Yi-Hsiang Hsu

Twenty bone-mineral-density loci identified by large-scale meta-analysis of genome-wide association studies

Bone mineral density (BMD) is a heritable complex trait used in the clinical diagnosis of osteoporosis and the assessment of fracture risk. We performed meta-analysis of five genome-wide association studies of femoral neck and lumbar spine BMD in 19,195 subjects of Northern European descent. We identified 20 BMD loci that reached genome-wide significance (GWS; P < 5 × 10−8), of which 13 map to regions not previously associated with this trait: 1p31.3 (GPR177), 2p21 (SPTBN1), 3p22 (CTNNB1), 4q21.1 (MEPE), 5q14 (MEF2C), 7p14 (STARD3NL), 7q21.3 (FLJ42280), 11p11.2 (LRP4, ARHGAP1, F2), 11p14.1 (DCDC5), 11p15 (SOX6), 16q24 (FOXL1), 17q21 (HDAC5) and 17q12 (CRHR1). The meta-analysis also confirmed at GWS level seven known BMD loci on 1p36 (ZBTB40), 6q25 (ESR1), 8q24 (TNFRSF11B), 11q13.4 (LRP5), 12q13 (SP7), 13q14 (TNFSF11) and 18q21 (TNFRSF11A). The many SNPs associated with BMD map to genes in signaling pathways with relevance to bone metabolism and highlight the complex genetic architecture that underlies osteoporosis and variation in BMD.

FTO Obesity Variant Circuitry and Adipocyte Browning in Humans

BACKGROUND Genomewide association studies can be used to identify disease-relevant genomic regions, but interpretation of the data is challenging. The FTO region harbors the strongest genetic association with obesity, yet the mechanistic basis of this association remains elusive. METHODS We examined epigenomic data, allelic activity, motif conservation, regulator expression, and gene coexpression patterns, with the aim of dissecting the regulatory circuitry and mechanistic basis of the association between the FTO region and obesity. We validated our predictions with the use of directed perturbations in samples from patients and from mice and with endogenous CRISPR-Cas9 genome editing in samples from patients. RESULTS Our data indicate that the FTO allele associated with obesity represses mitochondrial thermogenesis in adipocyte precursor cells in a tissue-autonomous manner. The rs1421085 T-to-C single-nucleotide variant disrupts a conserved motif for the ARID5B repressor, which leads to derepression of a potent preadipocyte enhancer and a doubling of IRX3 and IRX5 expression during early adipocyte differentiation. This results in a cell-autonomous developmental shift from energy-dissipating beige (brite) adipocytes to energy-storing white adipocytes, with a reduction in mitochondrial thermogenesis by a factor of 5, as well as an increase in lipid storage. Inhibition of Irx3 in adipose tissue in mice reduced body weight and increased energy dissipation without a change in physical activity or appetite. Knockdown of IRX3 or IRX5 in primary adipocytes from participants with the risk allele restored thermogenesis, increasing it by a factor of 7, and overexpression of these genes had the opposite effect in adipocytes from nonrisk-allele carriers. Repair of the ARID5B motif by CRISPR-Cas9 editing of rs1421085 in primary adipocytes from a patient with the risk allele restored IRX3 and IRX5 repression, activated browning expression programs, and restored thermogenesis, increasing it by a factor of 7. CONCLUSIONS Our results point to a pathway for adipocyte thermogenesis regulation involving ARID5B, rs1421085, IRX3, and IRX5, which, when manipulated, had pronounced pro-obesity and anti-obesity effects. (Funded by the German Research Center for Environmental Health and others.).

An Integration of Genome-Wide Association Study and Gene Expression Profiling to Prioritize the Discovery of Novel Susceptibility Loci for Osteoporosis-Related Traits

Osteoporosis is a complex disorder and commonly leads to fractures in elderly persons. Genome-wide association studies (GWAS) have become an unbiased approach to identify variations in the genome that potentially affect health. However, the genetic variants identified so far only explain a small proportion of the heritability for complex traits. Due to the modest genetic effect size and inadequate power, true association signals may not be revealed based on a stringent genome-wide significance threshold. Here, we take advantage of SNP and transcript arrays and integrate GWAS and expression signature profiling relevant to the skeletal system in cellular and animal models to prioritize the discovery of novel candidate genes for osteoporosis-related traits, including bone mineral density (BMD) at the lumbar spine (LS) and femoral neck (FN), as well as geometric indices of the hip (femoral neck-shaft angle, NSA; femoral neck length, NL; and narrow-neck width, NW). A two-stage meta-analysis of GWAS from 7,633 Caucasian women and 3,657 men, revealed three novel loci associated with osteoporosis-related traits, including chromosome 1p13.2 (RAP1A, p = 3.6×10−8), 2q11.2 (TBC1D8), and 18q11.2 (OSBPL1A), and confirmed a previously reported region near TNFRSF11B/OPG gene. We also prioritized 16 suggestive genome-wide significant candidate genes based on their potential involvement in skeletal metabolism. Among them, 3 candidate genes were associated with BMD in women. Notably, 2 out of these 3 genes (GPR177, p = 2.6×10−13; SOX6, p = 6.4×10−10) associated with BMD in women have been successfully replicated in a large-scale meta-analysis of BMD, but none of the non-prioritized candidates (associated with BMD) did. Our results support the concept of our prioritization strategy. In the absence of direct biological support for identified genes, we highlighted the efficiency of subsequent functional characterization using publicly available expression profiling relevant to the skeletal system in cellular or whole animal models to prioritize candidate genes for further functional validation.

Variation in genes involved in the RANKL/RANK/OPG bone remodeling pathway are associated with bone mineral density at different skeletal sites in men

In order to assess the contribution of polymorphisms in the RANKL (TNFSF11), RANK (TNFRSF11A) and OPG (TNFRSF11B) genes to variations in bone mineral density (BMD), a population-based cohort with 1,120 extreme low hip BMD cases or extreme high hip BMD controls was genotyped on five SNPs. We further explored the associations between these genetic variations and forearm BMDs by genotyping 266 offspring and 309 available parents from 160 nuclear families. A family-based association test was used. Significantly positive associations were found for A163G polymorphisms in the promoter regions of the OPG gene, a missense substitution in exon 7 (Ala192Val) of the RANK gene and rs9594782 SNP in the 5′ UTR of the RANKL gene with BMD in men only. Men with TC/CC genotypes of the rs9594782 SNP had a 2.1 times higher risk of extremely low hip BMD (P=0.004), and lower whole body BMD (P<0.001). Subjects with the TC genotype of the Ala192Val polymorphism had a 40% reduced risk of having extremely low hip BMD (P<0.01), and higher whole body BMD (P<0.01). Subjects with the GG genotype of the A163G polymorphism had a 70% reduced risk of having extremely low hip BMD (P<0.05), and higher whole body BMD (P<0.01). Significant gene–gene interactions were also observed among the OPG, RANK and RANKL genes. Our findings suggest that genetic variation in genes involved in the RANKL/RANK/OPG bone remodeling pathway are strongly associated with BMD at different skeletal sites in adult men, but not in women.

FTO Polymorphisms Are Associated With Obesity but Not Diabetes Risk in Postmenopausal Women

The FTO gene was recently identified as a susceptibility locus for both obesity and type 2 diabetes by whole‐genome association analyses of several European populations. We tested for an association between FTO risk alleles and obesity and diabetes in a well‐characterized multiethnic cohort of postmenopausal women in the United States. We genotyped two most significantly associated single‐nucleotide polymorphisms (SNPs) (rs9939609 and rs8050136) in intron 1 of FTO gene in a nested case‐control study of 1,517 diabetes cases and 2,123 controls from the Womens Health Initiative‐Observational Study (WHI‐OS). The allelic frequencies of either rs9939609 or rs8050136 differed widely across four ethnic groups. The frequency of the rare allele A of rs9939609 among controls was much lower in Asians/Pacific Islanders (17%) than in blacks (45%), whites (40%), and Hispanics (31%). We found significant associations of rs9939609 with BMI and waist circumference in white and Hispanic women, but not among black and Asian/Pacific Islander women. On average, each copy of the risk‐allele A at rs9939609 was significantly associated with 0.45 kg/m2 increase in BMI (95% confidence interval (CI): 0.16–0.74; P = 0.004) and 0.97 cm increase in waist circumference (95% CI: 0.21–0.65; P = 0.0002). Similar results were observed for rs8050136. However, we found no significant genetic associations with diabetes risk, either within the full study sample or in any ethnic group. In conclusion, common genetic variants in the intron 1 of FTO gene may confer a modest susceptibility to obesity in an ethnicity‐specific manner, but may be unlikely to contribute to a clinically significant diabetes risk.

Prevalence and Physical Determinants of Low Back Pain in a Rural Chinese Population

Study Design. This was a community-based cross-sectional study that is part of an ongoing study of genetics and osteoporosis. Objectives. To estimate the 1-year self-reported prevalence of low back pain (LBP); and to assess the association between physical exposures (occupation, physical stress, and vibration) and LBP in a rural Chinese population. This study also explored how these associations change when individuals with LBP report additional pains in other parts of the body. Summary of Background Data. Little is known about the prevalence and determinants of LBP in rural developing populations, even though approximately half of the world’s population, mostly from the developing world, is engaged in agriculture, a known strenuous activity. Methods. Families with at least three participating siblings residing in the surrounding rural region of Anqing city, Anhui Province, China, were invited to participate in the study through public announcements and direct invitations by village physicians. A questionnaire including demographics, physical exposures, and musculoskeletal pain was administered to 13,965 men and women (age, 25–64 years). Generalized estimating equations were used to estimate LBP prevalence and examine associations between LBP and physical exposures. Results. The 1-year prevalence of LBP was 64%. Twenty-five percent reported no pain at any body site. Women had higher prevalence of LBP than men across all age groups. Being a farmer, reporting moderate or heavy physical stress, and having had former or current exposures to vibration were positively associated with LBP (P < 0.05). Physical exposures were also associated with cases of LBP combined with other musculoskeletal pains, and generally, the more pains individuals reported, the more likely it was that they were farmers and were exposed to vibration. Conclusion. A high prevalence of LBP and LBP with additional musculoskeletal pain existed in this rural Chinese sample. We found evidence of a link between physical exposures and LBP, and LBP with additional musculoskeletal pain. Further understanding of the characteristics and risk factors of LBP in rural developing areas is needed.

Genome-Wide Association Studies of Skeletal Phenotypes: What We Have Learned and Where We Are Headed

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 skeleton, particularly Wnt signaling (AXIN1, LRP5, CTNNB1, DKK1, FOXC2, HOXC6, LRP4, MEF2C, PTHLH, RSPO3, SFRP4, TGFBR3, WLS, WNT3, WNT4, WNT5B, WNT16), bone development: ossification (CLCN7, CSF1, MEF2C, MEPE, PKDCC, PTHLH, RUNX2, SOX6, SOX9, SPP1, SP7), mesenchymal-stem-cell differentiation (FAM3C, MEF2C, RUNX2, SOX4, SOX9, SP7), osteoclast differentiation (JAG1, RUNX2), and TGF-signaling (FOXL1, SPTBN1, TGFBR3). There are still 30 BMD GWAS loci without prior molecular or biological evidence of their involvement in skeletal phenotypes. Other skeletal phenotypes that either have been or are being studied include hip geometry, bone ultrasound, quantitative computed tomography, high-resolution peripheral quantitative computed tomography, biochemical markers, and fractures such as vertebral, nonvertebral, hip, and forearm. CONCLUSIONS Although several challenges lie ahead as GWAS moves into the next generation, there are prospects of new discoveries in skeletal biology. This review integrates findings from previous GWAS and provides a roadmap for future directions building on current GWAS successes.

Common genetic variants of the ion channel transient receptor potential membrane melastatin 6 and 7 (TRPM6 and TRPM7), magnesium intake, and risk of type 2 diabetes in women

BackgroundIon channel transient receptor potential membrane melastatin 6 and 7 (TRPM6 and TRPM7) play a central role in magnesium homeostasis, which is critical for maintaining glucose and insulin metabolism. However, it is unclear whether common genetic variation in TRPM6 and TRPM7 contributes to risk of type 2 diabetes.MethodsWe conducted a nested case-control study in the Womens Health Study. During a median of 10 years of follow-up, 359 incident diabetes cases were diagnosed and matched by age and ethnicity with 359 controls. We analyzed 20 haplotype-tagging single nucleotide polymorphisms (SNPs) in TRPM6 and 5 common SNPs in TRPM7 for their association with diabetes risk.ResultsOverall, there was no robust and significant association between any single SNP and diabetes risk. Neither was there any evidence of association between common TRPM6 and TRPM7 haplotypes and diabetes risk. Our haplotype analyses suggested a significant risk of type 2 diabetes among carriers of both the rare alleles from two non-synomous SNPs in TRPM6 (Val1393Ile in exon 26 [rs3750425] and Lys1584Glu in exon 27 [rs2274924]) when their magnesium intake was lower than 250 mg per day. Compared with non-carriers, women who were carriers of the haplotype 1393Ile-1584Glu had an increased risk of type 2 diabetes (OR, 4.92, 95% CI, 1.05–23.0) only when they had low magnesium intake (<250 mg/day).ConclusionOur results provide suggestive evidence that two common non-synonymous TRPM6 coding region variants, Ile1393Val and Lys1584Glu polymorphisms, might confer susceptibility to type 2 diabetes in women with low magnesium intake. Further replication in large-scale studies is warranted.

Pleiotropic genes for metabolic syndrome and inflammation

Metabolic syndrome (MetS) has become a health and financial burden worldwide. The MetS definition captures clustering of risk factors that predict higher risk for diabetes mellitus and cardiovascular disease. Our study hypothesis is that additional to genes influencing individual MetS risk factors, genetic variants exist that influence MetS and inflammatory markers forming a predisposing MetS genetic network. To test this hypothesis a staged approach was undertaken. (a) We analyzed 17 metabolic and inflammatory traits in more than 85,500 participants from 14 large epidemiological studies within the Cross Consortia Pleiotropy Group. Individuals classified with MetS (NCEP definition), versus those without, showed on average significantly different levels for most inflammatory markers studied. (b) Paired average correlations between 8 metabolic traits and 9 inflammatory markers from the same studies as above, estimated with two methods, and factor analyses on large simulated data, helped in identifying 8 combinations of traits for follow-up in meta-analyses, out of 130,305 possible combinations between metabolic traits and inflammatory markers studied. (c) We performed correlated meta-analyses for 8 metabolic traits and 6 inflammatory markers by using existing GWAS published genetic summary results, with about 2.5 million SNPs from twelve predominantly largest GWAS consortia. These analyses yielded 130 unique SNPs/genes with pleiotropic associations (a SNP/gene associating at least one metabolic trait and one inflammatory marker). Of them twenty-five variants (seven loci newly reported) are proposed as MetS candidates. They map to genes MACF1, KIAA0754, GCKR, GRB14, COBLL1, LOC646736-IRS1, SLC39A8, NELFE, SKIV2L, STK19, TFAP2B, BAZ1B, BCL7B, TBL2, MLXIPL, LPL, TRIB1, ATXN2, HECTD4, PTPN11, ZNF664, PDXDC1, FTO, MC4R and TOMM40. Based on large data evidence, we conclude that inflammation is a feature of MetS and several gene variants show pleiotropic genetic associations across phenotypes and might explain a part of MetS correlated genetic architecture. These findings warrant further functional investigation.

PPARG by dietary fat interaction influences bone mass in mice and humans

Adult BMD, an important risk factor for fracture, is the result of genetic and environmental interactions. A quantitative trait locus (QTL) for the phenotype of volumetric BMD (vBMD), named Bmd8, was found on mid‐distal chromosome (Chr) 6 in mice. This region is homologous to human Chr 3p25. The B6.C3H‐6T (6T) congenic mouse was previously created to study this QTL. Using block haplotyping of the 6T congenic region, expression analysis in the mouse, and examination of nonsynonymous SNPs, peroxisome proliferator activated receptor γ (Pparg) was determined to be the most likely candidate gene for the Bmd8 QTL of the 630 genes located in the congenic region. Furthermore, in the C3H/HeJ (C3H) strain, which is the donor strain for the 6T congenic, several polymorphisms were found in the Pparg gene. On challenge with a high‐fat diet, we found that the 6T mouse has a lower areal BMD (aBMD) and volume fraction of trabecular bone (BV/TV%) of the distal femur compared with B6 mice. Interactions between SNPs in the PPARG gene and dietary fat for the phenotype of BMD were examined in the Framingham Offspring Cohort. This analysis showed that there was a similar interaction of the PPARG gene and diet (fat intake) on aBMD in both men and women. These findings suggest that dietary fat has a significant influence on BMD that is dependent on the alleles present for the PPARG gene.