Osteoporosis, a highly heritable disease, is characterized mainly by low bone-mineral density (BMD), poor bone geometry, and/or osteoporotic fractures (OF). Copy-number variation (CNV) has been shown to be associated with complex human diseases. The contribution of CNV to osteoporosis has not been determined yet. We conducted case-control genome-wide CNV analyses, using the Affymetrix 500K Array Set, in 700 elderly Chinese individuals comprising 350 cases with homogeneous hip OF and 350 matched controls. We constructed a genomic map containing 727 CNV regions in Chinese individuals. We found that CNV 4q13.2 was strongly associated with OF (p = 2.0 × 10−4, Bonferroni-corrected p = 0.02, odds ratio = 1.73). Validation experiments using PCR and electrophoresis, as well as real-time PCR, further identified a deletion variant of UGT2B17 in CNV 4q13.2. Importantly, the association between CNV of UGT2B17 and OF was successfully replicated in an independent Chinese sample containing 399 cases with hip OF and 400 controls. We further examined this CNV's relevance to major risk factors for OF (i.e., hip BMD and femoral-neck bone geometry) in both Chinese (689 subjects) and white (1000 subjects) samples and found consistently significant results (p = 5.0 × 10−4 −0.021). Because UGT2B17 encodes an enzyme catabolizing steroid hormones, we measured the concentrations of serum testosterone and estradiol for 236 young Chinese males and assessed their UGT2B17 copy number. Subjects without UGT2B17 had significantly higher concentrations of testosterone and estradiol. Our findings suggest the important contribution of CNV of UGT2B17 to the pathogenesis of osteoporosis.

Genome-wide Copy-Number-Variation Study Identified a Susceptibility Gene, UGT2B17, for Osteoporosis

To identify and validate genes associated with bone mineral density (BMD), which is a prominent osteoporosis risk factor, we tested 379,319 SNPs in 1000 unrelated white U.S. subjects for associations with BMD. For replication, we genotyped the most significant SNPs in 593 white U.S. families (1972 subjects), a Chinese hip fracture (HF) sample (350 cases, 350 controls), a Chinese BMD sample (2955 subjects), and a Tobago cohort of African ancestry (908 males). Publicly available Framingham genome-wide association study (GWAS) data (2953 whites) were also used for in silico replication. The GWAS detected two BMD candidate genes, ADAMTS18 (ADAM metallopeptidase with thrombospondin type 1 motif, 18) and TGFBR3 (transforming growth factor, beta receptor III). Replication studies verified the significant findings by GWAS. We also detected significant associations with hip fracture for ADAMTS18 SNPs in the Chinese HF sample. Meta-analyses supported the significant associations of ADAMTS18 and TGFBR3 with BMD (p values: 2.56 × 10−5 to 2.13 × 10−8; total sample size: n = 5925 to 9828). Electrophoretic mobility shift assay suggested that the minor allele of one significant ADAMTS18 SNP might promote binding of the TEL2 factor, which may repress ADAMTS18 expression. The data from NCBI GEO expression profiles also showed that ADAMTS18 and TGFBR3 genes were differentially expressed in subjects with normal skeletal fracture versus subjects with nonunion skeletal fracture. Overall, the evidence supports that ADAMTS18 and TGFBR3 might underlie BMD determination in the major human ethnic groups.

https://www.cell.com/ajhg/pdf/S0002-9297(09)00059-7.pdf

Genome-wide Association and Follow-Up Replication Studies Identified ADAMTS18 and TGFBR3 as Bone Mass Candidate Genes in Different Ethnic Groups

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, q(fdr) = 0.043, p(fwer) = 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.

/pdf/pathway-based-genome-wide-association-analysis-identified-4j4j95d6hp.pdf

Pathway-Based Genome-Wide Association Analysis Identified the Importance of Regulation-of-Autophagy Pathway for Ultradistal Radius BMD

Osteoporosis is characterized by low bone mineral density and structural deterioration of bone tissue, leading to an increased risk of fractures. It is the most common metabolic bone disorder worldwide, affecting one in three women and one in eight men over the age of 50. In the past 15 years, a large number of genes have been reported as being associated with osteoporosis. However, only in the past 4 years we have witnessed an accelerated pace in identifying and validating osteoporosis susceptibility loci. This increase in pace is mostly due to large-scale association studies, meta-analyses, and genome-wide association studies of both single nucleotide polymorphisms and copy number variations. A comprehensive review of these developments revealed that, to date, at least 15 genes (VDR, ESR1, ESR2, LRP5, LRP4, SOST, GRP177, OPG, RANK, RANKL, COLIA1, SPP1, ITGA1, SP7, and SOX6) can be reasonably assigned as confirmed osteoporosis susceptibility genes, whereas, another >30 genes are promising candidate genes. Notably, confirmed and promising genes are clustered in three biological pathways, the estrogen endocrine pathway, the Wnt/β-catenin signaling pathway, and the RANKL/RANK/OPG pathway. New biological pathways will certainly emerge when more osteoporosis genes are identified and validated. These genetic findings may provide new routes toward improved therapeutic and preventive interventions of this complex disease.

Genetics of osteoporosis: accelerating pace in gene identification and validation

Context: The primary goals of genome-wide association studies (GWAS) are to discover new molecular and biological pathways involved in the regulation of bone metabolism that can be leveraged for drug development. In addition, the identified genetic determinants may be used to enhance current risk factor profiles. Evidence Acquisition: There have been more than 40 published GWAS on skeletal phenotypes, predominantly focused on dual-energy x-ray absorptiometry-derived bone mineral density (BMD) of the hip and spine. Evidence Synthesis: Sixty-six BMD loci have been replicated across all the published GWAS, confirming the highly polygenic nature of BMD variation. Only seven of the 66 previously reported genes (LRP5, SOST, ESR1, TNFRSF11B, TNFRSF11A, TNFSF11, PTH) from candidate gene association studies have been confirmed by GWAS. Among 59 novel BMD GWAS loci that have not been reported by previous candidate gene association studies, some have been shown to be involved in key biological pathways involving the...

/pdf/clinical-review-genome-wide-association-studies-of-skeletal-1scjj1xn0m.pdf

Clinical review: Genome-wide association studies of skeletal phenotypes: what we have learned and where we are headed.

Osteoporosis, the most prevalent metabolic bone disease among older people, increases risk for low trauma hip fractures (HF) that are associated with high morbidity and mortality. Hip bone size (BS) has been identified as one of the key measurable risk factors for HF. Although hip BS is highly genetically determined, genetic factors underlying the trait are still poorly defined. Here, we performed the first genome-wide association study (GWAS) of hip BS interrogating ∼380,000 SNPs on the Affymetrix platform in 1,000 homogeneous unrelated Caucasian subjects, including 501 females and 499 males. We identified a gene, PLCL1 (phospholipase c-like 1), that had four SNPs associated with hip BS at, or approaching, a genome-wide significance level in our female subjects; the most significant SNP, rs7595412, achieved a p value of 3.72×10−7. The gene's importance to hip BS was replicated using the Illumina genotyping platform in an independent UK cohort containing 1,216 Caucasian females. Two SNPs of the PLCL1 gene, rs892515 and rs9789480, surrounded by the four SNPs identified in our GWAS, achieved p values of 8.62×10−3 and 2.44×10−3, respectively, for association with hip BS. Imputation analyses on our GWAS and the UK samples further confirmed the replication signals; eight SNPs of the gene achieved combined imputed p values<10−5 in the two samples. The PLCL1 gene's relevance to HF was also observed in a Chinese sample containing 403 females, including 266 with HF and 177 control subjects. A SNP of the PLCL1 gene, rs3771362 that is only ∼0.6 kb apart from the most significant SNP detected in our GWAS (rs7595412), achieved a p value of 7.66×10−3 (odds ratio = 0.26) for association with HF. Additional biological support for the role of PLCL1 in BS comes from previous demonstrations that the PLCL1 protein inhibits IP3 (inositol 1,4,5-trisphosphate)-mediated calcium signaling, an important pathway regulating mechanical sensing of bone cells. Our findings suggest that PLCL1 is a novel gene associated with variation in hip BS, and provide new insights into the pathogenesis of HF.

/pdf/identification-of-plcl1-gene-for-hip-bone-size-variation-in-3e2n7z5uvj.pdf

Identification of PLCL1 gene for hip bone size variation in females in a genome-wide association study.

Identification of PLCL1 Gene for Hip Bone Size Variation in Females in a Genomewide Association Study

BACKGROUND & AIMS: Early reports suggest that both fecal shedding and dysbiosis of the gut microbiome are associated to disease severity in COVID-19 patients. We investigated the gut microbiome as well as the prevalence of SARS-CoV-2 in stool samples from two French populations: exposed healthcare workers and elderly hospitalized COVID-19 patients. The predictive power of bacterial loss of diversity and detection of SARS-CoV-2 in stool was assessed at 4 weeks against clinical outcomes in the patient group. METHODS: 79 healthcare workers in contact with COVID-19 patients and 64 elderly patients hospitalised in a COVID-19 unit in France were included in the EDIFICE trial from April 2020 until May 2021. Stool samples were collected at inclusion. Loss of bacterial diversity was diagnosed based on 16S rRNA gene sequencing. Stool positivity to SARS-CoV-2 was determined by RT-PCR. Clinical outcomes were recorded at a 4 weeks follow up visit. In particular, these include whether the patient had been put under oxygen during the 4 weeks follow up, whether he had been discharged with or without aggravation from initial symptoms or whether the patient had died. The primary end point was to validate the hypothesis that hospitalized COVID-19 patients had more often lost their bacterial diversity than highly exposed active healthcare workers. RESULTS: Elderly hospitalised patients with COVID-19 had more frequently lost their bacterial diversity when compared to exposed healthcare workers (p-value = 0.005), their severe dysbiosis was characterized by enrichment of the family Erysipelotrichaceae and depletion of beneficial bacteria at the genus level such as butyrate producers (Butyrivibrio, Roseburia, Faecalibacterium) and Bifidobacterium. The virus was detected in 61% of hospitalized patients and in only one healthcare workers (2%) who had previously been diagnosed with COVID-19 (p-value<0.001). No significant difference in the gut microbiome composition at the genus level of patients that tested positive in stool versus patients that tested negative was observed. Neither bacterial loss of diversity nor positivity to SARS-CoV-2 were associated to clinical outcome at 4 weeks. CONCLUSIONS: We report findings of the first French trial investigating the clinical interest of stool based diagnosis of SARS-CoV-2 and loss of bacterial diversity in a population of elderly hospitalised COVID-19 patients and highly exposed healthcare workers. Our findings of reduced bacterial diversity and a strong gut dysbiosis in elderly hospitalized COVID-19 patients are highly consistent with previous reports mostly from Chinese populations. A major limitation is that observed differences in the gut microbiome between the two studied groups cannot be attributed to COVID-19 per se given the large number of confounding factors. SARS-CoV-2 was detected in the stool of the majority of hospitalized patients even several weeks after initial diagnosis by nasopharyngeal swabs. This high prevalence warrants further investigation by the scientific community into mechanism.

Alesandra Cervino

Papers

Identification of PLCL1 gene for hip bone size variation in females in a genome-wide association study.

Identification of PLCL1 Gene for Hip Bone Size Variation in Females in a Genomewide Association Study

Results from EDIFICE : A French pilot study on COVID-19 and the gut microbiome in a hospital environment