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Large-scale gene sequencing studies for complex traits have the potential to identify causal genes with therapeutic implications. We performed gene-based association testing of blood lipid levels with rare (minor allele frequency170,000 individuals from multiple ancestries: 97,493 European, 30,025 South Asian, 16,507 African, 16,440 Hispanic/Latino, 10,420 East Asian, and 1,182 Samoan. We identified 35 genes associated with circulating lipid levels. Ten of these: ALB, SRSF2, JAK2, CREB3L3, TMEM136, VARS, NR1H3, PLA2G12A, PPARG and STAB1 have not been implicated for lipid levels using rare coding variation in population-based samples. We prioritize 32 genes identified in array-based genome-wide association study (GWAS) loci based on gene-based associations, of which three: EVI5, SH2B3, and PLIN1, had no prior evidence of rare coding variant associations. Most of the associated genes showed evidence of association in multiple ancestries. Also, we observed an enrichment of gene-based associations for low-density lipoprotein cholesterol drug target genes, and for genes closest to GWAS index single nucleotide polymorphisms (SNP). Our results demonstrate that gene-based associations can be beneficial for drug target development and provide evidence that the gene closest to the array-based GWAS index SNP is often the functional gene for blood lipid levels.

Autosomal genetic analyses of blood lipids have yielded key insights for coronary heart disease (CHD). However, X chromosome genetic variation is understudied for blood lipids in large sample sizes. We now analyze genetic and blood lipid data in a high-coverage whole X chromosome sequencing study of 65,322 multi-ancestry participants and perform replication among 456,893 European participants. Common alleles on chromosome Xq23 are strongly associated with reduced total cholesterol, LDL cholesterol, and triglycerides (min P = 8.5 × 10−72), with similar effects for males and females. Chromosome Xq23 lipid-lowering alleles are associated with reduced odds for CHD among 42,545 cases and 591,247 controls (P = 1.7 × 10−4), and reduced odds for diabetes mellitus type 2 among 54,095 cases and 573,885 controls (P = 1.4 × 10−5). Although we observe an association with increased BMI, waist-to-hip ratio adjusted for BMI is reduced, bioimpedance analyses indicate increased gluteofemoral fat, and abdominal MRI analyses indicate reduced visceral adiposity. Co-localization analyses strongly correlate increased CHRDL1 gene expression, particularly in adipose tissue, with reduced concentrations of blood lipids., The influence of X chromosome genetic variation on blood lipids and coronary heart disease (CHD) is not well understood. Here, the authors analyse X chromosome sequencing data across 65,322 multi-ancestry individuals, identifying associations of the Xq23 locus with lipid changes and reduced risk of CHD and diabetes mellitus.

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.

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 &gt, 0.5 cm than that in very small PTMCs (&le, 0.5 cm) and decreased again in PTCs &gt, 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.

DHFRgene amplification is present in methotrexate (MTX)-resistant colon cancer cells and acute lymphoblastic leukemia. However, little is known aboutDHFRgene amplification due to difficulties in quantifying amplification size and recognizing the repetitive rearrangements involved in the process. In this study, we have 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). Amplification of theDHFRgene was optimized to generate homogenously amplified patterns. The amplification units of 11 genes, from theDHFRgene to theATP6AP1Lgene position on chromosome 5 (~2.2Mbp), and a twenty-fold tandemly amplified region were verified using long-range genome and RNA sequencing data. In doing so, a novel inversion at the start and end positions of the amplified region as well as frameshift insertions in most of theMSHandMLHgenes were detected. These might stimulate chromosomal breakage and cause the dysregulation of mismatch repair pathways. Using Hi-C technology, high adjusted interaction frequencies were detected on the amplified unit and unsuspected position on 5q, which could have a complex network of spatial contacts to harbor gene amplification. Characterizing the tandem gene-amplified unit and genomic variants as well as chromosomal interactions on intra-chromosome 5 can be critical in identifying the mechanisms behind genomic rearrangements. These findings may give new insight into the mechanisms underlying the amplification process and evolution of drug resistance.

Novel coronavirus (SARS-CoV-2) is found to cause a large outbreak started from Wuhan since December 2019 in China and SARS-CoV-2 infections have been reported with epidemiological linkage to China in 25 countries until now. We isolated SARS-CoV-2 from the oropharyngeal sample obtained from the patient with the first laboratory-confirmed SARS-CoV-2 infection in Korea. Cytopathic effects of SARS-CoV-2 in the Vero cell cultures were confluent 3 days after the first blind passage of the sample. Coronavirus was confirmed with spherical particle having a fringe reminiscent of crown on transmission electron microscopy. Phylogenetic analyses of whole genome sequences showed that it clustered with other SARS-CoV-2 reported from Wuhan.

Recent work has demonstrated that two archaic human groups (Neanderthals and Denisovans) interbred with modern humans and contributed to the contemporary human gene pool. These findings relied on the availability of high-coverage genomes from both Neanderthals and Denisovans. Here we search for evidence of archaic admixture from a worldwide panel of 1,667 individuals using an approach that does not require the presence of an archaic human reference genome. We find no evidence for archaic admixture in the Andaman Islands, as previously claimed, or on the island of Flores, where Homo floresiensis fossils have been found. However, we do find evidence for at least one archaic admixture event in sub-Saharan Africa, with the strongest signal in Khoesan and Pygmy individuals from Southern and Central Africa. The locations of these putative archaic admixture tracts are weighted against functional regions of the genome, consistent with the long-term effects of purifying selection against introgressed genetic material.

Objective The aim of this study was to develop a common targeted massively parallel sequencing platform for the noninvasive prenatal diagnosis (NIPD) of multiple X-linked diseases. Method The custom capture probe was designed to target 33 genes and recombination hotspots. We tested the carrier mother and male proband pair of 6 families. Plasma DNA of the pregnant carrier mother was collected at different gestational weeks and sequenced. The fetal genotype of each family was determined by estimating the imbalance between the 2 maternal haplotypes constructed using a common custom-designed platform. Results The targeted sequencing of the maternal, proband, and fetal genomic DNAs and maternal plasma DNAs resulted in uniform coverage across the target region. Three to 5 recombination points were observed in each sample. However, these recombination points did not affect the haplotype dosage analysis for fetal genotype prediction. Consequently, all fetal genotypes in the 6 families obtained from haplotype dosage analysis of maternal plasma sequencing data were predicted correctly. Conclusions Since a single platform that covers multiple diseases may prevent the need for disease-specific probes for the NIPD of individual disorders, this approach may provide a practical advantage for clinically implementing the NIPD of X-linked diseases.

To elucidate the genetics of coronary artery disease (CAD) in the Japanese population, we conducted a large-scale genome-wide association study (GWAS) of 168,228 Japanese (25,892 cases and 142,336 controls) with genotype imputation using a newly developed reference panel of Japanese haplotypes including 1,782 CAD cases and 3,148 controls. We detected 9 novel disease-susceptibility loci and Japanese-specific rare variants contributing to disease severity and increased cardiovascular mortality. We then conducted a transethnic meta-analysis and discovered 37 additional novel loci. Using the result of the meta-analysis, we derived a polygenic risk score (PRS) for CAD, which outperformed those derived from either Japanese or European GWAS. The PRS prioritized risk factors among various clinical parameters and segregated individuals with increased risk of long-term cardiovascular mortality. Our data improves clinical characterization of CAD genetics and suggests the utility of transethnic meta-analysis for PRS derivation in non-European populations.

There are various factors that alter physiological characteristics in skin. Elucidating the underlying mechanism of transcriptional alterations by intrinsic and extrinsic factors may lead us to understand the aging process of skin. To identify the transcriptomic changes of the aging skin, we analyzed publicly available RNA sequencing data from Genotype-Tissue Expression (GTEx) project. GTEx provided RNA sequencing data of suprapubic (n=228) and lower leg (n=349) skins, which are photo-protected and photo-damaged. Using differentially expressed gene analysis and weighted gene co-expression network analysis, we characterized transcriptomic changes due to UV exposure and aging. Genes involved in skin development such as epidermal differentiation complex component (SPRR and LCE families), vasculature development (TGFBR1, TGFBR2, TGFBR3, KDR, FGF2, and VEGFC), and matrix metalloproteinase (MMP2, MMP3, MMP8, MMP10, and MMP13) were up-regulated by UV exposure. Also, down-regulated lipid metabolism and mitochondrial biogenesis were observed in photo-damaged skin. Moreover, wound healing process was universally down-regulated in suprapubic and lower leg with aging and further down-regulation of lipid metabolism and up-regulation of vasculature development were found as photo-aging signatures. In this study, dynamic transcriptomic alterations were observed in aged skin. Hence, our findings may help to discover a potential therapeutic target for skin rejuvenation.

The immune microenvironment in lung squamous cell carcinoma (LUSC) is not well understood, with interactions between the host immune system and the tumor, as well as the molecular pathogenesis of LUSC, awaiting better characterization. To date, no molecularly targeted agents have been developed for LUSC treatment. Identification of predictive and prognostic biomarkers for LUSC could help optimize therapy decisions. We sequenced whole exomes and RNA from 101 tumors and matched noncancer control Korean samples. We used the information to predict subtype-specific interactions within the LUSC microenvironment and to connect genomic alterations with immune signatures. Hierarchical clustering based on gene expression and mutational profiling revealed subtypes that were either immune defective or immune competent. We analyzed infiltrating stromal and immune cells to further characterize the tumor microenvironment. Elevated expression of macrophage 2 signature genes in the immune competent subtype confirmed that tumor-associated macrophages (TAM) linked inflammation and mutation-driven cancer. A negative correlation was evident between the immune score and the amount of somatic copy-number variation (SCNV) of immune genes (r = −0.58). The SCNVs showed a potential detrimental effect on immunity in the immune-deficient subtype. Knowledge of the genomic alterations in the tumor microenvironment could be used to guide design of immunotherapy options that are appropriate for patients with certain cancer subtypes. Cancer Immunol Res; 6(7); 848–59. ©2018 AACR.

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 (H

Studies have shown that the current human reference genome (GRCh38) might miss information for some populations, but “exactly what we miss” is still elusive due to the lower contiguity of non-reference genomes. We juxtaposed the GRCh38 with high contiguity genome assemblies, AK1, to show that ∼1.8% (∼53.4 Mbp) of AK1 sequences missed in GRCh38 with ∼0.76% (∼22.2 Mbp) of ectopic chromosomes. The unique AK1 sequences harbored ∼1,390 putative coding elements. We found that ∼5.3Mb (∼0.2%) of the AK1 sequences aligned and recovered the “unmapped” reads of fourteen individuals (5 East-Asians, 4 Europeans, and 5 Africans) as a reference. The regions that “unmapped” reads aligned included 110 common (shared between ≥2 individuals) and 38 globally (≥7 individuals) missing regions with 25 candidate coding elements. We verified that many of the common missing regions exist in multiple populations and chimpanzee’s DNA. Our study illuminates not only the discovery of missing information but the use of highly precise ethnic genomes in understanding human genetics.

For the noninvasive prenatal diagnosis (NIPD) of X-linked recessive diseases such as Duchenne muscular dystrophy (DMD), maternal haplotype phasing is a critical step for dosage analysis of the inherited allele. Until recently, the proband-based indirect haplotyping method has been preferred despite its limitations for use in clinical practice. Here, we describe a method for directly determining the maternal haplotype without requiring the proband’s DNA in DMD families. We used targeted linked-read deep sequencing (mean coverage of 692×) of gDNA from 5 mothers to resolve their haplotypes and predict the mutation status of the fetus. The haplotype of DMD alleles in the carrier mother was successfully phased through a targeted linked-read sequencing platform. Compared with the proband-based phasing method, linked-read sequencing was more accurate in differentiating whether the recombination events occurred in the proband or in the fetus. The predicted inheritance of the DMD mutation was diagnosed correctly in all 5 families in which the mutation had been confirmed using amniocentesis or chorionic villus sampling. Direct haplotyping by this targeted linked-read sequencing method could be used as a phasing method for the NIPD of DMD, especially when the genomic DNA of the proband is unavailable.

Background: Elucidating aging-related transcriptomic changes in human organs is necessary to understand the aging physiology and mechanisms, but little is known regarding the thyroid gland. We investigated aging-related transcriptomic alterations in the human thyroid gland and characterized the related molecular functions. Methods: Publicly available RNA sequencing data of 322 thyroid tissue samples from the Genotype-Tissue Expression project were analyzed. In addition, our own 64 RNA sequencing data of normal thyroid tissue samples were used as a validation set. To comprehensively evaluate the associations between aging and transcriptomic changes, we performed a weighted gene coexpression network analysis and pathway enrichment analysis. The thyroid differentiation score was then used for further analysis, defining the correlations between thyroid differentiation and aging. Results: The most significant aging-related transcriptomic change in thyroid was the downregulation of genes related to the mitochondrial and proteasomal functions (p = 3 × 10−6). Moreover, genes that are associated with immune processes were significantly upregulated with age (p = 3 × 10−4), and all of them overlapped with the upregulated genes in the thyroid glands affected by lymphocytic thyroiditis. Furthermore, these aging-related changes were not significantly different according to sex, but in terms of the thyroid differentiation, females were more susceptible to aging-related changes (p for trend = 0.03). Conclusions: Aging-related transcriptomic changes in the thyroid gland were associated with mitochondrial and proteasomal dysfunction, loss of differentiation, and activation of autoimmune processes. Our results provide clues to better understanding the age-related decline in thyroid function and higher susceptibility to autoimmune thyroid disease.

In the original article, part of Table 1 headings and entries were missing. The correct Table is as shown below. The original article has been corrected.

Objective To analyze whole exome sequencing (WES) data on ovarian clear cell carcinoma (OCCC) in Korean patients via the technique of next generation sequencing (NGS). Genomic profiles were compared between endometriosis-associated OCCC (EMS-OCCC) and Non-EMS-OCCC. Methods We used serum samples and cancer tissues, stored at the Seoul National University Hospital Human Biobank, that were initially collected from women diagnosed with OCCC between 2012 and 2016. In total, 15 patients were enrolled: 5 with pathologically confirmed EMS-OCCC and 10 with Non-EMS-OCCC. We performed NGS WES on 15 fresh frozen OCCC tissues and matched serum samples, enabling comprehensive genomic characterization of OCCC. Results OCCC was characterized by complex genomic alterations, with a median of 178 exonic mutations (range, 111-25,798) and a median of 343 somatic copy number variations (range, 43-1,820) per tumor sample. In all, 54 somatic mutations were discovered across 14 genes, including PIK3CA (40%), ARID1A (40%), and KRAS (20%) in the 15 Korean OCCCs. Copy number gains in NTRK1 (33%), MYC (40%), and GNAS (47%) and copy number losses in TET2 (73%), TSC1 (67%), BRCA2 (60%), and SMAD4 (47%) were frequent. The significantly altered pathways were associated with proliferation and survival (including the PI3K/AKT, TP53, and ERBB2 pathways) in 87% of OCCCs and with chromatin remodeling in 47% of OCCCs. No significant differences in frequencies of genetic alterations were detected between EMS-OCCC and Non-EMS-OCCC groups. Conclusion We successfully characterized the genomic landscape of 15 Korean patients with OCCC. We identified potential therapeutic targets for the treatment of this malignancy.

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.

Although various chemokines have pro-tumorigenic actions in cancers, the effects of CXCL16 remain controversial. The aim of this study was to investigate the molecular characteristics of CXCL16-expressing papillary thyroid cancers (PTCs). CXCL16 expressions were significantly higher in PTCs than benign or normal thyroid tissues. In the TCGA dataset for PTCs, a higher CXCL16 expression was associated with M2 macrophage- and angiogenesis-related genes and poor prognostic factors including a higher TNM staging and the BRAFV600E mutation. PTCs with a higher expression of 3-gene panel including CXCL16, AHNAK2, and THBS2 showed poor recurrence-free survivals than that of the lower expression group. Next, shCXCL16 was introduced into BHP10-3SCp cells to deplete the endogenous CXCL16, and then, the cells were subcutaneously injected to athymic mice. Tumors from the BHP10-3SCpshCXCL16 exhibited a delayed tumor growth with decreased numbers of ERG+ endothelial cells and F4/80+ macrophages than those from the BHP10-3SCpcontrol. CXCL16-related genes including AHNAK2 and THBS2 were downregulated in the tumors from the BHP10-3SCpshCXCL16 compared with that from the BHP10-3SCpcontrol. In conclusion, a higher CXCL16 expression was associated with macrophage- and angiogenesis-related genes and aggressive phenotypes in PTC. Targeting CXCL16 may be a good therapeutic strategy for advanced thyroid cancer.

Dutasteride, a dual inhibitor of both type I and II 5α-reductases, is used to treat male pattern hair loss (MPHL). However, patient response to dutasteride varies in each individual, the cause of which is yet to be identified. To identify genetic variants associated with response to dutasteride treatment for MPHL, a total of 42 men with moderate MPHL who had been treated with dutasteride for 6 months were genotyped and analysed by quantitative linear regression, case-control association tests, and Fisher’s exact test. The synonymous single nucleotide polymorphism (SNP) rs72623193 in DHRS9 was most significantly associated with response to dutasteride, followed by the non-synonymous SNP rs2241057 in CYP26B1. Additionally, variants in ESR1, SRD5A1, CYP19A1, and RXRG are suggested to be associated with response to dutasteride. Cumulative effect and interaction among these SNPs were presented in both additive and non-additive models.

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.

Recent reports have identified differences in the mutational spectra across human populations. While 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 is 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 continues to contaminate modern studies in hidden ways. It may be time to retire or upgrade such legacy sequencing data.

Genotype imputation using the reference panel is a cost-effective strategy to fill millions of missing genotypes for the purpose of various genetic analyses. Here, we present the Northeast Asian Reference Database (NARD), including whole-genome sequencing data of 1,781 individuals from Korea, Mongolia, Japan, China, and Hong Kong. NARD provides the genetic diversities of Korean (n=850) and Mongolian (n=386) ancestries that were not present in the 1000 Genomes Project Phase 3 (1KGP3). We combined and re-phased the genotypes from NARD and 1KGP3 to construct a union set of haplotypes. This approach established a robust imputation reference panel for the Northeast Asian populations, which yields the greatest imputation accuracy of rare and low-frequency variants compared with the existing panels. Also, we illustrate that NARD can potentially improve disease variant discovery by reducing pathogenic candidates. Overall, this study provides a decent reference panel for the genetic studies in Northeast Asia.

Summary paragraphThe Trans-Omics for Precision Medicine (TOPMed) program seeks to elucidate the genetic architecture and disease biology of heart, lung, blood, and sleep disorders, with the ultimate goal of improving diagnosis, treatment, and prevention. The initial phases of the program focus on whole genome sequencing of individuals with rich phenotypic data and diverse backgrounds. Here, we describe TOPMed goals and design as well as resources and early insights from the sequence data. The resources include a variant browser, a genotype imputation panel, and sharing of genomic and phenotypic data via dbGaP. In 53,581 TOPMed samples, >400 million single-nucleotide and insertion/deletion variants were detected by alignment with the reference genome. Additional novel variants are detectable through assembly of unmapped reads and customized analysis in highly variable loci. Among the >400 million variants detected, 97% have frequency

Additional file 1: Figure S1. Geographic map of the study area in the NARD. Figure S2. Correlation between the sequencing depth and number of variants. Figure S3. Transition to transversion ratio of the populations in the NARD. Figure S4. Heterozygous to homozygous ratio of the global populations. Figure S5. Number of loss-of-function variants. Figure S6. Hardy-Weinberg Equilibrium test of variants in the NARD. Figure S7. Novel variant statistics. Figure S8. Differential genetic composition of the two MNG groups. Figure S9. Imputation performance evaluation of FRA individuals. Figure S10. Imputation performance evaluation of CHN and JPN individuals. Figure S11. Length distribution of shared IBD tracts between the two individuals in each population. Figure S12. The flow chart of the pipeline consisting of four major steps for NARD imputation server. Figure S13. The cross-validation error inferred by ADMIXTURE algorithm.

Triple-negative breast cancer (TNBC) accounts for 10–20% of all diagnosed BCs and it is enriched in BRCA1 mutation. Guidelines for Western countries suggest that BRCA 1/2 genetic testing should be done for patients with TNBC diagnosed less than 60 years, but there is lack of evidence supporting genetic testing in Asian populations. We determined the prevalence of germline BRCA 1/2 mutations among unselected Korean patients with TNBC and analyzed oncologic outcomes. From among 1628 women with TNBC who underwent surgery at Samsung Medical Center (SMC) between Jul 2008 and Jan 2016, 999 samples were available in the SMC biobank for testing germline BRCA 1/2 mutations using next-generation DNA sequencing. Overall, 131 Korean patients (13.1%) had BRCA 1/2 mutations: 97 (9.7%) were in BRCA 1, and 35 (3.5%) were in BRCA 2. One patient had both BRCA 1 and BRCA 2 mutations. Overall, 68 distinct pathologic or likely pathogenic variants (43 BRCA1 and 25 BRCA2) were found. Among those diagnosed at ≤ 60 years, the prevalence of BRCA 1/2 mutation was 14.5%. The mean age of diagnosis of BRCA1/2 mutation carriers was significantly younger than that of non-carriers (45.6 vs. 50.1 years, p

BACKGROUND Recent reports suggest that mutations in the promoter of the gene encoding telomerase reverse transcriptase (TERT) affect thyroid cancer outcomes. METHODS In all, 551 patients with differentiated thyroid cancer (DTC) enrolled in this study. The median follow-up duration was 4.8 years (interquartile range, 3.4-10.6 years). RESULTS TERT promoter mutations were detected in 25 DTCs (4.5%): 2.8% in neither BRAF-mutated nor RAS-mutated tumors, 4.8% in BRAF-mutated tumors, and 11.3% in RAS-mutated tumors. Moreover, they were frequently observed in American Thyroid Association (ATA) high-risk and TNM stage III/IV groups (9.1% and 12.9%, respectively). The coexistence of BRAF or RAS with TERT promoter mutations increased aggressive clinicopathologic features, recurrence (hazard ratio [HR] for BRAF, 4.64; 95% confidence interval [CI], 1.42-15.18; HR for RAS, 5.36; 95% CI, 1.20-24.02), and mortality (HR for BRAF, 15.13; 95% CI, 1.55-148.23; HR for RAS, 14.75; 95% CI, 1.30-167.00), even after adjustments for the age at diagnosis and sex, although the significance was lost after additional adjustments for pathologic characteristics. Furthermore, TERT promoter mutations significantly increased the risk of both recurrence and mortality in the ATA high-risk (HR for recurrence, 5.79; 95% CI, 2.07-16.18; HR for mortality, 16.16; 95% CI, 2.10-124.15) and TNM stage III/IV groups (HR for recurrence, 3.60; 95% CI, 1.19-10.85; HR for mortality, 9.06; 95% CI, 2.09-39.26). CONCLUSIONS The coexistence of BRAF or RAS mutations enhanced the prognostic effects of TERT promoter mutations. Furthermore, TERT promoter mutations strengthened the predictions of mortality and recurrence by the ATA and TNM staging systems, particularly for high-risk patients with DTC. Cancer 2016. © 2016 American Cancer Society.

The identification and clinical validation of cancer driver genes are essential to accelerate the translational transition of cancer genomics, as well as to find clinically confident targets for the therapeutic intervention of cancers. Here we identified recurrent LMNA-NTRK1 and TPM3-NTRK1 fusions in Korean patients with colon cancer (3 out of 147, 2%) through next-generation RNA sequencing (RNA-seq). NTRK1 fusions were mutually exclusive oncogenic drivers of colon cancer that were accompanied with in vitro potential of colony formation and in vivo tumorigenicity comparable to KM12, a human colon cancer cell line harboring TPM3-NTRK1 fusion. NTRK1-encoded TrkA protein was prevalent in 11 out of 216 Korean (5.1%) and 28 out of 472 Chinese patients (5.9%) from independent cohorts, respectively. The expression level of TrkA was significantly correlated with NTRK1 fusion (p = 0.0192), which was verified by a fluorescence in situ hybridization (FISH). Korean patients with TrkA-positive colon cancer had a marginal but significant shorter overall survival time than TrkA-negative colon cancer [hazard ratio (HR) = 0.5346, 95% confidential interval (CI) = 0.2548-0.9722, p = 0.0411]. In addition, KM12 cell line was sensitive to selective TrkA inhibitors. These results demonstrate that NTRK1 fusion is granted as a clinically relevant target for therapeutic intervention of colon cancer.

We report a novel strategy to directly convert adult human dermal fibroblasts (aHDFs) into authentic endothelial cells (ECs). Lentiviral vectors encoding endothelial transcription factors (TFs) were constructed, used to transduce aHDFs. We examined whether 5 TFs (FOXO1, ER71, KLF2, TAL1, and LMO2

Among subtypes of follicular variant of papillary thyroid carcinoma (FVPTC), encapsulated FVPTC (EFVPTC) shows more indolent behavior than infiltrative FVPTC (IFVPTC). In particular, noninvasive EFVPTC, now designated as noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP), tends to have an excellent prognosis. However, it remains unclear whether the molecular pathogenesis or signature of the various forms of FVPTC is different. By massively parallel sequencing analysis, this study comprehensively characterized the transcriptional and mutational landscape of FVPTC and established correlations with phenotypic subtypes.This study included 48 FVPTCs: 17 NIFTPs, 13 invasive EFVPTCs (I-EFVPTCs), and 18 IFVPTCs. For comparison, 55 classical papillary thyroid carcinomas (cPTCs) harboring a BRAFIn NIFTP, the BRAFMolecular profiles of NIFTP and I-EFVPTC may be shared with FA/miFTC, while IFVPTC seems to be associated with a similar profile as cPTC. Activation of cell adhesion and invasion pathways may play a key role in the development of invasive phenotypes of FVPTC.

Characterizing the molecular immune subtype and micro-environment of lung cancer is necessary to understand immunogenic interactions between infiltrating immune and stromal cells, and how tumor cells overcome immune checkpoint blockades. This study seeks to identify computational methodologies for subtyping gene expression-based tumor-immune micro-environment interactions, which differentiate non-small cell lung cancer (NSCLC) into immune-defective and immune-competent subtypes. Here, 101 lung squamous cell carcinomas (LUSCs) and 87 lung adenocarcinomas (LUADs) tumor samples have been analyzed. Several micro-environmental factors differentially induce LUAD or LUSC immune subtypes, as well as immune checkpoint expression. In particular, tumor-associated macrophages (TAMs) are key immune cells play a vital role in inflammation and cancer micro-environments of LUSCs; whereas, regulatory B cells are immunosuppressive and tumorigenic in LUADs. Additionally, cytolytic activity upon CD8+ T cell activation is decreased by the abundance of B cells and macrophages in immune-competent subtypes. Therefore, identifying immune subtypes in lung cancer and their impact on tumor micro-environment will lead to clinical tools for assessing LUADs and LUSCs in patients, as well as maximize the efficacy of immune checkpoint inhibitors.

Pluripotency is defined by the ability of a cell to differentiate to the derivatives of all the three embryonic germ layers: ectoderm, mesoderm and endoderm. Pluripotent cells can be captured via the archetypal derivation of embryonic stem cells or via somatic cell reprogramming. Somatic cells are induced to acquire a pluripotent stem cell (iPSC) state through the forced expression of key transcription factors, and in the mouse these cells can fulfil the strictest of all developmental assays for pluripotent cells by generating completely iPSC-derived embryos and mice. However, it is not known whether there are additional classes of pluripotent cells, or what the spectrum of reprogrammed phenotypes encompasses. Here we explore alternative outcomes of somatic reprogramming by fully characterizing reprogrammed cells independent of preconceived definitions of iPSC states. We demonstrate that by maintaining elevated reprogramming factor expression levels, mouse embryonic fibroblasts go through unique epigenetic modifications to arrive at a stable, Nanog-positive, alternative pluripotent state. In doing so, we prove that the pluripotent spectrum can encompass multiple, unique cell states.

Cancer cells undergo uncontrolled proliferation, and aberrant mitochondrial alterations. Tumor necrosis factor receptorassociated protein 1 (TRAP1) is a mitochondrial heat shock protein. TRAP1 mRNA is highly expressed in some cancer cell lines and tumor tissues. However, the effects of its overexpression on mitochondria are unclear. In this study, we assessed mitochondrial changes accompanying TRAP1 overexpression, in a mouse cell line, NIH/3T3. We found that overexpression of TRAP1 leads to a series of mitochondrial aberrations, including increase in basal ROS levels, and decrease in mitochondrial biogenesis, together with a decrease in peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) mRNA levels. We also observed increased extracellular signal-regulated kinase (ERK) phosphorylation, and enhanced proliferation of TRAP1 overexpressing cells. This study suggests that overexpression of TRAP1 might be a critical link between mitochondrial disturbances and carcinogenesis. [BMB Reports 2014; 47(5): 280-285]

Objective Although genome-wide association studies (GWAS) have substantially contributed to understanding the genetic architecture, unidentified variants for complex traits remain an issue. One of the efficient approaches is the improvement of the power of GWAS scan by weighting P values with prior linkage signals. Our objective was to identify the novel candidates for obesity in Asian populations by using genemapping strategies that combine linkage and association analyses. Design and Methods To obtain linkage information for body mass index (BMI) and waist circumference (WC), we performed a multipoint genome-wide linkage study in an isolated Mongolian sample of 1,049 individuals from 74 families. Next, a family-based GWAS, which integrates within- and between-family components, was performed using the genotype data of 756 individuals of the Mongolian sample, and P values for association were weighted using linkage information obtained previously. Results For both BMI (LOD = 3.3) and WC (LOD = 2.6), the highest linkage peak was discovered at chromosome 10q11.22. In family-based GWAS combined with linkage information, six single-nucleotide polymorphisms (SNPs) for BMI and five SNPs for WC reached a significant level of association (linkage weighted P < 1 × 10-5). Of these, only one of the SNPs associated with WC (rs1704198) was replicated in 327 Korean families comprising 1,301 individuals. This SNP was located in the proximity of the prosperorelated homeobox 1 (PROX1) gene, the function of which was validated previously in a mouse model. Conclusion Our powerful strategic analysis enabled the discovery of a novel candidate gene, PROX1, associated with WC in an Asian population.

Genetic studies on facial morphology targeting healthy populations are fundamental in understanding the specific genetic influences involved; yet, most studies to date, if not all, have been focused on congenital diseases accompanied by facial anomalies. To study the specific genetic cues determining facial morphology, we estimated familial correlations and heritabilities of 14 facial measurements and 3 latent factors inferred from a factor analysis in a subset of the Korean population. The study included a total of 229 individuals from 38 families. We evaluated a total of 14 facial measurements using 2D digital photographs. We performed factor analysis to infer common latent variables. The heritabilities of 13 facial measurements were statistically significant (p < 0.05) and ranged from 0.25 to 0.61. Of these, the heritability of intercanthal width in the orbital region was found to be the highest (h (2) = 0.61, SE = 0.14). Three factors (lower face portion, orbital region, and vertical length) were obtained through factor analysis, where the heritability values ranged from 0.45 to 0.55. The heritability values for each factor were higher than the mean heritability value of individual original measurements. We have confirmed the genetic influence on facial anthropometric traits and suggest a potential way to categorize and analyze the facial portions into different groups.

Targeted resequencing using next-generation sequencing technology is being rapidly applied to the molecular diagnosis of human genetic diseases. The group of muscular dystrophies may be an appropriate candidate for this approach because these diseases exhibit genotype–phenotype heterogeneity. To perform a proof-of-concept study, we selected four patients with congenital muscular dystrophies with defective glycosylation of alpha-dystroglycan. A custom-solution-based target enrichment kit was designed to capture whole-genic regions of the 26 muscular-dystrophy-related genes, including six genes implicated in alpha-dystroglycanopathies. Although approximately 95% of both coding and noncoding regions were covered with at least 15-read depth, parts of the coding exons of FKRP and POMT2 were insufficiently covered. Homozygous and compound heterozygous POMGnT1 mutations were found in two patients. Two novel noncoding variants of FKTN were identified in one patient who had a retrotransposon insertion mutation of FKTN in only one allele. The current targeted resequencing strategy yielded promising results for the extension of this method to other muscular dystrophies. As suboptimal coverage in a small subset of coding regions may affect the sensitivity of the method, complementary Sanger sequencing may be required.

The estimated glomerular filtration rate is a well-known measure of renal function and is widely used to follow the course of disease. Although there have been several investigations establishing the genetic background contributing to renal function, Asian populations have rarely been used in these genome-wide studies. Here, we aimed to find candidate genetic determinants of renal function in 1007 individuals from 73 extended families of Mongolian origin. Linkage analysis found two suggestive regions near 9q21 (logarithm of odds (LOD) 2.82) and 15q15 (LOD 2.70). The subsequent family-based association study found 2 and 10 significant single-nucleotide polymorphisms (SNPs) in each region, respectively. The strongest SNPs on chromosome 9 and 15 were rs17400257 and rs1153831 with P -values of 7.21 × 10 -9 and 2.47 × 10 -11 , respectively. Genes located near these SNPs are considered candidates for determining renal function and include FRMD3 , GATM , and SPATA5L1 . Thus, we identified possible loci that determine renal function in an isolated Asian population. Consistent with previous reports, our study found genes linked and associated with renal function in other populations.

De novo assembly and phasing of the genome of an individual from Korea using a combination of different sequencing approaches provides a useful population-specific reference genome and represents the most contiguous human genome assembly so far. Jeong-Sun Seo and colleagues report de novo assembly and phasing of the genome of an individual from Korea using a combination of PacBio long-read sequencing, Illumina short-read sequencing, 10X Genomics linked reads, bacterial artificial chromosome (BAC) sequencing and BioNano Genomics optical mapping. This provides a useful population-specific reference genome and represents the most contiguous human genome assembly to date. The authors use this to close gaps in the human reference genome and map structural variation. Advances in genome assembly and phasing provide an opportunity to investigate the diploid architecture of the human genome and reveal the full range of structural variation across population groups. Here we report the de novo assembly and haplotype phasing of the Korean individual AK1 (ref. 1) using single-molecule real-time sequencing2, next-generation mapping3, microfluidics-based linked reads4, and bacterial artificial chromosome (BAC) sequencing approaches. Single-molecule sequencing coupled with next-generation mapping generated a highly contiguous assembly, with a contig N50 size of 17.9 Mb and a scaffold N50 size of 44.8 Mb, resolving 8 chromosomal arms into single scaffolds. The de novo assembly, along with local assemblies and spanning long reads, closes 105 and extends into 72 out of 190 euchromatic gaps in the reference genome, adding 1.03 Mb of previously intractable sequence. High concordance between the assembly and paired-end sequences from 62,758 BAC clones provides strong support for the robustness of the assembly. We identify 18,210 structural variants by direct comparison of the assembly with the human reference, identifying thousands of breakpoints that, to our knowledge, have not been reported before. Many of the insertions are reflected in the transcriptome and are shared across the Asian population. We performed haplotype phasing of the assembly with short reads, long reads and linked reads from whole-genome sequencing and with short reads from 31,719 BAC clones, thereby achieving phased blocks with an N50 size of 11.6 Mb. Haplotigs assembled from single-molecule real-time reads assigned to haplotypes on phased blocks covered 89% of genes. The haplotigs accurately characterized the hypervariable major histocompatability complex region as well as demonstrating allele configuration in clinically relevant genes such as CYP2D6. This work presents the most contiguous diploid human genome assembly so far, with extensive investigation of unreported and Asian-specific structural variants, and high-quality haplotyping of clinically relevant alleles for precision medicine.

Nasopharyngeal carcinoma (NPC) is an aggressive head and neck cancer characterized by Epstein-Barr virus (EBV) infection and dense lymphocyte infiltration. The scarcity of NPC genomic data hinders the understanding of NPC biology, disease progression and rational therapy design. Here we performed whole-exome sequencing (WES) on 111 micro-dissected EBV-positive NPCs, with 15 cases subjected to further whole-genome sequencing (WGS), to determine its mutational landscape. We identified enrichment for genomic aberrations of multiple negative regulators of the NF-κB pathway, including CYLD, TRAF3, NFKBIA and NLRC5, in a total of 41% of cases. Functional analysis confirmed inactivating CYLD mutations as drivers for NPC cell growth. The EBV oncoprotein latent membrane protein 1 (LMP1) functions to constitutively activate NF-κB signalling, and we observed mutual exclusivity among tumours with somatic NF-κB pathway aberrations and LMP1-overexpression, suggesting that NF-κB activation is selected for by both somatic and viral events during NPC pathogenesis., Nasopharyngeal cancer is frequently characterized by Epstein-Barr virus infection. Here, using genomic analyses, the authors find that the tumours harbour mutations in genes involved in the NF-κB signalling pathway or overexpress a viral oncoprotein, latent membrane protein 1.

All cancers harbor molecular alterations in their genomes. The transcriptional consequences of these somatic mutations have not yet been comprehensively explored in lung cancer. Here we present the first large scale RNA sequencing study of lung adenocarcinoma, demonstrating its power to identify somatic point mutations as well as transcriptional variants such as gene fusions, alternative splicing events, and expression outliers. Our results reveal the genetic basis of 200 lung adenocarcinomas in Koreans including deep characterization of 87 surgical specimens by transcriptome sequencing. We identified driver somatic mutations in cancer genes including EGFR, KRAS, NRAS, BRAF, PIK3CA, MET, and CTNNB1. Candidates for novel driver mutations were also identified in genes newly implicated in lung adenocarcinoma such as LMTK2, ARID1A, NOTCH2, and SMARCA4. We found 45 fusion genes, eight of which were chimeric tyrosine kinases involving ALK, RET, ROS1, FGFR2, AXL, and PDGFRA. Among 17 recurrent alternative splicing events, we identified exon 14 skipping in the proto-oncogene MET as highly likely to be a cancer driver. The number of somatic mutations and expression outliers varied markedly between individual cancers and was strongly correlated with smoking history of patients. We identified genomic blocks within which gene expression levels were consistently increased or decreased that could be explained by copy number alterations in samples. We also found an association between lymph node metastasis and somatic mutations in TP53. These findings broaden our understanding of lung adenocarcinoma and may also lead to new diagnostic and therapeutic approaches.

Purpose The purpose of this study was to demonstrate a negative association between intraocular pressure (IOP) and age in 2 Asian populations. In addition, we evaluated genetic and nongenetic factors associated with IOP. Design Family-based cohort study. Participants Study subjects >10 years of age from one Korean ( The Healthy Twin ; n=1431) and 2 Mongolian populations ( The GENDISCAN ; n=859 and 806) with IOP values. Methods The IOP values were measured with a noncontact tonometer. Each participant received a standard health examination and received questionnaires, which include candidate risk factors on IOP. Mixed models were used to identify risk factors for IOP. Variance-component methods were applied to estimate the heritability of IOP. Main Outcome Measures The negative trend of IOP with aging and evaluation of impact of genetic and nongenetic components on IOP. Results The mean ages were 43.6, 34.1, and 36.3 years for the Korean, Orhontuul, and Dashbalbar populations, respectively. The mean IOPs were 14.4 mmHg (95% confidence interval [CI], 14.3–14.6) in the Koreans and 14.1 mmHg (95% CI, 13.9–14.3) and 12.6 mmHg (95% CI, 12.4–12.9) in the Orhontuul and Dashbalbar populations, respectively. In the 3 populations, the IOP decreased as age increased. We replicated an association of systolic blood pressure (SBP) with IOP. In addition, components of the metabolic syndrome (MS), such as plasma glucose, lipid level, and body mass index, showed positive associations with IOP, after adjusting for age and SBP. The IOP also had strong genetic contributions in all populations (heritability, 0.47–0.51). Conclusions Negative associations between age and IOP were observed in all 3 populations, which cannot be explained by the increasing prevalence of myopia in the younger generation. The different age trend in IOP may in part be responsible for differences in the prevalence of glaucoma subtypes. Our findings suggest that associations between IOP and MS components were independent of established risk factors such as SBP or age. In addition, the importance of inherited risks requires further genetic dissection of IOP determinants for biological understandings of underlying pathophysiology. Financial Disclosure(s) The authors have no proprietary or commercial interest in any materials discussed in this article.

Rapid advances in DNA sequencing promise to enable new diagnostics and individualized therapies. Achieving personalized medicine, however, will require extensive research on highly reidentifiable, integrated datasets of genomic and health information. To assist with this, participants in the Personal Genome Project choose to forgo privacy via our institutional review board- approved “open consent” process. The contribution of public data and samples facilitates both scientific discovery and standardization of methods. We present our findings after enrollment of more than 1,800 participants, including whole-genome sequencing of 10 pilot participant genomes (the PGP-10). We introduce the Genome-Environment-Trait Evidence (GET-Evidence) system. This tool automatically processes genomes and prioritizes both published and novel variants for interpretation. In the process of reviewing the presumed healthy PGP-10 genomes, we find numerous literature references implying serious disease. Although it is sometimes impossible to rule out a late-onset effect, stringent evidence requirements can address the high rate of incidental findings. To that end we develop a peer production system for recording and organizing variant evaluations according to standard evidence guidelines, creating a public forum for reaching consensus on interpretation of clinically relevant variants. Genome analysis becomes a two-step process: using a prioritized list to record variant evaluations, then automatically sorting reviewed variants using these annotations. Genome data, health and trait information, participant samples, and variant interpretations are all shared in the public domain—we invite others to review our results using our participant samples and contribute to our interpretations. We offer our public resource and methods to further personalized medical research.

The identification of the molecular events that drive cancer transformation is essential to the development of targeted agents that improve the clinical outcome of lung cancer. Many studies have reported genomic driver mutations in non-small-cell lung cancers (NSCLCs) over the past decade; however, the molecular pathogenesis of >40% of NSCLCs is still unknown. To identify new molecular targets in NSCLCs, we performed the combined analysis of massively parallel whole-genome and transcriptome sequencing for cancer and paired normal tissue of a 33-yr-old lung adenocarcinoma patient, who is a never-smoker and has no familial cancer history. The cancer showed no known driver mutation in EGFR or KRAS and no EML4-ALK fusion. Here we report a novel fusion gene between KIF5B and the RET proto-oncogene caused by a pericentric inversion of 10p11.22–q11.21. This fusion gene overexpresses chimeric RET receptor tyrosine kinase, which could spontaneously induce cellular transformation. We identified the KIF5B-RET fusion in two more cases out of 20 primary lung adenocarcinomas in the replication study. Our data demonstrate that a subset of NSCLCs could be caused by a fusion of KIF5B and RET, and suggest the chimeric oncogene as a promising molecular target for the personalized diagnosis and treatment of lung cancer.

To elucidate the genes responsible for constitutive human skin color, we measured the extent of skin pigmentation in the buttock, representative of lifelong non-sun-exposed skin, and conducted a gene mapping study on skin color in an isolated Mongolian population composed of 344 individuals from 59 families who lived in Dashbalbar, Mongolia. The heritability of constitutive skin color was 0.82, indicating significant genetic association on this trait. Through the linkage analysis using 1,039 short tandem repeat (STR) microsatellite markers, we identified a novel genomic region regulating constitutive skin color on 11q24.2 with an logarithm of odds (LOD) score of 3.39. In addition, we also found other candidate regions on 17q23.2, 6q25.1, and 13q33.2 (LOD ≥ 2). Family-based association tests on these regions with suggestive linkage peaks revealed ten and two significant single nucleotide polymorphisms (SNPs) on the linkage regions of chromosome 11 and 17, respectively. We were able to discover four possible candidate genes that would be implicated to regulate human skin color: ETS1, UBASH3B, ASAM, and CLTC.

Background Duchenne muscular dystrophy or Becker muscular dystrophy might be a suitable candidate disease for application of next-generation sequencing in the genetic diagnosis because the complex mutational spectrum and the large size of the dystrophin gene require two or more analytical methods and have a high cost. The authors tested whether large deletions/duplications or small mutations, such as point mutations or short insertions/deletions of the dystrophin gene, could be predicted accurately in a single platform using next-generation sequencing technology. Methods A custom solution-based target enrichment kit was designed to capture whole genomic regions of the dystrophin gene and other muscular-dystrophy-related genes. A multiplexing strategy, wherein four differently bar-coded samples were captured and sequenced together in a single lane of the Illumina Genome Analyser, was applied. The study subjects were 25 patients: 16 with deficient dystrophin expression without a large deletion/duplication and 9 with a known large deletion/duplication. Results Nearly 100% of the exonic region of the dystrophin gene was covered by at least eight reads with a mean read depth of 107. Pathogenic small mutations were identified in 15 of the 16 patients without a large deletion/duplication. Using these 16 patients as the standard, the authors' method accurately predicted the deleted or duplicated exons in the 9 patients with known mutations. Inclusion of non-coding regions and paired-end sequence analysis enabled accurate identification by increasing the read depth and providing information about the breakpoint junction. Conclusions The current method has an advantage for the genetic diagnosis of Duchenne muscular dystrophy and Becker muscular dystrophy wherein a comprehensive mutational search may be feasible using a single platform.