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

Abstract

SUMMARY The extent to which low-frequency (minor allele frequency [MAF] between 1–5%) and rare (MAF ≤ 1%) variants contribute to complex traits and disease in the general population is largely unknown. Bone mineral density (BMD) is highly heritable, is a major predictor of osteoporotic fractures and has been previously associated with common genetic variants1–8, and rare, population-specific, coding variants9. 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), whole-exome sequencing (n= 3,549), deep imputation of genotyped samples using a combined UK10K/1000Genomes 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 4-fold larger than the mean of previously reported common variants for lumbar spine BMD8 (rs11692564[T], MAF = 1.7%, replication effect size = +0.20 standard deviations [SD], Pmeta = 2×10−14), which was also associated with a decreased risk of fracture (OR = 0.85; P = 2×10−11; ncases = 98,742 and ncontrols = 409,511). Using an En1Cre/flox mouse model, we observed that conditional loss of En1 results in low bone mass, likely as a consequence of high bone turn-over. We also identified a novel low-frequency non-coding variant with large effects on BMD near WNT16 (rs148771817[T], MAF = 1.1%, replication effect size = +0.39 SD, Pmeta = 1×10−11). 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.

Low-Frequency Synonymous Coding Variation in CYP2R1 Has Large Effects on Vitamin D Levels and Risk of Multiple Sclerosis

“…Participating studies separately genotyped samples and imputed them to WGS-based reference panels. The most recent imputation panels, such as the UK10K and 1000 Genomes Project (v.3) combined panel (7,562 haplotypes from the UK10K project and 2,184 haplotypes from the 1000 Genomes Project 9 ) and the Haplotype Reference Consortium (HRC) panel (64,976 haplotypes 15 ), enabled more accurate imputation of low-frequency variants than the UK10K or 1000 Genomes reference panel alone. 9 Specifically, 11 of the 17 participating cohorts were imputed to the combined UK10K and 1000 Genomes reference panel (total number of imputed individuals included in the meta-analysis ¼ 25,589).…”

“…WGS for the ALSPAC and TUK cohorts has been described in detail in a previous publication from our group. 7 Table S8 summarizes the data-generation method for sequencing-based cohorts. Participating studies separately genotyped samples and imputed them to WGS-based reference panels.…”

Genetics of osteoporosis: searching for candidate genes for bone fragility

“…Using this approach, the novel candidate gene EN1 was identified, significantly related to both BMD and fracture risk. Animal models and in vitro studies indicate a possible role for EN1 in osteoblasts, offering an exciting opportunity for the discovery of new mechanisms in bone formation (53). Finally, this study also suggests that lower frequency variants may have higher impact on BMD and fractures, warranting further studies.…”

“…In 2015, a breakthrough GWAS based on wholegenome sequencing was published by Zheng and cols., with enough power to detect the effects of lowfrequency variants (minor allele frequency [MAF] between 1-5%), which are usually not contemplated by genotype-based GWAS (53). Using this approach, the novel candidate gene EN1 was identified, significantly related to both BMD and fracture risk.…”

Genetic Studies of Endophenotypes From Spine CT Scans Provide Novel Insights Into the Contribution of Mechanosensory Pathways to Vertebral Fractures and Spinal Curvature

“…DXA-based studies have led the way in identifying genetic influences on the skeleton, exemplified by a recent genome wide association study (GWAS) followed by replication which identified allelic variation at the EN1 locus as a novel, but low frequency determinant of bone mineral density (BMD) and fracture risk (1) . One of the main advantages of DXA scans is that due to their wide availability and low radiation dose, large scale GWAS meta-analyses involving many thousands of individuals are possible, providing the requisite power for studies of this type.…”