Synthesizing genome-wide association studies and expression microarray reveals novel genes that act in the human growth plate to modulate height

Abstract

Previous meta-analysis of genome-wide association (GWA) studies has identified 180 loci that influence adult height. However, each GWA locus typically comprises a set of contiguous genes, only one of which presumably modulates height. We reasoned that many of the causative genes within these loci influence height because they are expressed in and function in the growth plate, a cartilaginous structure that causes bone elongation and thus determines stature. Therefore, we used expression microarray studies of mouse and rat growth plate, human disease databases and a mouse knockout phenotype database to identify genes within the GWAS loci that are likely required for normal growth plate function. Each of these approaches identified significantly more genes within the GWA height loci than at random genomic locations (P < 0.0001 each), supporting the validity of the approach. The combined analysis strongly implicates 78 genes in growth plate function, including multiple genes that participate in PTHrP-IHH, BMP and CNP signaling, and many genes that have not previously been implicated in the growth plate. Thus, this analysis reveals a large number of novel genes that regulate human growth plate chondrogenesis and thereby contribute to the normal variations in human adult height. The analytic approach developed for this study may be applied to GWA studies for other common polygenic traits and diseases, thus providing a new general strategy to identify causative genes within GWA loci and to translate genetic associations into mechanistic biological insights.

Figure 1.

Venn diagram showing the number of genes with expression that is growth plate-specific, spatially regulated in growth plate or temporally regulated in growth plate by microarray. ‘Growth plate specific’ indicates expression in growth plate ≥2.0-fold greater than kidney, lung and heart in 1-week-old mouse (n = 5, FDR < 1%, Genome Array 430 2.0, Affymetrix). ‘Spatially regulated’ indicates up- or downregulated from resting to proliferative zone (≥2.0-fold, FDR < 5%) or from proliferative to hypertrophic zone (≥2.0-fold, FDR < 5%) in 1-week-old rat (n = 6, Genome Array 230 2.0, Affymetrix). ‘Temporally regulated’ indicates expression that is up- or downregulated from 3 weeks to 12 weeks of age in rat proliferative zone (≥2.0-fold, FDR < 5%, n = 6, Rat Genome Array 230 2.0, Affymetrix,). Examples of genes in each overlapping subset are shown in boxes.

Figure 2.

Observed and expected positional distribution of 78 growth plate-related genes relative to SNP. Seventy-eight genes in the GWA loci were implicated in growth plate function by expression analysis, mouse phenotype or human phenotype. Of these 78, 46 are located closest to the height SNP. In 24 of these cases, the strongly associated height SNP is located within the growth plate gene itself. This distribution differs significantly (P < 0.0001, χ² test) from a random allocation calculated based on the overall distribution of 833 genes in 207 loci. Thus, the growth plate-related genes tend to lie closer to the SNP identified by GWA than would be expected by chance.

Figure 3.

Examples of genes from GWA analysis that have a well-established role in chondrogenesis. PTHrP (encoded by PTHLH) produced in the resting zone inhibits differentiation of proliferative chondrocytes. The production of PTHrP is in turn stimulated by IHH from the hypertrophic zone. IHH stimulates chondrocyte proliferation in the proliferative zone and the expression of HHIP, which inhibits and modulates the action of IHH in the resting zone. Together, IHH and PTHrP form a negative feedback loop that determines the height of the proliferative zone. BMP2 and BMP6, produced by hypertrophic chondrocytes, stimulate proliferation and differentiation of chondrocytes, which is partially antagonized by an inhibitory effect of FGFs, such as FGF18. RUNX2 and RUNX3 are expressed in the hypertrophic zone and stimulate hypertrophic differentiation through one of their downstream targets MEF2C, which is also highly expressed in the hypertrophic zone. RUNX2 also interacts with the IHH-PTHrP feedback loop by inducing IHH expression. +, stimulatory effect; −, inhibitory effect.