Yan Wen

CRISPR/Cas9 for genome editing: progress, implications and challenges

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) protein 9 system provides a robust and multiplexable genome editing tool, enabling researchers to precisely manipulate specific genomic elements, and facilitating the elucidation of target gene function in biology and diseases. CRISPR/Cas9 comprises of a nonspecific Cas9 nuclease and a set of programmable sequence-specific CRISPR RNA (crRNA), which can guide Cas9 to cleave DNA and generate double-strand breaks at target sites. Subsequent cellular DNA repair process leads to desired insertions, deletions or substitutions at target sites. The specificity of CRISPR/Cas9-mediated DNA cleavage requires target sequences matching crRNA and a protospacer adjacent motif locating at downstream of target sequences. Here, we review the molecular mechanism, applications and challenges of CRISPR/Cas9-mediated genome editing and clinical therapeutic potential of CRISPR/Cas9 in future.

Genome-wide association study identifies ITPR2 as a susceptibility gene for Kashin-Beck disease in Han Chinese.

Kashin‐Beck disease (KBD) is a chronic osteochondropathy, the pathogenesis of which remains elusive. The aim of this study was to identify susceptibility genes for KBD by conducting a 2‐stage genome‐wide association study (GWAS).

Brief Report: Genome-Wide Association Study Identifies ITPR2 as a Susceptibility Gene for Kashin-Beck Disease in Han Chinese

Kashin‐Beck disease (KBD) is a chronic osteochondropathy, the pathogenesis of which remains elusive. The aim of this study was to identify susceptibility genes for KBD by conducting a 2‐stage genome‐wide association study (GWAS).

Exome Sequencing and Functional Analysis Identifies a Novel Mutation in EXT1 Gene That Causes Multiple Osteochondromas

Multiple osteochondromas (MO) is an inherited skeletal disorder, and the molecular mechanism of MO remains elusive. Exome sequencing has high chromosomal coverage and accuracy, and has recently been successfully used to identify pathogenic gene mutations. In this study, exome sequencing followed by Sanger sequencing validation was first used to screen gene mutations in two representative MO patients from a Chinese family. After filtering the data from the 1000 Genome Project and the dbSNP database (build 132), the detected candidate gene mutations were further validated via Sanger sequencing of four other members of the same MO family and 200 unrelated healthy subjects. Immunohistochemisty and multiple sequence alignment were performed to evaluate the importance of the identified causal mutation. A novel frameshift mutation, c.1457insG at codon 486 of exon 6 of EXT1 gene, was identified, which truncated the glycosyltransferase domain of EXT1 gene. Multiple sequence alignment showed that codon 486 of EXT1 gene was highly conserved across various vertebrates. Immunohistochemisty demonstrated that the chondrocytes with functional EXT1 in MO were less than those in extragenetic solitary chondromas. The novel c.1457insG deleterious mutation of EXT1 gene reported in this study expands the causal mutation spectrum of MO, and may be helpful for prenatal genetic screening and early diagnosis of MO.

COL9A1 Gene Polymorphism Is Associated with Kashin-Beck Disease in a Northwest Chinese Han Population

Objective We sought to determine whether genomic polymorphism in collagen IX genes (COL9A) was associated with Kashin-Beck disease (KBD). Methods Twenty seven single nucleotide polymorphisms (SNPs) in COL9AI, COL9A2 and COL9A3 were genotyped in 274 KBD cases and 248 healthy controls using the Sequenom MassARRAY system. Associations between the COL9A polymorphism and KBD risk were detected using an unconditional logistic regression model. Linkage disequilibrium (LD) and haplotypes analysis were performed with the Haploview software. Results After Bonferroni correction, the frequency distribution of genotypes in rs6910140 in COL9A1 was significantly different between the KBD and the control groups (X 2 = 16.74, df = 2, P = 0.0002). Regression analysis showed that the allele “C” in SNP rs6910140 had a significant protective effect on KBD [odds ratio (OR) = 0.49, 95% confidence interval (CI) = 0.34–0.70, P = 0.0001]. The frequencies of alleles and genotypes in rs6910140 were significantly different among subjects of different KBD stages (allele: X 2 = 7.82, df = 2, P = 0.02, genotype: X 2 = 14.81, df = 4, P = 0.005). However, haplotype analysis did not detect any significant association between KBD and COL9A1, COL9A2 and COL9A3. Conclusions We observed a significant association between rs6910140 of COL9A1 and KBD, suggesting a role of COL9A1 in the development of KBD.

Comparative analysis of gene expression profiles of hip articular cartilage between non-traumatic necrosis and osteoarthritis.

Hip cartilage destruction is consistently observed in the non-traumatic osteonecrosis of femoral head (NOFH) and accelerates its bone necrosis. The molecular mechanism underlying the cartilage damage of NOFH remains elusive. In this study, we conducted a systematically comparative study of gene expression profiles between NOFH and osteoarthritis (OA). Hip articular cartilage specimens were collected from 12 NOFH patients and 12 controls with traumatic femoral neck fracture for microarray (n=4) and quantitative real-time PCR validation experiments (n=8). Gene expression profiling of articular cartilage was performed using Agilent Human 4×44K Microarray chip. The accuracy of microarray experiment was further validated by qRT-PCR. Gene expression results of OA hip cartilage were derived from previously published study. Significance Analysis of Microarrays (SAM) software was applied for identifying differently expressed genes. Gene ontology (GO) and pathway enrichment analysis were conducted by Gene Set Enrichment Analysis software and DAVID tool, respectively. Totally, 27 differently expressed genes were identified for NOFH. Comparing the gene expression profiles of NOFH cartilage and OA cartilage detected 8 common differently expressed genes, including COL5A1, OGN, ANGPTL4, CRIP1, NFIL3, METRNL, ID2 and STEAP1. GO comparative analysis identified 10 common significant GO terms, mainly implicated in apoptosis and development process. Pathway comparative analysis observed that ECM-receptor interaction pathway and focal adhesion pathway were enriched in the differently expressed genes of both NOFH and hip OA. In conclusion, we identified a set of differently expressed genes, GO and pathways for NOFH articular destruction, some of which were also involved in the hip OA. Our study results may help to reveal the pathogenetic similarities and differences of cartilage damage of NOFH and hip OA.

Genome-wide pathway-based association study implicates complement system in the development of Kashin-Beck disease in Han Chinese

Kashin-Beck disease (KBD) is a chronic osteochondropathy. The pathogenesis of KBD remains unknown. To identify relevant biological pathways for KBD, we conducted a genome-wide pathway-based association study (GWPAS) following by replication analysis, totally using 2743 Chinese Han adults. A modified gene set enrichment algorithm was used to detect association between KBD and 963 biological pathways. Cartilage gene expression analysis and serum complement measurement were performed to evaluate the functional relevance of identified pathway with KBD. We found that the Complement and Coagulation Cascades (CACC) pathway was significantly associated with KBD (P value=3.09×10(-5), false-discovery rate=0.042). Within the CACC pathway, the most significant association was observed at rs1656966 (P value=1.97×10(-4)) of KNG1 gene. Further replication study observed that rs1656966 (P value=0.037) was significantly associated with KBD in an independent validation sample of 1026 subjects. Gene expression analysis observed that CFD (ratio=3.39±2.68), A2M (ratio=3.67±5.63), C5 (ratio=2.65±2.52) and CD46 (ratio=2.29±137) genes of the CACC pathway were up-regulated in KBD articular cartilage compared to healthy articular cartilage. The serum level of complement C5 in KBD patients were significantly higher than that in healthy controls (P value=0.038). Our study is the first to suggest that complement system-related CACC pathway contributed to the development of KBD.

Tissue-specific pathway association analysis using genome-wide association study summaries

Motivation: Pathway association analysis has made great achievements in elucidating the genetic basis of human complex diseases. However, current pathway association analysis approaches fail to consider tissue-specificity. Results: We developed a tissue-specific pathway interaction enrichment analysis algorithm (TPIEA). TPIEA was applied to two large Caucasian and Chinese genome-wide association study summary datasets of bone mineral density (BMD). TPIEA identified several significant pathways for BMD [false discovery rate (FDR) < 0.05], such as KEGG FOCAL ADHESION and KEGG AXON GUIDANCE, which had been demonstrated to be involved in the development of osteoporosis. We also compared the performance of TPIEA and classical pathway enrichment analysis, and TPIEA presented improved performance in recognizing disease relevant pathways. TPIEA may help to fill the gap of classic pathway association analysis approaches by considering tissue specificity. Availability and Implementation: The online web tool of TPIEA is available at https://sourceforge.net/projects/tpieav1/files. Contact: fzhxjtu@mail.xjtu.edu.cn Supplementary information: Supplementary data are available at Bioinformatics online.

Genome-wide DNA methylation profiling of articular cartilage reveals significant epigenetic alterations in Kashin-Beck disease and osteoarthritis

OBJECTIVE To determine genome-wide DNA methylation profiles of knee cartilage from patients with Kashin-Beck disease (KBD) and osteoarthritis (OA). METHOD Knee cartilage was collected from 14 grade III KBD patients, 5 primary OA patients and 13 healthy subjects. The genome-wide methylation profiles of 5 KBD cartilage, 5 OA cartilage and 5 normal cartilage were determined by Illumina HumanMethylation450 array. Illumina Methylation Analyzer package was employed for identifying differentially methylated CpG sites. Functional annotation and enrichment analysis of differentially methylated genes (DMG) were conducted using GeneRIF database, Ingenuity Pathway Analysis (IPA) and The Database for Annotation, Visualization and Integrated Discovery (DAVID). Mass spectrometry (MS) and immunohistochemistry (IHC) were conducted to validate the functional relevance of identified KBD associated gene. RESULTS We identified a total of 1212 differentially methylated CpG sites in KBD vs Normal, annotated to 264 hypermethylated and 368 hypomethylated genes. Comparing the DNA methylation profiles of KBD vs Normal and OA vs Normal detected overlap of 367 differentially methylated CpG sites (annotated to 182 genes) as well as 845 KBD-specific differentially methylated CpG sites (annotated to 471 unique genes). MS and IHC confirmed the hypermethylation status and decreased protein expression of HAPLN1 gene in KBD cartilage. CONCLUSION Our data implicate epigenetic dysregulation of a host of genes in KBD and OA. Furthermore, we observed common causal epigenetic changes shared by KBD and OA.

Genome-wide association studies and gene expression profiles of rheumatoid arthritis: An analysis

Objectives The molecular mechanism of rheumatoid arthritis (RA) remains elusive. We conducted a protein-protein interaction network-based integrative analysis of genome-wide association studies (GWAS) and gene expression profiles of RA. Methods We first performed a dense search of RA-associated gene modules by integrating a large GWAS meta-analysis dataset (containing 5539 RA patients and 20 169 healthy controls), protein interaction network and gene expression profiles of RA synovium and peripheral blood mononuclear cells (PBMCs). Gene ontology (GO) enrichment analysis was conducted by DAVID. The protein association networks of gene modules were generated by STRING. Results For RA synovium, the top-ranked gene module is HLA-A, containing TAP2, HLA-A, HLA-C, TAPBP and LILRB1 genes. For RA PBMCs, the top-ranked gene module is GRB7, consisting of HLA-DRB5, HLA-DRA, GRB7, CD63 and KIT genes. Functional enrichment analysis identified three significant GO terms for RA synovium, including antigen processing and presentation of peptide antigen via major histocompatibility complex class I (false discovery rate (FDR) = 4.86 × 10 – 4), antigen processing and presentation of peptide antigen (FDR = 2.33 × 10 – 3) and eukaryotic translation initiation factor 4F complex (FDR = 2.52 × 10 – 2). Conclusion This study reported several RA-associated gene modules and their functional association networks. Cite this article: X. Xiao, J. Hao, Y. Wen, W. Wang, X. Guo, F. Zhang. Genome-wide association studies and gene expression profiles of rheumatoid arthritis: an analysis. Bone Joint Res 2016;5:314–319. DOI: 10.1302/2046-3758.57.2000502.