Identification of IDUA and WNT16 phosphorylation-related non-synonymous polymorphisms for bone mineral density in meta-analyses of genome-wide association studies

Protein phosphorylation regulates a wide variety of cellular processes. Thus, we hypothesize that single-nucleotide polymorphisms (SNPs) that may modulate protein phosphorylation could affect osteoporosis risk. Based on a previous conventional genome-wide association (GWA) study, we conducted a three-stage meta-analysis targeting phosphorylation-related SNPs (phosSNPs) for femoral neck (FN)-bone mineral density (BMD), total hip (HIP)-BMD, and lumbar spine (LS)-BMD phenotypes. In stage 1, 9593 phosSNPs were meta-analyzed in 11,140 individuals of various ancestries. Genome-wide significance (GWS) and suggestive significance were defined by α = 5.21 × 10–6 (0.05/9593) and 1.00 × 10–4, respectively. In stage 2, nine stage 1–discovered phosSNPs (based on α = 1.00 × 10–4) were in silico meta-analyzed in Dutch, Korean, and Australian cohorts. In stage 3, four phosSNPs that replicated in stage 2 (based on α = 5.56 × 10–3, 0.05/9) were de novo genotyped in two independent cohorts. IDUA rs3755955 and rs6831280, and WNT16 rs2707466 were associated with BMD phenotypes in each respective stage, and in three stages combined, achieving GWS for both FN-BMD (p = 8.36 × 10–10, p = 5.26 × 10–10, and p = 3.01 × 10–10, respectively) and HIP-BMD (p = 3.26 × 10–6, p = 1.97 × 10–6, and p = 1.63 × 10–12, respectively). Although in vitro studies demonstrated no differences in expressions of wild-type and mutant forms of IDUA and WNT16B proteins, in silico analyses predicts that WNT16 rs2707466 directly abolishes a phosphorylation site, which could cause a deleterious effect on WNT16 protein, and that IDUA phosSNPs rs3755955 and rs6831280 could exert indirect effects on nearby phosphorylation sites. Further studies will be required to determine the detailed and specific molecular effects of these BMD-associated non-synonymous variants. © 2015 American Society for Bone and Mineral Research.

Identification of a novel FGFRL1 MicroRNA target site polymorphism for bone mineral density in meta-analyses of genome-wide association studies

MicroRNAs (miRNAs) are critical post-transcriptional regulators. Based on a previous genome-wide association (GWA) scan, we conducted a polymorphism in microRNAs' Target Sites (poly-miRTS)-centric multistage meta-analysis for lumbar spine (LS)-, total hip (HIP)-, and femoral neck (FN)-bone mineral density (BMD). In stage I, 41,102 poly-miRTSs were meta-analyzed in 7 cohorts with a genome-wide significance (GWS) α=0.05/41,102=1.22×10-6. By applying α=5×10-5 (suggestive significance), 11 poly-miRTSs were selected, with FGFRL1 rs4647940 and PRR5 rs3213550 as top signals for FN-BMD (P-value=7.67×10-6 and 1.58×10-5) in gender-combined sample. In stage II in silico replication (two cohorts), FGFRL1 rs4647940 was the only signal marginally replicated for FN-BMD (P-value=5.08×10-3) at α=0.10/11=9.09×10-3. PRR5 rs3213550 was also selected based on biological significance. In stage III de novo genotyping replication (two cohorts), FGFRL1 rs4647940 was the only signal significantly replicated for FN-BMD (P-value=7.55×10-6) at α=0.05/2=0.025 in gender-combined sample. Aggregating three stages, FGFRL1 rs4647940 was the single stage I-discovered and stages II- and III-replicated signal attaining GWS for FN-BMD (P-value=8.87×10-12). Dual-luciferase reporter assays demonstrated that FGFRL1 3' untranslated region harboring rs4647940 appears to be hsa-miR-140-5p's target site. In a zebrafish microinjection experiment, dre-miR-140-5p is shown to exert a dramatic impact on craniofacial skeleton formation. Taken together, we provided functional evidence for a novel FGFRL1 poly-miRTS rs4647940 in a previously known 4p16.3 locus, and experimental and clinical genetics studies have shown both FGFRL1 and hsa-miR-140-5p are important for bone formation. © The Author 2015. Published by Oxford University Press. All rights reserved.

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 5 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. © The Author 2013. Published by Oxford University Press.

Whole-genome sequencing identifies EN1 as a determinant of bone density and fracture

The extent to which low-frequency (minor allele frequency (MAF) between 1-5%) and rare (MAF density (BMD) is highly heritable, a major predictor of osteoporotic fractures, and has been previously associated with common genetic variants, as well as rare, population-specific, coding variants. Here we identify novel non-coding genetic variants with large effects on BMD (ntotal = 53,236) and fracture (ntotal = 508,253) in individuals of European ancestry from the general population. Associations for BMD were derived from whole-genome sequencing (n = 2,882 from UK10K (ref. 10); a population-based genome sequencing consortium), whole-exome sequencing (n = 3,549), deep imputation of genotyped samples using a combined UK10K/1000 Genomes reference panel (n = 26,534), and de novo replication genotyping (n = 20,271). We identified a low-frequency non-coding variant near a novel locus, EN1, with an effect size fourfold larger than the mean of previously reported common variants for lumbar spine BMD (rs11692564(T), MAF = 1.6%, replication effect size = +0.20 s.d., Pmeta = 2 x 10(-14)), which was also associated with a decreased risk of fracture (odds ratio = 0.85; P = 2 x 10(-11); ncases = 98,742 and ncontrols = 409,511). Using an En1(cre/flox) mouse model, we observed that conditional loss of En1 results in low bone mass, probably as a consequence of high bone turnover. We also identified a novel low-frequency non-coding variant with large effects on BMD near WNT16 (rs148771817(T), MAF = 1.2%, replication effect size = +0.41 s.d., Pmeta = 1 x 10(-11)). In general, there was an excess of association signals arising from deleterious coding and conserved non-coding variants. These findings provide evidence that low-frequency non-coding variants have large effects on BMD and fracture, thereby providing rationale for whole-genome sequencing and improved imputation reference panels to study the genetic architecture of complex traits and disease in the general population.

Directional dominance on stature and cognition in diverse human populations

Homozygosity has long been associated with rare, often devastating, Mendelian disorders1, and Darwin was one of the first to recognize that inbreeding reduces evolutionary fitness2. However, the effect of the more distant parental relatedness that is common in modern human populations is less well understood. Genomic data now allow us to investigate the effects of homozygosity on traits of public health importance by observing contiguous homozygous segments (runs of homozygosity), which are inferred to be homozygous along their complete length. Given the low levels of genome-wide homozygosity prevalent in most human populations, information is required on very large numbers of people to provide sufficient power3, 4. Here we use runs of homozygosity to study 16 health-related quantitative traits in 354,224 individuals from 102 cohorts, and find statistically significant associations between summed runs of homozygosity and four complex traits: height, forced expiratory lung volume in one second, general cognitive ability and educational attainment (P < 1 × 10−300, 2.1 × 10−6, 2.5 × 10−10 and 1.8 × 10−10, respectively). In each case, increased homozygosity was associated with decreased trait value, equivalent to the offspring of first cousins being 1.2 cm shorter and having 10 months’ less education. Similar effect sizes were found across four continental groups and populations with different degrees of genome-wide homozygosity, providing evidence that homozygosity, rather than confounding, directly contributes to phenotypic variance. Contrary to earlier reports in substantially smaller samples5, 6, no evidence was seen of an influence of genome-wide homozygosity on blood pressure and low density lipoprotein cholesterol, or ten other cardio-metabolic traits. Since directional dominance is predicted for traits under directional evolutionary selection7, this study provides evidence that increased stature and cognitive function have been positively selected in human evolution, whereas many important risk factors for late-onset complex diseases may not have been.

Position preference and diffusion path of an oxygen ion in apatite-type lanthanum silicate La9.33Si6O26: A density functional study

Using density functional theory, we investigated the position preference and diffusion mechanisms of interstitial oxygen ions in lanthanum silicate La9.33Si6O26, which is an apatite-structured oxide and a promising candidate electrolyte material for solid oxide fuel cells. The reported lanthanum vacancies were explicitly taken into account by theoretically determining their arrangement with a supercell model. The most stable structures and the formation energies of oxygen interstitials were determined for each charged state. It was found that the double-negatively charged state is stable over a wide range of the Fermi level, and that the excess oxygen ions form split interstitials with the original oxygen ions, while the neutral and the single-negatively charged states preferably form molecular oxygen. These species were found near the lanthanum vacancy site. The theoretically determined migration pathway along the c-axis essentially follows an interstitialcy mechanism. The obtained migration barrier is sensitive to the charge state, and is also affected by the lanthanum vacancy. The barrier height of the double-negatively charged state was calculated to be 0.58 eV for the model structure, which is consistent with the measured activation energy.