Li-Shu Zhang

Multistage genome-wide association meta-analyses identified two new loci for bone mineral density

Aiming to identify novel genetic variants and to confirm previously identified genetic variants associated with bone mineral density (BMD), we conducted a three-stage genome-wide association (GWA) meta-analysis in 27 061 study subjects. Stage 1 meta-analyzed seven GWA samples and 11 140 subjects for BMDs at the lumbar spine, hip and femoral neck, followed by a Stage 2 in silico replication of 33 SNPs in 9258 subjects, and by a Stage 3 de novo validation of three SNPs in 6663 subjects. Combining evidence from all the stages, we have identified two novel loci that have not been reported previously at the genome-wide significance (GWS; 5.0 × 10(-8)) level: 14q24.2 (rs227425, P-value 3.98 × 10(-13), SMOC1) in the combined sample of males and females and 21q22.13 (rs170183, P-value 4.15 × 10(-9), CLDN14) in the female-specific sample. The two newly identified SNPs were also significant in the GEnetic Factors for OSteoporosis consortium (GEFOS, n = 32 960) summary results. We have also independently confirmed 13 previously reported loci at the GWS level: 1p36.12 (ZBTB40), 1p31.3 (GPR177), 4p16.3 (FGFRL1), 4q22.1 (MEPE), 5q14.3 (MEF2C), 6q25.1 (C6orf97, ESR1), 7q21.3 (FLJ42280, SHFM1), 7q31.31 (FAM3C, WNT16), 8q24.12 (TNFRSF11B), 11p15.3 (SOX6), 11q13.4 (LRP5), 13q14.11 (AKAP11) and 16q24 (FOXL1). Gene expression analysis in osteogenic cells implied potential functional association of the two candidate genes (SMOC1 and CLDN14) in bone metabolism. Our findings independently confirm previously identified biological pathways underlying bone metabolism and contribute to the discovery of novel pathways, thus providing valuable insights into the intervention and treatment of osteoporosis.

Pathway-based genome-wide association analysis identified the importance of regulation-of-autophagy pathway for ultradistal radius BMD.

Wrist fracture is not only one of the most common osteoporotic fractures but also a predictor of future fractures at other sites. Wrist bone mineral density (BMD) is an important determinant of wrist fracture risk, with high heritability. Specific genes underlying wrist BMD variation are largely unknown. Most published genome‐wide association studies (GWASs) have focused only on a few top‐ranking single‐nucleotide polymorphisms (SNPs)/genes and considered each of the identified SNPs/genes independently. To identify biologic pathways important to wrist BMD variation, we used a novel pathway‐based analysis approach in our GWAS of wrist ultradistal radius (UD) BMD, examining approximately 500,000 SNPs genome‐wide from 984 unrelated whites. A total of 963 biologic pathways/gene sets were analyzed. We identified the regulation‐of‐autophagy (ROA) pathway that achieved the most significant result (p = .005, qfdr = 0.043, pfwer = 0.016) for association with UD BMD. The ROA pathway also showed significant association with arm BMD in the Framingham Heart Study sample containing 2187 subjects, which further confirmed our findings in the discovery cohort. Earlier studies indicated that during endochondral ossification, autophagy occurs prior to apoptosis of hypertrophic chondrocytes, and it also has been shown that some genes in the ROA pathway (e.g., INFG) may play important roles in osteoblastogenesis or osteoclastogenesis. Our study supports the potential role of the ROA pathway in human wrist BMD variation and osteoporosis. Further functional evaluation of this pathway to determine the mechanism by which it regulates wrist BMD should be pursued to provide new insights into the pathogenesis of wrist osteoporosis.

Biological Pathway‐Based Genome‐Wide Association Analysis Identified the Vasoactive Intestinal Peptide (VIP) Pathway Important for Obesity

Recent genome‐wide association (GWA) studies have identified a number of novel genes/variants predisposing to obesity. However, most GWA studies have focused on individual single‐nucleotide polymorphism (SNPs)/genes with a strong statistical association with a phenotypic trait without considering potential biological interplay of the tested genes. In this study, we performed biological pathway‐based GWA analysis for BMI and body fat mass. We used individual level genotype data generated from 1,000 unrelated US whites that were genotyped for ∼500,000 SNPs. Statistical analysis of pathways was performed using a modification of the Gene Set Enrichment Algorithm. A total of 963 pathways extracted from the BioCarta, Kyoto Encyclopedia of Genes and Genomes (KEGG), Ambion GeneAssist, and Gene Ontology (GO) databases were analyzed. Among all of the pathways analyzed, the vasoactive intestinal peptide (VIP) pathway was most strongly associated with fat mass (nominal P = 0.0009) and was the third most strongly associated pathway with BMI (nominal P = 0.0006). After multiple testing correction, the VIP pathway achieved false‐discovery rate (FDR) q values of 0.042 and 0.120 for fat mass and BMI, respectively. Our study is the first to demonstrate that the VIP pathway may play an important role in development of obesity. The study also highlights the importance of pathway‐based GWA analysis in identification of additional genes/variants for complex human diseases.

Impaired osteoblast function in GPRC6A null mice

GPRC6A is a widely expressed orphan G protein–coupled receptor that senses extracellular amino acids, osteocalcin, and divalent cations in vitro. GPRC6A null (GPRC6A−/−) mice exhibit multiple metabolic abnormalities including osteopenia. To investigate whether the osseous abnormalities are a direct function of GPRC6A in osteoblasts, we examined the function of primary osteoblasts and bone marrow stromal cell cultures (BMSCs) in GPRC6A−/− mice. We confirmed that GPRC6A−/− mice exhibited a decrease in bone mineral density (BMD) associated with reduced expression of osteocalcin, ALP, osteoprotegerin, and Runx2‐II transcripts in bone. Osteoblasts and BMSCs derived from GPRC6A−/− mice exhibited an attenuated response to extracellular calcium‐stimulated extracellular signal‐related kinase (ERK) activation, diminished alkaline phosphatase (ALP) expression, and impaired mineralization ex vivo. In addition, siRNA‐mediated knockdown of GPRC6A in MC3T3 osteoblasts also resulted in a reduction in extracellular calcium‐stimulated ERK activity. To explore the potential relevance of GPRC6A function in humans, we looked for an association between GPRC6A gene polymorphisms and BMD in a sample of 1000 unrelated American Caucasians. We found that GPRC6A gene polymorphisms were significantly associated with human spine BMD. These data indicate that GRPC6A directly participates in the regulation of osteoblast‐mediated bone mineralization and may mediate the anabolic effects of extracellular amino acids, osteocalcin, and divalent cations in bone.

IL21R and PTH may underlie variation of femoral neck bone mineral density as revealed by a genome-wide association study

Bone mineral density (BMD) measured at the femoral neck (FN) is the most important risk phenotype for osteoporosis and has been used as a reference standard for describing osteoporosis. The specific genes influencing FN BMD remain largely unknown. To identify such genes, we first performed a genome‐wide association (GWA) analysis for FN BMD in a discovery sample consisting of 983 unrelated white subjects. We then tested the top significant single‐nucleotide polymorphisms (SNPs; 175 SNPs with p < 5 × 10−4) for replication in a family‐based sample of 2557 white subjects. Combing results from these two samples, we found that two genes, parathyroid hormone (PTH) and interleukin 21 receptor (IL21R), achieved consistent association results in both the discovery and replication samples. The PTH gene SNPs, rs9630182, rs2036417, and rs7125774, achieved p values of 1.10 × 10−4, 3.24 × 10−4, and 3.06 × 10−4, respectively, in the discovery sample; p values of 6.50 × 10−4, 5.08 × 10−3, and 5.68 × 10−3, respectively, in the replication sample; and combined p values of 3.98 × 10−7, 9.52 × 10−6, and 1.05 × 10−5, respectively, in the total sample. The IL21R gene SNPs, rs8057551, rs8061992, and rs7199138, achieved p values of 1.51 × 10−4, 1.53 × 10−4, and 3.88 × 10−4, respectively, in the discovery sample; p values of 2.36 × 10−3, 6.74 × 10−3, and 6.41 × 10−3, respectively, in the replication sample; and combined p values of 2.31 × 10−6, 8.62 × 10−6, and 1.41 × 10−5, respectively, in the total sample. The effect size of each SNP was approximately 0.11 SD estimated in the discovery sample. PTH and IL21R both have potential biologic functions important to bone metabolism. Overall, our findings provide some new clues to the understanding of the genetic architecture of osteoporosis.

A new permutation strategy of pathway-based approach for genome-wide association study

BackgroundRecently introduced pathway-based approach is promising and advantageous to improve the efficiency of analyzing genome-wide association scan (GWAS) data to identify disease variants by jointly considering variants of the genes that belong to the same biological pathway. However, the current available pathway-based approaches for analyzing GWAS have limited power and efficiency.ResultsWe proposed a new and efficient permutation strategy based on SNP randomization for determining significance in pathway analysis of GWAS. The developed permutation strategy was evaluated and compared to two previously available methods, i.e. sample permutation and gene permutation, through simulation studies and a study on a real dataset. Results showed that the proposed permutation strategy is more powerful and efficient with greatly reducing the computational complexity.ConclusionOur findings indicate the improved performance of SNP permutation and thus render pathway-based analysis of GWAS more applicable and attractive.

HMGA2 Is Confirmed To Be Associated with Human Adult Height

Recent genome‐wide association studies have identified a novel polymorphism, rs1042725, in the HMGA2 gene for human adult height, a highly heritable complex trait. Replications in independent populations are needed to evaluate a positive finding and determine its generality. Thus, we performed a replication study to examine the associations between polymorphisms in HMGA2 and adult height in two US Caucasian populations (an unrelated sample of 998 subjects and a family‐based sample of 8385 subjects) and a Chinese population (1638 unrelated Han subjects). We confirmed the association between rs1042725 in HMGA2 and adult height both in the unrelated and family‐based Caucasian populations (overall P= 4.25 × 10−9). Another two SNPs (rs7968902 and rs7968682), which were in high linkage disequilibrium with rs1042725, also achieved the significance level in both Caucasian populations (overall P= 6.34 × 10−7, and 2.72 × 10−9, respectively). Our results provide strong support to the initial finding. Moreover, SNP rs1042725 was firstly found to be associated with adult height (P= 0.008) in the Chinese population, and the effect is in the same direction as in the Caucasian populations, suggesting that it is a common variant across different populations. Our study further highlights the importance of the HMGA2 genes involvement in normal growth.

A follow-up association study of two genetic variants for bone mineral density variation in Caucasians.

SummaryWe tested whether two genetic variants were associated with BMD at multiple clinically relevant skeletal sites in Caucasians. We found that variant rs7776725 is consistently associated with hip, spine, wrist and whole-body BMD, which highlights the potential importance of this variant or linked variants for osteoporosis.IntroductionA recent genome-wide association study identified two single nucleotide polymorphisms (SNPs), rs7776725 and rs1721400, that were associated with bone mineral density (BMD) variation at the radius, tibia and calcaneus in a Korean population. In this study, we aimed to test whether the association of these two genetic variants can be replicated in Caucasians and whether their association with BMD can be extended to other clinically relevant skeletal sites.MethodsWe performed this study in two large cohorts of unrelated US Caucasians. Area BMD at the hip, spine, wrist (ultra-distal radius) and whole body were measured with Hologic dual-energy X-ray absorptiometer. SNPs were genotyped with Affymetrix human genome-wide genotyping arrays. Association analyses were performed using PLINK.ResultsWe detected highly significant association (combined p = 1.42 × 10−16) of rs7776725 with wrist BMD but only borderline association signal (combined p = 0.017) for rs1721400 with wrist BMD. In addition, we found that rs7776725 was associated with BMD at the hip, spine and whole body. At the FAM3C gene locus where rs7776725 was located, we identified several other SNPs (rs4727922, rs1803389, rs718766 and rs7793554) that were also associated with BMD.ConclusionsThis is the first follow-up association study of rs7776725 and rs1721400 with BMD. The rs7776725 showed consistent association with BMD at multiple clinically important skeletal sites, which highlighted the potential importance of rs7776725 or linked SNPs for risk of osteoporosis. Further in-depth re-sequencing studies and functional assays are necessary to elucidate the underlying mechanisms.

Host cellular signaling induced by influenza virus.

A wide range of host cellular signal transduction pathways can be stimulated by influenza virus infection. Some of these signal transduction pathways induce the host cell’s innate immune response against influenza virus, while others are essential for efficient influenza virus replication. This review examines the cellular signaling induced by influenza virus infection in host cells, including host pattern recognition receptor (PRR)-related signaling, protein kinase C (PKC), Raf/MEK/ERK and phosphatidy-linositol-3-kinase (PI3K)/Akt signaling, and the corresponding effects on the host cell and/or virus, such as recognition of virus by the host cell, viral absorption and entry, viral ribonucleoprotein (vRNP) export, translation control of cellular and viral proteins, and virus-induced cell apoptosis. Research into influenza virus-induced cell signaling promotes a clearer understanding of influenza virus-host interactions and assists in the identification of novel antiviral targets and antiviral strategies.

A genome wide association study between copy number variation (CNV) and human height in Chinese population.

Copy number variation (CNV) is a type of genetic variation which may have important roles in phenotypic variability and disease susceptibility. To hunt for genetic variants underlying human height variation, we performed a genome wide CNV association study for human height in 618 Chinese unrelated subjects using Affymetrix 500K array set. After adjusting for age and sex, we found that four CNVs at 6p21.3, 8p23.3-23.2, 9p23 and 16p12.1 were associated with human height (with borderline significant p value: 0.013, 0.011, 0.024, 0.049; respectively). However, after multiple tests correction, none of them was associated with human height. We observed that the gain of copy number (more than 2 copies) at 8p23.3-23.2 was associated with lower height (normal copy number vs. gain of copy number: 161.2 cm vs. 153.7 cm, p = 0.011), which accounted for 0.9% of height variation. Loss of copy number (less than 2 copies) at 6p21.3 was associated with 0.8% lower height (loss of copy number vs. normal copy number: 154.5 cm vs. 161.1 cm, p = 0.013). Since no important genes influencing height located in CNVs at loci of 8p23.3-23.2 and 6p21.3, the two CNVs may cause the structural rearrangements of neighbored important candidate genes, thus regulates the variation of height. Our results expand our knowledge of the genetic factors underlying height variation and the biological regulation of human height.