Association weight matrix for the genetic dissection of puberty in beef cattle Marina R. S. Fortesa,b,c, Antonio Revertera,b, Yuandan Zhanga,d, Eliza Collisa,b, Shivashankar H. Nagarajb,NickN.Jonssona,c,e, Kishore C. Prayagaa,b,1, Wes Barrisa,b, and Rachel J. Hawkena,b,2 aCooperative Research Centre for Beef Genetic Technologies; bCommonwealth Scientific and Industrial Research Organization, division of Livestock Industries, Queensland Bioscience Precinct, Brisbane QLD 4067, Australia; cThe University of Queensland, School of Veterinary Science, Gatton QLD 4343, Australia; dAnimal Genetics and Breeding Unit, University of New England, Armidale NSW 2351, Australia; and eFaculty of Veterinary Medicine, University of Glasgow, Glasgow G61 1QH, United Kingdom Edited by George Seidel, Colorado State University, Fort Collins, CO, and approved June 21, 2010 (received for review February 23, 2010) We describe a systems biology approach for the genetic dissection tional data on traits related to puberty are available. For example, of complex traits based on applying gene network theory to the re- weight and condition score are often measured on occasions sults from genome-wide associations. The associations of single- throughout an animal’s development. Hence, understanding ge- nucleotide polymorphisms (SNP) that were individually associated netics of cattle puberty and its biology serves two purposes: as with a primary phenotype of interest, age at puberty in our study, a strategy to develop efficient livestock resources and as a model were explored across 22 related traits. Genomic regions were sur- for human biology. veyed for genes harboring the selected SNP. As a result, an asso- The focus of this work is to demonstrate a unique systems ap- ciation weight matrix (AWM) was constructed with as many rows proach, which we call an association weight matrix (AWM), ap- as genes and as many columns as traits. Each {i, j} cell value in the propriated for GWASs of complex traits. We examined cattle AWM corresponds to the z-score normalized additive effect of the ith puberty from a GWAS based on ∼50,000 single-nucleotide poly- gene (via its neighboring SNP) on the jth trait. Columnwise, the AWM morphisms (SNPs) and 22 traits as an example of a typical complex recovered the genetic correlations estimated via pedigree-based phenotype. We designed the AWM with elements corresponding restricted maximum-likelihood methods. Rowwise, a combination to the standardized additive effect of the ith SNP (in rows) on the of hierarchical clustering, gene network, and pathway analyses iden- jth trait (in columns). Rowwise, the AWM explored gene-to-gene tified genetic drivers that would have been missed by standard interactions for cattle puberty across the genome; columnwise, it genome-wide association studies. Finally, the promoter regions of estimated correlations between traits influencing puberty. the AWM-predicted targets of three key transcription factors (TFs), estrogen-related receptor γ (ESRRG), Pal3 motif, bound by a PPAR-γ Results homodimer, IR3 sites (PPARG), and Prophet of Pit 1, PROP paired-like Commonly, GWASs are single-trait–single-SNP analyses. This homeobox 1 (PROP1), were surveyed to identify binding sites cor- analysis for our main pubertal trait, the age of occurrence of the responding to those TFs. Applied to our case, the AWM results re- first corpus luteum (AGECL), resulted in more associated SNPs capitulate the known biology of puberty, captured experimentally than the number expected by chance alone: 2,799 SNPs at P < 0.05, validated binding sites, and identified candidate genes and gene– 588 SNPs at P < 0.01, and 69 SNPs at P < 0.001. We report the gene interactions for further investigation. results of the single-trait–single-SNP association analyses for later comparison against those from the AWM approach. bovine | complex traits | fertility | reproduction | systems biology Each SNP effect, from the total set of 50,070 SNPs, was used as a data point in the calculation of all pairwise correlations between he analysis of genome-wide association studies (GWASs) ap- the 22 traits. As a result, AGECL correlated with weight at first R plied to complex traits remains a challenge (1). Addressing corpus luteum (WTCL) ( = 0.64) and with postpartum anoestrus T R a complex trait by a single, often binary, phenotypic measure is interval (PPAI) ( = 0.31) among other traits (Table S1). When common practice but is limiting. It is not easy to find the right selecting SNPs to build the AWM, we considered all 22 traits and the balance between applying a conservative significance threshold SNP-to-gene distance (as per Fig. S1). With increasing SNP-to-gene fi that gives rise to a small number of strong and hopefully biologically distance, we observed decay in the SNP signi cance, as measured by P value (Fig. S2). We selected 3,159 SNPs to build the AWM, where meaningful associations and applying a relaxed threshold yielding A numerous associations, many of which are new but potentially false. SNPs were rows and 22 related traits were columns (Fig. S3 ). Columnwise, the AWM was used to calculate correlations be- In addition, an increase in sample size coupled with a denser chip tween traits. This result is visualized as a hierarchical tree where results in a larger number of associations that, on average, have AGECL clusters with WTCL and both are close to PPAI (Fig. a much smaller effect (2). Accepting a large number of associations S3B). On the hierarchical tree cluster, a strong positive correlation while simultaneously reducing the number of false positives would is displayed as proximity, whereas a strong negative correlation is be ideal. It is reasonable to propose that a holistic approach applied fi displayed as a large distance. To observe negative and positive to a relaxed signi cance threshold could be the solution. Such correlations equally, we developed the quantitative trait network a strategy would be particularly useful when investigating the ge- netic basis of complex traits that, by definition, are influenced by numerous genes and pathways. Author contributions: M.R.S.F., A.R., K.C.P., and R.J.H. designed research; M.R.S.F. and E.C. In our study, age at puberty was the complex phenotype con- performed research; S.H.N. and W.B. contributed new reagents/analytic tools; M.R.S.F., sidered. Puberty, or the progression to sexual maturity, is a de- A.R., Y.Z., and S.H.N. analyzed data; and M.R.S.F., A.R., N.N.J., and R.J.H. wrote the paper. velopmental process with genetic drivers conserved among species The authors declare no conflict of interest. (3). It is an important phenotype for the beef industry because late This article is a PNAS Direct Submission. puberty has negative effects on reproduction rates and profitability Freely available online through the PNAS open access option. (4). Age at puberty is moderately heritable, with estimates of 1Present address: Pfizer Animal Genetics, Albion, Brisbane, QLD 4010, Australia. heritability in cattle ranging from 0.16 to 0.57 (5, 6). In humans, 2To whom correspondence should be addressed. E-mail: [email protected]. ∼ 50% of the variation in age of puberty is genetic (7, 8). An ad- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. vantage to working with cattle as a model species is that observa- 1073/pnas.1002044107/-/DCSupplemental. 13642–13647 | PNAS | August 3, 2010 | vol. 107 | no. 31 www.pnas.org/cgi/doi/10.1073/pnas.1002044107 Downloaded by guest on September 27, 2021 (QTN) from AWM SNPs, which shows a high degree of interaction GO term and pathway analyses applied to the AWM network among all 22 traits (Fig. S3C). For visual comparison, we also identified biological processes and pathways that are relevant for present a QTN (Fig. S3D) based on published genetic correlations cattle puberty. (5, 9). A formal comparison between these genetic correlations To test the likelihood of AWM predictions being random we and SNP-based correlations showed a moderate agreement (R2 = built a control gene network. As expected, the control gene network 0.6439) between the two approaches (Fig. 1). When all SNPs were had the topology of a random network with the majority of the considered, the trait correlations were closer to the genetic esti- genes having an average number of connections (10). The distri- mates (R2 = 0.7034; Fig. 1). The same comparison using the 3,159 bution of number of connections per gene in the control network SNPs included in the AWM (or 6.2% of the total) captures 64% of was different from that in the AWM network (P < 0.0001), so the variation estimated by genetic correlations (Fig. 1). So AWM is AWM was not random. equivalent to 91% (or 64/70) of the variation that was captured To test the likelihood of AWM predictions being simply a re- using the entire SNP chip. Fig. 1 also reveals a pattern of increasing flection of LD between SNPs from the initial GWAS, we calculated regression coefficients with decreasing number of SNPs: 0.5697 all pairwise D′ and R2. Whereas some AWM predictions were un- (whole chip) to 0.7415 (AWM) to 1.0389 (top 71 SNPs). Hence, derlined by strong LD, most were not. This result was expected the more stringent we are at selecting SNPs, the more unbiased the considering the average distance between AWM-selected SNPs recovery becomes (regression coefficient closer to unity). There- (825 kb ± 1 Mb). fore, the criteria to include SNPs in the AWM resulted in en- We selected 3 key transcription factors (TFs) from 34 available richment with nonredundant genetic information, implying that (see Methods for details and SI Text for TF list). Key TFs, Prophet of the AWM could be used for estimating genetic correlations, al- Pit 1, PROP paired-like homeobox 1 (PROP1), Pal3 motif, bound by though it was not developed with this aim.