Drosophila Models Reveal Properties of Mutant Lamins That Give Rise to Distinct Diseases.

Mutations in the LMNA gene cause a collection of diseases known as laminopathies, including muscular dystrophies, lipodystrophies, and early-onset aging syndromes. The LMNA gene encodes A-type lamins, lamins A/C, intermediate filaments that form a meshwork underlying the inner nuclear membrane. Lamins have a conserved domain structure consisting of a head, coiled-coil rod, and C-terminal tail domain possessing an Ig-like fold. This study identified differences between two mutant lamins that cause distinct clinical diseases. One of the LMNA mutations encodes lamin A/C p.R527P and the other codes lamin A/C p.R482W, which are typically associated with muscular dystrophy and lipodystrophy, respectively. To determine how these mutations differentially affect muscle, we generated the equivalent mutations in the Drosophila Lamin C (LamC) gene, an orthologue of human LMNA. The muscle-specific expression of the R527P equivalent showed cytoplasmic aggregation of LamC, a reduced larval muscle size, decreased larval motility, and cardiac defects resulting in a reduced adult lifespan. By contrast, the muscle-specific expression of the R482W equivalent caused an abnormal nuclear shape without a change in larval muscle size, larval motility, and adult lifespan compared to controls. Collectively, these studies identified fundamental differences in the properties of mutant lamins that cause clinically distinct phenotypes, providing insights into disease mechanisms.

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.

Copy number variation in the complement factor H-related genes and age-related macular degeneration.

PURPOSE: To determine the contribution of copy number variation (CNV) in the regulation of complement activation (RCA) locus to the development of age-related macular degeneration (AMD). METHODS: A multiplex ligation-dependent probe amplification assay was developed to quantify the number of copies of CFH, CFHR3, CFHR1, CFHR4, CFHR2, and CFHR5 in humans. Subjects with (451) and without (362) AMD were genotyped using the assay, and the impact on AMD risk was evaluated. RESULTS: Eight unique combinations of copy number variation were observed in the 813 subjects. Combined deletion of CFHR3 and CFHR1 was protective (OR=0.47, 95% confidence interval 0.36-0.62) against AMD and was observed in 88 (82 [18.6%] with one deletion, 6 [1.4%] with two deletions) subjects with AMD and 127 (108 [30.7%] with one deletion, 19 [5.4%] with two deletions) subjects without AMD. Other deletions were much less common: CFH intron 1 (n=2), CFH exon 18 (n=2), combined CFH exon 18 and CFHR3 (n=1), CFHR3 (n=2), CFHR1 (n=1), combined CFHR1 and CFHR4 (n=15), and CFHR2 deletion (n=7, 0.9%). The combined CFHR3 and CFHR1 deletion was observed on a common protective haplotype, while the others appeared to have arisen on multiple different haplotypes. CONCLUSIONS: We found copy number variations of CFHR3, CFHR1, CFHR4, and CFHR2. Combined deletion of CFHR3 and CFHR1 was associated with a decreased risk of developing AMD. Other deletions were not sufficiently common to have a statistically detectable impact on the risk of AMD, and duplications were not observed.

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.

Anterior keratocyte depletion in fuchs endothelial dystrophy.

OBJECTIVE: To determine if keratocyte populations are different in corneas with Fuchs dystrophy compared with control corneas. METHODS: Eleven corneas excised during penetrating keratoplasty for Fuchs dystrophy and 5 control corneas of eyes enucleated for choroidal melanoma were examined using light microscopy. Twenty control corneas age-matched to the corneas with Fuchs dystrophy were examined using confocal microscopy in vivo. The number of keratocytes in a full-thickness column of central stroma with frontal area of 1 mm(2), determined using histologic and confocal methods, was compared between corneas with Fuchs dystrophy and controls. RESULTS: By histology, the mean (SD) number of cells in a full-thickness column of stroma in Fuchs dystrophy (12 215 [1394] cells) was less than in control corneas (15 628 [710] cells; P < .001). The mean (SD) number of keratocytes in the anterior 10% of the stroma of corneas with Fuchs dystrophy (682 [274] cells) was less than in the control corneas measured using histology (1858 [404] cells; P < .001) and confocal microscopy (1481 [397] cells; P < .001). CONCLUSIONS: Keratocytes are depleted by 54% to 63% in the anterior 10% of the stroma of corneas that require penetrating keratoplasty for Fuchs dystrophy. Keratocyte loss might contribute to anterior stromal changes that persist and degrade vision after endothelial keratoplasty.

Characterization of the retinal proteome during rod photoreceptor genesis.

BACKGROUND: The 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. FINDINGS: A 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. CONCLUSIONS: Our 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.

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.

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.