Molecular Genetics of Age-related Macular Degeneration

Age-related macular degeneration (AMD; OMIM # 603075) is an eye disease of the elderly, signs of which appear after the age of 50. In the Western world it is a leading cause of permanent visual loss with a prevalence of 8.5% in persons under 54 years of age and of 37% in persons over 75 years of age. Early forms of AMD may be asymptomatic, but in the late forms usually a central scotoma in the visual field follows severely complicating daily tasks. Smoking, age, and genetic predisposition are known risk factors for AMD. Until recently no true susceptibility genes had been identified though the composition of drusen deposits, the hallmarks of AMD, has suggested that the complement system might play a role in the pathogenesis of AMD. When four groups reported in March 2005, that, on chromosome 1q32, a Y402H variant in the complement factor H (CFH) gene confers risk for AMD in independent Caucasian samples, a new period in the field of genetic research of AMD started. CFH is a key regulator of the complement system. Thus, it is logical to speculate, that it plays a role in the pathogenesis of AMD. We performed a case-control association study to analyse whether the CFH Y402H variant contain a risk for AMD in the Finnish population. Although the population of Finland represents a genetic isolate, the CFH Y402H polymorphism was associated with AMD also in our patient sample with similar risk allele frequencies as in the other Caucasian populations. We further evaluated the effects of this variant, but no association between lesion subtype (predominantly classic, minimally classic or occult lesion) or lesion size of neovascular AMD and the CFH Y402H variant was detected. Neither did the variant have an effect on the photodynamic therapy (PDT) outcome. The patients that respond to PDT carried the risk genotype as frequently as those who did not respond, and no difference was found in the number of PDT sessions needed in patients with or without the risk genotypes of CFH Y402H. Functional analyses, however, showed that the binding of C-reactive protein (CRP) to CFH was significantly reduced in patients with the risk genotype of Y402H. In the past two years, the LOC387715/ high-temperature requirement factor A1 (HTRA1) locus on 10q26 has also been repeatedly associated with AMD in several populations. The recent discovery of the LOC387715 protein on the mitochondrial outer membrane suggests that the LOC387715 gene, not HTRA1, is the true predisposing gene in this region, although its biological function is still unknown. In our Finnish patient material, patients with AMD carried the A69S risk genotype of LOC387715 more frequently than the controls. Also, for the first time, an interaction between the CFH Y402H and the LOC387715 A69S variants was found. The most recently detected susceptibilty gene of AMD, the complement component 3 (C3) gene, encodes the central component of the complement system, C3. In our Finnish sample, an additive gene effect for the C3 locus was detected, though weaker than the effects for the two main loci, CFH and LOC387715. Instead, the hemicentin-1 or the elongation of very long chain fatty acids-like 4 genes that have also been suggested as candidate genes for AMD did not carry a risk for AMD in the Finnish population. This was the first series of molecular genetic study of AMD in Finland. We showed that two common risk variants, CFH Y402H and LOC387715 A69S, represent a high risk of AMD also in the isolated Finnish population, and furthermore, that they had a statistical interaction. It was demonstrated that the CFH Y402H risk genotype affects the binding of CFH to CRP thus suggesting that complement indeed plays an important role in the pathogenesis of AMD.

Uniparental DNA markers and forensic genetics in Finland

Over the years, a wide range of methods to verify identity have been developed. Molecular markers have been used for identification since the 1920s, commencing with blood types and culminating with the advent of DNA techniques in the 1980s. Identification is required by authorities in many occasions, e.g. in disputed paternity cases, identification of deceased, or crime investigation. To clarify maternal and paternal lineages, uniparental DNA markers in mtDNA and Y-chromosome can be utilized. These markers have several advantages: male specific Y-chromosome can be used to identify a male from a mixture of male and female cells, e.g. in rape cases. MtDNA is durable and has a high copy number, allowing analyses even from old or degraded samples. However, both markers are lineage-specific, not individualizing, and susceptible to genetic drift. Prior to the application of any DNA marker in forensic casework, it is of utmost importance to investigate its qualities and peculiarities in the target population. Earlier studies on the Finnish population have shown reduced variation in the Y-chromosome, but in mtDNA results have been ambiguous. The obtained results confirmed the low diversity in Y-chromosome in Finland. Detailed population analysis revealed large regional differences, and extremely reduced diversity especially in East Finland. Analysis of the qualities affecting Y-chromosomal short tandem repeat (Y-STR) variation and mutation frequencies, and search of new polymorphic markers resulted a set of Y-STRs with especially high diversity in Finland. Contrary to Y-chromosome, neither reduced diversity nor regional differences were found in mtDNA within Finland. In fact, mtDNA diversity was found similar to other European populations. The revealed peculiarities in the uniparental markers are a legacy of the Finnish population history. The obtained results challenge the traditional explanation which emphasizes relatively recent founder effects creating the observed east-west patterns. Uniparentally inherited markers, both mtDNA and Y-chromosome, are applicable for identification purposes in Finland. By adjusting the analysed Y marker set to meet the characteristics of Finnish population, Y-chromosomal diversity increases and the regional differentiation decreases, resulting increase in discrimination power and thus usefulness of Y-chromosomal analysis in forensic casework.

Intraspecific variation in brain size and architecture : population divergence and phenotypic plasticity

Brain size and architecture exhibit great evolutionary and ontogenetic variation. Yet, studies on population variation (within a single species) in brain size and architecture, or in brain plasticity induced by ecologically relevant biotic factors have been largely overlooked. Here, I address the following questions: (i) do locally adapted populations differ in brain size and architecture, (ii) can the biotic environment induce brain plasticity, and (iii) do locally adapted populations differ in levels of brain plasticity? In the first two chapters I report large variation in both absolute and relative brain size, as well as in the relative sizes of brain parts, among divergent nine-spined stickleback (Pungitius pungitius) populations. Some traits show habitat-dependent divergence, implying natural selection being responsible for the observed patterns. Namely, marine sticklebacks have relatively larger bulbi olfactorii (chemosensory centre) and telencephala (involved in learning) than pond sticklebacks. Further, I demonstrate the importance of common garden studies in drawing firm evolutionary conclusions. In the following three chapters I show how the social environment and perceived predation risk shapes brain development. In common frog (Rana temporaria) tadpoles, I demonstrate that under the highest per capita predation risk, tadpoles develop smaller brains than in less risky situations, while high tadpole density results in enlarged tectum opticum (visual brain centre). Visual contact with conspecifics induces enlarged tecta optica in nine-spined sticklebacks, whereas when only olfactory cues from conspecifics are available, bulbus olfactorius become enlarged.Perceived predation risk results in smaller hypothalami (complex function) in sticklebacks. Further, group-living has a negative effect on relative brain size in the competition-adapted pond sticklebacks, but not in the predation-adapted marine sticklebacks. Perceived predation risk induces enlargement of bulbus olfactorius in pond sticklebacks, but not in marine sticklebacks who have larger bulbi olfactorii than pond fish regardless of predation. In sum, my studies demonstrate how applying a microevolutionary approach can help us to understand the enormous variation observed in the brains of wild animals a point-of-view which I high-light in the closing review chapter of my thesis.

Transposable element-medicated evolution of sex: A population genetic model

For a population made up of individuals capable of sexual as well as asexual modes of reproduction, conditions for the spread of a transposable element are explored using a one-locus, two-haplotype model. The analysis is then extended to include the possibility that the transposable element can modulate the probability of sexual reproduction, thus casting Hickey’s (1982,Genetics 101: 519–531) suggestion in a population genetics framework. The model explicitly includes the cost of sexual reproduction, fitness disadvantage to the transposable element, probability of transposition, and the predisposition for sexual reproduction in the presence and absence of the transposable element. The model predicts several kinds of outcome, including initial frequency dependence and stable polymorphism. More importantly, it is seen that for a wide range of parameter values, the transposable element can go to fixation. Therefore it is able to convert the population from a predominantly asexual to a predominantly sexual mode of reproduction. Viewed in conjunction with recent results implicating short stretches of apparently non-coding DNA in sex determination (McCoubreyet al. 1988,Science 242: 1146–1151), the model hints at the important role this mechanism could have played in the evolution of sexuality.

Sibling rivalry between seeds within a fruit: Some population genetic models

Competition between seeds within a fruit for parental resources is described using one-locus-two-allele models. While a �normal� allele leads to an equitable distribution of resources between seeds (a situation which also corresponds to the parental optimum), the �selfish� allele is assumed to cause the seed carrying it to usurp a higher proportion of the resources. The outcome of competition between �selfish� alleles is also assumed to lead to an asymmetric distribution of resources, the �winner� being chosen randomly. Conditions for the spread of an initially rare selfish allele and the optimal resource allocation corresponding to the evolutionarily stable strategy, derived for species with n-seeded fruits, are in accordance with expectations based on Hamilton�s inclusive fitness criteria. Competition between seeds is seen to be most intense when there are only two seeds, and decreases with increasing number of seeds, suggesting that two-seeded fruits would be rarer than one-seeded or many-seeded ones. Available data from a large number of plant species are consistent with this prediction of the model.

A finite population model of mlecular evolution: theory and computation

This article is concerned with the evolution of haploid organisms that reproduce asexually. In a seminal piece of work, Eigen and coauthors proposed the quasispecies model in an attempt to understand such an evolutionary process. Their work has impacted antiviral treatment and vaccine design strategies. Yet, predictions of the quasispecies model are at best viewed as a guideline, primarily because it assumes an infinite population size, whereas realistic population sizes can be quite small. In this paper we consider a population genetics-based model aimed at understanding the evolution of such organisms with finite population sizes and present a rigorous study of the convergence and computational issues that arise therein. Our first result is structural and shows that, at any time during the evolution, as the population size tends to infinity, the distribution of genomes predicted by our model converges to that predicted by the quasispecies model. This justifies the continued use of the quasispecies model to derive guidelines for intervention. While the stationary state in the quasispecies model is readily obtained, due to the explosion of the state space in our model, exact computations are prohibitive. Our second set of results are computational in nature and address this issue. We derive conditions on the parameters of evolution under which our stochastic model mixes rapidly. Further, for a class of widely used fitness landscapes we give a fast deterministic algorithm which computes the stationary distribution of our model. These computational tools are expected to serve as a framework for the modeling of strategies for the deployment of mutagenic drugs.

Variation at Diabetes- and Obesity-Associated Loci May Mirror Neutral Patterns of Human Population Diversity and Diabetes Prevalence in India

South Asian populations harbor a high degree of genetic diversity, due in part to demographic history. Two studies on genome-wide variation in Indian populations have shown that most Indian populations show varying degrees of admixture between ancestral north Indian and ancestral south Indian components. As a result of this structure, genetic variation in India appears to follow a geographic cline. Similarly, Indian populations seem to show detectable differences in diabetes and obesity prevalence between different geographic regions of the country. We tested the hypothesis that genetic variation at diabetes-and obesity-associated loci may be potentially related to different genetic ancestries. We genotyped 2977 individuals from 61 populations across India for 18 SNPs in genes implicated in T2D and obesity. We examined patterns of variation in allele frequency across different geographical gradients and considered state of origin and language affiliation. Our results show that most of the 18 SNPs show no significant correlation with latitude, the geographic cline reported in previous studies, or by language family. Exceptions include KCNQ1 with latitude and THADA and JAK1 with language, which suggests that genetic variation at previously ascertained diabetes-associated loci may only partly mirror geographic patterns of genome-wide diversity in Indian populations.

Effects of Withania somnifera and Tinospora cordifolia Extracts on the Side Population Phenotype of Human Epithelial Cancer Cells: Toward Targeting Multidrug Resistance in Cancer

Recent reports suggest the existence of a subpopulation of stem-like cancer cells, termed as cancer stem cells (CSCs), which bear functional and phenotypic resemblance with the adult, tissue-resident stem cells. Side population (SP) assay based on differential efflux of Hoechst 33342 has been effectively used for the isolation of CSCs. The drug resistance properties of SP cells are typically due to the increased expression of ABC transporters leading to drug efflux. Conventionally used chemotherapeutic drugs may often leads to an enrichment of SP, revealing their inability to target the drug-resistant SP and CSCs. Thus, identification of agents that can reduce the SP phenotype is currently in vogue in cancer therapeutics. Withania somnifera (WS) and Tinospora cordifolia (TC) have been used in Ayurveda for treating various diseases, including cancer. In the current study, we have investigated the effects of ethanolic (ET) extracts of WS and TC on the cancer SP phenotype. Interestingly, we found significant decrease in SP on treatment with TC-ET, but not with WS-ET. The SP-inhibitory TC-ET was further fractionated into petroleum ether (TC-PET), dichloromethane (TC-DCM), and n-butyl alcohol (TC-nBT) fractions using bioactivity-guided fractionation. Our data revealed that TC-PET and TC-DCM, but not TC-nBT, significantly inhibited SP in a dose-dependent manner. Furthermore, flow cytometry-based functional assays revealed that TC-PET and TC-DCM significantly inhibited ABC-B1 and ABC-G2 transporters and sensitized cancer cells toward chemotherapeutic drug-mediated cytotoxicity. Thus, the TC-PET and TC-DCM may harbor phytochemicals with the potential to reverse the drug-resistant phenotype, thus improving the efficacy of cancer chemotherapy.

DNA methylation analysis of phenotype specific stratified Indian population

Background: DNA methylation and its perturbations are an established attribute to a wide spectrum of phenotypic variations and disease conditions. Indian traditional system practices personalized medicine through indigenous concept of distinctly descriptive physiological, psychological and anatomical features known as prakriti. Here we attempted to establish DNA methylation differences in these three prakriti phenotypes. Methods: Following structured and objective measurement of 3416 subjects, whole blood DNA of 147 healthy male individuals belonging to defined prakriti (Vata, Pitta and Kapha) between the age group of 20-30years were subjected to methylated DNA immunoprecipitation (MeDIP) and microarray analysis. After data analysis, prakriti specific signatures were validated through bisulfite DNA sequencing. Results: Differentially methylated regions in CpG islands and shores were significantly enriched in promoters/UTRs and gene body regions. Phenotypes characterized by higher metabolism (Pitta prakriti) in individuals showed distinct promoter (34) and gene body methylation (204), followed by Vata prakriti which correlates to motion showed DNA methylation in 52 promoters and 139 CpG islands and finally individuals with structural attributes (Kapha prakriti) with 23 and 19 promoters and CpG islands respectively. Bisulfite DNA sequencing of prakriti specific multiple CpG sites in promoters and 5'-UTR such as; LHX1 (Vata prakriti), SOX11 (Pitta prakriti) and CDH22 (Kapha prakriti) were validated. Kapha prakriti specific CDH22 5'-UTR CpG methylation was also found to be associated with higher body mass index (BMI). Conclusion: Differential DNA methylation signatures in three distinct prakriti phenotypes demonstrate the epigenetic basis of Indian traditional human classification which may have relevance to personalized medicine.

Phylogeography and non-invasive landscape genetics of the European pine marten ("martes martes" L. 1758) : insights into ancient and contemporary processes shaping genetic variation

283 p. : graf., map.

A Comparison of the SNP Variation in the Calcification PMCA Gene of Lottia gigantea between a Santa Barbara Population and a More Acidic Monterey Bay Population

As the atmospheric levels of CO2 rise from human activity, the carbonic acid levels of the ocean increase, causing ocean acidification. This increase in acidity breaks down the calcified bodies that many marine organisms depend upon. Upwelling regions such as Monterey Bay in California have pH levels that are not expected to reach the open ocean for a few decades. This study reviews one of the common intertidal animals of the California coast, the Owl Limpet Lottia gigantea, and its genetic variation of the plasma membrane Ca2+ ATPase (PMCA) in relation to the acidity of its environment. The PMCA protein functions in the calcification process of many organisms. Specifically in limpets, this gene functions to form its protective shell. Single-nucleotide polymorphisms (SNPs) were found among five sections of the gene to determine variation between the acidic environment population in Monterey, California and the non-acidic environment population in Santa Barbara, California. While some variation was determined, the Monterey Bay and Santa Barbara Lottia gigantea populations are not significantly distinct at the PMCA gene. Sections B, C, and D were found to be linked. Only one location in Section B was found to have an amino acid change within an exon. Section A has the strongest connection to the sampling location. Monterey individuals were seen to be more genetically recognizable, while Santa Barbara individuals showed slightly more variation. Understanding the trends of ocean acidification, upwelling region activities, and population genetics will assist in determining how the ocean environment will behave in the future.

Landscape Genetics for the Empirical Assessment of Resistance Surfaces: The European Pine Marten (Martes martes) as a Target-Species of a Regional Ecological Network

Coherent ecological networks (EN) composed of core areas linked by ecological corridors are being developed worldwide with the goal of promoting landscape connectivity and biodiversity conservation. However, empirical assessment of the performance of EN designs is critical to evaluate the utility of these networks to mitigate effects of habitat loss and fragmentation. Landscape genetics provides a particularly valuable framework to address the question of functional connectivity by providing a direct means to investigate the effects of landscape structure on gene flow. The goals of this study are (1) to evaluate the landscape features that drive gene flow of an EN target species (European pine marten), and (2) evaluate the optimality of a regional EN design in providing connectivity for this species within the Basque Country (North Spain). Using partial Mantel tests in a reciprocal causal modeling framework we competed 59 alternative models, including isolation by distance and the regional EN. Our analysis indicated that the regional EN was among the most supported resistance models for the pine marten, but was not the best supported model. Gene flow of pine marten in northern Spain is facilitated by natural vegetation, and is resisted by anthropogenic landcover types and roads. Our results suggest that the regional EN design being implemented in the Basque Country will effectively facilitate gene flow of forest dwelling species at regional scale.

Inferring Population Genetic Structure in Widely and Continuously Distributed Carnivores: The Stone Marten (Martes foina) as a Case Study

The stone marten is a widely distributed mustelid in the Palaearctic region that exhibits variable habitat preferences in different parts of its range. The species is a Holocene immigrant from southwest Asia which, according to fossil remains, followed the expansion of the Neolithic farming cultures into Europe and possibly colonized the Iberian Peninsula during the Early Neolithic (ca. 7,000 years BP). However, the population genetic structure and historical biogeography of this generalist carnivore remains essentially unknown. In this study we have combined mitochondrial DNA (mtDNA) sequencing (621 bp) and microsatellite genotyping (23 polymorphic markers) to infer the population genetic structure of the stone marten within the Iberian Peninsula. The mtDNA data revealed low haplotype and nucleotide diversities and a lack of phylogeographic structure, most likely due to a recent colonization of the Iberian Peninsula by a few mtDNA lineages during the Early Neolithic. The microsatellite data set was analysed with a) spatial and non-spatial Bayesian individual-based clustering (IBC) approaches (STRUCTURE, TESS, BAPS and GENELAND), and b) multivariate methods [discriminant analysis of principal components (DAPC) and spatial principal component analysis (sPCA)]. Additionally, because isolation by distance (IBD) is a common spatial genetic pattern in mobile and continuously distributed species and it may represent a challenge to the performance of the above methods, the microsatellite data set was tested for its presence. Overall, the genetic structure of the stone marten in the Iberian Peninsula was characterized by a NE-SW spatial pattern of IBD, and this may explain the observed disagreement between clustering solutions obtained by the different IBC methods. However, there was significant indication for contemporary genetic structuring, albeit weak, into at least three different subpopulations. The detected subdivision could be attributed to the influence of the rivers Ebro, Tagus and Guadiana, suggesting that main watercourses in the Iberian Peninsula may act as semi-permeable barriers to gene flow in stone martens. To our knowledge, this is the first phylogeographic and population genetic study of the species at a broad regional scale. We also wanted to make the case for the importance and benefits of using and comparing multiple different clustering and multivariate methods in spatial genetic analyses of mobile and continuously distributed species.

Perspectives from agriculture: advances in livestock breeding - implications for aquaculture genetics

In this paper we present livestock breeding developments that could be taken into consideration in the genetic improvement of farmed aquaculture species, especially in freshwater fish. Firstly, the current breeding objective in aquatic species has focused almost exclusively on the improvement of body weight at harvest or on growth related traits. This is unlikely to be sufficient to meet the future needs of the aquaculture industry. To meet future demands breeding programs will most likely have to include additional traits, such as fitness related ones (survival, disease resistance), feed efficiency, or flesh quality, rather than only growth performance. In order to select for a multi-trait breeding objective, genetic variation in traits of interest and the genetic relationships among them need to be estimated. In addition, economic values for these traits will be required. Generally, there is a paucity of data on variable and fixed production costs in aquaculture, and this could be a major constraint in the further expansion of the breeding objectives. Secondly, genetic evaluation systems using the restricted maximum likelihood method (REML) and best linear unbiased prediction (BLUP) in a framework of mixed model methodology could be widely adopted to replace the more commonly used method of mass selection based on phenotypic performance. The BLUP method increases the accuracy of selection and also allows the management of inbreeding and estimation of genetic trends. BLUP is an improvement over the classic selection index approach, which was used in the success story of the genetically improved farmed tilapia (GIFT) in the Philippines, with genetic gains from 10 to 20 per cent per generation of selection. In parallel with BLUP, optimal genetic contribution theory can be applied to maximize genetic gain while constraining inbreeding in the long run in selection programs. Thirdly, by using advanced statistical methods, genetic selection can be carried out not only at the nucleus level but also in lower tiers of the pyramid breeding structure. Large scale across population genetic evaluation through genetic connectedness using cryopreserved sperm enables the comparison and ranking of genetic merit of all animals across populations, countries or years, and thus the genetically superior brood stock can be identified and widely used and exchanged to increase the rate of genetic progress in the population as a whole. It is concluded that sound genetic programs need to be established for aquaculture species. In addition to being very effective, fully pedigreed breeding programs would also enable the exploration of possibilities of integrating molecular markers (e.g., genetic tagging using DNA fingerprinting, marker (gene) assisted selection) and reproductive technologies such as in-vitro fertilization using cryopreserved spermatozoa.