Laura A. Hecker

Genetic control of the alternative pathway of complement in humans and age-related macular degeneration

Activation of the alternative pathway of complement is implicated in common neurodegenerative diseases including age-related macular degeneration (AMD). We explored the impact of common variation in genes encoding proteins of the alternative pathway on complement activation in human blood and in AMD. Genetic variation across the genes encoding complement factor H (CFH), factor B (CFB) and component 3 (C3) was determined. The influence of common haplotypes defining transcriptional and translational units on complement activation in blood was determined in a quantitative genomic association study. Individual haplotypes in CFH and CFB were associated with distinct and novel effects on plasma levels of precursors, regulators and activation products of the alternative pathway of complement in human blood. Further, genetic variation in CFH thought to influence cell surface regulation of complement did not alter plasma complement levels in human blood. Plasma markers of chronic activation (split-products Ba and C3d) and an activating enzyme (factor D) were elevated in AMD subjects. Most of the elevation in AMD was accounted for by the genetic variation controlling complement activation in human blood. Activation of the alternative pathway of complement in blood is under genetic control and increases with age. The genetic variation associated with increased activation of complement in human blood also increased the risk of AMD. Our data are consistent with a disease model in which genetic variation in the complement system increases the risk of AMD by a combination of systemic complement activation and abnormal regulation of complement activation in local tissues.

Contribution of copy number variation in the regulation of complement activation locus to development of age-related macular degeneration.

PURPOSE To develop an assay for determining the number of copies of the genes encoding complement factor H related 3 (CFHR3) and 1 (CFHR1) and determine the contribution of copy number variation (CNV) at CFHR3 and CFHR1 to the development of age-related macular degeneration (AMD). METHODS A multiplex ligation-dependent probe amplification (MLPA) assay was developed to quantify the number of copies of CFHR3 and CFHR1 in humans. Subjects with (n = 252) and without (n = 249) AMD were genotyped using the assay, and the impact on AMD risk was evaluated. RESULTS The MLPA assay provided a consistent estimate of the number of copies of CFHR3 and CFHR1 in 500 of the 501 samples. Four different combinations of CNVs were observed with frequencies as follows: both CFHR3 and CFHR1 deletion (14%), CFHR3-only deletion (0.4%), CFHR1-only deletion (1.1%), and CFHR1 duplication (0.1%). Deletion of both copies of CFHR3 and CFHR1 decreased the odds of having AMD eightfold (95% CI 2-36) and always occurred on a protective haplotype, never on the risk haplotype tagged by the Y402H risk allele in CFH. The protection conferred by deletion of CFHR3 and CFHR1 could not be distinguished from the absence of the risk haplotype. CONCLUSIONS Both deletions and duplications of genes in the regulation of complement activation locus segregated in Caucasians. Deletion of CFHR3 and CFHR1 protected against the development of AMD at least in part because the deletion tagged a protective haplotype and did not occur on the risk haplotype.

Genetic Control of Complement Activation in Humans and Age Related Macular Degeneration

The major focus of our research is to understand how age-related macular degeneration (AMD) develops. It is known that genetic variation can explain much of the risk of developing AMD. However, we do not know what controls the transition between a normal fundus and the extensive accumulation of subretinal inflammatory material that we recognize as drusen in AMD. We do know that the accumulation of this inflammatory material that characterizes the maculopathy underlying AMD is by far the most important predictor of late AMD. Late or advanced forms of AMD include geographic atrophy in which there is patchy death of the retina and exudation in which abnormal neovascularization invades the subretinal or subretinal pigment epithelial space. Thus, preventing the accumulation of the inflammatory debris underneath the retina could be expected to alleviate much of the vision loss from this devastating disease.

Characterization of the retinal proteome during rod photoreceptor genesis

BackgroundThe process of rod photoreceptor genesis, cell fate determination and differentiation is complex and multi-factorial. Previous studies have defined a model of photoreceptor differentiation that relies on intrinsic changes within the presumptive photoreceptor cells as well as changes in surrounding tissue that are extrinsic to the cell. We have used a proteomics approach to identify proteins that are dynamically expressed in the mouse retina during rod genesis and differentiation.FindingsA series of six developmental ages from E13 to P5 were used to define changes in retinal protein expression during rod photoreceptor genesis and early differentiation. Retinal proteins were separated by isoelectric focus point and molecular weight. Gels were analyzed for changes in protein spot intensity across developmental time. Protein spots that peaked in expression at E17, P0 and P5 were picked from gels for identification. There were 239 spots that were picked for identification based on their dynamic expression during the developmental period of maximal rod photoreceptor genesis and differentiation. Of the 239 spots, 60 of them were reliably identified and represented a single protein. Ten proteins were represented by multiple spots, suggesting they were post-translationally modified. Of the 42 unique dynamically expressed proteins identified, 16 had been previously reported to be associated with the developing retina.ConclusionsOur results represent the first proteomics study of the developing mouse retina that includes prenatal development. We identified 26 dynamically expressed proteins in the developing mouse retina whose expression had not been previously associated with retinal development.

Using a Seed-Network to Query Multiple Large-Scale Gene Expression Datasets from the Developing Retina in Order to Identify and Prioritize Experimental Targets

Understanding the gene networks that orchestrate the differentiation of retinal progenitors into photoreceptors in the developing retina is important not only due to its therapeutic applications in treating retinal degeneration but also because the developing retina provides an excellent model for studying CNS development. Although several studies have profiled changes in gene expression during normal retinal development, these studies offer at best only a starting point for functional studies focused on a smaller subset of genes. The large number of genes profiled at comparatively few time points makes it extremely difficult to reliably infer gene networks from a gene expression dataset. We describe a novel approach to identify and prioritize from multiple gene expression datasets, a small subset of the genes that are likely to be good candidates for further experimental investigation. We report progress on addressing this problem using a novel approach to querying multiple large-scale expression datasets using a ‘seed network’ consisting of a small set of genes that are implicated by published studies in rod photoreceptor differentiation. We use the seed network to identify and sort a list of genes whose expression levels are highly correlated with those of multiple seed network genes in at least two of the five gene expression datasets. The fact that several of the genes in this list have been demonstrated, through experimental studies reported in the literature, to be important in rod photoreceptor function provides support for the utility of this approach in prioritizing experimental targets for further experimental investigation. Based on Gene Ontology and KEGG pathway annotations for the list of genes obtained in the context of other information available in the literature, we identified seven genes or groups of genes for possible inclusion in the gene network involved in differentiation of retinal progenitor cells into rod photoreceptors. Our approach to querying multiple gene expression datasets using a seed network constructed from known interactions between specific genes of interest provides a promising strategy for focusing hypothesis-driven experiments using large-scale ‘omics’ data.