Spectral Tuning and Adaptation to Different Light Environments of Mysid Visual Pigments

In the present thesis, questions of spectral tuning, the relation of spectral and thermal properties of visual pigments, and evolutionary adaptation to different light environments were addressed using a group of small crustaceans of the genus Mysis as a model. The study was based on microspectrophotometric measurements of visual pigment absorbance spectra, electrophysiological measurements of spectral sensitivities of dark-adapted eyes, and sequencing of the opsin gene retrieved through PCR. The spectral properties were related to the spectral transmission of the respective light environments, as well as to the phylogentic histories of the species. The photoactivation energy (Ea) was estimated from temperature effects on spectral sensitivity in the long-wavelength range, and calculations were made for optimal quantum catch and optimal signal-to-noise ratio in the different light environments. The opsin amino acid sequences of spectrally characterized individuals were compared to find candidate residues for spectral tuning. The general purpose was to clarify to what extent and on what time scale adaptive evolution has driven the functional properties of (mysid) visual pigments towards optimal performance in different light environments. An ultimate goal was to find the molecular mechanisms underlying the spectral tuning and to understand the balance between evolutionary adaptation and molecular constraints. The totally consistent segregation of absorption maxima (λmax) into (shorter-wavelength) marine and (longer-wavelength) freshwater populations suggests that truly adaptive evolution is involved in tuning the visual pigment for optimal performance, driven by selection for high absolute visual sensitivity. On the other hand, the similarity in λmax and opsin sequence between several populations of freshwater M. relicta in spectrally different lakes highlights the limits to adaptation set by evolutionary history and time. A strong inverse correlation between Ea and λmax was found among all visual pigments studied in these respects, including those of M. relicta and 10 species of vertebrate pigments, and this was used to infer thermal noise. The conceptual signal-to-noise ratios thus calculated for pigments with different λmax in the Baltic Sea and Lake Pääjärvi light environments supported the notion that spectral adaptation works towards maximizing the signal-to-noise ratio rather than quantum catch as such. Judged by the shape of absorbance spectra, the visual pigments of all populations of M. relicta and M. salemaai used exclusively the A2 chromophore (3, 4-dehydroretinal). A comparison of amino acid substitutions between M. relicta and M. salemaai indicated that mysid shrimps have a small number of readily available tuning sites to shift between a shorter - and a longer -wavelength opsin. However, phylogenetic history seems to have prevented marine M. relicta from converting back to the (presumably) ancestral opsin form, and thus the more recent reinvention of marine spectral sensitivity has been accomplished by some other novel mechanism, yet to be found

Nephrin-associated molecules in human glomerular diseases

The glomerular epithelial cells and their intercellular junctions, termed slit diaphragms, are essential components of the filtration barrier in the kidney glomerulus. Nephrin is a transmembrane adhesion protein of the slit diaphragm and a signalling molecule regulating podocyte physiology. In congenital nephrotic syndrome of the Finnish type, mutation of nephrin leads to disruption of the permeability barrier and leakage of plasma proteins into the urine. This doctoral thesis hypothesises that novel nephrin-associated molecules are involved in the function of the filtration barrier in health and disease. Bioinformatics tools were utilized to identify novel nephrin-like molecules in genomic databases, and their distribution in the kidney and other tissues was investigated. Filtrin, a novel nephrin homologue, is expressed in the glomerular podocytes and, according to immunoelectron microscopy, localizes at the slit diaphragm. Interestingly, the nephrin and filtrin genes, NPHS1 and KIRREL2, locate in a head-to-head orientation on chromosome 19q13.12. Another nephrin-like molecule, Nphs1as was cloned in mouse, however, no expression was detected in the kidney but instead in the brain and lymphoid tissue. Notably, Nphs1as is transcribed from the nephrin locus in an antisense orientation. The glomerular mRNA and protein levels of filtrin were measured in kidney biopsies of patients with proteinuric diseases, and marked reduction of filtrin mRNA levels was detected in the proteinuric samples as compared to controls. In addition, altered distribution of filtrin in injured glomeruli was observed, with the most prominent decrease of the expression in focal segmental glomerulosclerosis. The role of the slit diaphragm-associated genes for the development of diabetic nephropathy was investigated by analysing single nucleotide polymorphisms. The genes encoding filtrin, densin-180, NEPH1, podocin, and alpha-actinin-4 were analysed, and polymorphisms at the alpha-actinin-4 gene were associated with diabetic nephropathy in a gender-dependent manner. Filtrin is a novel podocyte-expressed protein with localization at the slit diaphragm, and the downregulation of filtrin seems to be characteristic for human proteinuric diseases. In the context of the crucial role of nephrin for the glomerular filter, filtrin appears to be a potential candidate molecule for proteinuria. Although not expressed in the kidney, the nephrin antisense Nphs1as may regulate the expression of nephrin in extrarenal tissues. The genetic association analysis suggested that the alpha-actinin-4 gene, encoding an actin-filament cross-linking protein of the podocytes, may contribute to susceptibility for diabetic nephropathy.

Molecular genetics of X-linked cone-rod dystrophy and Åland Island eye disease

Inherited retinal diseases are the most common cause of vision loss among the working population in Western countries. It is estimated that ~1 of the people worldwide suffer from vision loss due to inherited retinal diseases. The severity of these diseases varies from partial vision loss to total blindness, and at the moment no effective cure exists. To date, nearly 200 mapped loci, including 140 cloned genes for inherited retinal diseases have been identified. By a rough estimation 50% of the retinal dystrophy genes still await discovery. In this thesis we aimed to study the genetic background of two inherited retinal diseases, X-linked cone-rod dystrophy and Åland Island eye disease. X-linked cone-rod dystrophy (CORDX) is characterized by progressive loss of visual function in school age or early adulthood. Affected males show reduced visual acuity, photophobia, myopia, color vision defects, central scotomas, and variable changes in fundus. The disease is genetically heterogeneous and two disease loci, CORDX1 and CORDX2, were known prior to the present thesis work. CORDX1, located on Xp21.1-11.4, is caused by mutations in the RPGR gene. CORDX2 is located on Xq27-28 but the causative gene is still unknown. Åland Island eye disease (AIED), originally described in a family living in Åland Islands, is a congenital retinal disease characterized by decreased visual acuity, fundus hypopigmentation, nystagmus, astigmatism, red color vision defect, myopia, and defective night vision. AIED shares similarities with another retinal disease, congenital stationary night blindness (CSNB2). Mutations in the L-type calcium channel α1F-subunit gene, CACNA1F, are known to cause CSNB2, as well as AIED-like disease. The disease locus of the original AIED family maps to the same genetic interval as the CACNA1F gene, but efforts to reveal CACNA1F mutations in patients of the original AIED family have been unsuccessful. The specific aims of this study were to map the disease gene in a large Finnish family with X-linked cone-rod dystrophy and to identify the disease-causing genes in the patients of the Finnish cone-rod dystrophy family and the original AIED family. With the help of linkage and haplotype analyses, we could localize the disease gene of the Finnish cone-rod dystrophy family to the Xp11.4-Xq13.1 region, and thus establish a new genetic X-linked cone-rod dystrophy locus, CORDX3. Mutation analyses of candidate genes revealed three novel CACNA1F gene mutations: IVS28-1 GCGTC>TGG in CORDX3 patients, a 425 bp deletion, comprising exon 30 and flanking intronic regions in AIED patients, and IVS16+2T>C in an additional Finnish patient with a CSNB2-like phenotype. All three novel mutations altered splice sites of the CACNA1F gene, and resulted in defective pre-mRNA splicing suggesting altered or absent channel function as a disease mechanism. The analyses of CACNA1F mRNA also revealed novel alternative wt splice variants, which may enhance channel diversity or regulate the overall expression level of the channel. The results of our studies may be utilized in genetic counseling of the families, and they provide a basis for studies on the pathogenesis of these diseases. In the future, the knowledge of the genetic defects may be used in the identification of specific therapies for the patients.

SPECT-aided diagnostics and genetic risk factors in Parkinson's disease

Hypokinesia, rigidity, tremor, and postural instability are the cardinal symptoms of Parkinson s disease (PD). Since these symptoms are not specific to PD the diagnosis may be uncertain in early PD. Etiology and pathogenesis of PD remain unclear. There is no neuroprotective therapy. Genetic findings are expected to reveal metabolic routes in PD pathogenesis and thereby eventually lead to therapeutic innovations. In this thesis, we first aimed to study the usefulness and accuracy of 123I-b-CIT SPECT in the diagnosis of PD in a consecutive clinic-based material including various movement disorders. We subsequently a genetic project to identify genetic risk factors for sporadic PD using a candidate gene approach in a case-control setting including 147 sporadic PD patients and 137 spouse controls. Dopamine transporter imaging by 123I-b-CIT SPECT could distinguish PD from essential tremor, drug-induced parkinsonism, dystonia and psychogenic parkinsonism. However, b-CIT uptake in Parkinson plus syndromes (PSP and multiple system atrophy) and dementia with Lewy bodies was not significantly different from PD. 123I-b-CIT SPECT could not reliably differentiate PD from vascular parkinsonism. 123I-b-CIT SPECT was 100% sensitive and specific in the diagnosis of PD in patients younger than 55 years but less specific in older patients, due to differential distribution of the above conditions in the younger and older age groups. 123I-b-CIT SPECT correlated with symptoms and detected bilateral nigrostriatal defect in patients whose PD was still in unilateral stage. Thus, in addition to as a differential diagnostic aid, 123I-b-CIT SPECT may be used to detect PD early, even pre-symptomatically in at-risk individuals. 123I-b-CIT SPECT was used to aid in the collection of patients to the genetic studies. In the genetic part of this thesis we found an association between PD and a polymorphic CAG-repeat in POLG1 gene encoding the catalytic subunit of mitochondrial polymerase gamma. The CAG-repeat encodes a polyglutamine tract (polyQ), the two most common lengths of which are 10Q (86-90%) and 11Q. In our Finnish material, the rarer non-10Q or non-11Q length variants (6Q-9Q, 12Q-14Q, 4R+9Q) were more frequent in patients than in spouse controls (10% vs. 3.5 %, p=0.003), or population controls (p=0.001). Therefore, we performed a replication study in 652 North American PD patients and 292 controls. Non-10/11Q alleles were more common in the US PD patients compared to the controls but the difference did not reach statistical significance (p=0.07). This larger data suggested our original definition of variant length allele might need reconsideration. Most previous studies on phenotypic effects of POLG1 polyQ have defined 10Q as the only normal allele. Non-10Q alleles were significantly more common in patients compared to the controls (17.3% vs. 12.3 %, p= 0.005). This association between non-10Q length variants and PD remained significant when compared to a larger set of 1541 literature controls (p=0.00005). In conclusion, POLG1 polyQ alleles other than 10Q may predispose to PD. We did not find association between PD and parkin or DJ-1, genes underlying autosomal recessive parkinsonism. The functional Val158Met polymorphism, which affects the catalytic effect of COMT enzyme, and another coding polymorphism in COMT were not associated with PD in our patient material. The APOE e2/3/4 polymorphism modifies risk for Alzheimer s disease and prognosis of for example brain trauma. APOE promoter and enhancer polymorphisms 219G/T and +113G/C, and APOE e3 haplotypes, have also been shown to modify the risk of Alzheimer s disease but not reported in PD. No association was found between PD and APOE e2/3/4 polymorphism, the promoter or enhancer polymorphisms, or the e3 haplotypes.

ATM, RAD50 and p53 in breast cancer

Breast cancer is the most commonly occurring cancer among women, and its incidence is increasing worldwide. Positive family history is a well established risk factor for breast cancer, and it is suggested that the proportion of breast cancer that can be attributed to genetic factors may be as high as 30%. However, all the currently known breast cancer susceptibility genes are estimated to account for 20-30% of familial breast cancer, and only 5% of the total breast cancer incidence. It is thus likely that there are still other breast cancer susceptibility genes to be found. Cellular responses to DNA damage are crucial for maintaining genomic integrity and preventing the development of cancer. The genes operating in DNA damage response signaling network are thus good candidates for breast cancer susceptibility genes. The aim of this study was to evaluate the role of three DNA damage response associated genes, ATM, RAD50, and p53, in breast cancer. ATM, a gene causative for ataxia telangiectasia (A-T), has long been a strong candidate for a breast cancer susceptibility gene because of its function as a key DNA damage signal transducer. We analyzed the prevalence of known Finnish A-T related ATM mutations in large series of familial and unselected breast cancer cases from different geographical regions in Finland. Of the seven A-T related mutations, two were observed in the studied familial breast cancer patients. Additionally, a third mutation previously associated with breast cancer susceptibility was also detected. These founder mutations may be responsible for excess familial breast cancer regionally in Northern and Central Finland, but in Southern Finland our results suggest only a minor effect, if any, of any ATM genetic variants on familial breast cancer. We also screened the entire coding region of the ATM gene in 47 familial breast cancer patients from Southern Finland, and evaluated the identified variants in additional cases and controls. All the identified variants were too rare to significantly contribute to breast cancer susceptibility. However, the role of ATM in cancer development and progression was supported by the results of the immunohistochemical studies of ATM expression, as reduced ATM expression in breast carcinomas was found to correlate with tumor differentiation and hormone receptor status. Aberrant ATM expression was also a feature shared by the BRCA1/2 and the difficult-to-treat ER/PR/ERBB2-triple-negative breast carcinomas. From the clinical point of view, identification of phenotypic and genetic similarities between the BRCA1/2 and the triple-negative breast tumors could have an implication in designing novel targeted therapies to which both of these classes of breast cancer might be exceptionally sensitive. Mutations of another plausible breast cancer susceptibility gene, RAD50, were found to be very rare, and RAD50 can only be making a minor contribution to familial breast cancer predisposition in UK and Southern Finland. The Finnish founder mutation RAD50 687delT seems to be a null allele and may carry a small increased risk of breast cancer. RAD50 is not acting as a classical tumor suppressor gene, but it is possible that RAD50 haploinsufficiency is contributing to cancer. In addition to relatively rare breast cancer susceptibility alleles, common polymorphisms may also be associated with increased breast cancer risk. Furthermore, these polymorphisms may have an impact on the progression and outcome of the disease. Our results suggest no effect of the common p53 R72P polymorphism on familial breast cancer risk or breast cancer risk in the population, but R72P seems to be associated with histopathologic features of the tumors and survival of the patients; 72P homozygous genotype was an independent prognostic factor among the unselected breast cancer patients, with a two-fold increased risk of death. These results present important novel findings also with clinical significance, as codon 72 genotype could be a useful additional prognostic marker in breast cancer, especially among the subgroup of patients with wild-type p53 in their tumors.

Identification of genetic susceptibility loci for migraine

Migraine is the common cause of chronic episodic headache, affecting 12%-15% of the Caucasian population (41 million Europeans and some half a million Finns), and causes considerable loss of quality of life to its sufferers, as well as being linked to increased risk for a wide range of conditions, from depression to stroke. Migraine is the 19th most severe disease in terms of disability-adjusted life years, and 9th among women. It is characterized by attacks of headache accompanied by sensitivity to external stimuli lasting 4-72 hours, and in a third of cases by neurological aura symptoms, such as loss of vision, speech or muscle function. The underlying pathophysiology, including what triggers migraine attacks and why they occur in the first place, is largely unknown. The aim of this study was to identify genetic factors associated with the hereditary susceptibility to migraine, in order to gain a better understanding of migraine mechanisms. In this thesis, we report the results of genetic linkage and association analyses on a Finnish migraine patient collection as well as migraineurs from Australia, Denmark, Germany, Iceland and the Netherlands. Altogether we studied genetic information of nearly 7,000 migraine patients and over 50,000 population-matched controls. We also developed a new migraine analysis method called the trait component analysis, which is based on individual patient responses instead of the clinical diagnosis. Using this method, we detected a number of new genetic loci for migraine, including on chromosome 17p13 (HLOD 4.65) and 10q22-q23 (female-specific HLOD 7.68) with significant evidence of linkage, along with five other loci (2p12, 8q12, 4q28-q31, 18q12-q22, and Xp22) detected with suggestive evidence of linkage. The 10q22-q23 locus was the first genetic finding in migraine to show linkage to the same locus and markers in multiple populations, with consistent detection in six different scans. Traditionally, ion channels have been thought to play a role in migraine susceptibility, but we were able to exclude any significant role for common variants in a candidate gene study of 155 ion transport genes. This was followed up by the first genome-wide association study in migraine, conducted on 2,748 migraine patients and 10,747 matched controls followed by a replication in 3,209 patients and 40,062 controls. In this study, we found interesting results with genome-wide significance, providing targets for future genetic and functional studies. Overall, we found several promising genetic loci for migraine providing a promising base for future studies in migraine.

Studies on diamond-like carbon and novel diamond-like carbon polymer hybrid coatings deposited with filtered pulsed arc discharge method

The main obstacle for the application of high quality diamond-like carbon (DLC) coatings has been the lack of adhesion to the substrate as the coating thickness is increased. The aim of this study was to improve the filtered pulsed arc discharge (FPAD) method. With this method it is possible to achieve high DLC coating thicknesses necessary for practical applications. The energy of the carbon ions was measured with an optoelectronic time-of-flight method. An in situ cathode polishing system used for stabilizing the process yield and the carbon ion energies is presented. Simultaneously the quality of the coatings can be controlled. To optimise the quality of the deposition process a simple, fast and inexpensive method using silicon wafers as test substrates was developed. This method was used for evaluating the suitability of a simplified arc-discharge set-up for the deposition of the adhesion layer of DLC coatings. A whole new group of materials discovered by our research group, the diamond-like carbon polymer hybrid (DLC-p-h) coatings, is also presented. The parent polymers used in these novel coatings were polydimethylsiloxane (PDMS) and polytetrafluoroethylene (PTFE). The energy of the plasma ions was found to increase when the anode-cathode distance and the arc voltage were increased. A constant deposition rate for continuous coating runs was obtained with an in situ cathode polishing system. The novel DLC-p-h coatings were found to be water and oil repellent and harder than any polymers. The lowest sliding angle ever measured from a solid surface, 0.15 ± 0.03°, was measured on a DLC-PDMS-h coating. In the FPAD system carbon ions can be accelerated to high energies (≈ 1 keV) necessary for the optimal adhesion (the substrate is broken in the adhesion and quality test) of ultra thick (up to 200 µm) DLC coatings by increasing the anode-cathode distance and using high voltages (up to 4 kV). An excellent adhesion can also be obtained with the simplified arc-discharge device. To maintain high process yield (5µm/h over a surface area of 150 cm2) and to stabilize the carbon ion energies and the high quality (sp3 fraction up to 85%) of the resulting coating, an in situ cathode polishing system must be used. DLC-PDMS-h coating is the superior candidate coating material for anti-soiling applications where also hardness is required.

Genetic background of bipolar disorder and related cognitive impairments

Bipolar disorder (BP) is a complex psychiatric disorder characterized by episodes of mania and depression. BP affects approximately 1% of the world’s population and shows no difference in lifetime prevalence between males and females. BP arises from complex interactions among genetic, developmental and environmental factors, and it is likely that several predisposing genes are involved in BP. The genetic background of BP is still poorly understood, although intensive and long-lasting research has identified several chromosomal regions and genes involved in susceptibility to BP. This thesis work aims to identify the genetic variants that influence bipolar disorder in the Finnish population by candidate gene and genome-wide linkage analyses in families with many BP cases. In addition to diagnosis-based phenotypes, neuropsychological traits that can be seen as potential endophenotypes or intermediate traits for BP were analyzed. In the first part of the thesis, we examined the role of the allelic variants of the TSNAX/DISC1 gene cluster to psychotic and bipolar spectrum disorders and found association of distinct allelic haplotypes with these two groups of disorders. The haplotype at the 5’ end of the Disrupted-in-Schizophrenia-1 gene (DISC1) was over-transmitted to males with psychotic disorder (p = 0.008; for an extended haplotype p = 0.0007 with both genders), whereas haplotypes at the 3’ end of DISC1 associated with bipolar spectrum disorder (p = 0.0002; for an extended haplotype p = 0.0001). The variants of these haplotypes also showed association with different cognitive traits. The haplotypes at the 5’ end associated with perseverations and auditory attention, while the variants at the 3’ end associated with several cognitive traits including verbal fluency and psychomotor processing speed. Second, in our complete set of BP families with 723 individuals we studied six functional candidate genes from three distinct signalling systems: serotonin-related genes (SLC6A4 and TPH2), BDNF -related genes (BDNF, CREB1 and NTRK2) and one gene related to the inflammation and cytokine system (P2RX7). We replicated association of the functional variant Val66Met of BDNF with BP and better performance in retention. The variants at the 5’ end of SLC6A4 also showed some evidence of association among males (p = 0.004), but the widely studied functional variants did not yield any significant results. A protective four-variant haplotype on P2RX7 showed evidence of association with BP and executive functions: semantic and phonemic fluency (p = 0.006 and p = 0.0003, respectively). Third, we analyzed 23 bipolar families originating from the North-Eastern region of Finland. A genome-wide scan was performed using the 6K single nucleotide polymorphism (SNP) array. We identified susceptibility loci at chromosomes 7q31 with a LOD score of 3.20 and at 9p13.1 with a LOD score of 4.02. We followed up both linkage findings in the complete set of 179 Finnish bipolar families. The finding on chromosome 9p13 was supported (maximum LOD score of 3.02), but the susceptibility gene itself remains unclarified. In the fourth part of the thesis, we wanted to test the role of the allelic variants that have associated with bipolar disorder in recent genome-wide association studies (GWAS). We could confirm findings for the DFNB31, SORCS2, SCL39A3, and DGKH genes. The best signal in this study comes from DFNB31, which remained significant after multiple testing corrections. Two variants of SORCS2 were allelic replications and presented the same signal as the haplotype analysis. However, no association was detected with the PALB2 gene, which was the most significantly associated region in the previous GWAS. Our results indicate that BP is heterogeneous and its genetic background may accordingly vary in different populations. In order to fully understand the allelic heterogeneity that underlies common diseases such as BP, complete genome sequencing for many individuals with and without the disease is required. Identification of the specific risk variants will help us better understand the pathophysiology underlying BP and will lead to the development of treatments with specific biochemical targets. In addition, it will further facilitate the identification of environmental factors that alter risk, which will potentially provide improved occupational, social and psychological advice for individuals with high risk of BP.

Identification of loci for migraine with aura

Common migraine, i.e. migraine with (MA) or without aura (MO), is a chronic neurological disorder affecting about 10% of the Caucasian population. In MA, migraine headache is preceded by visual, sensoric and/or dysphasic reversible aura symptoms. Twin and family studies have suggested a multifactorial mode of inheritance for common migraine, and a stronger genetic component for MA than for MO. Since there is no biological or genetic marker to identify common migraine, aura symptoms provide a distinctive character to identify those suspected of suffering from migraine. The aim of this study was to identify MA susceptibility loci in well-phenotyped migraine samples with familial predisposition using different gene mapping methods. Genes coding for endothelin1 and its receptors EDNRA and ENDRB are potential candidate genes for cortical spreading depression (CSD), which is considered to be the underlying mechanism of migraine aura. The role of these genes in MA was studied in 850 Finnish migraine cases and 890 control individuals. Rare homozygous EDNRA SNPs showed nominal association with MA and with the age of onset trait (20 years). This result was also detected in the pooled analysis on 648 German MA cases and 651 control individuals when the test was adjusted for gender and sample origin. Evaluation of SNP genotyping reactions with two different DNA polymerase enzymes ensured that the genotype quality was high, and thus the discovered associations are considered reliable. The role of the 19p13 region was studied in a linkage analysis of 72 Finnish MA families. This region contains two migraine-associated genes: CACNA1A, which is associated with a predisposition to a rare Mendelian form of MA, familial hemiplegic migraine (FHM), and the insulin receptor gene (INSR) that is associated with common migraine. No evidence of linkage between the 19p13 and MA was detected. A novel visual aura locus was mapped to chromosome 9q21-q22 with significant evidence of linkage using a genome-wide linkage approach in 36 Finnish MA families. Five additional, potential loci were also detected. The 9q21-q22 region has previously been linked to occipitotemporal lobe epilepsy and MA, both of which involve prominent visual symptoms. Our result further supports a shared background for these episodic disorders.

Genetic studies on Finnish lupus erythematosus patients with cutaneous manifestations

Lupus erythematosus (LE) is a chronic, heterogeneous autoimmune disorder with abnormal immune responses, including production of autoantibodies and immune complexes. Clinical presentations of the disease range from mild cutaneous manifestations to a more generalised systemic involvement of internal organs. Cutaneous (CLE) forms are further subclassified into discoid LE (DLE), subacute cutaneous LE (SCLE) and acute cutaneous lupus erythematosus (ACLE), and may later progress to systemic disease (SLE). Both genetic and environmental factors contribute to the disease, although the precise aetiology is still elusive. Furthermore, complex gene-gene or gene-environment interactions may result in different subphenotypes of lupus. The genetic background of CLE is poorly known and only a few genes are confirmed, while the number of robust genetic associations in SLE exceeds 30. The aim of this thesis was to characterise the recruited patients clinically, and identify genetic variants conferring susceptibility to cutaneous variants of LE. Given that cutaneous and systemic disease may share underlying genetic factors, putative CLE candidate genes for genotyping were selected among those showing strong evidence of association in SLE. The correlation between relevant clinical manifestations and risk genotypes was investigated in order to find specific subphenotype associations. In addition, epistatic interactions in SLE were studied. Finally, the role of tissue degrading matrix metalloproteinases (MMP) in LE tissue injury was explored. These studies were conducted in Finnish case-control and family cohort, and Swedish case-control cohort. The clinical picture of the patients in terms of cutaneous, haematological and immunological findings resembled that described in the contemporary literature. However, the proportion of daily smokers was very high supporting the role of smoking in disease aetiology. The results confirmed that, even though clinically distinct entities, CLE and SLE share predisposing genetic factors. For the first time it was shown that known SLE susceptibility genes IRF5 and TYK2 also increase the risk of CLE. A tendency toward gene-gene interaction between these genes was found in SLE. As a remarkable novel finding, it was observed that ITGAM polymorphisms associated even more strongly to DLE than SLE, and the risk estimates were substantially higher than those reported for SLE. Several other recently identified SLE susceptibility genes showed signs of good or modest association especially in DLE. Subphenotype analyses indicated possible associations to clinical features, but marginally significant results reflected lack of sufficient power for these studies. Thorough immunohistochemical analyses of several MMPs demonstrated a role in epidermal changes and dermal tissue remodelling in diseased skin, and suggested that targeted action using selective MMP inhibitors may reduce lupus-induced damage in inflamed tissues. In conclusion, the results provide an insight into the genetics of CLE and demonstrate that genetic predisposition is at least in part shared between cutaneous and systemic variants of LE. This doctoral study has contributed IRF5, TYK2, ITGAM and several other novel genes to the so far short list of genes implicated in CLE susceptibility. Detailed examination of the function of these genes in CLE pathogenesis warrants further studies. Furthermore, the results support the need of subphenotype analysis with sample sizes large enough to reveal possible specific disease associations in order to better understand the heterogeneous nature and clinical specificities of the disease. Comprehensive analysis of clinical data suggests that smoking is an environmental triggering factor.

Major histocompatibility complex and coronary artery disease: Special emphasis on Chlamydia pneumoniae, and periodontitis

Most of the genes in the MHC region are involveed in adaptive and innate immunity, with essential function in inflammatory reactions and in protection against infections. These genes might serve as a candidate region for infection and inflammation associated diseases. CAD is an inflammatory disease. The present set of studies was performed to assess whether the MHC region harbors genetic markers for CAD, and whether these genetic markers explain the CAD risk factors: e.g., C. pneumoniae, periodontitis, and periodontal pathogens. Study I was performed using two separate patient materials and age- and sex-matched healthy controls, categorizing them into two independent studies: the HTx and ACS studies. Both studies consistently showed the HLA-A3– B35– DR1 (35 ancestral haplotype) haplotype as a susceptible MHC genetic marker for CAD. HLA-DR1 alone was associated not only with CAD, but also with CAD risk factor diseases, e.g., diabetes mellitus, and hyperlipidemia. The ACS study further showed the HLA-B*07 and -DRB1*15 -related haplotype as a protective MHC haplotype for CAD. Study II showed that patients with CAD showed signs of chronic C. pneumoniae infection when compared to age- and sex-matched healthy controls. HLA-B*35 or -related haplotypes associated with the C. pneumoniae infection markers. Among these haplotype carriers, males and smokers associated with elevated C. pneumoniae infection markers. Study III showed that CAD patients with periodontitis had elevated serum markers of P. gingivalis and occurrence of the pathogen in saliva. LTA+496C strongly associated with periodontitis, while HLA-DRB1*01 with periodontitis and with the elevated serum antibodies of P. gingivalis. Study IV showed that the increased level of C3/C4 ratio was a new risk factor and was associated with recurrent cardiovascular end-points. The increased C3 and decreased C4 concentrations in serum explained the increased level of the C3/C4 ratio. Both the higher than cut-off value (4.53) and the highest quartile of the C3/C4 ratio were also associated with worst survival, increased end-points, and C4 null alleles. The presence of C4 null alleles associated with decreased serum C4 concentration, and increased C3/C4 ratio. In conclusion, the present studies show that the CAD susceptibility haplotype (HLA-A3− B35− DR1 -related haplotypes, Study I) partially explains the development of CAD in patients possessing several recognized and novel risk factors: diabetes mellitus, increased LDL, smoking, C4B*Q0, C. pneumnoiae, periodontitis, P. gingivalis, and complement C3/C4 ratio (Study II, III, and IV).

Common and rare variants of the renin-angiotensin system and their relation to antihypertensive drug responses

Most of the diseases affecting public health, like hypertension, are multifactorial by etiology. Hypertension is influenced by genetic, life style and environmental factors. Estimation of the influence of genes to the risk of essential hypertension varies from 30 to 50%. It is plausible that in most of the cases susceptibility to hypertension is determined by the action of more than one gene. Although the exact molecular mechanism underlying essential hypertension remains obscure, several monogenic forms of hypertension have been identified. Since common genetic variations may predict, not only to susceptibility to hypertension, but also response to antihypertensive drug therapy, pharmacogenetic approaches may provide useful markers in finding relations between candidate genes and phenotypes of hypertension. The aim of this study was to identify genetic mutations and polymorphisms contributing to human hypertension, and examine their relationships to intermediate phenotypes of hypertension, such as blood pressure (BP) responses to antihypertensive drugs or biochemical laboratory values. Two groups of patients were investigated in the present study. The first group was collected from the database of patients investigated in the Hypertension Outpatient Ward, Helsinki University Central Hospital, and consisted of 399 subjects considered to have essential hypertension. Frequncies of the mutant or variant alleles were compared with those in two reference groups, healthy blood donors (n = 301) and normotensive males (n = 175). The second group of subjects with hypertension was collected prospectively. The study subjects (n=313) underwent a protocol lasting eight months, including four one-month drug treatment periods with antihypertensive medications (thiazide diuretic, β-blocker, calcium channel antagonist, and an angiotensin II receptor antagonist). BP responses and laboratory values were related to polymorphims of several candidate genes of the renin-angiotensin system (RAS). In addition, two patients with typical features of Liddle’s syndrome were screened for mutations in kidney epithelial sodium channel (ENaC) subunits. Two novel mutations causing Liddle’s syndrome were identified. The first mutation identified located in the beta-subunit of ENaC and the second mutation found located in the gamma-subunit, constituting the first identified Liddle mutation locating in the extracellular domain. This mutation showed 2-fold increase in channel activity in vitro. Three gene variants, of which two are novel, were identified in ENaC subunits. The prevalence of the variants was three times higher in hypertensive patients (9%) than in reference groups (3%). The variant carriers had increased daily urinary potassium excretion rate in relation to their renin levels compared with controls suggesting increased ENaC activity, although in vitro they did not show increased channel activity. Of the common polymorphisms of the RAS studied, angiotensin II receptor type I (AGTR1) 1166 A/C polymorphism was associated with modest changes in RAS activity. Thus, patients homozygous for the C allele tended to have increased aldosterone and decreased renin levels. In vitro functional studies using transfected HEK293 cells provided additional evidence that the AGTR1 1166 C allele may be associated with increased expression of the AGTR1. Common polymorphisms of the alpha-adducin and the RAS genes did not significantly predict BP responses to one-month monotherapies with hydroclorothiazide, bisoprolol, amlodipin, or losartan. In conclusion, two novel mutations of ENaC subunits causing Liddle’s syndrome were identified. In addition, three common ENaC polymorphisms were shown to be associated with occurrence of essential hypertension, but their exact functional and clinical consequences remain to be explored. The AGTR1 1166 C allele may modify the endocrine phenotype of hypertensive patients, when present in homozygous form. Certain widely studied polymorphisms of the ACE, angiotensinogen, AGTR1 and alpha-adducin genes did not significantly affect responses to a thiazide, β-blocker, calcium channel antagonist, and angiotensin II receptor antagonist.

Prognostic markers in head and neck carcinoma

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide. Well-known risk factors include tobacco smoking and alcohol consumption. Overall survival has improved, but is still low especially in developing countries. One reason for this is the often advanced stage of the disease at the time of diagnosis, but also lack of reliable prognostic tools to enable individualized patient treatment to improve outcome. To date, the TNM classification still serves as the best disease evaluation criterion, although it does not take into account the molecular basis of the tumor. The need for surrogate molecular markers for more accurate disease prediction has increased research interests in this field. We investigated the prevalence, physical status, and viral load of human papillomavirus (HPV) in HNSCC to determine the impact of HPV on head and neck carcinogenesis. The prevalence and genotyping of HPV were assessed with an SPF10 PCR microtiter plate-based hybridization assay (DEIA), followed by a line probe-based genotyping assay. More than half of the patients had HPV DNA in their tumor specimens. Oncogenic HPV-16 was the most common type, and coinfections with other oncogenic and benign associated types also existed. HPV-16 viral load was unevenly distributed among different tumor sites; the tonsils harbored significantly greater amounts of virus than other sites. Episomal location of HPV-16 was associated with large tumors, and both integrated and mixed forms of viral DNA were detected. In this series, we could not show that the presence of HPV DNA correlated with survival. In addition, we investigated the prevalence and genotype of HPV in laryngeal carcinoma patients in a prospective Nordic multicenter study based on fresh-frozen laryngeal tumor samples to determine whether the tumors were HPV-associated. These patients were also examined and interviewed at diagnosis for known risk factors, such as tobacco smoking and alcohol consumption, and for several other habituations to elucidate their effects on patient survival. HPV analysis was performed with the same protocols as in the first study. Only 4% of the specimens harbored HPV DNA. Heavy drinking was associated with poor survival. Heavy drinking patients were also younger than nonheavy drinkers and had a more advanced stage of disease at diagnosis. Heavy drinkers had worse oral hygiene than nonheavy drinkers; however, poor oral hygiene did not have prognostic significance. History of chronic laryngitis, gastroesophageal reflux disease, and orogenital sex contacts were rare in this series. To clarify why vocal cord carcinomas seldom metastasize, we determined tumor lymph vessel (LVD) and blood vessel (BVD) densities in HNSCC patients. We used a novel lymphatic vessel endothelial marker (LYVE-1 antibody) to locate the lymphatic vessels in HNSCC samples and CD31 to detect the blood microvessels. We found carcinomas of the vocal cords to harbor less lymphatic and blood microvessels than carcinomas arising from sites other than vocal cords. The lymphatic and blood microvessel densities did not correlate with tumor size. High BVD was strongly correlated with high LVD. Neither BVD nor LVD showed any association with survival in our series. The immune system plays an important role in tumorigenesis, as neoplastic cells have to escape the cytotoxic lymphocytes in order to survive. Several candidate HLA class II alleles have been reported to be prognostic in cervical carcinomas, an epithelial malignancy resembling HNSCC. These alleles may have an impact on head and neck carcinomas as well. We determined HLA-DRB1* and -DQB1* alleles in HNSCC patients. Healthy organ donors served as controls. The Inno-LiPA reverse dot-blot kit was used to identify alleles in patient samples. No single haplotype was found to be predictive of either the risk for head and neck cancer, or the clinical course of the disease. However, alleles observed to be prognostic in cervical carcinomas showed a similar tendency in our series. DRB1*03 was associated with node-negative disease at diagnosis. DRB1*08 and DRB1*13 were associated with early-stage disease; DRB1*04 had a lower risk for tumor relapse; and DQB1*03 and DQB1*0502 were more frequent in controls than in patients. However, these associations reached only borderline significance in our HNSCC patients.

Characterization of LRRTM and NGR gene families: Expression and functions

Some leucine-rich repeat (LRR) -containing membrane proteins are known regulators of neuronal growth and synapse formation. In this work I characterize two gene families encoding neuronal LRR membrane proteins, namely the LRRTM (leucine-rich repeat, transmembrane neuronal) and NGR (Nogo-66 receptor) families. I studied LRRTM and NGR family member's mRNA tissue distribution by RT-PCR and by in situ hybridization. Subcellular localization of LRRTM1 protein was studied in neurons and in non-neuronal cells. I discovered that LRRTM and NGR family mRNAs are predominantly expressed in the nervous system, and that each gene possesses a specific expression pattern. I also established that LRRTM and NGR family mRNAs are expressed by neurons, and not by glial cells. Within neurons, LRRTM1 protein is not transported to the plasma membrane; rather it localizes to endoplasmic reticulum. Nogo-A (RTN4), MAG, and OMgp are myelin-associated proteins that bind to NgR1 to limit axonal regeneration after central nervous system injury. To better understand the functions of NgR2 and NgR3, and to explore the possible redundancy in the signaling of myelin inhibitors of neurite growth, I mapped the interactions between NgR family and the known and candidate NgR1 ligands. I identified high-affinity interactions between RTN2-66, RTN3-66 and NgR1. I also demonstrate that Rtn3 mRNA is expressed in the same glial cell population of mouse spinal cord white matter as Nogo-A mRNA, and thus it could have a role in myelin inhibition of axonal growth. To understand how NgR1 interacts with multiple structurally divergent ligands, I aimed first to map in more detail the nature of Nogo-A:NgR1 interactions, and then to systematically map the binding sites of multiple myelin ligands in NgR1 by using a library of NgR1 expression constructs encoding proteins with one or multiple surface residues mutated to alanine. My analysis of the Nogo-A:NgR1 -interactions revealed a novel interaction site between the proteins, suggesting a trivalent Nogo-A:NgR1-interaction. Our analysis also defined a central binding region on the concave side of NgR1's LRR domain that is required for the binding of all known ligands, and a surrounding region critical for binding MAG and OMgp. To better understand the biological role of LRRTMs, I generated Lrrtm1 and Lrrtm3 knock out mice. I show here that reporter genes expressed from the targeted loci can be used for maping the neuronal connections of Lrrtm1 and Lrrtm3 expressing neurons in finer detail. With regard to LRRTM1's role in humans, we found a strong association between a 70 kb-spanning haplotype in the proposed promoter region of LRRTM1 gene and two possibly related phenotypes: left-handedness and schizophrenia. Interestingly, the responsible haplotype was linked to phenotypic variability only when paternally inherited. In summary, I identified two families of neuronal receptor-like proteins, and mapped their expression and certain protein-protein interactions. The identification of a central binding region in NgR1 shared by multiple ligands may facilitate the design and development of small molecule therapeutics blocking binding of all NgR1 ligands. Additionally, the genetic association data suggests that allelic variation upstream of LRRTM1 may play a role in the development of left-right brain asymmetry in humans. Lrrtm1 and Lrrtm3 knock out mice developed as a part of this study will likely be useful for schizophrenia and Alzheimer s disease research.

Genetic Variation and Clinical Manifestations in Inflammatory Bowel Disease

Crohn s disease (CD) and ulcerative colitis (UC), collectively known as inflammatory bowel disease (IBD), are characterised by chronic inflammation of the gastrointestinal tract. IBD prevalence in Finland is approximately 3-4 per 1000 inhabitants with a peak incidence in adolescence. The symptoms of IBD include diarrhoea, abdominal pain, fever, and weight loss. The precise aetiology of IBD is unknown but interplay of environmental risk factors and immunologic changes trigger the disease in a genetically susceptible individual. Twin and family studies have provided strong evidence for genetic factors in IBD susceptibility, and genetic factors may be more prominent in CD than UC. The first CD susceptibility gene was identified in 2001. Three common mutations R702W, G908R, and 1007fs of the CARD15/NOD2 gene are shown to associate independently with CD but the magnitude of association varies between different populations. The present study aimed at identifying mutations and genetic variations in IBD susceptibility and candidate genes. In addition, correlation to phenotype was also assessed. One of the main objectives of this study was to evaluate the role of CARD15 in a Finnish CD cohort. 271 CD patients were studied for the three common mutations and the results showed a lower mutation frequency than in other Caucasian populations. Only 16% of the patients carried one of the three mutations. Ileal location as well as stricturing and penetrating behaviour of the disease were associated with occurrence of the mutations. The whole protein coding region of CARD15 was screened for possible Finnish founder mutations. In addition to several sequence variants, five novel mutations (R38M, W355X, P727L, W907R, and R1019X) were identified in five patients. Functional consequences of these novel variants were studied in vitro, and these studies demonstrated a profound impairment of MDP response. Investigation of CARD15 mutation frequency in healthy people across three continents showed a large geographic fluctuation. No simple correlation between mutation frequency and disease incidence was seen in populations studied. The occurrence of double mutant carriers in healthy controls suggested that the penetrance of risk alleles is low. Other main objectives aimed at identifying other genetic variations that are involved in the susceptibility to IBD. We investigated the most plausible IBD candidate genes including TRAF6, SLC22A4, SLC22A5, DLG5, TLR4, TNFRSF1A, ABCB1/MDR1, IL23R, and ATG16L1. The marker for a chromosome 5 risk haplotype and the rare HLA-DRB1*0103 allele were also studied. The study cohort consisted of 699 IBD patients (240 CD and 459 UC), of which 23% had a first-degree relative with IBD. Of the several candidate genes studied, IL23R was associated with CD susceptibility, and TNFRSF1A as well as the HLA-DRB1*0103 allele with UC susceptibility. IL23R variants also showed association with the stricturing phenotype and longer disease duration in CD patients. In addition, TNFRSF1A variants were more common among familial UC and ileocolonic CD. In conclusion, the common CARD15 mutations were shown to account for 16% of CD cases in Finland. Novel CARD15 variants identified in the present study are most likely disease-causing mutations, as judged by the results of in vitro studies. The present study also confirms the IL23R association with CD susceptibility and, in addition, TNFRSF1A and HLA-DRB1*0103 allele association with UC of specific clinical phenotypes.