Alzheimer's disease (AD) has a complex pattern of inheritance and many genes have recently been reported to contribute to the disease susceptibility. We selected 106 SNPs within 16 candidate genes and performed a multistage association study using 4 sample sets consisting of 731 AD patients and 738 control subjects to identify genetic factors for AD in Han Chinese. A single nucleotide polymorphism (SNP) in the insulin degrading enzyme gene (IDE), rs3781239, showed a significant association with AD. The C allele increased the risk of AD 1.72-fold than the G allele (odds ratio [OR] = 1.72, 95% confidence interval [CI] = 1.17-2.53, p = 0.006) and CC carriers had a 4.89-fold higher risk for AD than that of the carriers with CG and GG genotypes (odds ratio = 4.89, 95% CI = 1.85-12.91, p = 0.001). Moreover, the CC genotype was significantly associated with earlier age at onset (p = 0.001, hazard ratio [HR] = 2.09, 95% CI = 1.38-3.18). Our data suggest that the polymorphism of IDE is associated with susceptibility to Alzheimer's disease in Han Chinese.

Hypertension is a heritable and major contributor to the global burden of disease. The sum of rare and common genetic variants robustly identified so far explain only 1%-2% of the population variation in BP and hypertension. This suggests the existence of more undiscovered common variants. We conducted a genome-wide association study in 1,621 hypertensive cases and 1,699 controls and follow-up validation analyses in 19,845 cases and 16,541 controls using an extreme case-control design. We identified a locus on chromosome 16 in the 5' region of Uromodulin (UMOD; rs13333226, combined P value of 3.6 × 10⁻¹¹). The minor G allele is associated with a lower risk of hypertension (OR [95%CI]: 0.87 [0.84-0.91]), reduced urinary uromodulin excretion, better renal function; and each copy of the G allele is associated with a 7.7% reduction in risk of CVD events after adjusting for age, sex, BMI, and smoking status (H.R. = 0.923, 95% CI 0.860-0.991; p = 0.027). In a subset of 13,446 individuals with estimated glomerular filtration rate (eGFR) measurements, we show that rs13333226 is independently associated with hypertension (unadjusted for eGFR: 0.89 [0.83-0.96], p = 0.004; after eGFR adjustment: 0.89 [0.83-0.96], p = 0.003). In clinical functional studies, we also consistently show the minor G allele is associated with lower urinary uromodulin excretion. The exclusive expression of uromodulin in the thick portion of the ascending limb of Henle suggests a putative role of this variant in hypertension through an effect on sodium homeostasis. The newly discovered UMOD locus for hypertension has the potential to give new insights into the role of uromodulin in BP regulation and to identify novel drugable targets for reducing cardiovascular risk.

X-linked congenital generalized hypertrichosis (CGH), an extremely rare condition characterized by universal overgrowth of terminal hair, was first mapped to chromosome Xq24-q27.1 in a Mexican family. However, the underlying genetic defect remains unknown. We ascertained a large Chinese family with an X-linked congenital hypertrichosis syndrome combining CGH, scoliosis, and spina bifida and mapped the disease locus to a 5.6 Mb critical region within the interval defined by the previously reported Mexican family. Through the combination of a high-resolution copy-number variation (CNV) scan and targeted genomic sequencing, we identified an interchromosomal insertion at Xq27.1 of a 125,577 bp intragenic fragment of COL23A1 on 5q35.3, with one X breakpoint within and the other very close to a human-specific short palindromic sequence located 82 kb downstream of SOX3. In the Mexican family, we found an interchromosomal insertion at the same Xq27.1 site of a 300,036 bp genomic fragment on 4q31.2, encompassing PRMT10 and TMEM184C and involving parts of ARHGAP10 and EDNRA. Notably, both of the two X breakpoints were within the short palindrome. The two palindrome-mediated insertions fully segregate with the CGH phenotype in each of the families, and the CNV gains of the respective autosomal genomic segments are not present in the public database and were not found in 1274 control individuals. Analysis of control individuals revealed deletions ranging from 173 bp to 9104 bp at the site of the insertions with no phenotypic consequence. Taken together, our results strongly support the pathogenicity of the identified insertions and establish X-linked congenital hypertrichosis syndrome as a genomic disorder.

Population structure is an important cause leading to inconsistent results in population-based association studies (PBAS) of human diseases. Various statistical methods have been proposed to reduce the negative impact of population structure on PBAS. Due to lack of structural information in real populations, it is difficult to evaluate the impact of population structure on PBAS in real populations.

Since their arrival in the Tibetan Plateau during the Neolithic Age, Tibetans have been well-adapted to extreme environmental conditions and possess genetic variation that reflect their living environment and migratory history. To investigate the origin of Tibetans and the genetic basis of adaptation in a rigorous environment, we genotyped 30 Tibetan individuals with more than one million SNP markers. Our findings suggested that Tibetans, together with the Yi people, were descendants of Tibeto-Burmans who diverged from ancient settlers of East Asia. The valleys of the Hengduan Mountain range may be a major migration route. We also identified a set of positively-selected genes that belong to functional classes of the embryonic, female gonad, and blood vessel developments, as well as response to hypoxia. Most of these genes were highly correlated with population-specific and beneficial phenotypes, such as high infant survival rate and the absence of chronic mountain sickness.

The proteins of the Inhibitor of Growth (ING) family are involved in multiple cellular functions such as cell cycle regulation, apoptosis, and chromatin remodeling. For ING5, its actual role in growth suppression and the necessary partners are not known. In a yeast-two-hybrid approach with human bone marrow derived cDNA, we identified ING5 as well as several other proteins as interaction partners of Inhibitor of cyclin A1 (INCA1) that we previously characterized as a novel interaction partner of cyclin A1/CDK2. ING5 expression in leukemic AML blasts was severely reduced compared to normal bone marrow. In line, ING5 inhibited bone marrow colony formation upon retroviral transduction. However, Inca1(-/-) bone marrow colony formation was not suppressed by ING5. In murine embryonic fibroblast (MEF) cells from Inca1(+/+) and Inca1(-/-) mice, overexpression of ING5 suppressed cell proliferation only in the presence of INCA1, while ING5 had no effect in Inca1(-/-) MEFs. ING5 overexpression induced a delay in S-phase progression, which required INCA1. Finally, ING5 overexpression enhanced Fas-induced apoptosis in Inca1(+/+) MEFs, while Inca1(-/-) MEFs were protected from Fas antibody-induced apoptosis. Taken together, these results indicate that ING5 is a growth suppressor with suppressed expression in AML whose functions depend on its interaction with INCA1.

Kashin-Beck Disease (KBD) is an endemic osteochondropathy, the pathogenesis of which remains unclear now. In this study, we compared gene expression profiles of articular cartilage derived respectively from KBD patients and normal controls. Total RNA were isolated, amplified, labeled and hybridized to Agilent human 1A 22 k whole genome microarray chip. qRT-PCR was conducted to validate our microarray data. We detected 57 up-regulated genes (ratios ≥2.0) and 24 down-regulated genes (ratios ≤0.5) in KBD cartilage. To further identify the key genes involved in the pathogenesis of KBD, Bayesian analysis of variance for microarrays (BAM) software was applied and identified 12 potential key genes with an average ratio 6.64, involved in apoptosis, metabolism, cytokine & growth factor and cytoskeleton & cell movement. Gene Set Enrichment Analysis (GSEA) software was used to identify differently expressed gene ontology categories and pathways. GSEA found that a set of apoptosis, hypoxia and mitochondrial function related gene ontology categories and pathways were significantly up-regulated in KBD compared to normal controls. Based on the results of this study, we suggest that chronic hypoxia-induced mitochondrial damage and apoptosis might play an important role in the pathogenesis of KBD. Our efforts may help to understand the pathogenesis of KBD as well as other osteoarthrosis with similar articular cartilage lesions.

CCAAT/enhancer binding protein (C/EBP)-homologous protein (CHOP) has been shown to be a key molecule in endoplasmic reticulum (ER) stress-mediated apoptosis. ER oxidoreductin 1-α (ERO1α), a target of CHOP, is an important oxidizing enzyme that regulates reactive oxygen species (ROS), which play a prominent role in hepatocellular death during acute liver failure (ALF). However, little is known about how CHOP facilitates ROS-induced hepatocellular injury. The present study was designed to investigate the roles and molecular mechanisms of CHOP in ALF. In the liver tissues from ALF patients, the expression of CHOP was significantly increased, which was accompanied by increased expression of dsRNA-dependent protein kinase (PKR)-like ER kinase (PERK) signalling, activating transcription factor 4 (ATF6) signalling, inositol-requiring enzyme-1 (IRE1) signalling and ERO1α, as compared with healthy controls. In the mouse model of galactosamine (GaIN)/lipopolysaccharide (LPS)-induced ALF, the hepatocellular injury was accompanied by up-regulated PERK signalling, ATF6 signalling, IRE1 signalling, CHOP and ERO1α. In contrast, CHOP deficiency decreased hepatocellular apoptosis/necrosis and increased animal survival. Furthermore, disruption of CHOP decreased ERO1α expression leading to reducing ROS-induced cell death in vivo and in vitro. Interestingly, ERO1α overexpression restored GaIN/LPS-induced hepatocellular injury in CHOP-deficient mice. Our studies demonstrate for the first time that CHOP promotes liver damage during ALF through activation of ERO1α, a key mediator to link ER stress and ROS. Therefore, targeting CHOP/ERO1α signalling could be a novel therapeutic approach during ALF.

The absence of a productive cell culture system hampered detailed analysis of the structure and protein composition of the hepatitis E virion. In this study, hepatitis E virus from a robust HEV cell culture system and from the feces of infected monkeys at the peak of virus excretion was purified by ultra-centrifugation. The common feature of the two samples after ultracentrifugation was that the ORF2 protein mainly remained in the top fractions. The ORF2 protein from cell culture system was glycosylated, with an apparent molecular weight of 88 kDa, and was not infectious in PLC/PRF/5 cells. The ORF2 protein in this fraction can bind to and protect HEV RNA from digestion by RNase A. The RNA-ORF2 product has a similar sedimentation coefficient to the virus from feces. The viral RNA in the cell culture supernatant was mainly in the fraction of 1.15 g/cm3 but that from the feces was mainly in the fraction of 1.21 g/cm3. Both were infectious in PLC/PRF/5 cells. And the fraction in the middle of the gradient (1.06 g/cm3) from the cell culture supernatant,but not that from the feces, also has ORF2 protein and HEV RNA but was not infectious in PLC/PRF/5.The infectious RNA-rich fraction from the cell culture contained ORF3 protein and lipid but the corresponding fraction from feces had no lipid and little ORF3 protein. The lipid on the surface of the virus has no effect on its binding to cells but the ORF3 protein interferes with binding. The result suggests that most of the secreted ORF2 protein is not associated with HEV RNA and that hepatitis E virus produced in cell culture differs in structure from the virus found in feces in that it has a lipid envelope.

The CRISPR/Cas9 system has revolutionized mammalian somatic cell genetics. Genome-wide functional screens using CRISPR/Cas9-mediated knockout or dCas9 fusion-mediated inhibition/activation (CRISPRi/a) are powerful techniques for discovering phenotype-associated gene function. We systematically assessed the DNA sequence features that contribute to single guide RNA (sgRNA) efficiency in CRISPR-based screens. Leveraging the information from multiple designs, we derived a new sequence model for predicting sgRNA efficiency in CRISPR/Cas9 knockout experiments. Our model confirmed known features and suggested new features including a preference for cytosine at the cleavage site. The model was experimentally validated for sgRNA-mediated mutation rate and protein knockout efficiency. Tested on independent data sets, the model achieved significant results in both positive and negative selection conditions and outperformed existing models. We also found that the sequence preference for CRISPRi/a is substantially different from that for CRISPR/Cas9 knockout and propose a new model for predicting sgRNA efficiency in CRISPRi/a experiments. These results facilitate the genome-wide design of improved sgRNA for both knockout and CRISPRi/a studies.

In previous studies, we reported that the sortilin-related receptor, L (DLR class) A repeats containing (SORL1) gene single nucleotide polymorphisms (SNPs) are associated with the risk of sporadic Alzheimer's disease (SAD) in the Han Chinese population. To further explore the relationships between SORL1 genetic variants and SAD, we conducted a two-step study. Sequencing analysis in 50 case samples identified 14 SNPs within the promoter and untranslated region of the SORL1 gene. Subsequent genotyping analysis in 106 patients with SAD and 179 healthy controls detected a significant association between the "G" allele of SNP rs1133174 in the 3' untranslated region of the SORL1 gene and SAD risk (odds ratio =1.92, 95% confidence interval [95% CI] =1.28-2.90, adjusted P=0.028). In addition, "G" allele carriers of rs1133174 (GA + GG) have a 2.15-fold increased risk of SAD compared to noncarriers (AA) (adjusted P=0.042). However, no significant positive associations were observed in the other 13 SNPs within the SORL1 gene. These preliminary findings suggest that the SORL1 SNP rs1133174 may be a potential risk locus for SAD in the Han Chinese population.

Lung cancer is an aggressive malignancy with high morbidity and mortality. Chemotherapy has always been the principal treatment measure, but its acquired resistance becomes a critical problem. In the current study, we established a new docetaxel-resistant human non-small lung cancer (NSCLC) cell line A549/Docetaxel. The resistance index (RI) of A549/Docetaxel cells and A549 induced by TGF-β to docetaxel were 8.91 and 11.5, respectively. Compared to the parental A549 cells, the multiplication time of A549/Docetaxel was prolonged, the proportion of the cell cycle in the S phase decreased while that in the G1 phase increased, and apoptotic rate was much lower. The morphology of the resistant cells eventuated epithelial-mesenchymal transition (EMT), which was confirmed by the higher expression of fibronectin, vimentin (mesenchymal markers), and lower expression of E-cadherin (epithelial marker) at mRNA and proteins levels. Furthermore, the representative markers for docetaxel resistance were examined, including ABCB1 (MDR1), Bcl-2, Bax, and tubulin, to figure out the mechanisms of the resistance of A549/Docetaxel. In summary, we have established a typical docetaxel-resistant human NSCLC cell line A549/Docetaxel, and it was suggested that the multidrug resistance of A549/Docetaxel was related to EMT.

Besides single-nucleotide variants in the human genome, large-scale genomic variants, such as copy number variations (CNVs), are being increasingly discovered as a genetic source of human diversity and the pathogenic factors of diseases. Recent experimental findings have shed light on the links between different genome architectures and CNV mutagenesis. In this review, we summarize various genomic features and discuss their contributions to CNV formation. Genomic repeats, including both low-copy and high-copy repeats, play important roles in CNV instability, which was initially known as DNA recombination events. Furthermore, it has been found that human genomic repeats can also induce DNA replication errors and consequently result in CNV mutations. Some recent studies showed that DNA replication timing, which reflects the high-order information of genomic organization, is involved in human CNV mutations. Our review highlights that genome architecture, from DNA sequence to high-order genomic organization, is an important molecular factor in CNV mutagenesis and human genomic instability.

Genetic screens are powerful tools for identifying genes responsible for diverse phenotypes. Here we describe a genome-wide CRISPR/Cas9-mediated loss-of-function screen in tumor growth and metastasis. We mutagenized a non-metastatic mouse cancer cell line using a genome-scale library with 67,405 single-guide RNAs (sgRNAs). The mutant cell pool rapidly generates metastases when transplanted into immunocompromised mice. Enriched sgRNAs in lung metastases and late-stage primary tumors were found to target a small set of genes, suggesting that specific loss-of-function mutations drive tumor growth and metastasis. Individual sgRNAs and a small pool of 624 sgRNAs targeting the top-scoring genes from the primary screen dramatically accelerate metastasis. In all of these experiments, the effect of mutations on primary tumor growth positively correlates with the development of metastases. Our study demonstrates Cas9-based screening as a robust method to systematically assay gene phenotypes in cancer evolution in vivo.

We sought to determine whether genomic polymorphism in collagen IX genes (COL9A) was associated with Kashin-Beck disease (KBD).

Systematic interrogation of gene function requires the ability to perturb gene expression in a robust and generalizable manner. Here we describe structure-guided engineering of a CRISPR-Cas9 complex to mediate efficient transcriptional activation at endogenous genomic loci. We used these engineered Cas9 activation complexes to investigate single-guide RNA (sgRNA) targeting rules for effective transcriptional activation, to demonstrate multiplexed activation of ten genes simultaneously, and to upregulate long intergenic non-coding RNA (lincRNA) transcripts. We also synthesized a library consisting of 70,290 guides targeting all human RefSeq coding isoforms to screen for genes that, upon activation, confer resistance to a BRAF inhibitor. The top hits included genes previously shown to be able to confer resistance, and novel candidates were validated using individual sgRNA and complementary DNA overexpression. A gene expression signature based on the top screening hits correlated with markers of BRAF inhibitor resistance in cell lines and patient-derived samples. These results collectively demonstrate the potential of Cas9-based activators as a powerful genetic perturbation technology.

This study aimed to identify the optimal neural progenitor cell transplantation time for spinal cord injury in rats via the subarachnoid space. Cultured neural progenitor cells from 14-day embryonic rats, constitutively expressing enhanced green fluorescence protein, or media alone, were injected into the subarachnoid space of adult rats at 1 hour (acute stage), 7 days (subacute stage) and 28 days (chronic stage) after contusive spinal cord injury. Results showed that grafted neural progenitor cells migrated and aggregated around the blood vessels of the injured region, and infiltrated the spinal cord parenchyma along the tissue spaces in the acute stage transplantation group. However, this was not observed in subacute and chronic stage transplantation groups. O4- and glial fibrillary acidic protein-positive cells, representing oligodendrocytes and astrocytes respectively, were detected in the core of the grafted cluster attached to the cauda equina pia surface in the chronic stage transplantation group 8 weeks after transplantation. Both acute and subacute stage transplantation groups were negative for O4 and glial fibrillary acidic protein cells. Basso, Beattie and Bresnahan scale score comparisons indicated that rat hind limb locomotor activity showed better recovery after acute stage transplantation than after subacute and chronic transplantation. Our experimental findings suggest that the subarachnoid route could be useful for transplantation of neural progenitor cells at the acute stage of spinal cord injury. Although grafted cells survived only for a short time and did not differentiate into astrocytes or neurons, they were able to reach the parenchyma of the injured spinal cord and improve neurological function in rats. Transplantation efficacy was enhanced at the acute stage in comparison with subacute and chronic stages.

MicroRNAs delicately regulate the balance of angiogenesis. Here we show that depletion of all microRNAs suppresses tumor angiogenesis. We generated microRNA-deficient tumors by knocking out Dicer1. These tumors are highly hypoxic but poorly vascularized, suggestive of deficient angiogenesis signaling. Expression profiling revealed that angiogenesis genes were significantly down-regulated as a result of the microRNA deficiency. Factor inhibiting hypoxia-inducible factor 1 (HIF-1), FIH1, is derepressed under these conditions and suppresses HIF transcription. Knocking out FIH1 using CRISPR/Cas9-mediated genome engineering reversed the phenotypes of microRNA-deficient cells in HIF transcriptional activity, VEGF production, tumor hypoxia, and tumor angiogenesis. Using multiplexed CRISPR/Cas9, we deleted regions in FIH1 3' untranslated regions (UTRs) that contain microRNA-binding sites, which derepresses FIH1 protein and represses hypoxia response. These data suggest that microRNAs promote tumor responses to hypoxia and angiogenesis by repressing FIH1.

CRISPR-Cas9 is a versatile genome editing technology for studying the functions of genetic elements. To broadly enable the application of Cas9 in vivo, we established a Cre-dependent Cas9 knockin mouse. We demonstrated in vivo as well as ex vivo genome editing using adeno-associated virus (AAV)-, lentivirus-, or particle-mediated delivery of guide RNA in neurons, immune cells, and endothelial cells. Using these mice, we simultaneously modeled the dynamics of KRAS, p53, and LKB1, the top three significantly mutated genes in lung adenocarcinoma. Delivery of a single AAV vector in the lung generated loss-of-function mutations in p53 and Lkb1, as well as homology-directed repair-mediated Kras(G12D) mutations, leading to macroscopic tumors of adenocarcinoma pathology. Together, these results suggest that Cas9 mice empower a wide range of biological and disease modeling applications.

Clinical evidence shows that visceral fat accumulation decreases whereas sc fat increases in patients treated with thiazolidinediones (TZDs), a type of peroxisome proliferator-activated receptor (PPAR)γ agonist. To clarify the molecular mechanism of the differential effects of PPARγ agonists on sc and visceral adipose, we investigated expression profiling of PPARγ-regulated micro-RNAs (miRNAs) using miRNA microarray. The level of 182 miRNAs changed in human sc adipose treated with pioglitazone, whereas only 46 miRNAs changed in visceral adipose. Among these miRNAs, 27 miRNAs changed in both human sc and visceral adipocytes. Specifically, 7 miRNAs changed at the same direction in sc and visceral adipocytes, whereas 20 miRNAs changed at opposite directions in these two fat depots. Bioinformatics analysis showed that these miRNAs and the predicted target genes were involved in TGF-β-, Wnt/β-catenin-, and insulin-signaling pathways and related to metabolic regulation or cell cycle. Among the miRNAs changed at the same direction in sc and visceral adipocytes, miR-378, located in the first intron of PPARγ coactivator 1β (PGC1β), was coordinately expressed with PGC1β during adipogenesis. Moreover, miR-378 and PGC1β were both up-regulated by PPARγ agonist. We also provided evidence that miR-378 promoted adipogenesis in sc fat, but not in visceral fat. These results display miRNAs expression profiling altered in sc and visceral adipogenesis regulated by PPARγ and suggest a potential mechanism underlying the differential effects of TZDs on the 2 fat depot accumulations.