Identification of genetic modifiers of age-at-onset for familial Parkinson's disease.

Parkinson's disease (PD) is the most common cause of neurodegenerative movement disorder and the second most common cause of dementia. Genes are thought to have a stronger effect on age-at-onset of PD than on risk, yet there has been a phenomenal success in identifying risk loci but not age-at-onset modifiers. We conducted a genome-wide study for age-at-onset. We analysed familial and non-familial PD separately, per prior evidence for strong genetic effect on age-at-onset in familial PD. GWAS was conducted in 431 unrelated PD individuals with at least one affected relative (familial PD) and 1544 non-familial PD from the NeuroGenetics Research Consortium (NGRC); an additional 737 familial PD and 2363 non-familial PD were used for replication. In familial PD, two signals were detected and replicated robustly: one mapped to LHFPL2 on 5q14.1 (PNGRC = 3E-8, PReplication = 2E-5, PNGRC + Replication = 1E-11), the second mapped to TPM1 on 15q22.2 (PNGRC = 8E-9, PReplication = 2E-4, PNGRC + Replication = 9E-11). The variants that were associated with accelerated onset had low frequencies (<0.02). The LHFPL2 variant was associated with earlier onset by 12.33 [95% CI: 6.2; 18.45] years in NGRC, 8.03 [2.95; 13.11] years in replication, and 9.79 [5.88; 13.70] years in the combined data. The TPM1 variant was associated with earlier onset by 15.30 [8.10; 22.49] years in NGRC, 9.29 [1.79; 16.79] years in replication, and 12.42 [7.23; 17.61] years in the combined data. Neither LHFPL2 nor TPM1 was associated with age-at-onset in non-familial PD. LHFPL2 (function unknown) is overexpressed in brain tumours. TPM1 encodes a highly conserved protein that regulates muscle contraction, and is a tumour-suppressor gene.

CFH haplotypes without the Y402H coding variant show strong association with susceptibility to age-related macular degeneration.

In developed countries, age-related macular degeneration is a common cause of blindness in the elderly. A common polymorphism, encoding the sequence variation Y402H in complement factor H (CFH), has been strongly associated with disease susceptibility. Here, we examined 84 polymorphisms in and around CFH in 726 affected individuals (including 544 unrelated individuals) and 268 unrelated controls. In this sample, 20 of these polymorphisms showed stronger association with disease susceptibility than the Y402H variant. Further, no single polymorphism could account for the contribution of the CFH locus to disease susceptibility. Instead, multiple polymorphisms defined a set of four common haplotypes (of which two were associated with disease susceptibility and two seemed to be protective) and multiple rare haplotypes (associated with increased susceptibility in aggregate). Our results suggest that there are multiple disease susceptibility alleles in the region and that noncoding CFH variants play a role in disease susceptibility.

Primary open angle glaucoma in a Caucasian population is associated with the p53 codon 72 polymorphism.

PURPOSE: Apoptosis has been implicated as the mechanism for retinal ganglion cell death in primary open-angle glaucoma (POAG), a complex neurodegenerative disease. There have been inconsistent reports regarding increased risk of POAG and a polymorphism (Arg72Pro) within the tumor suppressor gene, p53. The goal of this study was to examine the role of this polymorphism in susceptibility to POAG in a Caucasian population from the United States. METHODS: We generated genotypes in 191 unrelated Caucasian POAG patients and 167 unrelated Caucasian controls for the following polymorphisms within p53: rs1042522 (Arg72Pro), rs17878362 (16 bp Ins/Del), and rs1800371 (Pro47Ser) by PCR amplification followed by restriction digestion and sequence analysis. RESULTS: There was a significant difference in genotypic frequencies for rs1042522 (Arg72Pro) between POAG patients and controls (chi(2)= 9.56, p=0.008). Individuals who were homozygous for the arginine allele have a 1.9 fold significantly increased risk of developing glaucoma (95%CI: 1.16-2.82, p=0.01). Interestingly, we found that the frequency of the arginine allele was even higher in the normal-tension glaucoma (NTG) subtype compared to high-tension POAG (0.81 versus 0.76). CONCLUSIONS: Our preliminary results indicate that the arginine variant of rs1042522 within p53 is associated with increased risk of POAG. This variant has increased apoptotic potential, thus the retinal ganglion cells in carriers of the arginine allele may have greater susceptibility to apoptosis.

Extraocular muscle morphogenesis and gene expression are regulated by Pitx2 gene dose.

PURPOSE: PITX2 gene dose plays a central role in Axenfeld-Rieger syndrome. The purpose of this study was to test the hypothesis that the effects of Pitx2 gene dose on eye development can be molecularly dissected in available Pitx2 mutant mice. METHODS: A panel of mice with Pitx2 gene dose ranging from wild-type (+/+) to none (-/-) was generated. Eye morphogenesis was assessed in animals with each Pitx2 gene dose. We also compared global gene expression in eye primordia taken from e12.5 Pitx2+/+, Pitx2+/-, Pitx2-/- embryos using gene microarrays. The validity of microarray results was confirmed by qRT-PCR. RESULTS: Morphogenesis of all extraocular muscle bundles correlated highly with Pitx2 gene dose, but there were some differences in sensitivity among muscle groups. Superior and inferior oblique muscles were most sensitive and disappeared before the four rectus muscles. Expression of muscle-specific genes was globally sensitive to Pitx2 gene dose, including the muscle-specific transcription factor genes Myf5, Myog, Myod1, Smyd1, Msc, and Csrp3. CONCLUSIONS: Pitx2 gene dose regulates both morphogenesis and gene expression in developing extraocular muscles. The expression of key muscle-specific transcription factor genes is regulated by Pitx2 gene dose, suggesting that sufficient levels of PITX2 protein are essential for early initiation of the myogenic regulatory cascade in extraocular muscles. These results document the first ocular tissue affected by Pitx2 gene dose in a model organism, where the underlying mechanisms can be analyzed, and provide a paradigm for future experiments designed to elucidate additional effects of Pitx2 gene dose during eye development.

Familial aggregation of Parkinson disease: a comparative study of early-onset and late-onset disease.

CONTEXT: It is unclear whether late-onset Parkinson disease (PD), which is the most typical and most common form of the disease, has a familial component. Evidence for familial aggregation is key to whether research should focus on gene discovery or search for environmental factors. OBJECTIVE: To investigate familial aggregation of early-onset and late-onset PD separately. METHODS: Using survival methods, age-specific risk of PD was calculated and compared for 525 parents and siblings of 117 patients with early-onset PD, 1642 parents and siblings of 343 patients with late-onset PD, and 522 parents and siblings of 114 controls. The index patients were ascertained from a movement disorder clinic. Spouses and friends served as controls. RESULTS: Compared with the relatives of controls, age-specific risk of PD was increased 7.76-fold in the relatives of patients with early-onset disease (P<.001) and 2.95-fold in the relatives of those with late-onset disease (P =.02). CONCLUSIONS: Late-onset PD has a significant familial component. The magnitude of recurrence risk to relatives suggests a genetic etiology, without ruling out the possibility of a coexisting environmental component.

Association of apolipoprotein E alleles with susceptibility to age-related macular degeneration in a large cohort from a single center.

PURPOSE: To examine the effect of apolipoprotein E (APOE) alleles on age-related macular degeneration (AMD) risk and on age at diagnosis of AMD in a large patient cohort recruited from a single center. METHODS: The frequency of APOE alleles was analyzed in 632 unrelated AMD patients and 206 unrelated controls, all of whom were of white ancestry. The presence or absence of disease symptoms in all patients and controls was based on clinical examination and/or ophthalmic records. The association with APOE was explored in the context of AMD subtypes, family history status, possible interaction with smoking, and distribution of age at diagnosis of AMD. RESULTS: The frequency of the epsilon4 allele was significantly reduced in patients compared with controls (0.10 vs. 0.14, P < or = 0.02). Gender- and age-adjusted odds ratios indicated that epsilon4-carriers have significantly lower risk of developing AMD compared to epsilon3epsilon3 subjects (OR = 0.55, 95% CI: 0.37-0.82, P = 0.004). In the cohort, AMD patients with a positive family history exhibited a significant 3.5 years earlier age at diagnosis (P = 0.001); however, APOE alleles did not appear to modulate the age at diagnosis of AMD. CONCLUSIONS: The association between the APOE-epsilon4 allele and a reduced risk of AMD was established in a large cohort with sufficient statistical power. How distinct APOE alleles affect AMD susceptibility warrants further investigation.

Gene expression profile of native human retinal pigment epithelium.

PURPOSE: To generate a profile of genes expressed in the native human retinal pigment epithelium and identify candidate genes for retinal and macular diseases. METHODS: Two cDNA libraries (one amplified, the other unamplified) were constructed using RNA isolated from native human RPE sheets. The sequence from the 5' end was obtained for randomly selected clones from the two libraries. Of these, more than 2000 expressed sequence tags (ESTs) were analyzed for similarity to sequences and gene clusters in public databases. RESULTS: EST analysis revealed several known RPE-expressed genes and more than 500 genes that have been characterized previously but were not known to be expressed in the RPE. Transthyretin and 90-kDa heat shock protein represent the most abundant transcripts identified in these RPE libraries. More than 200 novel ESTs and putative proteins were identified. An additional 344 sequences matched only the human genomic sequence. CONCLUSIONS: High-complexity cDNA libraries were generated from native human RPE. Analysis of ESTs generated from these libraries has yielded a profile of genes expressed in the native RPE. Several of the identified genes are known to play a significant role in the RPE. Novel ESTs, putative proteins, and genomic hits may represent as yet unidentified RPE-expressed genes and many of these, mapping in the region of retinal disease loci, may serve as candidate genes. In addition, the nonredundant set of more than 1100 genes and ESTs described herein will be a valuable resource for generating gene microarrays, which can assist in delineating RPE expression profiles during human disease pathogenesis.

Evaluation and optimization of procedures for target labeling and hybridization of cDNA microarrays.

PURPOSE: To evaluate and optimize methods of target labeling and microarray hybridization using eye gene microarrays. Standardized protocols that consistently produce low background and high intensity hybridization with small amounts of starting RNA are needed to extract differentially expressed genes from a pool of thousands of unaltered genes. METHODS: Two identical aliquots of RNA from P19 cell line were labeled with Cy3 or Cy5 dyes using four different methods and self-against-self hybridization was performed on mouse eye gene arrays. The validity and reproducibility of these protocols were further examined using target RNAs isolated from wild-type or neural retinal leucine zipper (Nrl) knockout mouse retinas. Hybridizations were also carried out on human gene array slides with different amounts of starting RNA from human retina. RESULTS: Using self-against-self hybridization, we optimized the protocols for direct labeling (R-square = 0.93), aminoallyl indirect labeling (R-square = 0.97), Genisphere 3DNA labeling (R-square = 0.96), and for microarray hybridization and washing. Although small amounts of initial RNA can be used in TSA method, inconsistent labeling was encountered under our experimental conditions. When retinal RNA targets from Nrl+/+ and Nrl-/- mice were tested by direct and aminoallyl indirect labeling protocols, both produced varying hybridization results with low intensity spots and non-uniform backgrounds. However, the Genisphere 3DNA labeling procedure consistently yielded strong hybridization and R-square values of 0.92 or higher. Furthermore, expression profiles were compatible with prior knowledge of this mouse model. Serial analysis of hybridizations with various starting amounts of RNA showed that the Genisphere 3DNA protocol could produce reliable signal intensity with 3 microgram of total RNA. CONCLUSIONS: We have systematically evaluated and optimized methods for target labeling, microarray hybridization and washing. These procedures have been used for expression profiling with 3 microgram of starting RNA. Our studies should encourage further use of microarray technology for gene profiling during eye development and in retinal diseases.

Age at onset of Parkinson disease and apolipoprotein E genotypes.

Several lines of evidence suggest that the variable age at onset of Parkinson disease (PD) is likely influenced by genes. The apolipoprotein E (APOE) gene is associated with onset of Alzheimer disease, and possibly other neurodegenerative disorders. APOE has been investigated in relation to onset of PD, but results have been inconsistent. The aim of the present study was to determine if APOE genotypes are associated with onset age of PD, using a patient population large enough to assure sufficient power. We studied 521 unrelated Caucasian patients with idiopathic PD from movement disorder clinics in Oregon and Washington. Genotyping and statistical analyses were carried out using standard methods. Age at onset of PD was significantly earlier in patients with the varepsilon3varepsilon4/varepsilon4varepsilon4 genotype than in patients with the varepsilon3varepsilon3 genotype (56.1 +/- 10.9 vs. 59.6 +/- 11.0, P = 0.003). The significantly earlier onset of PD was not influenced by the possible effects of recruitment site, family history and gender. The effect of the varepsilon2varepsilon3 genotype on onset of PD differed between the two recruitment sites. There was a trend for earlier onset of PD in varepsilon2varepsilon3 patients than in varepsilon3varepsilon3 patients only in the Oregon sample. In conclusion, APOE is associated with age at onset of PD.

Complement factor H genetic variant and age-related macular degeneration: effect size, modifiers and relationship to disease subtype.

BACKGROUND: Variation in the complement factor H gene (CFH) is associated with risk of late age-related macular degeneration (AMD). Previous studies have been case-control studies in populations of European ancestry with little differentiation in AMD subtype, and insufficient power to confirm or refute effect modification by smoking. METHODS: To precisely quantify the association of the single nucleotide polymorphism (SNP rs1061170, 'Y402H') with risk of AMD among studies with differing study designs, participant ancestry and AMD grade and to investigate effect modification by smoking, we report two unpublished genetic association studies (n = 2759) combined with data from 24 published studies (26 studies, 26,494 individuals, including 14,174 cases of AMD) of European ancestry, 10 of which provided individual-level data used to test gene-smoking interaction; and 16 published studies from non-European ancestry. RESULTS: In individuals of European ancestry, there was a significant association between Y402H and late-AMD with a per-allele odds ratio (OR) of 2.27 [95% confidence interval (CI) 2.10-2.45; P = 1.1 x 10(-161)]. There was no evidence of effect modification by smoking (P = 0.75). The frequency of Y402H varied by ancestral origin and the association with AMD in non-Europeans was less clear, limited by paucity of studies. CONCLUSION: The Y402H variant confers a 2-fold higher risk of late-AMD per copy in individuals of European descent. This was stable to stratification by study design and AMD classification and not modified by smoking. The lack of association in non-Europeans requires further verification. These findings are of direct relevance for disease prediction. New research is needed to ascertain if differences in circulating levels, expression or activity of factor H protein explain the genetic association.

Distinct signature of altered homeostasis in aging rod photoreceptors: implications for retinal diseases.

BACKGROUND: Advanced age contributes to clinical manifestations of many retinopathies and represents a major risk factor for age-related macular degeneration, a leading cause of visual impairment and blindness in the elderly. Rod photoreceptors are especially vulnerable to genetic defects and changes in microenvironment, and are among the first neurons to die in normal aging and in many retinal degenerative diseases. The molecular mechanisms underlying rod photoreceptor vulnerability and potential biomarkers of the aging process in this highly specialized cell type are unknown. METHODOLOGY/PRINCIPAL FINDINGS: To discover aging-associated adaptations that may influence rod function, we have generated gene expression profiles of purified rod photoreceptors from mouse retina at young adult to early stages of aging (1.5, 5, and 12 month old mice). We identified 375 genes that showed differential expression in rods from 5 and 12 month old mouse retina compared to that of 1.5 month old retina. Quantitative RT-PCR experiments validated expression change for a majority of the 25 genes that were examined. Macroanalysis of differentially expressed genes using gene class testing and protein interaction networks revealed overrepresentation of cellular pathways that are potentially photoreceptor-specific (angiogenesis and lipid/retinoid metabolism), in addition to age-related pathways previously described in several tissue types (oxidative phosphorylation, stress and immune response). CONCLUSIONS/SIGNIFICANCE: Our study suggests a progressive shift in cellular homeostasis that may underlie aging-associated functional decline in rod photoreceptors and contribute to a more permissive state for pathological processes involved in retinal diseases.

Targeting of GFP to newborn rods by Nrl promoter and temporal expression profiling of flow-sorted photoreceptors.

The Maf-family transcription factor Nrl is a key regulator of photoreceptor differentiation in mammals. Ablation of the Nrl gene in mice leads to functional cones at the expense of rods. We show that a 2.5-kb Nrl promoter segment directs the expression of enhanced GFP specifically to rod photoreceptors and the pineal gland of transgenic mice. GFP is detected shortly after terminal cell division, corresponding to the timing of rod genesis revealed by birthdating studies. In Nrl-/- retinas, the GFP+ photoreceptors express S-opsin, consistent with the transformation of rod precursors into cones. We report the gene profiles of freshly isolated flow-sorted GFP+ photoreceptors from wild-type and Nrl-/- retinas at five distinct developmental stages. Our results provide a framework for establishing gene regulatory networks that lead to mature functional photoreceptors from postmitotic precursors. Differentially expressed rod and cone genes are excellent candidates for retinopathies.

Strong association of the Y402H variant in complement factor H at 1q32 with susceptibility to age-related macular degeneration.

Using a large sample of cases and controls from a single center, we show that a T-->C substitution in exon 9 (Y402H) of the complement factor H gene is strongly associated with susceptibility to age-related macular degeneration, the most common cause of blindness in the elderly. Frequency of the C allele was 0.61 in cases, versus 0.34 in age-matched controls (P<1x10(-24)). Genotype frequencies also differ markedly between cases and controls (chi2=112.68 [2 degrees of freedom]; P<1x10(-24)). A multiplicative model fits the data well, and we estimate the population frequency of the high-risk C allele to be 0.39 (95% confidence interval 0.36-0.42) and the genotype relative risk to be 2.44 (95% confidence interval 2.08-2.83) for TC heterozygotes and 5.93 (95% confidence interval 4.33-8.02) for CC homozygotes.

Toll-like receptor 4 variant D299G is associated with susceptibility to age-related macular degeneration.

Age-related macular degeneration (AMD) is a genetically heterogeneous disease that leads to progressive and irreversible vision loss among the elderly. Inflammation, oxidative damage, cholesterol metabolism and/or impaired function of retinal pigment epithelium (RPE) have been implicated in AMD pathogenesis. We examined toll-like receptor 4 (TLR4) as a candidate gene for AMD susceptibility because: (i) the TLR4 gene is located on chromosome 9q32-33, a region exhibiting evidence of linkage to AMD in three independent reports; (ii) the TLR4-D299G variant is associated with reduced risk of atherosclerosis, a chronic inflammatory disease with subendothelial accumulation; (iii) the TLR4 is not only a key mediator of proinflammatory signaling pathways but also linked to regulation of cholesterol efflux and (iv) the TLR4 participates in phagocytosis of photoreceptor outer segments by the RPE. We examined D299G and T399I variants of TLR4 in a sample of 667 unrelated AMD patients and 439 unrelated controls, all of Caucasian ancestry. Multiple logistic regression demonstrated an increased risk of AMD in carriers of the G allele at TLR4 residue 299 (odds ratio=2.65, P=0.025), but lack of an independent effect by T399I variant. TLR4-D299G showed an additive effect on AMD risk (odds ratio=4.13, P=0.002) with allelic variants of apolipoprotein E (APOE) and ATP-binding cassette transporter-1 (ABCA1), two genes involved in cholesterol efflux. Interestingly, the effect of TLR4, APOE and ABCA1 variants on AMD susceptibility was opposite to that of association with atherosclerosis risk. Our data provide evidence of a link between multiple diverse mechanisms underlying AMD pathogenesis.

Meta-analysis of genome scans of age-related macular degeneration.

A genetic contribution to the development of age-related macular degeneration (AMD) is well established. Several genome-wide linkage studies have identified a number of putative susceptibility loci for AMD but only a few of these regions have been replicated in independent studies. Here, we perform a meta-analysis of six AMD genome screens using the genome-scan meta-analysis method, which allows linkage results from several studies to be combined, providing greater power to identify regions that show only weak evidence for linkage in individual studies. Results from non-parametric analysis for a broad AMD clinical phenotype (including two studies with quantitative traits) were extracted. For each study, 120 genomic bins of approximately 30 cM were defined and ranked according to maximum evidence for linkage within each bin. Bin ranks were weighted according to study size and summed across all studies; the summed rank (SR) for each bin was assessed empirically for significance using permutation methods. A high SR indicates a region with consistent evidence for linkage across studies. The strongest evidence for an AMD susceptibility locus was found on chromosome 10q26 where genome-wide significant linkage was observed (P=0.00025). Several other regions met the empirical significance criteria for bins likely to contain linked loci including adjacent pairs of bins on chromosomes 1q, 2p, 3p and 16. Several of the regions identified here showed only weak evidence for linkage in the individual studies. These results will help prioritize regions for future positional and functional candidate gene studies in AMD.

Mutation in a short-chain collagen gene, CTRP5, results in extracellular deposit formation in late-onset retinal degeneration: a genetic model for age-related macular degeneration.

A primary feature of age-related macular degeneration (AMD) is the presence of extracellular deposits between the retinal pigment epithelium (RPE) and underlying Bruch's membrane, leading to RPE dysfunction, photoreceptor death and severe visual loss. AMD accounts for about 50% of blind registrations in Western countries and is a common, genetically complex disorder. Very little is known regarding its molecular basis. Late-onset retinal degeneration (L-ORD) is an autosomal dominant disorder with striking clinical and pathological similarity to AMD. Here we show that L-ORD is genetically heterogeneous and that a proposed founder mutation in the CTRP5 (C1QTNF5) gene, which encodes a novel short-chain collagen, changes a highly conserved serine to arginine (Ser163Arg) in 7/14 L-ORD families and 0/1000 control individuals. The mutation occurs in the gC1q domain of CTRP5 and results in abnormal high molecular weight aggregate formation which may alter its higher-order structure and interactions. These results indicate a novel disease mechanism involving abnormal adhesion between RPE and Bruch's membrane.

Age-related macular degeneration: a high-resolution genome scan for susceptibility loci in a population enriched for late-stage disease.

Age-related macular degeneration (AMD) is a complex multifactorial disease that affects the central region of the retina. AMD is clinically heterogeneous, leading to geographic atrophy (GA) and/or choroidal neovascularization (CNV) at advanced stages. Considerable data exists in support of a genetic predisposition for AMD. Recent linkage studies have provided evidence in favor of several AMD susceptibility loci. We have performed a high-resolution (5-cM) genome scan of 412 affected relative pairs that were enriched for late-stage disease (GA and/or CNV). Nonparametric linkage analysis was performed using two different diagnostic criteria and also by dividing the affected individuals according to GA or CNV phenotype. Our results demonstrate evidence of linkage in regions that were suggested in at least one previous study at chromosomes 1q (236-240 cM in the Marshfield genetic map), 5p (40-50 cM), and 9q (111 cM). Multipoint analysis of affected relatives with CNV provided evidence of additional susceptibility loci on chromosomes 2p (10 cM) and 22q (25 cM). A recently identified Gln5345Arg change in HEMICENTIN-1 on chromosome 1q25 was not detected in 274 affected members in the restricted group with AMD, 346 additional patients with AMD, and 237 unaffected controls. Our results consolidate the chromosomal locations of several AMD susceptibility loci and, together with previous reports, should facilitate the search for disease-associated sequence variants.