Genetic Heterogeneity in Autism Spectrum Disorders in a Population Isolate

Positional cloning has enabled hypothesis-free, genome-wide scans for genetic factors contributing to disorders or traits. Traditionally linkage analysis has been used to identify regions of interest, followed by meticulous fine mapping and candidate gene screening using association methods and finally sequencing of regions of interest. More recently, genome-wide association analysis has enabled a more direct approach to identify specific genetic variants explaining a part of the variance of the phenotype of interest. Autism spectrum disorders (ASDs) are a group of childhood onset neuropsychiatric disorders with shared core symptoms but varying severity. Although a strong genetic component has been established in ASDs, genetic susceptibility factors have largely eluded characterization. Here, we have utilized modern molecular genetic methods combined with the advantages provided by the special population structure in Finland to identify genetic risk factors for ASDs. The results of this study show that numerous genetic risk factors exist for ASDs even within a population isolate. Stratification based on clinical phenotype resulted in encouraging results, as previously identified linkage to 3p14-p24 was replicated in an independent family set of families with Asperger syndrome, but no other ASDs. Fine-mapping of the previously identified linkage peak for ASDs at 3q25-q27 revealed association between autism and a subunit of the 5-hydroxytryptamine receptor 3C (HTR3C). We also used dense, genome-wide single nucleotide polymorphism (SNP) data to characterize the population structure of Finns. We observed significant population substructure which correlates with the known history of multiple consecutive bottle-necks experienced by the Finnish population. We used this information to ascertain a genetically homogenous subset of autism families to identify possible rare, enriched risk variants using genome-wide SNP data. No rare enriched genetic risk factors were identified in this dataset, although a subset of families could be genealogically linked to form two extended pedigrees. The lack of founder mutations in this isolated population suggests that the majority of genetic risk factors are rare, de novo mutations unique to individual nuclear families. The results of this study are consistent with others in the field. The underlying genetic architecture for this group of disorders appears highly heterogeneous, with common variants accounting for only a subset of genetic risk. The majority of identified risk factors have turned out to be exceedingly rare, and only explain a subset of the genetic risk in the general population in spite of their high penetrance within individual families. The results of this study, together with other results obtained in this field, indicate that family specific linkage, homozygosity mapping and resequencing efforts are needed to identify these rare genetic risk factors.

Molecular Tuning of Rhodopsins for High Visual Sensitivity in Different Light Environments : Variation in Absorbance Spectrum and Opsin Sequence within and between Species

Visual pigments of different animal species must have evolved at some stage to match the prevailing light environments, since all visual functions depend on their ability to absorb available photons and transduce the event into a reliable neural signal. There is a large literature on correlation between the light environment and spectral sensitivity between different fish species. However, little work has been done on evolutionary adaptation between separated populations within species. More generally, little is known about the rate of evolutionary adaptation to changing spectral environments. The objective of this thesis is to illuminate the constraints under which the evolutionary tuning of visual pigments works as evident in: scope, tempo, available molecular routes, and signal/noise trade-offs. Aquatic environments offer Nature s own laboratories for research on visual pigment properties, as naturally occurring light environments offer an enormous range of variation in both spectral composition and intensity. The present thesis focuses on the visual pigments that serve dim-light vision in two groups of model species, teleost fishes and mysid crustaceans. The geographical emphasis is in the brackish Baltic Sea area with its well-known postglacial isolation history and its aquatic fauna of both marine and fresh-water origin. The absorbance spectrum of the (single) dim-light visual pigment were recorded by microspectrophotometry (MSP) in single rods of 26 fish species and single rhabdoms of 8 opossum shrimp populations of the genus Mysis inhabiting marine, brackish or freshwater environments. Additionally, spectral sensitivity was determined from six Mysis populations by electroretinogram (ERG) recording. The rod opsin gene was sequenced in individuals of four allopatric populations of the sand goby (Pomatoschistus minutus). Rod opsins of two other goby species were investigated as outgroups for comparison. Rod absorbance spectra of the Baltic subspecies or populations of the primarily marine species herring (Clupea harengus membras), sand goby (P. minutus), and flounder (Platichthys flesus) were long-wavelength-shifted compared to their marine populations. The spectral shifts are consistent with adaptation for improved quantum catch (QC) as well as improved signal-to-noise ratio (SNR) of vision in the Baltic light environment. Since the chromophore of the pigment was pure A1 in all cases, this has apparently been achieved by evolutionary tuning of the opsin visual pigment. By contrast, no opsin-based differences were evident between lake and sea populations of species of fresh-water origin, which can tune their pigment by varying chromophore ratios. A more detailed analysis of differences in absorbance spectra and opsin sequence between and within populations was conducted using the sand goby as model species. Four allopatric populations from the Baltic Sea (B), Swedish west coast (S), English Channel (E), and Adriatic Sea (A) were examined. Rod absorbance spectra, characterized by the wavelength of maximum absorbance (λmax), differed between populations and correlated with differences in the spectral light transmission of the respective water bodies. The greatest λmax shift as well as the greatest opsin sequence difference was between the Baltic and the Adriatic populations. The significant within-population variation of the Baltic λmax values (506-511 nm) was analyzed on the level of individuals and was shown to correlate well with opsin sequence substitutions. The sequences of individuals with λmax at shorter wavelengths were identical to that of the Swedish population, whereas those with λmax at longer wavelengths additionally had substitution F261F/Y in the sixth transmembrane helix of the protein. This substitution (Y261) was also present in the Baltic common gobies and is known to redshift spectra. The tuning mechanism of the long-wavelength type Baltic sand gobies is assumed to be the co-expression of F261 and Y261 in all rods to produce ≈ 5 nm redshift. The polymorphism of the Baltic sand goby population possibly indicates ambiguous selection pressures in the Baltic Sea. The visual pigments of all lake populations of the opossum shrimp (Mysis relicta) were red-shifted by 25 nm compared with all Baltic Sea populations. This is calculated to confer a significant advantage in both QC and SNR in many humus-rich lakes with reddish water. Since only A2 chromophore was present, the differences obviously reflect evolutionary tuning of the visual protein, the opsin. The changes have occurred within the ca. 9000 years that the lakes have been isolated from the Sea after the most recent glaciation. At present, it seems that the mechanism explaining the spectral differences between lake and sea populations is not an amino acid substitution at any other conventional tuning site, but the mechanism is yet to be found.

Primordial Isocurvature Perturbation in Light of CMB and LSS Data

The increased accuracy in the cosmological observations, especially in the measurements of the comic microwave background, allow us to study the primordial perturbations in grater detail. In this thesis, we allow the possibility for a correlated isocurvature perturbations alongside the usual adiabatic perturbations. Thus far the simplest six parameter \Lambda CDM model has been able to accommodate all the observational data rather well. However, we find that the 3-year WMAP data and the 2006 Boomerang data favour a nonzero nonadiabatic contribution to the CMB angular power sprctrum. This is primordial isocurvature perturbation that is positively correlated with the primordial curvature perturbation. Compared with the adiabatic \Lambda CMD model we have four additional parameters describing the increased complexity if the primordial perturbations. Our best-fit model has a 4% nonadiabatic contribution to the CMB temperature variance and the fit is improved by \Delta\chi^2 = 9.7. We can attribute this preference for isocurvature to a feature in the peak structure of the angular power spectrum, namely, the widths of the second and third acoustic peak. Along the way, we have improved our analysis methods by identifying some issues with the parametrisation of the primordial perturbation spectra and suggesting ways to handle these. Due to the improvements, the convergence of our Markov chains is improved. The change of parametrisation has an effect on the MCMC analysis because of the change in priors. We have checked our results against this and find only marginal differences between our parametrisation.

Non-Gaussian Cosmological Perturbations from Hybrid Inflation and Preheating

This thesis consists of four research papers and an introduction providing some background. The structure in the universe is generally considered to originate from quantum fluctuations in the very early universe. The standard lore of cosmology states that the primordial perturbations are almost scale-invariant, adiabatic, and Gaussian. A snapshot of the structure from the time when the universe became transparent can be seen in the cosmic microwave background (CMB). For a long time mainly the power spectrum of the CMB temperature fluctuations has been used to obtain observational constraints, especially on deviations from scale-invariance and pure adiabacity. Non-Gaussian perturbations provide a novel and very promising way to test theoretical predictions. They probe beyond the power spectrum, or two point correlator, since non-Gaussianity involves higher order statistics. The thesis concentrates on the non-Gaussian perturbations arising in several situations involving two scalar fields, namely, hybrid inflation and various forms of preheating. First we go through some basic concepts -- such as the cosmological inflation, reheating and preheating, and the role of scalar fields during inflation -- which are necessary for the understanding of the research papers. We also review the standard linear cosmological perturbation theory. The second order perturbation theory formalism for two scalar fields is developed. We explain what is meant by non-Gaussian perturbations, and discuss some difficulties in parametrisation and observation. In particular, we concentrate on the nonlinearity parameter. The prospects of observing non-Gaussianity are briefly discussed. We apply the formalism and calculate the evolution of the second order curvature perturbation during hybrid inflation. We estimate the amount of non-Gaussianity in the model and find that there is a possibility for an observational effect. The non-Gaussianity arising in preheating is also studied. We find that the level produced by the simplest model of instant preheating is insignificant, whereas standard preheating with parametric resonance as well as tachyonic preheating are prone to easily saturate and even exceed the observational limits. We also mention other approaches to the study of primordial non-Gaussianities, which differ from the perturbation theory method chosen in the thesis work.

Measuring the Cosmic Microwave Background : Topics along the analysis pipeline

The first quarter of the 20th century witnessed a rebirth of cosmology, study of our Universe, as a field of scientific research with testable theoretical predictions. The amount of available cosmological data grew slowly from a few galaxy redshift measurements, rotation curves and local light element abundances into the first detection of the cos- mic microwave background (CMB) in 1965. By the turn of the century the amount of data exploded incorporating fields of new, exciting cosmological observables such as lensing, Lyman alpha forests, type Ia supernovae, baryon acoustic oscillations and Sunyaev-Zeldovich regions to name a few. -- CMB, the ubiquitous afterglow of the Big Bang, carries with it a wealth of cosmological information. Unfortunately, that information, delicate intensity variations, turned out hard to extract from the overall temperature. Since the first detection, it took nearly 30 years before first evidence of fluctuations on the microwave background were presented. At present, high precision cosmology is solidly based on precise measurements of the CMB anisotropy making it possible to pinpoint cosmological parameters to one-in-a-hundred level precision. The progress has made it possible to build and test models of the Universe that differ in the way the cosmos evolved some fraction of the first second since the Big Bang. -- This thesis is concerned with the high precision CMB observations. It presents three selected topics along a CMB experiment analysis pipeline. Map-making and residual noise estimation are studied using an approach called destriping. The studied approximate methods are invaluable for the large datasets of any modern CMB experiment and will undoubtedly become even more so when the next generation of experiments reach the operational stage. -- We begin with a brief overview of cosmological observations and describe the general relativistic perturbation theory. Next we discuss the map-making problem of a CMB experiment and the characterization of residual noise present in the maps. In the end, the use of modern cosmological data is presented in the study of an extended cosmological model, the correlated isocurvature fluctuations. Current available data is shown to indicate that future experiments are certainly needed to provide more information on these extra degrees of freedom. Any solid evidence of the isocurvature modes would have a considerable impact due to their power in model selection.

Genetic Mechanisms Underlying Autism Spectrum Disorders

Autism is a childhood-onset developmental disorder characterized by deficits in reciprocal social interaction, verbal and non-verbal communication, and dependence on routines and rituals. It belongs to a spectrum of disorders (autism spectrum disorders, ASDs) which share core symptoms but show considerable variation in severity. The whole spectrum affects 0.6-0.7% of children worldwide, inducing a substantial public health burden and causing suffering to the affected families. Despite having a very high heritability, ASDs have shown exceptional genetic heterogeneity, which has complicated the identification of risk variants and left the etiology largely unknown. However, recent studies suggest that rare, family-specific factors contribute significantly to the genetic basis of ASDs. In this study, we investigated the role of DISC1 (Disrupted-in-schizophrenia-1) in ASDs, and identified association with markers and haplotypes previously associated with psychiatric phenotypes. We identified four polymorphic micro-RNA target sites in the 3 UTR of DISC1, and showed that hsa-miR-559 regulates DISC1 expression in vitro in an allele-specific manner. We also analyzed an extended autism pedigree with genealogical roots in Central Finland reaching back to the 17th century. To take advantage of the beneficial characteristics of population isolates to gene mapping and reduced genetic heterogeneity observed in distantly related individuals, we performed a microsatellite-based genome-wide screen for linkage and linkage disequilibrium in this pedigree. We identified a putative autism susceptibility locus on chromosome 19p13.3 and obtained further support for previously reported loci at 1q23 and 15q11-q13. To follow-up these findings, we extended our study sample from the same sub-isolate and initiated a genome-wide analysis of homozygosity and allelic sharing using high-density SNP markers. We identified a small number of haplotypes shared by different subsets of the genealogically connected cases, along with convergent biological pathways from SNP and gene expression data, which highlighted axon guidance molecules in the pathogenesis of ASDs. In conclusion, the results obtained in this thesis show that multiple distinct genetic variants are responsible for the ASD phenotype even within single pedigrees from an isolated population. We suggest that targeted resequencing of the shared haplotypes, linkage regions, and other susceptibility loci is essential to identify the causal variants. We also report a possible micro-RNA mediated regulatory mechanism, which might partially explain the wide-range neurobiological effects of the DISC1 gene.

Evidence for a Narrow Near-Threshold Structure in the J/ψϕ Mass Spectrum in B+→J/ψϕK+ Decays

Evidence is reported for a narrow structure near the $J/\psi\phi$ threshold in exclusive $B^+\to J/\psi\phi K^+$ decays produced in $\bar{p} p $ collisions at $\sqrt{s}=1.96 \TeV$. A signal of $14\pm5$ events, with statistical significance in excess of 3.8 standard deviations, is observed in a data sample corresponding to an integrated luminosity of $2.7 \ifb$, collected by the CDF II detector. The mass and natural width of the structure are measured to be $4143.0\pm2.9(\mathrm{stat})\pm1.2(\mathrm{syst}) \MeVcc$ and $11.7^{+8.3}_{-5.0}(\mathrm{stat})\pm3.7(\mathrm{syst}) \MeVcc$.

Detection, epidemiology and host spectrum of cowpox and Borna disease virus infections

Several orthopoxviruses (OPV) and Borna disease virus (BDV) are enveloped, zoonotic viruses with a wide geographical distribution. OPV antibodies cross-react, and former smallpox vaccination has therefore protected human populations from another OPV infection, rodent-borne cowpox virus (CPXV). Cowpox in humans and cats usually manifests as a mild, self-limiting dermatitis and constitutional symptoms, but it can be severe and even life-threatening in the immunocompromised. Classical Borna disease is a progressive meningoencephalomyelitis in horses and sheep known in central Europe for centuries. Nowadays the virus or its close relative infects humans and also several other species in central Europe and elsewhere, but the existence of human Borna disease with its suspected neuropsychiatric symptoms is controversial. The epidemiology of BDV is largely unknown, and the present situation is even more intriguing following the recent detection of several-million-year-old, endogenized BDV genes in primate and various other vertebrate genomes. The aims of this study were to elucidate the importance of CPXV and BDV in Finland and in possible host species, and particularly to 1) establish relevant methods for the detection of CPXV and other OPVs as well as BDV in Finland, 2) determine whether CPXV and BDV exist in Finland, 3) discover how common OPV immunity is in different age groups in Finland, 4) characterize possible disease cases and clarify their epidemiological context, 5) establish the hosts and possible reservoir species of these viruses and their geographical distribution in wild rodents, and 6) elucidate the infection kinetics of BDV in the bank vole. An indirect immunofluorescence assay and avidity measurement were established for the detection, timing and verification of OPV or BDV antibodies in thousands of blood samples from humans, horses, ruminants, lynxes, gallinaceous birds, dogs, cats and rodents. The mostly vaccine-derived OPV seroprevalence was found to decrease gradually according to the year of birth of the sampled human subjects from 100% to 10% in those born after 1977. On the other hand, OPV antibodies indicating natural contact with CPXV or other OPVs were commonly found in domestic and wild animals: the horse, cow, lynx, dog, cat and, with a prevalence occasionally even as high as 92%, in wild rodents, including some previously undetected species and new regions. Antibodies to BDV were detected in humans, horses, a dog, cats, and for the first time in wild rodents, such as bank voles (Myodes glareolus). Because of the controversy within the human Borna disease field, extra verification methods were established for BDV antibody findings: recombinant nucleocapsid and phosphoproteins were produced in Escherichia coli and in a baculovirus system, and peptide arrays were additionally applied. With these verification assays, Finnish human, equine, feline and rodent BDV infections were confirmed. Taken together, wide host spectra were evident for both OPV and BDV infections based on the antibody findings, and OPV infections were found to be geographically broadly distributed. PCR amplification methods were utilised for hundreds of blood and tissue samples. The methods included conventional, nested and real-time PCRs with or without the reverse transcription step and detecting four or two genes of OPVs and BDV, respectively. OPV DNA could be amplified from two human patients and three bank voles, whereas no BDV RNA was detected in naturally infected individuals. Based on the phylogenetic analyses, the Finnish OPV sequences were closely related although not identical to a Russian CPXV isolate, and clearly different from other CPXV strains. Moreover, the Finnish sequences only equalled each other, but the short amplicons obtained from German rodents were identical to monkeypox virus, in addition to German CPXV variants. This reflects the close relationship of all OPVs. In summary, RNA of the Finnish BDV variant could not be detected with the available PCR methods, but OPV DNA infrequently could. The OPV species infecting the patients of this study was proven to be CPXV, which is most probably also responsible for the rodent infections. Multiple cell lines and some newborn rodents were utilised in the isolation of CPXV and BDV from patient and wildlife samples. CPXV could be isolated from a child with severe, generalised cowpox. BDV isolation attempts from rodents were unsuccessful in this study. However, in parallel studies, a transient BDV infection of cells inoculated with equine brain material was detected, and BDV antigens discovered in archival animal brains using established immunohistology. Thus, based on several independent methods, both CPXV and BDV (or a closely related agent) were shown to be present in Finland. Bank voles could be productively infected with BDV. This experimental infection did not result in notable pathological findings or symptoms, despite the intense spread of the virus in the central and peripheral nervous system. Infected voles commonly excreted the virus in urine and faeces, which emphasises their possible role as a BDV reservoir. Moreover, BDV RNA was regularly reverse transcribed into DNA in bank voles, which was detected by amplifying DNA by PCR without reverse transcription, and verified with nuclease treatments. This finding indicates that BDV genes could be endogenized during an acute infection. Although further transmission studies are needed, this experimental infection demonstrated that the bank vole can function as a potential BDV reservoir. In summary, multiple methods were established and applied in large panels to detect two zoonoses novel to Finland: cowpox virus and Borna disease virus. Moreover, new information was obtained on their geographical distribution, host spectrum, epidemiology and infection kinetics.