Methods Development Virtually all of the theoretical work during the past year has focused on the development of Tiled regression, linear regression based methods for intra-familial tests of association for quantitative traits that address non-independence both at the marker and observational level. Tiled regression uses both multiple and stepwise regression methods in predefined segments of the genome, defined by hotspot blocks, to identify independent genetic variants responsible for the variation or susceptibility in quantitative and qualitative traits, respectively. Multiple and stepwise methods are used to test for associations on the sequence variants in each tile to select the independent markers within each tile. Higher order stepwise regressions are then used to identify significant variant across tiles, chromosomes and the entire genome. Quantitative and qualitative traits can be analyzed. With this approach, it becomes practical to analyze hundreds of thousands or millions of markers and their significant gene x gene interaction terms. This approach can substantially reduce the total number of tests to a number closer to the number of tiles rather than the number of markers. Furthermore, the tiled approach can be incorporated into a linear regression framework that allows for non-independence between observations incorporating features from the Regression of Offspring on Mid-Parant (ROMP) and Generalized Estimating Equations approaches. The tiled regression methodology has been implemented in TRAP, a software package written in the freely available R language. Functions are provided for assigning SNPs to hotspot-based tiles, data input, analysis and output of results. The package is structured modularly, so that it may be used as a single program or with user-written functions to allow for alternate tile definition or data format. This approach has been applied to both SNP data from fine mapping SNP studies with the scoliosis data in collaboration with Dr. Nancy Miller (U of Colorado), and two targeted candidate gene sequencing projects, an NF1 project in collaboration with Dr. Douglas Stewart and the ClinSeq project, in collaboration with Dr. Les Biesecker. Collaborations Familial Idiopathic Scoliosis Several analyses focusing on candidate regions and phenotypic subsets have been completed and manuscripts have either been submitted or are in preparation. These include: 1) In this study of susceptibility loci in FIS families with at least one individual with a triple curve, candidate regions have been identified on chromosomes 6 and 10 Marosy et al. 2010. 2) Statistical genetic analysis of two sets of families with familial idiopathic scoliosis with characteristics nearly identical to those of the sample analyzed in Miller et al. 2005. Linkage analysis and tests of association were performed in two regions on chromosome 1, previously identified as primary candidate regions. We have identified several regions of interest for subsequent nextgen sequencing Behnemann, doctoral thesis. 3) Targeted sequencing of the IRX gene family in families with kyphoscoliosis. We have identified an association between kyphoscoliosis and a sequence variant in an upstream conserved region of one of the IRX genes. Association analysis resulted in 12 SNPs with p-values <0.025, of which 11 are 500 kb from IRX1, including the most significant SNP (p = 0.000382). One of these SNPs is in a HCNR sharing 87% sequence identity with a HCNR upstream from IRX3 on 16q12 Justice, in preparation. 4) Statistical genetic analysis of STRPs and SNPs on chromosomes 9 and 16. Fine mapping on chromosomes 9 and 16 was performed to narrow previously identified candidate regions. Linkage and association studies identified several highly significant regions that are candidates for nextgen sequencing Miller et al., in preparation. 5) A study based on the presence of males with severe scoliosis Miller et al., submitted. The males with severe curve subset was comprised of 25 families (207 individuals) in which at least one male was diagnosed in adolescence with a ≥30 lateral curvature. The genome-wide linkage analysis for the qualitative and quantitative traits resulted in significant p-values (2 adjacent markers with p-values <0.01) on chromosomes 2, 16 and 22. Significant SNPs lie primarily in the introns of the LARGE gene, integral to the development and maintenance of skeletal muscle, and SFI1, responsible for the integrity of the chromosomal centromere complex. Other large ongoing collaborations include: 1) Clinical characterization of NF1 (Dr. Douglas Stewart, NIH/NCI) 2) the ClinSeq project (Les Biesecker, NIH/NHGRI) 3) the GeneSTAR project (Drs. Diane and Lewis Becker, Johns Hopkins University School of Medicine) Mathias et al., 2010 4) the India Diabetes Project (Dr. Rasika Mathias, Johns Hopkins University School of Medicine) 5) Variation in metabolites in the Irish (Dr. Larry Brody, NIH/NHGRI)
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