Screening and breeding for genetic resistance to anthracnose in mango

Postharvest diseases remain a significant constraint to the transport, storage and marketing of mangoes. The two main ones are anthracnose and stem end rot. Anthracnose caused by Colletotrichum gloeosporioides is the more wide-spread of the two. Varieties within Mangifera indica are known to vary in their level of reactions to anthracnose; however, the best tolerance in current commercial cultivars is not sufficient to eliminate the need for pre- and postharvest fungicides treatments. A screening program was initiated in mango accessions in the Australian National Mango Genebank to look for any significant resistance to C. gloeosporioides in fruit as they ripened. Screening was conducted by rating reactions to natural infection of anthracnose and reactions to artificially inoculating fruit with virulent isolates of C. gloeosporioides. A range of reactions to the pathogen were identified, with strong resistance found in one accession of the species M. laurina. This accession was used as the pollen parent in a controlled crossing program with a M. indica hybrid from the Australian Mango Breeding Program (AMBP). Sixty successful hybrids between the species have been generated. The hybrid population will be screened for resistance to anthracnose and used for gene discovery investigations to identify markers for anthracnose resistance.

The Genetic Architecture of Climatic Adaptation of Tropical Cattle

Adaptation of global food systems to climate change is essential to feed the world. Tropical cattle production, a mainstay of profitability for farmers in the developing world, is dominated by heat, lack of water, poor quality feedstuffs, parasites, and tropical diseases. In these systems European cattle suffer significant stock loss, and the cross breeding of taurine x indicine cattle is unpredictable due to the dilution of adaptation to heat and tropical diseases. We explored the genetic architecture of ten traits of tropical cattle production using genome wide association studies of 4,662 animals varying from 0% to 100% indicine. We show that nine of the ten have genetic architectures that include genes of major effect, and in one case, a single location that accounted for more than 71% of the genetic variation. One genetic region in particular had effects on parasite resistance, yearling weight, body condition score, coat colour and penile sheath score. This region, extending 20 Mb on BTA5, appeared to be under genetic selection possibly through maintenance of haplotypes by breeders. We found that the amount of genetic variation and the genetic correlations between traits did not depend upon the degree of indicine content in the animals. Climate change is expected to expand some conditions of the tropics to more temperate environments, which may impact negatively on global livestock health and production. Our results point to several important genes that have large effects on adaptation that could be introduced into more temperate cattle without detrimental effects on productivity.

Increased virulence of rabbit haemorrhagic disease virus associated with genetic resistance in wild Australian rabbits (Oryctolagus cuniculus)

The release of myxoma virus (MYXV) and Rabbit Haemorrhagic Disease Virus (RHDV) in Australia with the aim of controlling overabundant rabbits has provided a unique opportunity to study the initial spread and establishment of emerging pathogens, as well as their co-evolution with their mammalian hosts. In contrast to MYXV, which attenuated shortly after its introduction, rapid attenuation of RHDV has not been observed. By studying the change in virulence of recent field isolates at a single field site we show, for the first time, that RHDV virulence has increased through time, likely because of selection to overcome developing genetic resistance in Australian wild rabbits. High virulence also appears to be favoured as rabbit carcasses, rather than diseased animals, are the likely source of mechanical insect transmission. These findings not only help elucidate the co-evolutionary interaction between rabbits and RHDV, but reveal some of the key factors shaping virulence evolution.

Spot form of net blotch resistance in barley is under complex genetic control

Key message: Evaluation of resistance toPyrenophora teresf.maculatain barley breeding populations via association mapping revealed a complex genetic architecture comprising a mixture of major and minor effect genes. Abstract: In the search for stable resistance to spot form of net blotch (Pyrenophora teres f. maculata, SFNB), association mapping was conducted on four independent barley (Hordeum vulgare L.) breeding populations comprising a total of 898 unique elite breeding lines from the Northern Region Barley Breeding Program in Australia for discovery of quantitative trait loci (QTL) influencing resistance at seedling and adult plant growth stages. A total of 29 significant QTL were validated across multiple breeding populations, with 22 conferring resistance at both seedling and adult plant growth stages. The remaining 7 QTL conferred resistance at either seedling (2 QTL) or adult plant (5 QTL) growth stages only. These 29 QTL represented 24 unique genomic regions, of which five were found to co-locate with previously identified QTL for SFNB. The results indicated that SFNB resistance is controlled by a large number of QTL varying in effect size with large effects QTL on chromosome 7H. A large proportion of the QTL acted in the same direction for both seedling and adult responses, suggesting that phenotypic selection for SFNB resistance performed at either growth stage could achieve adequate levels of resistance. However, the accumulation of specific resistance alleles on several chromosomes must be considered in molecular breeding selection strategies.

Genetic characterization of field-evolved resistance to phosphine in the rusty grain beetle, Cryptolestes ferrugineus (Laemophloeidae: Coleoptera)

Inheritance of resistance to phosphine fumigant was investigated in three field-collected strains of rusty grain beetle, Cryptolestes ferrugineus, Susceptible (S-strain), Weakly Resistant (Weak-R) and Strongly Resistant (Strong-R). The strains were purified for susceptibility, weak resistance and strong resistance to phosphine, respectively, to ensure homozygosity of resistance genotype. Crosses were established between S-strain × Weak-R, S-strain × Strong-R and Weak-R × Strong-R, and the dose mortality responses to phosphine of these strains and their F1, F2 and F1-backcross progeny were obtained. The fumigations were undertaken at 25 °C and 55% RH for 72 h. Weak-R and Strong-R showed resistance factors of 6.3 × and 505 × compared with S-strain at the LC50. Both weak and strong resistances were expressed as incompletely recessive with degrees of dominance of − 0.48 and − 0.43 at the LC50, respectively. Responses of F2 and F1-backcross progeny indicated the existence of one major gene in Weak-R, and at least two major genes in Strong-R, one of which was allelic with the major factor in Weak-R. Phenotypic variance analyses also estimated that the number of independently segregating genes conferring weak resistance was 1 (nE = 0.89) whereas there were two genes controlling strong resistance (nE = 1.2). The second gene, unique to Strong-R, interacted synergistically with the first gene to confer a very high level of resistance (~ 80 ×). Neither of the two major resistance genes was sex linked. Despite the similarity of the genetics of resistance to that previously observed in other pest species, a significant proportion (~ 15 to 30%) of F1 individuals survived at phosphine concentrations higher than predicted. Thus it is likely that additional dominant heritable factors, present in some individuals in the population, also influenced the resistance phenotype. Our results will help in understanding the process of selection for phosphine resistance in the field which will inform resistance management strategies. In addition, this information will provide a basis for the identification of the resistance genes.

Genetic characterization of field-evolved resistance to phosphine in the rusty grain beetle, Cryptolestes ferrugineus (Laemophloeidae: Coleoptera)

Inheritance of resistance to phosphine fumigant was investigated in three field-collected strains of rusty grain beetle, Cryptolestes ferrugineus, Susceptible (S-strain), Weakly Resistant (Weak-R) and Strongly Resistant (Strong-R). The strains were purified for susceptibility, weak resistance and strong resistance to phosphine, respectively, to ensure homozygosity of resistance genotype. Crosses were established between S-strain × Weak-R, S-strain × Strong-R and Weak-R × Strong-R, and the dose mortality responses to phosphine of these strains and their F1, F2 and F1-backcross progeny were obtained. The fumigations were undertaken at 25 °C and 55% RH for 72 h. Weak-R and Strong-R showed resistance factors of 6.3 × and 505 × compared with S-strain at the LC50. Both weak and strong resistances were expressed as incompletely recessive with degrees of dominance of − 0.48 and − 0.43 at the LC50, respectively. Responses of F2 and F1-backcross progeny indicated the existence of one major gene in Weak-R, and at least two major genes in Strong-R, one of which was allelic with the major factor in Weak-R. Phenotypic variance analyses also estimated that the number of independently segregating genes conferring weak resistance was 1 (nE = 0.89) whereas there were two genes controlling strong resistance (nE = 1.2). The second gene, unique to Strong-R, interacted synergistically with the first gene to confer a very high level of resistance (~ 80 ×). Neither of the two major resistance genes was sex linked. Despite the similarity of the genetics of resistance to that previously observed in other pest species, a significant proportion (~ 15 to 30%) of F1 individuals survived at phosphine concentrations higher than predicted. Thus it is likely that additional dominant heritable factors, present in some individuals in the population, also influenced the resistance phenotype. Our results will help in understanding the process of selection for phosphine resistance in the field which will inform resistance management strategies. In addition, this information will provide a basis for the identification of the resistance genes.

Epidemiology and genetic diversity of Tobacco streak virus and related subgroup 1 ilarviruses

A quarter of Australia’s sunflower production is from the central highlands region of Queensland and is currently worth six million dollars ($AUD) annually. From the early 2000s a severe necrosis disorder of unknown aetiology was affecting large areas of sunflower crops in central Queensland, leading to annual losses of up to 20%. Other crops such as mung bean and cotton were also affected. This PhD study was undertaken to determine if the causal agent of the necrosis disorder was of viral origin and, if so, to characterise its genetic diversity, biology and disease cycle, and to develop effective control strategies. The research described in this thesis identified Tobacco streak virus (TSV; genus Ilarvirus, family Bromoviridae) as the causal agent of the previously unidentified necrosis disorder of sunflower in central Queensland. TSV was also the cause of commonly found diseases in a range of other crops in the same region including cotton, chickpea and mung bean. This was the first report from Australia of natural field infections of TSV from these four crops. TSV strains have previously been reported from other regions of Australia in several hosts based on serological and host range studies. In order to determine the relatedness of previously reported TSV strains with TSV from central Queensland, we characterised the genetic diversity of the known TSV strains from Australia. We identified two genetically distinct TSV strains from central Queensland and named them based on their major alternative hosts, TSV-parthenium from Parthenium hysterophorus and TSV-crownbeard from Verbesina encelioides. They share only 81 % total-genome nucleotide sequence identity. In addition to TSV-parthenium and TSV-crownbeard from central Queensland, we also described the complete genomes of two other ilarvirus species. This proved that previously reported TSV strains, TSV-S isolated from strawberry and TSV-Ag from Ageratum houstonianum, were actually the first record of Strawberry necrotic shock virus from Australia, and a new subgroup 1 ilarvirus, Ageratum latent virus. Our results confirmed that the TSV strains found in central Queensland were not related to previously described strains from Australia and may represent new incursions. This is the first report of the genetic diversity within subgroup 1 ilarviruses from Australia. Based on field observations we hypothesised that parthenium and crownbeard were acting as symptomless hosts of TSV-parthenium and TSV-crownbeard, respectively. We developed strain-specific multiplex PCRs for the three RNA segments to accurately characterise the range of naturally infected hosts across central Queensland. Results described in this thesis show compelling evidence that parthenium and crownbeard are the major (symptomless) alternative hosts of TSV-parthenium and TSV-crownbeard. While both TSV strains had wide natural host ranges, the geographical distribution of each strain was closely associated with the respective distribution of their major alternative hosts. Both TSV strains were commonly found across large areas of central Queensland, but we only found strong evidence for the TSV-parthenium strain being associated with major disease outbreaks in nearby crops. The findings from this study demonstrate that both TSV-parthenium and TSV-crownbeard have similar life cycles but some critical differences. We found both TSV strains to be highly seed transmitted from their respective major alternative hosts from naturally infected mother plants and survived in seed for more than 2 years. We conclusively demonstrated that both TSV strains were readily transmitted via virus-infected pollen taken from the major alternative hosts. This transmission was facilitated by the most commonly collected thrips species, Frankliniella schultzei and Microcephalothrips abdominalis. These results illustrate the importance of seed transmission and efficient thrips vector species for the effective survival of these TSV strains in an often harsh environment and enables the rapid development of TSV disease epidemics in surrounding crops. Results from field surveys and inoculation tests indicate that parthenium is a poor host of TSV-crownbeard. By contrast, crownbeard was naturally infected by, and an experimental host of TSV-parthenium. However, this infection combination resulted in non-viable crownbeard seed. These differences appear to be an effective biological barrier that largely restricts these two TSV strains to their respective major alternative hosts. Based on our field observations we hypothesised that there were differences in relative tolerance to TSV infection between different sunflower hybrids and that seasonal variation in disease levels was related to rainfall in the critical early crop stage. Results from our field trials conducted over multiple years conclusively demonstrated significant differences in tolerance to natural infections of TSV-parthenium in a wide range of sunflower hybrids. Glasshouse tests indicate the resistance to TSV-parthenium identified in the sunflower hybrids is also likely to be effective against TSV-crownbeard. We found a significant negative association between TSV disease incidence in sunflowers and accumulated rainfall in the months of March and April with increasing rainfall resulting in reduced levels of disease. Our results indicate that the use of tolerant sunflower germplasm will be a critical strategy to minimise the risk of TSV epidemics in sunflower.

Genetic diversity and epidemiology of Tobacco streak virus and related subgroup 1 Ilarviruses

A quarter of Australia’s sunflower production is from the central highlands region of Queensland and is currently worth six million dollars ($AUD) annually. From the early 2000s a severe necrosis disorder of unknown aetiology was affecting large areas of sunflower crops in central Queensland, leading to annual losses of up to 20%. Other crops such as mung bean and cotton were also affected. This PhD study was undertaken to determine if the causal agent of the necrosis disorder was of viral origin and, if so, to characterise its genetic diversity, biology and disease cycle, and to develop effective control strategies. The research described in this thesis identified Tobacco streak virus (TSV; genus Ilarvirus, family Bromoviridae) as the causal agent of the previously unidentified necrosis disorder of sunflower in central Queensland. TSV was also the cause of commonly found diseases in a range of other crops in the same region including cotton, chickpea and mung bean. This was the first report from Australia of natural field infections of TSV from these four crops. TSV strains have previously been reported from other regions of Australia in several hosts based on serological and host range studies. In order to determine the relatedness of previously reported TSV strains with TSV from central Queensland, we characterised the genetic diversity of the known TSV strains from Australia. We identified two genetically distinct TSV strains from central Queensland and named them based on their major alternative hosts, TSV-parthenium from Parthenium hysterophorus and TSV-crownbeard from Verbesina encelioides. They share only 81 % total-genome nucleotide sequence identity. In addition to TSV-parthenium and TSV-crownbeard from central Queensland, we also described the complete genomes of two other ilarvirus species. This proved that previously reported TSV strains, TSV-S isolated from strawberry and TSV-Ag from Ageratum houstonianum, were actually the first record of Strawberry necrotic shock virus from Australia, and a new subgroup 1 ilarvirus, Ageratum latent virus. Our results confirmed that the TSV strains found in central Queensland were not related to previously described strains from Australia and may represent new incursions. This is the first report of the genetic diversity within subgroup 1 ilarviruses from Australia. Based on field observations we hypothesised that parthenium and crownbeard were acting as symptomless hosts of TSV-parthenium and TSV-crownbeard, respectively. We developed strain-specific multiplex PCRs for the three RNA segments to accurately characterise the range of naturally infected hosts across central Queensland. Results described in this thesis show compelling evidence that parthenium and crownbeard are the major (symptomless) alternative hosts of TSV-parthenium and TSV-crownbeard. While both TSV strains had wide natural host ranges, the geographical distribution of each strain was closely associated with the respective distribution of their major alternative hosts. Both TSV strains were commonly found across large areas of central Queensland, but we only found strong evidence for the TSV-parthenium strain being associated with major disease outbreaks in nearby crops. The findings from this study demonstrate that both TSV-parthenium and TSV-crownbeard have similar life cycles but some critical differences. We found both TSV strains to be highly seed transmitted from their respective major alternative hosts from naturally infected mother plants and survived in seed for more than 2 years. We conclusively demonstrated that both TSV strains were readily transmitted via virus-infected pollen taken from the major alternative hosts. This transmission was facilitated by the most commonly collected thrips species, Frankliniella schultzei and Microcephalothrips abdominalis. These results illustrate the importance of seed transmission and efficient thrips vector species for the effective survival of these TSV strains in an often harsh environment and enables the rapid development of TSV disease epidemics in surrounding crops. Results from field surveys and inoculation tests indicate that parthenium is a poor host of TSV-crownbeard. By contrast, crownbeard was naturally infected by, and an experimental host of TSV-parthenium. However, this infection combination resulted in non-viable crownbeard seed. These differences appear to be an effective biological barrier that largely restricts these two TSV strains to their respective major alternative hosts. Based on our field observations we hypothesised that there were differences in relative tolerance to TSV infection between different sunflower hybrids and that seasonal variation in disease levels was related to rainfall in the critical early crop stage. Results from our field trials conducted over multiple years conclusively demonstrated significant differences in tolerance to natural infections of TSV-parthenium in a wide range of sunflower hybrids. Glasshouse tests indicate the resistance to TSV-parthenium identified in the sunflower hybrids is also likely to be effective against TSV-crownbeard. We found a significant negative association between TSV disease incidence in sunflowers and accumulated rainfall in the months of March and April with increasing rainfall resulting in reduced levels of disease. Our results indicate that the use of tolerant sunflower germplasm will be a critical strategy to minimise the risk of TSV epidemics in sunflower.

Spatiotemporal aspects of Hendra Virus infection in Pteropid Bats (Flying-Foxes) in Eastern Australia

Hendra virus (HeV) causes highly lethal disease in horses and humans in the eastern Australian states of Queensland (QLD) and New South Wales (NSW), with multiple equine cases now reported on an annual basis. Infection and excretion dynamics in pteropid bats (flying-foxes), the recognised natural reservoir, are incompletely understood. We sought to identify key spatial and temporal factors associated with excretion in flying-foxes over a 2300 km latitudinal gradient from northern QLD to southern NSW which encompassed all known equine case locations. The aim was to strengthen knowledge of Hendra virus ecology in flying-foxes to improve spillover risk prediction and exposure risk mitigation strategies, and thus better protect horses and humans. Monthly pooled urine samples were collected from under roosting flying-foxes over a three-year period and screened for HeV RNA by quantitative RT-PCR. A generalised linear model was employed to investigate spatiotemporal associations with HeV detection in 13,968 samples from 27 roosts. There was a non-linear relationship between mean HeV excretion prevalence and five latitudinal regions, with excretion moderate in northern and central QLD, highest in southern QLD/northern NSW, moderate in central NSW, and negligible in southern NSW. Highest HeV positivity occurred where black or spectacled flying-foxes were present; nil or very low positivity rates occurred in exclusive grey-headed flying-fox roosts. Similarly, little red flying-foxes are evidently not a significant source of virus, as their periodic extreme increase in numbers at some roosts was not associated with any concurrent increase in HeV detection. There was a consistent, strong winter seasonality to excretion in the southern QLD/northern NSW and central NSW regions. This new information allows risk management strategies to be refined and targeted, mindful of the potential for spatial risk profiles to shift over time with changes in flying-fox species distribution.

Methicillin-resistant Staphylococcus aureus in Finland: recent changes in the epidemiology, long-term facility aspects, and phenotypic and molecular detection of isolates

Staphylococcus aureus is one of the most important bacteria that cause disease in humans, and methicillin-resistant S. aureus (MRSA) has become the most commonly identified antibiotic-resistant pathogen in many parts of the world. MRSA rates have been stable for many years in the Nordic countries and the Netherlands with a low MRSA prevalence in Europe, but in the recent decades, MRSA rates have increased in those low-prevalence countries as well. MRSA has been established as a major hospital pathogen, but has also been found increasingly in long-term facilities (LTF) and in communities of persons with no connections to the health-care setting. In Finland, the annual number of MRSA isolates reported to the National Infectious Disease Register (NIDR) has constantly increased, especially outside the Helsinki metropolitan area. Molecular typing has revealed numerous outbreak strains of MRSA, some of which have previously been associated with community acquisition. In this work, data on MRSA cases notified to the NIDR and on MRSA strain types identified with pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and staphylococcal cassette chromosome mec (SCCmec) typing at the National Reference Laboratory (NRL) in Finland from 1997 to 2004 were analyzed. An increasing trend in MRSA incidence in Finland from 1997 to 2004 was shown. In addition, non-multi-drug resistant (NMDR) MRSA isolates, especially those resistant only to methicillin/oxacillin, showed an emerging trend. The predominant MRSA strains changed over time and place, but two internationally spread epidemic strains of MRSA, FIN-16 and FIN-21, were related to the increase detected most recently. Those strains were also one cause of the strikingly increasing invasive MRSA findings. The rise of MRSA strains with SCCmec types IV or V, possible community-acquired MRSA was also detected. With questionnaires, the diagnostic methods used for MRSA identification in Finnish microbiology laboratories and the number of MRSA screening specimens studied were reviewed. Surveys, which focused on the MRSA situation in long-term facilities in 2001 and on the background information of MRSA-positive persons in 2001-2003, were also carried out. The rates of MRSA and screening practices varied widely across geographic regions. Part of the NMDR MRSA strains could remain undetected in some laboratories because of insufficient diagnostic techniques used. The increasing proportion of elderly population carrying MRSA suggests that MRSA is an emerging problem in Finnish long-term facilities. Among the patients, 50% of the specimens were taken on a clinical basis, 43% on a screening basis after exposure to MRSA, 3% on a screening basis because of hospital contact abroad, and 4% for other reasons. In response to an outbreak of MRSA possessing a new genotype that occurred in a health care ward and in an associated nursing home of a small municipality in Northern Finland in autumn 2003, a point-prevalence survey was performed six months later. In the same study, the molecular epidemiology of MRSA and methicillin-sensitive S. aureus (MSSA) strains were also assessed, the results to the national strain collection compared, and the difficulties of MRSA screening with low-level oxacillin-resistant isolates encountered. The original MRSA outbreak in LTF, which consisted of isolates possessing a nationally new PFGE profile (FIN-22) and internationally rare MLST type (ST-27), was confined. Another previously unrecognized MRSA strain was found with additional screening, possibly indicating that current routine MRSA screening methods may be insufficiently sensitive for strains possessing low-level oxacillin resistance. Most of the MSSA strains found were genotypically related to the epidemic MRSA strains, but only a few of them had received the SCCmec element, and all those strains possessed the new SCCmec type V. In the second largest nursing home in Finland, the colonization of S. aureus and MRSA, and the role of screening sites along with broth enrichment culture on the sensitivity to detect S. aureus were studied. Combining the use of enrichment broth and perineal swabbing, in addition to nostrils and skin lesions swabbing, may be an alternative for throat swabs in the nursing home setting, especially when residents are uncooperative. Finally, in order to evaluate adequate phenotypic and genotypic methods needed for reliable laboratory diagnostics of MRSA, oxacillin disk diffusion and MIC tests to the cefoxitin disk diffusion method at both +35°C and +30°C, both with or without an addition of sodium chloride (NaCl) to the Müller Hinton test medium, and in-house PCR to two commercial molecular methods (the GenoType® MRSA test and the EVIGENETM MRSA Detection test) with different bacterial species in addition to S. aureus were compared. The cefoxitin disk diffusion method was superior to that of oxacillin disk diffusion and to the MIC tests in predicting mecA-mediated resistance in S. aureus when incubating at +35°C with or without the addition of NaCl to the test medium. Both the Geno Type® MRSA and EVIGENETM MRSA Detection tests are usable, accurate, cost-effective, and sufficiently fast methods for rapid MRSA confirmation from a pure culture.

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.

Genetic analysis of chromosomal regions 2q33, 7q32 and 19q13 in multiple sclerosis susceptibility

Multiple sclerosis (MS) is an immune-mediated demyelinating disorder of the central nervous system (CNS) affecting 0.1-0.2% of Northern European descent population. MS is considered to be a multifactorial disease, both environment and genetics play a role in its pathogenesis. Despite several decades of intense research, the etiological and pathogenic mechanisms underlying MS remain still largely unknown and no curative treatment exists. The genetic architecture underlying MS is complex with multiple genes involved. The strongest and the best characterized predisposing genetic factors for MS are located, as in other immune-mediated diseases, in the major histocompatibility complex (MHC) on chromosome 6. In humans MHC is called human leukocyte antigen (HLA). Alleles of the HLA locus have been found to associate strongly with MS and remained for many years the only consistently replicable genetic associations. However, recently other genes located outside the MHC region have been proposed as strong candidates for susceptibility to MS in several studies. In this thesis a new genetic locus located on chromosome 7q32, interferon regulatory factor 5 (IRF5), was identified in the susceptibility to MS. In particular, we found that common variation of the gene was associated with the disease in three different populations, Spanish, Swedish and Finnish. We also suggested a possible functional role for one of the risk alleles with impact on the expression of the IRF5 locus. Previous studies have pointed out a possible role played by chromosome 2q33 in the susceptibility to MS and other autoimmune disorders. The work described here also investigated the involvement of this chromosomal region in MS predisposition. After the detection of genetic association with 2q33 (article-1), we extended our analysis through fine-scale single nucleotide polymorphism (SNP) mapping to define further the contribution of this genomic area to disease pathogenesis (article-4). We found a trend (p=0.04) for association to MS with an intronic SNP located in the inducible T-cell co-stimulator (ICOS) gene, an important player in the co-stimulatory pathway of the immune system. Expression analysis of ICOS revealed a novel, previously uncharacterized, alternatively spliced isoform, lacking the extracellular domain that is needed for ligand binding. The stability of the newly-identified transcript variant and its subcellular localization were analyzed. These studies indicated that the novel isoform is stable and shows different subcellular localization as compared to full-length ICOS. The novel isoform might have a regulatory function, but further studies are required to elucidate its function. Chromosome 19q13 has been previously suggested as one of the genomic areas involved in MS predisposition. In several populations, suggestive linkage signals between MS predisposition and 19q13 have been obtained. Here, we analysed the role of allelic variation in 19q13 by family based association analysis in 782 MS families collected from Finland. In this dataset, we were not able to detect any statistically significant associations, although several previously suggested markers were included to the analysis. Replication of the previous findings on the basis of linkage disequilibrium between marker allele and disease/risk allele appears notoriously difficult because of limitations such as allelic heterogeneity. Re-sequencing based approaches may be required for elucidating the role of chromosome 19q13 with MS. This thesis has resulted in the identification of a new MS susceptibility locus (IRF5) previously associated with other inflammatory or autoimmune disorders, such as SLE. IRF5 is one of the mediators of interferons biological function. In addition to providing new insight in the possible pathogenetic pathway of the disease, this finding suggests that there might be common mechanisms between different immune-mediated disorders. Furthermore the work presented here has uncovered a novel isoform of ICOS, which may play a role in regulatory mechanisms of ICOS, an important mediator of lymphocyte activation. Further work is required to uncover its functions and possible involvement of the ICOS locus in MS susceptibility.

Clinical aspects of exhaled nitric oxide in adults : Associations with atopy, bronchial hyperresponsiveness, smoking and chronic obstruction

Measurement of fractional exhaled nitric oxide (FENO) has proven useful in assessment of patients with respiratory symptoms, especially in predicting steroid response. The objective of these studies was to clarify issues relevant for the clinical use of FENO. The influence of allergic sensitization per se on FENO in healthy asymptomatic subjects was studied, the association between airway inflammation and bronchial hyperresponsiveness (BHR) in steroid-naive subjects with symptoms suggesting asthma was examined, as well as the possible difference in this association between atopic and nonatopic subjects. Influence of smoking on FENO was compared between atopic and nonatopic steroid-naive asthmatics and healthy subjects. The short-term repeatability of FENO in COPD patients was examined in order to assess whether the degree of chronic obstruction influences the repeatability. For these purposes, we studied a random sample of 248 citizens of Helsinki, 227 army conscripts with current symptoms suggesting asthma, 19 COPD patients, and 39 healthy subjects. FENO measurement, spirometry and bronchodilatation test, structured interview. skin prick tests, and histamine and exercise challenges were performed. Among healthy subjects with no signs of airway diseases, median FENO was similar in skin prick test-positive and –negative subjects, and the upper normal limit of FENO was 30 ppb. In atopic and nonatopic subjects with symptoms suggesting asthma, FENO associated with severity of exercise- or histamine-induced BHR only in atopic patients. FENO in smokers with steroid-naive asthma was significantly higher than in healthy smokers and nonsmokers. Among atopic asthmatics, FENO was significantly lower in smokers than in nonsmokers, whereas no difference appeared among nonatopic asthmatics. The 24-h repeatability of FENO was equally good in COPD patients as in healthy subjects. These findings indicate that allergic sensitization per se does not influence FENO, supporting the view that elevated FENO indicates NO-producing airway inflammation, and that same reference range can be applied to both skin prick test-positive and -negative subjects. The significant correlation between FENO and degree of BHR only in atopic steroid-naive subjects with current asthmatic symptoms supports the view that pathogenesis of BHR in atopic asthma is strongly involved in NO-producing airway inflammation, whereas in development of BHR in nonatopic asthma other mechanisms may dominate. Attenuation of FENO only in atopic but not in nonatopic smokers with steroid-naive asthma may result from differences in mechanisms of FENO formation as well as in sensitivity of these mechanisms to smoking in atopic and nonatopic asthma. The results suggest, however, that in young adult smokers, FENO measurement may prove useful in assessment of airway inflammation. The short-term repeatability of FENO in COPD patients with moderate to very severe disease and in healthy subjects was equally good.

Prediction of individual genetic risk to prostate cancer using a polygenic score

BACKGROUND Polygenic risk scores comprising established susceptibility variants have shown to be informative classifiers for several complex diseases including prostate cancer. For prostate cancer it is unknown if inclusion of genetic markers that have so far not been associated with prostate cancer risk at a genome-wide significant level will improve disease prediction. METHODS We built polygenic risk scores in a large training set comprising over 25,000 individuals. Initially 65 established prostate cancer susceptibility variants were selected. After LD pruning additional variants were prioritized based on their association with prostate cancer. Six-fold cross validation was performed to assess genetic risk scores and optimize the number of additional variants to be included. The final model was evaluated in an independent study population including 1,370 cases and 1,239 controls. RESULTS The polygenic risk score with 65 established susceptibility variants provided an area under the curve (AUC) of 0.67. Adding an additional 68 novel variants significantly increased the AUC to 0.68 (P = 0.0012) and the net reclassification index with 0.21 (P = 8.5E-08). All novel variants were located in genomic regions established as associated with prostate cancer risk. CONCLUSIONS Inclusion of additional genetic variants from established prostate cancer susceptibility regions improves disease prediction. Prostate 75:1467–1474, 2015. © 2015 Wiley Periodicals, Inc.

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.