp3) for Japanese samples, and IKJPN showed similar imputation rates for the TMM and other Japanese samples for SNPs with minor allele frequencies (MAFs) higher than 1%. CONCLUSIONS: 1KJPN covered most of the variants found in the samples from areas of the Japanese mainland outside the Miyagi region, implying 1KJPN is representative of the Japanese population's genomes. 1KJPN and successive reference panels are useful genome reference panels for the mainland Japanese population. Importantly, the addition of whole genome sequences not included in the 1KJPN panel improved imputation efficiencies for SNPs with MAFs under 1% for samples from most regions of the Japanese archipelago.

A mutation in transcription factor MAFB causes Focal Segmental Glomerulosclerosis with Duane Retraction Syndrome.

Focal segmental glomerulosclerosis (FSGS) is a leading cause of end-stage renal disease in children and adults. Genetic factors significantly contribute to early-onset FSGS, but the etiologies of most adult cases remain unknown. Genetic studies of monogenic syndromic FSGS exhibiting extra-renal manifestations have uncovered an unexpected biological role for genes in the development of both podocytes and other cellular lineages. To help define these roles, we studied two unrelated families with FSGS associated with Duane Retraction Syndrome, characterized by impaired horizontal eye movement due to cranial nerve malformation. All four affected individuals developed FSGS and Duane Retraction Syndrome in their first to second decade of life, manifested as restricted abduction together with globe retraction and narrowed palpebral fissure on attempted adduction. Hypoplasia of the abducens nerves and hearing impairment occurred in severely affected individuals. Genetic analyses revealed that affected individuals harbor a rare heterozygous substitution (p.Leu239Pro) in MAFB, a leucine zipper transcription factor. Luciferase assays with cultured monocytes indicated that the substitution significantly reduced transactivation of the F4/80 promoter, the known MAFB recognition element. Additionally, immunohistochemistry indicated reduced MAFB expression in the podocytes of patients. Structural modeling suggested that the p.Leu239Pro substitution in the DNA-binding domain possibly interferes with the stability of the adjacent zinc finger. Lastly, podocytes in neonatal mice with p.Leu239Pro displayed impaired differentiation. Thus, MAFB mutations impair development and/or maintenance of podocytes, abducens neurons and the inner ear. The interactions between MAFB and regulatory elements in these developing organs are likely highly specific based on spatiotemporal requirements.

Exome Sequencing Landscape Analysis in Ovarian Clear Cell Carcinoma Shed Light on Key Chromosomal Regions and Mutation Gene Networks.

Previous studies have reported genome-wide mutation profile analyses in ovarian clear cell carcinomas (OCCCs). This study aims to identify specific novel molecular alterations by combined analyses of somatic mutation and copy number variation. We performed whole exome sequencing of 39 OCCC samples with 16 matching blood tissue samples. Four hundred twenty-six genes had recurrent somatic mutations. Among the 39 samples, ARID1A (62%) and PIK3CA (51%) were frequently mutated, as were genes such as KRAS (10%), PPP2R1A (10%), and PTEN (5%), that have been reported in previous OCCC studies. We also detected mutations in MLL3 (15%), ARID1B (10%), and PIK3R1 (8%), which are associations not previously reported. Gene interaction analysis and functional assessment revealed that mutated genes were clustered into groups pertaining to chromatin remodeling, cell proliferation, DNA repair and cell cycle checkpointing, and cytoskeletal organization. Copy number variation analysis identified frequent amplification in chr8q (64%), chr20q (54%), and chr17q (46%) loci as well as deletion in chr19p (41%), chr13q (28%), chr9q (21%), and chr18q (21%) loci. Integration of the analyses uncovered that frequently mutated or amplified/deleted genes were involved in the KRAS/phosphatidylinositol 3-kinase (82%) and MYC/retinoblastoma (75%) pathways as well as the critical chromatin remodeling complex switch/sucrose nonfermentable (85%). The individual and integrated analyses contribute details about the OCCC genomic landscape, which could lead to enhanced diagnostics and therapeutic options.

HLA-HD: An accurate HLA typing algorithm for next-generation sequencing data.

The accurate typing of human leukocyte antigen (HLA) alleles is critical for a variety of medical applications, such as genomic studies of multifactorial diseases, including immune system and inflammation-related disorders, and donor selection in organ transplantation and regenerative medicine. Here, we developed a new algorithm for determining HLA alleles using next-generation sequencing (NGS) results. The method consists of constructing an extensive dictionary of HLA alleles, precise mapping of the NGS reads, and calculating a score based on weighted read counts to select the most suitable pair of alleles. The developed algorithm compares the score of all allele pairs, taking into account variation not only in the domain for antigen presentation (G-DOMAIN), but also outside this domain. Using this method, HLA alleles could be determined with 6-digit precision. We showed that our method was more accurate than other NGS-based methods and revealed limitations of the conventional HLA typing technologies. Furthermore, we determined the complete genomic sequence of an HLA-A-like-pseudogene when we assembled NGS reads that had caused arguable typing, and found its identity with HLA-Y*02:01. The accuracy of the HLA-A allele typing was improved after the HLA-Y*02:01 sequence was included in the HLA allele dictionary.

A burden of rare variants in BMPR2 and KCNK3 contributes to a risk of familial pulmonary arterial hypertension.

BACKGROUND: Pulmonary arterial hypertension (PAH) is a severe lung disease with only few effective treatments available. Familial cases of PAH are usually recognized as an autosomal dominant disease, but incomplete penetrance of the disease makes it difficult to identify pathogenic variants in accordance with a Mendelian pattern of inheritance. METHODS: To elucidate the complex genetic basis of PAH, we obtained whole exome- or genome-sequencing data of 17 subjects from 9 families with heritable PAH and applied gene-based association analysis with 9 index patients and 300 PAH-free controls. RESULTS: A burden of rare variants in BMPR2 significantly contributed to the risk of the disease (p = 6.0 × 10-8). Eight of nine families carried four previously reported single nucleotide variants and four novel insertion/deletion variants in the gene. One of the novel variants was a large 6.5 kilobase-deletion. In the remaining one family, the patient carried a pathogenic variant in a member of potassium channels, KCNK3, which was the first replicative finding of channelopathy in an Asian population. CONCLUSIONS: The variety of rare pathogenic variants suggests that gene-based association analysis using genome-wide sequencing data from increased number of samples is essential to tracing the genetic heterogeneity and developing an appropriate panel for genetic testing.