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1.  Single-cell RNA sequencing reveals dynamic changes in A-to-I RNA editome during early human embryogenesis 
BMC Genomics  2016;17:766.
A-to-I RNA-editing mediated by ADAR (adenosine deaminase acting on RNA) enzymes that converts adenosine to inosine in RNA sequence can generate mutations and alter gene regulation in metazoans. Previous studies have shown that A-to-I RNA-editing plays vital roles in mouse embryogenesis. However, the RNA-editing activities in early human embryonic development have not been investigated.
Here, we characterized genome-wide A-to-I RNA-editing activities during human early embryogenesis by profiling 68 single cells from 29 human embryos spanning from oocyte to morula stages. We demonstrate dynamic changes in genome-wide RNA-editing during early human embryogenesis in a stage-specific fashion. In parallel with ADAR expression level changes, the genome-wide A-to-I RNA-editing levels in cells remained relatively stable until 4-cell stage, but dramatically decreased at 8-cell stage, continually decreased at morula stage. We detected 37 non-synonymously RNA-edited genes, of which 5 were frequently found in cells of multiple embryonic stages. Moreover, we found that A-to-I editings in miRNA-targeted regions of a substantial number of genes preferably occurred in one or two sequential stages.
Our single-cell analysis reveals dynamic changes in genome-wide RNA-editing during early human embryogenesis in a stage-specific fashion, and provides important insights into early human embryogenesis.
Electronic supplementary material
The online version of this article (doi:10.1186/s12864-016-3115-2) contains supplementary material, which is available to authorized users.
PMCID: PMC5043600  PMID: 27687780
RNA-editing; Single cell transcriptome; Embryogenesis
2.  RNA-seq analysis of the hypothalamic transcriptome reveals the networks regulating physiopathological progress in the diabetic GK rat 
Scientific Reports  2016;6:34138.
The Goto-Kakizaki (GK) rat is an animal model of non-obese type 2 diabetes (T2D). The GK rat was generated through the introduction of various genetic mutations from continuous inbreeding; these rats develop diabetes spontaneously. The mutated genes in GK rats may play key roles in the regulation of diabetes. The hypothalamus plays a central role in systematic energy homeostasis. Here, the hypothalamic transcriptomes in GK and Wistar rats at 4, 8 and 12 weeks were investigated by RNA-seq, and multiple variants and gene expression profiles were obtained. The number of variants identified from GK rats was significantly greater than that of Wistar rats, indicating that many variants were fixed and heritable in GK rats after selective inbreeding. The differential gene expression analysis indicated that GK rats had a dysfunctional hypothalamic melanocortin system and attenuation of the hypothalamic glucose-sensing pathway. In addition, we generated integrated gene network modules by combining the protein-protein interaction (PPI) network, co-expression network and mutations in GK and Wistar rats. In the modules, GK-specific genes, such as Bad, Map2k2, Adcy3, Adcy2 and Gstm6, may play key roles in hypothalamic regulation in GK rats. Our research provides a comprehensive map of the abnormalities in the GK rat hypothalamus, which reveals the new mechanisms of pathogenesis of T2D.
PMCID: PMC5039700  PMID: 27677945
3.  An Advanced Model to Precisely Estimate the Cell-Free Fetal DNA Concentration in Maternal Plasma 
PLoS ONE  2016;11(9):e0161928.
With the speedy development of sequencing technologies, noninvasive prenatal testing (NIPT) has been widely applied in clinical practice for testing for fetal aneuploidy. The cell-free fetal DNA (cffDNA) concentration in maternal plasma is the most critical parameter for this technology because it affects the accuracy of NIPT-based sequencing for fetal trisomies 21, 18 and 13. Several approaches have been developed to calculate the cffDNA fraction of the total cell-free DNA in the maternal plasma. However, most approaches depend on specific single nucleotide polymorphism (SNP) allele information or are restricted to male fetuses.
In this study, we present an innovative method to accurately deduce the concentration of the cffDNA fraction using only maternal plasma DNA. SNPs were classified into four maternal-fetal genotype combinations and three boundaries were added to capture effective SNP loci in which the mother was homozygous and the fetus was heterozygous. The median value of the concentration of the fetal DNA fraction was estimated using the effective SNPs. A depth-bias correction was performed using simulated data and corresponding regression equations for adjustments when the depth of the sequencing data was below 100-fold or the cffDNA fraction is less than 10%.
Using our approach, the median of the relative bias was 0.4% in 18 maternal plasma samples with a median sequencing depth of 125-fold. There was a significant association (r = 0.935) between our estimations and the estimations inferred from the Y chromosome. Furthermore, this approach could precisely estimate a cffDNA fraction as low as 3%, using only maternal plasma DNA at the targeted region with a sequencing depth of 65-fold. We also used PCR instead of parallel sequencing to calculate the cffDNA fraction. There was a significant association (r = 98.2%) between our estimations and those inferred from the Y chromosome.
PMCID: PMC5035032  PMID: 27662469
4.  The stepwise evolution of the exome during acquisition of docetaxel resistance in breast cancer cells 
BMC Genomics  2016;17:442.
Resistance to taxane-based therapy in breast cancer patients is a major clinical problem that may be addressed through insight of the genomic alterations leading to taxane resistance in breast cancer cells. In the current study we used whole exome sequencing to discover somatic genomic alterations, evolving across evolutionary stages during the acquisition of docetaxel resistance in breast cancer cell lines.
Two human breast cancer in vitro models (MCF-7 and MDA-MB-231) of the step-wise acquisition of docetaxel resistance were developed by exposing cells to 18 gradually increasing concentrations of docetaxel. Whole exome sequencing performed at five successive stages during this process was used to identify single point mutational events, insertions/deletions and copy number alterations associated with the acquisition of docetaxel resistance. Acquired coding variation undergoing positive selection and harboring characteristics likely to be functional were further prioritized using network-based approaches.
A number of genomic changes were found to be undergoing evolutionary selection, some of which were likely to be functional. Of the five stages of progression toward resistance, most resistance relevant genomic variation appeared to arise midway towards fully resistant cells corresponding to passage 31 (5 nM docetaxel) for MDA-MB-231 and passage 16 (1.2 nM docetaxel) for MCF-7, and where the cells also exhibited a period of reduced growth rate or arrest, respectively. MCF-7 cell acquired several copy number gains on chromosome 7, including ABC transporter genes, including ABCB1 and ABCB4, as well as DMTF1, CLDN12, CROT, and SRI. For MDA-MB-231 numerous copy number losses on chromosome X involving more than 30 genes was observed. Of these genes, CASK, POLA1, PRDX4, MED14 and PIGA were highly prioritized by the applied network-based gene ranking approach. At higher docetaxel concentration MCF-7 subclones exhibited a copy number loss in E2F4, and the gene encoding this important transcription factor was down-regulated in MCF-7 resistant cells.
Our study of the evolution of acquired docetaxel resistance identified several genomic changes that might explain development of docetaxel resistance. Interestingly, the most relevant resistance-associated changes appeared to originate midway through the evolution towards fully resistant cell lines. Our data suggest that no single genomic event sufficiently predicts resistance to docetaxel, but require genomic alterations affecting multiple pathways that in concert establish the final resistance stage.
Electronic supplementary material
The online version of this article (doi:10.1186/s12864-016-2749-4) contains supplementary material, which is available to authorized users.
PMCID: PMC4899892  PMID: 27277198
Breast cancer; Docetaxel resistance; Taxane; Exome sequencing
5.  A novel splice donor site mutation in EPHA2 caused congenital cataract in a Chinese family 
Indian Journal of Ophthalmology  2016;64(5):364-368.
Congenital cataract is a rare disorder characterized by crystallin denaturation, which becomes a major cause of childhood blindness. Although more than fifty pathogenic genes for congenital cataract have been reported, the genetic causes of many cataract patients remain unknown. In this study, the aim is to identify the genetic cause of a five-generation Chinese autosomal dominant congenital cataract family.
Whole exome sequencing (WES) was performed on three affected and one unaffected member of the family, known causative genes were scanned first. Sanger sequencing was used to validate co-segregation of the candidate variant in the family. The impact on the transcript and amino acid sequences of the variant was further analyzed.
We identified a novel splice donor site mutation c. 2825+1G >A in EPHA2 that was absent in public and in-house databases and showed co-segregation in the family. This variant resulted in an altered splice that led to protein truncation.
The mutation we identified was responsible for congenital cataract in our studied family. Our findings broaden the spectrum of causative mutations in EPHA2 gene for congenital cataract and suggest that WES is an efficient strategy to scan variants in known causative genes for genetically heterogeneous diseases.
PMCID: PMC4966373  PMID: 27380975
Autosomal dominant congenital cataract; EPHA2; splice donor site mutation; whole exome sequencing
6.  Novel Y-chromosomal microdeletions associated with non-obstructive azoospermia uncovered by high throughput sequencing of sequence-tagged sites (STSs) 
Scientific Reports  2016;6:21831.
Y-chromosomal microdeletion (YCM) serves as an important genetic factor in non-obstructive azoospermia (NOA). Multiplex polymerase chain reaction (PCR) is routinely used to detect YCMs by tracing sequence-tagged sites (STSs) in the Y chromosome. Here we introduce a novel methodology in which we sequence 1,787 (post-filtering) STSs distributed across the entire male-specific Y chromosome (MSY) in parallel to uncover known and novel YCMs. We validated this approach with 766 Chinese men with NOA and 683 ethnically matched healthy individuals and detected 481 and 98 STSs that were deleted in the NOA and control group, representing a substantial portion of novel YCMs which significantly influenced the functions of spermatogenic genes. The NOA patients tended to carry more and rarer deletions that were enriched in nearby intragenic regions. Haplogroup O2* was revealed to be a protective lineage for NOA, in which the enrichment of b1/b3 deletion in haplogroup C was also observed. In summary, our work provides a new high-resolution portrait of deletions in the Y chromosome.
PMCID: PMC4764820  PMID: 26907467
7.  A comparison of isolated circulating tumor cells and tissue biopsies using whole-genome sequencing in prostate cancer 
Oncotarget  2015;6(42):44781-44793.
Previous studies have demonstrated focal but limited molecular similarities between circulating tumor cells (CTCs) and biopsies using isolated genetic assays. We hypothesized that molecular similarity between CTCs and tissue exists at the single cell level when characterized by whole genome sequencing (WGS). By combining the NanoVelcro CTC Chip with laser capture microdissection (LCM), we developed a platform for single-CTC WGS. We performed this procedure on CTCs and tissue samples from a patient with advanced prostate cancer who had serial biopsies over the course of his clinical history. We achieved 30X depth and ≥ 95% coverage. Twenty-nine percent of the somatic single nucleotide variations (SSNVs) identified were founder mutations that were also identified in CTCs. In addition, 86% of the clonal mutations identified in CTCs could be traced back to either the primary or metastatic tumors. In this patient, we identified structural variations (SVs) including an intrachromosomal rearrangement in chr3 and an interchromosomal rearrangement between chr13 and chr15. These rearrangements were shared between tumor tissues and CTCs. At the same time, highly heterogeneous short structural variants were discovered in PTEN, RB1, and BRCA2 in all tumor and CTC samples. Using high-quality WGS on single-CTCs, we identified the shared genomic alterations between CTCs and tumor tissues. This approach yielded insight into the heterogeneity of the mutational landscape of SSNVs and SVs. It may be possible to use this approach to study heterogeneity and characterize the biological evolution of a cancer during the course of its natural history.
PMCID: PMC4792591  PMID: 26575023
circulating tumor cell; prostate cancer; whole genome sequencing; liquid biopsy; cancer heterogeneity
8.  Frequent alterations in cytoskeleton remodelling genes in primary and metastatic lung adenocarcinomas 
Nature Communications  2015;6:10131.
The landscape of genetic alterations in lung adenocarcinoma derived from Asian patients is largely uncharacterized. Here we present an integrated genomic and transcriptomic analysis of 335 primary lung adenocarcinomas and 35 corresponding lymph node metastases from Chinese patients. Altogether 13 significantly mutated genes are identified, including the most commonly mutated gene TP53 and novel mutation targets such as RHPN2, GLI3 and MRC2. TP53 mutations are furthermore significantly enriched in tumours from patients harbouring metastases. Genes regulating cytoskeleton remodelling processes are also frequently altered, especially in metastatic samples, of which the high expression level of IQGAP3 is identified as a marker for poor prognosis. Our study represents the first large-scale sequencing effort on lung adenocarcinoma in Asian patients and provides a comprehensive mutational landscape for both primary and metastatic tumours. This may thus form a basis for personalized medical care and shed light on the molecular pathogenesis of metastatic lung adenocarcinoma.
Despite lung adenocarcinoma having a high global mortality, the genetic mutations present in Asian patients are uncharacterized. Here the authors use genomic and transcriptomic analysis to identify thirteen significantly affected genes, including RHPN2, GLI3, MRC2, TP53 and IQGAP3.
PMCID: PMC4682110  PMID: 26647728
9.  Full-length single-cell RNA-seq applied to a viral human cancer: applications to HPV expression and splicing analysis in HeLa S3 cells 
GigaScience  2015;4:51.
Viral infection causes multiple forms of human cancer, and HPV infection is the primary factor in cervical carcinomas. Recent single-cell RNA-seq studies highlight the tumor heterogeneity present in most cancers, but virally induced tumors have not been studied. HeLa is a well characterized HPV+ cervical cancer cell line.
We developed a new high throughput platform to prepare single-cell RNA on a nanoliter scale based on a customized microwell chip. Using this method, we successfully amplified full-length transcripts of 669 single HeLa S3 cells and 40 of them were randomly selected to perform single-cell RNA sequencing. Based on these data, we obtained a comprehensive understanding of the heterogeneity of HeLa S3 cells in gene expression, alternative splicing and fusions. Furthermore, we identified a high diversity of HPV-18 expression and splicing at the single-cell level. By co-expression analysis we identified 283 E6, E7 co-regulated genes, including CDC25, PCNA, PLK4, BUB1B and IRF1 known to interact with HPV viral proteins.
Our results reveal the heterogeneity of a virus-infected cell line. It not only provides a transcriptome characterization of HeLa S3 cells at the single cell level, but is a demonstration of the power of single cell RNA-seq analysis of virally infected cells and cancers.
Electronic supplementary material
The online version of this article (doi:10.1186/s13742-015-0091-4) contains supplementary material, which is available to authorized users.
PMCID: PMC4635585  PMID: 26550473
Single-cell transcriptome; HeLa; HPV; Virus; Tumor heterogeneity; Cancer; RNA splicing
10.  Risk Factors and Early Predictors for Heterotopic Pregnancy after In Vitro Fertilization 
PLoS ONE  2015;10(10):e0139146.
This study investigated the risk factors and early predictors for heterotopic pregnancy (HP) after in vitro fertilization and embryo transfer (IVF-ET). From January 2008 to January 2013, 41 cases of HP and 72 cases of intrauterine twin pregnancy after IVF-ET were recruited and retrospectively analyzed. Compared with intrauterine twin pregnancy group, the HP group had a lower basal luteinizing hormone (LH) level (P = 0.005) and more cases had a history of hydrosalpinx (P = 0.008). After 14 days of IVF-ET, the serum β-HCG (β-human chorionic gonadotropin), E2 (Estradiol) and P (Progesterone) levels were lower in HP group (P<0.001, respectively). Moreover, vaginal bleeding and abdominal pain were the significant features of HP before diagnosis (P<0.001, respectively). Further by logistic regression, serum β-hCG, P levels on the 14th day after ET, and vaginal bleeding were identified as the independent factors of HP. These results indicate that when two or more embryos transferred in IVF procedure, β-hCG, P levels on the 14th day after ET, and vaginal bleeding could be taken as predictors for HP.
PMCID: PMC4624796  PMID: 26510008
11.  Whole Exome Sequencing Identifies Frequent Somatic Mutations in Cell-Cell Adhesion Genes in Chinese Patients with Lung Squamous Cell Carcinoma 
Scientific Reports  2015;5:14237.
Lung squamous cell carcinoma (SQCC) accounts for about 30% of all lung cancer cases. Understanding of mutational landscape for this subtype of lung cancer in Chinese patients is currently limited. We performed whole exome sequencing in samples from 100 patients with lung SQCCs to search for somatic mutations and the subsequent target capture sequencing in another 98 samples for validation. We identified 20 significantly mutated genes, including TP53, CDH10, NFE2L2 and PTEN. Pathways with frequently mutated genes included those of cell-cell adhesion/Wnt/Hippo in 76%, oxidative stress response in 21%, and phosphatidylinositol-3-OH kinase in 36% of the tested tumor samples. Mutations of Chromatin regulatory factor genes were identified at a lower frequency. In functional assays, we observed that knockdown of CDH10 promoted cell proliferation, soft-agar colony formation, cell migration and cell invasion, and overexpression of CDH10 inhibited cell proliferation. This mutational landscape of lung SQCC in Chinese patients improves our current understanding of lung carcinogenesis, early diagnosis and personalized therapy.
PMCID: PMC4621504  PMID: 26503331
12.  Global analysis of DNA methylation in hepatocellular carcinoma by a liquid hybridization capture-based bisulfite sequencing approach 
Clinical Epigenetics  2015;7(1):86.
Epigenetic alterations, such as aberrant DNA methylation of promoter and enhancer regions, which lead to atypical gene expression, have been associated with carcinogenesis. In hepatocellular carcinoma (HCC), genome-wide analysis of methylation has only recently been used. For a better understanding of hepatocarcinogenesis, we applied an even higher resolution analysis of the promoter methylome to identify previously unknown regions and genes differentially methylated in HCC.
Optimized liquid hybridization capture-based bisulfite sequencing (LHC-BS) was developed to quantitatively analyze 1.86 million CpG sites in individual samples from eight pairs of HCC and adjacent tissues. By linking the differentially methylated regions (DMRs) in promoters to the differentially expressed genes (DEGs), we identified 12 DMR-associated genes. We further utilized Illumina MiSeq combining the bisulfite sequencing PCR approach to validate the 12 candidate genes. Analysis of an additional 78 HCC pairs on the Illumina MiSeq platform confirmed that 7 genes showed either promoter hyper-methylation (SMAD6, IFITM1, LRRC4, CHST4, and TBX15) or hypo-methylation (CCL20 and NQO1) in HCC.
Novel methylome profiling provides a cost-efficient approach to identifying candidate genes in human HCC that may contribute to hepatocarcinogenesis. Our work provides further information critical for understanding the epigenetic processes underlying tumorigenesis and development of HCC.
Electronic supplementary material
The online version of this article (doi:10.1186/s13148-015-0121-1) contains supplementary material, which is available to authorized users.
PMCID: PMC4546208  PMID: 26300991
DNA methylation; Liquid hybridization capture-based bisulfite sequencing; Hepatocellular carcinoma
13.  Comparison of variations detection between whole-genome amplification methods used in single-cell resequencing 
GigaScience  2015;4:37.
Single-cell resequencing (SCRS) provides many biomedical advances in variations detection at the single-cell level, but it currently relies on whole genome amplification (WGA). Three methods are commonly used for WGA: multiple displacement amplification (MDA), degenerate-oligonucleotide-primed PCR (DOP-PCR) and multiple annealing and looping-based amplification cycles (MALBAC). However, a comprehensive comparison of variations detection performance between these WGA methods has not yet been performed.
We systematically compared the advantages and disadvantages of different WGA methods, focusing particularly on variations detection. Low-coverage whole-genome sequencing revealed that DOP-PCR had the highest duplication ratio, but an even read distribution and the best reproducibility and accuracy for detection of copy-number variations (CNVs). However, MDA had significantly higher genome recovery sensitivity (~84 %) than DOP-PCR (~6 %) and MALBAC (~52 %) at high sequencing depth. MALBAC and MDA had comparable single-nucleotide variations detection efficiency, false-positive ratio, and allele drop-out ratio. We further demonstrated that SCRS data amplified by either MDA or MALBAC from a gastric cancer cell line could accurately detect gastric cancer CNVs with comparable sensitivity and specificity, including amplifications of 12p11.22 (KRAS) and 9p24.1 (JAK2, CD274, and PDCD1LG2).
Our findings provide a comprehensive comparison of variations detection performance using SCRS amplified by different WGA methods. It will guide researchers to determine which WGA method is best suited to individual experimental needs at single-cell level.
Electronic supplementary material
The online version of this article (doi:10.1186/s13742-015-0068-3) contains supplementary material, which is available to authorized users.
PMCID: PMC4527218  PMID: 26251698
Whole genome amplification; Single-cell resequencing; Variations detection; DOP-PCR; MDA; MALBAC; Next-generation sequencing
14.  Family-based analysis of eight susceptibility loci in polycystic ovary syndrome 
Scientific Reports  2015;5:12619.
Polycystic ovary syndrome (PCOS) is a complex endocrine disorder that is proposed to have a genetic basis. A recent genome-wide association study (GWAS) identified eight new risk loci that are independently associated with PCOS. To further validate the findings, a total of 321 case-parent trios (963 participants) who had a proband affected with PCOS were recruited for the family-based study. The transmission disequilibrium test (TDT) was used to analyze associations between PCOS and ten single nucleotide polymorphisms (SNPs) mapped to eight new susceptibility loci. Significant differences in transmission were observed for the SNPs rs2349415 (located in the FSHR gene, P = 0.0001) and rs3802457 (located in the C9orf3 gene, P = 0.0001), even after correction for multiple testing bias. The present data provides further evidence for an association between two susceptibility loci, 2p16.3 and 9q22.32, and PCOS. Follow-up functional studies on the FSHR and C9orf3 genes are required to understand their roles in PCOS development.
PMCID: PMC4518258  PMID: 26220222
15.  Discovery of biclonal origin and a novel oncogene SLC12A5 in colon cancer by single-cell sequencing 
Cell Research  2014;24(6):701-712.
Single-cell sequencing is a powerful tool for delineating clonal relationship and identifying key driver genes for personalized cancer management. Here we performed single-cell sequencing analysis of a case of colon cancer. Population genetics analyses identified two independent clones in tumor cell population. The major tumor clone harbored APC and TP53 mutations as early oncogenic events, whereas the minor clone contained preponderant CDC27 and PABPC1 mutations. The absence of APC and TP53 mutations in the minor clone supports that these two clones were derived from two cellular origins. Examination of somatic mutation allele frequency spectra of additional 21 whole-tissue exome-sequenced cases revealed the heterogeneity of clonal origins in colon cancer. Next, we identified a mutated gene SLC12A5 that showed a high frequency of mutation at the single-cell level but exhibited low prevalence at the population level. Functional characterization of mutant SLC12A5 revealed its potential oncogenic effect in colon cancer. Our study provides the first exome-wide evidence at single-cell level supporting that colon cancer could be of a biclonal origin, and suggests that low-prevalence mutations in a cohort may also play important protumorigenic roles at the individual level.
PMCID: PMC4042168  PMID: 24699064
single-cell sequencing; colon cancer; SLC12A5; biclonal; oncogene
16.  Post-transcriptional Regulation of Keratinocyte Progenitor Cell Expansion, Differentiation and Hair Follicle Regression by miR-22 
PLoS Genetics  2015;11(5):e1005253.
Hair follicles (HF) undergo precisely regulated recurrent cycles of growth, cessation, and rest. The transitions from anagen (growth), to catagen (regression), to telogen (rest) involve a physiological involution of the HF. This process is likely coordinated by a variety of mechanisms including apoptosis and loss of growth factor signaling. However, the precise molecular mechanisms underlying follicle involution after hair keratinocyte differentiation and hair shaft assembly remain poorly understood. Here we demonstrate that a highly conserved microRNA, miR-22 is markedly upregulated during catagen and peaks in telogen. Using gain- and loss-of-function approaches in vivo, we find that miR-22 overexpression leads to hair loss by promoting anagen-to-catagen transition of the HF, and that deletion of miR-22 delays entry to catagen and accelerates the transition from telogen to anagen. Ectopic activation of miR-22 results in hair loss due to the repression a hair keratinocyte differentiation program and keratinocyte progenitor expansion, as well as promotion of apoptosis. At the molecular level, we demonstrate that miR-22 directly represses numerous transcription factors upstream of phenotypic keratin genes, including Dlx3, Foxn1, and Hoxc13. We conclude that miR-22 is a critical post-transcriptional regulator of the hair cycle and may represent a novel target for therapeutic modulation of hair growth.
Author Summary
Up to 60% people suffer from hair loss throughout their lifetime. Hair growth undergoes recurrent cycling of growth, regression, and resting phases with a defined periodicity. The main cause of human hair loss is due to the premature transition from growth to regression. Understanding of the molecular basis underlying hair regression is important to elucidate the mechanisms of hair loss. Here, we demonstrated that miR-22, a highly conserved microRNA, is critical for the transition from growth to regression of the hair follicle. Importantly, miR-22 could be a novel target for therapeutic therapy of hair loss disorders.
PMCID: PMC4447420  PMID: 26020521
17.  Molecular Signatures of Major Depression 
Current Biology  2015;25(9):1146-1156.
Adversity, particularly in early life, can cause illness. Clues to the responsible mechanisms may lie with the discovery of molecular signatures of stress, some of which include alterations to an individual’s somatic genome. Here, using genome sequences from 11,670 women, we observed a highly significant association between a stress-related disease, major depression, and the amount of mtDNA (p = 9.00 × 10−42, odds ratio 1.33 [95% confidence interval [CI] = 1.29–1.37]) and telomere length (p = 2.84 × 10−14, odds ratio 0.85 [95% CI = 0.81–0.89]). While both telomere length and mtDNA amount were associated with adverse life events, conditional regression analyses showed the molecular changes were contingent on the depressed state. We tested this hypothesis with experiments in mice, demonstrating that stress causes both molecular changes, which are partly reversible and can be elicited by the administration of corticosterone. Together, these results demonstrate that changes in the amount of mtDNA and telomere length are consequences of stress and entering a depressed state. These findings identify increased amounts of mtDNA as a molecular marker of MD and have important implications for understanding how stress causes the disease.
•Amount of mtDNA is increased, and telomeric DNA is shortened in major depression•Both changes can be induced with stress but are contingent on the depressed state•Changes are tissue specific and in part due to glucocorticoid secretion•Changes are in part reversible and represent switches in metabolic strategy
Cai et al. found increases in mtDNA and a reduction in telomeric DNA in cases of major depression using whole-genome sequencing. Both changes are depression state dependent. Mice exposed to chronic stress or glucorticoids showed that these changes reflect switches in metabolic strategy and are tissue specific and partial reversible.
PMCID: PMC4425463  PMID: 25913401
18.  An atypical form of AOA2 with myoclonus associated with mutations in SETX and AFG3L2 
BMC Medical Genetics  2015;16:16.
Hereditary ataxias are a heterogeneous group of neurodegenerative disorders, where exome sequencing may become an important diagnostic tool to solve clinically or genetically complex cases.
We describe an Italian family in which three sisters were affected by ataxia with postural/intentional myoclonus and involuntary movements at onset, which persisted during the disease. Oculomotor apraxia was absent. Clinical and genetic data did not allow us to exclude autosomal dominant or recessive inheritance and suggest a disease gene.
Exome sequencing identified a homozygous c.6292C > T (p.Arg2098*) mutation in SETX and a heterozygous c.346G > A (p.Gly116Arg) mutation in AFG3L2 shared by all three affected individuals. A fourth sister (II.7) had subclinical myoclonic jerks at proximal upper limbs and perioral district, confirmed by electrophysiology, and carried the p.Gly116Arg change. Three siblings were healthy.
Pathogenicity prediction and a yeast-functional assay suggested p.Gly116Arg impaired m-AAA (ATPases associated with various cellular activities) complex function.
Exome sequencing is a powerful tool in identifying disease genes. We identified an atypical form of Ataxia with Oculoapraxia type 2 (AOA2) with myoclonus at onset associated with the c.6292C > T (p.Arg2098*) homozygous mutation. Because the same genotype was described in six cases from a Tunisian family with a typical AOA2 without myoclonus, we speculate this latter feature is associated with a second mutated gene, namely AFG3L2 (p.Gly116Arg variant).
We suggest that variant phenotypes may be due to the combined effect of different mutated genes associated to ataxia or related disorders, that will become more apparent as the costs of exome sequencing progressively will reduce, amplifying its diagnostics use, and meanwhile proposing significant challenges in the interpretation of the data.
Electronic supplementary material
The online version of this article (doi:10.1186/s12881-015-0159-0) contains supplementary material, which is available to authorized users.
PMCID: PMC4422141  PMID: 25927548
AFG3L2; Exome sequencing; Senataxin; SETX; Modifier genes; SCAR1; Ataxia with Oculomotor Apraxia Type 2; Autosomal recessive ataxia; Myoclonus
19.  Excess of Rare Variants in Genes that are Key Epigenetic Regulators of Spermatogenesis in the Patients with Non-Obstructive Azoospermia 
Scientific Reports  2015;5:8785.
Non-obstructive azoospermia (NOA), a severe form of male infertility, is often suspected to be linked to currently undefined genetic abnormalities. To explore the genetic basis of this condition, we successfully sequenced ~650 infertility-related genes in 757 NOA patients and 709 fertile males. We evaluated the contributions of rare variants to the etiology of NOA by identifying individual genes showing nominal associations and testing the genetic burden of a given biological process as a whole. We found a significant excess of rare, non-silent variants in genes that are key epigenetic regulators of spermatogenesis, such as BRWD1, DNMT1, DNMT3B, RNF17, UBR2, USP1 and USP26, in NOA patients (P = 5.5 × 10−7), corresponding to a carrier frequency of 22.5% of patients and 13.7% of controls (P = 1.4 × 10−5). An accumulation of low-frequency variants was also identified in additional epigenetic genes (BRDT and MTHFR). Our study suggested the potential associations of genetic defects in genes that are epigenetic regulators with spermatogenic failure in human.
PMCID: PMC4350091  PMID: 25739334
21.  Clinical outcome of preimplantation genetic diagnosis and screening using next generation sequencing 
GigaScience  2014;3:30.
Next generation sequencing (NGS) is now being used for detecting chromosomal abnormalities in blastocyst trophectoderm (TE) cells from in vitro fertilized embryos. However, few data are available regarding the clinical outcome, which provides vital reference for further application of the methodology. Here, we present a clinical evaluation of NGS-based preimplantation genetic diagnosis/screening (PGD/PGS) compared with single nucleotide polymorphism (SNP) array-based PGD/PGS as a control.
A total of 395 couples participated. They were carriers of either translocation or inversion mutations, or were patients with recurrent miscarriage and/or advanced maternal age. A total of 1,512 blastocysts were biopsied on D5 after fertilization, with 1,058 blastocysts set aside for SNP array testing and 454 blastocysts for NGS testing. In the NGS cycles group, the implantation, clinical pregnancy and miscarriage rates were 52.6% (60/114), 61.3% (49/80) and 14.3% (7/49), respectively. In the SNP array cycles group, the implantation, clinical pregnancy and miscarriage rates were 47.6% (139/292), 56.7% (115/203) and 14.8% (17/115), respectively. The outcome measures of both the NGS and SNP array cycles were the same with insignificant differences. There were 150 blastocysts that underwent both NGS and SNP array analysis, of which seven blastocysts were found with inconsistent signals. All other signals obtained from NGS analysis were confirmed to be accurate by validation with qPCR. The relative copy number of mitochondrial DNA (mtDNA) for each blastocyst that underwent NGS testing was evaluated, and a significant difference was found between the copy number of mtDNA for the euploid and the chromosomally abnormal blastocysts. So far, out of 42 ongoing pregnancies, 24 babies were born in NGS cycles; all of these babies are healthy and free of any developmental problems.
This study provides the first evaluation of the clinical outcomes of NGS-based pre-implantation genetic diagnosis/screening, and shows the reliability of this method in a clinical and array-based laboratory setting. NGS provides an accurate approach to detect embryonic imbalanced segmental rearrangements, to avoid the potential risks of false signals from SNP array in this study.
Electronic supplementary material
The online version of this article (doi:10.1186/2047-217X-3-30) contains supplementary material, which is available to authorized users.
PMCID: PMC4326468  PMID: 25685330
Preimplantation genetic diagnosis/screening; Next generation sequencing; Blastocyst; Cryopreserved embryo transfer; Clinical outcome
22.  Integrated analyses of DNA methylation and hydroxymethylation reveal tumor suppressive roles of ECM1, ATF5, and EOMES in human hepatocellular carcinoma 
Genome Biology  2014;15(12):533.
Differences in 5-hydroxymethylcytosine, 5hmC, distributions may complicate previous observations of abnormal cytosine methylation statuses that are used for the identification of new tumor suppressor gene candidates that are relevant to human hepatocarcinogenesis. The simultaneous detection of 5-methylcytosine and 5-hydroxymethylcytosine is likely to stimulate the discovery of aberrantly methylated genes with increased accuracy in human hepatocellular carcinoma.
Here, we performed ultra-performance liquid chromatography/tandem mass spectrometry and single-base high-throughput sequencing, Hydroxymethylation and Methylation Sensitive Tag sequencing, HMST-seq, to synchronously measure these two modifications in human hepatocellular carcinoma samples. After identification of differentially methylated and hydroxymethylated genes in human hepatocellular carcinoma, we integrate DNA copy-number alterations, as determined using array-based comparative genomic hybridization data, with gene expression to identify genes that are potentially silenced by promoter hypermethylation.
We report a high enrichment of genes with epigenetic aberrations in cancer signaling pathways. Six genes were selected as tumor suppressor gene candidates, among which, ECM1, ATF5 and EOMES are confirmed via siRNA experiments to have potential anti-cancer functions.
Electronic supplementary material
The online version of this article (doi:10.1186/s13059-014-0533-9) contains supplementary material, which is available to authorized users.
PMCID: PMC4253612  PMID: 25517360
23.  Integrated detection of both 5-mC and 5-hmC by high-throughput tag sequencing technology highlights methylation reprogramming of bivalent genes during cellular differentiation 
Epigenetics  2013;8(4):421-430.
5-methylcytosine (5-mC) can be oxidized to 5-hydroxymethylcytosine (5-hmC). Genome-wide profiling of 5-hmC thus far indicates 5-hmC may not only be an intermediate form of DNA demethylation but could also constitute an epigenetic mark per se. Here we describe a cost-effective and selective method to detect both the hydroxymethylation and methylation status of cytosines in a subset of cytosines in the human genome. This method involves the selective glucosylation of 5-hmC residues, short-Sequence tag generation and high-throughput sequencing. We tested this method by screening H9 human embryonic stem cells and their differentiated embroid body cells, and found that differential hydroxymethylation preferentially occurs in bivalent genes during cellular differentiation. Especially, our results support hydroxymethylation can regulate key transcription regulators with bivalent marks through demethylation and affect cellular decision on choosing active or inactive state of these genes upon cellular differentiation. Future application of this technology would enable us to uncover the status of methylation and hydroxymethylation in dynamic biological processes and disease development in multiple biological samples.
PMCID: PMC3674051  PMID: 23502161
HMST-Seq; differentiation; embryonic stem cells; hydroxymethylation; methylation
24.  Complete Resequencing of 40 Genomes Reveals Domestication Events and Genes in Silkworm (Bombyx) 
Science (New York, N.Y.)  2009;326(5951):433-436.
A single–base pair resolution silkworm genetic variation map was constructed from 40 domesticated and wild silkworms, each sequenced to approximately threefold coverage, representing 99.88% of the genome. We identified ∼16 million single-nucleotide polymorphisms, many indels, and structural variations. We find that the domesticated silkworms are clearly genetically differentiated from the wild ones, but they have maintained large levels of genetic variability, suggesting a short domestication event involving a large number of individuals. We also identified signals of selection at 354 candidate genes that may have been important during domestication, some of which have enriched expression in the silk gland, midgut, and testis. These data add to our understanding of the domestication processes and may have applications in devising pest control strategies and advancing the use of silkworms as efficient bioreactors.
PMCID: PMC3951477  PMID: 19713493
25.  Ancient human genome sequence of an extinct Palaeo-Eskimo 
Nature  2010;463(7282):757-762.
We report here the genome sequence of an ancient human. Obtained from ∼4,000-year-old permafrost-preserved hair, the genome represents a male individual from the first known culture to settle in Greenland. Sequenced to an average depth of 20×, we recover 79% of the diploid genome, an amount close to the practical limit of current sequencing technologies. We identify 353,151 high-confidence single-nucleotide polymorphisms (SNPs), of which 6.8% have not been reported previously. We estimate raw read contamination to be no higher than 0.8%. We use functional SNP assessment to assign possible phenotypic characteristics of the individual that belonged to a culture whose location has yielded only trace human remains. We compare the high-confidence SNPs to those of contemporary populations to find the populations most closely related to the individual. This provides evidence for a migration from Siberia into the New World some 5,500 years ago, independent of that giving rise to the modern Native Americans and Inuit.
PMCID: PMC3951495  PMID: 20148029

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