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  • Bonetti, Alessandro (Helsingin yliopisto, 2009)
    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.
  • Suontama, Mari (Helsingin yliopisto, 2012)
    The objective of this thesis was to evaluate the usefulness of body measurements and functional and conformation traits of foals and studbook horses in the breeding programmes of Finnhorse and Standardbred. Thus, nongenetic effects, heritability and repeatability, and genetic correlations for foal and studbook traits were estimated. In addition, genetic parameters for trotting race performance and genetic correlations for foal and studbook traits with racing traits were assessed. Genetic response and accuracy were estimated using records of animal, half-sibs, and parents in selection scenarios for racing traits, for foal and racing traits, and for studbook and racing traits, and using records of animal, half-sibs, and parents for foal traits and racing traits of parents. Single racing time and annual earnings were the breeding objectives in the selection scenarios. High heritability estimates (0.50 to 0.80) indicated that most part of the variation for body measurements of foals and studbook horses is additive genetic. High genetic correlations (0.74 to 0.99) between body measurements of foals and studbook horses indicated that the traits are determined by the same additive genes at all ages. Low to moderate genetic correlations between body measurements and racing traits suggested that selection favours a larger body size at all ages. Heritability of conformation and functional traits in foals were low to moderate (0.08 to 0.46), being generally higher than studbook traits (0.06 to 0.21). Leg stances of Finnhorse foals, type, body conformation, and overall grade of foals, and leg quality of studbook Finnhorses were of moderate heritability. Genetic correlations among the traits were from low to moderate. Partially based on the previous study, there were less unfavourable genetic correlations among foal than studbook traits. Genetic correlations between the same traits in foals and studbook horses were relatively high (over 0.60), indicating that scant new information would be received through studbook traits in genetic evaluation. In addition, the foal traits of type, trot, and overall grade were moderately to highly genetically correlated (0.50 to 0.70) with several studbook traits. Being mainly favourable for the breeding objective, genetic correlations for conformation and functional traits with racing traits were highest for the foal traits of type, trot, and overall grade and for the studbook traits of character and movements (0.37 to 0.90). Conformation traits in Finnhorse mainly had low genetic correlations with racing traits, whereas in Standardbred leg quality of studbook horses, body conformation, leg stances, and hooves of foals and studbook horses had moderate to high genetic correlations with several racing traits. The foal trait of walk is of little value in genetic evaluation of trotters due to a low heritability and low or unfavourable genetic correlations with racing traits. The most effective way to make genetic progress for the breeding objective is direct selection for racing time and earnings. No more than the individual records of one racing year are necessary for reasonable accuracy if the average number of racing records from different information sources is available for selection. The conformation and functional traits in foals are useful in genetic evaluation because they are less pre-selected than the traits in studbook horses, which are mostly determined by selection based on racing records. The most beneficial for the breeding objective is to use the foal traits of moderate heritability and genetic correlations with racing traits (type, trot, overall grade) in selection before a racing career in the index with racing records of relatives. The greatest gain in this scenario is due to a shorter generation interval. It is possible to implement for AI stallions. Functional and conformation traits in foals would also benefit from multi-trait selection in terms of prediction accuracy in the combined index with racing traits. The use of multivariate models for whole trait sets in estimation of covariance components would further correct for selection, resulting in more accurate estimates of genetic correlations than in this study. Investigating the economic value of foal traits and their relationship with veterinary records would elucidate the relevance of sound conformation and movements in breeding programmes. To have more effective conformation traits in selection for breeding programmes, judging based on biological variation for a specific trait using linear scores or all-or-none variables is recommended.
  • Törnwall, Outi (Helsingin yliopisto, 2013)
    Chemosensory perceptions have biological relevance in aiding the recognition of nutrients and toxins, and initiating physiological processes that affect the digestion system. Food is first and foremost a source of nutrition affecting our well-being, but it is also characterized by one s culture and is a great source of pleasure and satisfaction. Affective responses to sensory characteristics of foods are the best predictor of human food choice in the absence of economic and availability constraints. The aim of this work was to elucidate the impact of genes and environment on responses and preference for astringent, pungent (sensory burn caused by spicy foods) and sour perceptions. Furthermore, a coherent picture of the chemosensory preferences and their underlying causes in young adulthood was achieved. Extensive data from 331 Finnish twins (21-25 years, 146 men, 185 women) were collected as part of FinnTwin12-study including pleasantness and intensity responses to samples spiked with sour, pungent, umami, and astringent stimuli (subgroup for astringency n=194, 96 men, 98 women). Liking and use-frequency responses for variety of food and beverage names representing these chemosensory qualities were collected. In addition, eating behavior related traits (e.g. food neophobia) were measured with validated scales. The heritabilities of chemosensory traits and food preferences were estimated. Furthermore, subgroups of respondents were identified based on preferences to sour, pungent and umami foods. Differences in food behavior and sensory responses, and the impact of genes were explored in the obtained subgroups. Sensory pleasantness and intensity responses to sourness and pungency showed modest heritabilities (12-31%) whereas astringency perception showed no inheritance. However, suggestive evidence that salivary secretion related to astringent stimulation is under genetic control was observed (heritability 43-51%). The preference for foods with sour and spicy flavor qualities showed a clearer and higher impact of genes (heritability 45-50%). Two subgroups of respondents were identified. The basic group was less dramatic in their flavor preferences, whereas the adventurous favored foods with sour and pungent qualities. The adventurous, were also less food neophobic and exhibited more tolerance for the sensory burn caused by capsaicin compared to the basic subgroup. The influence of genes underlying the subgrouping was discovered (heritability 66%). Being adventurous was suggestively found to be in linkage with umami and sour coding taste receptor genes: TAS1R1 and PKD1L3, respectively. This is the first work to demonstrate the role of genetics and environment in perceptions and preference for astringent and pungent flavor qualities, and one of the few to study the heritability of sour taste. It was demonstrated that preference for sour and pungent flavors was partially inherited. Furthermore, two subgroups of individuals were found differing in their flavor preferences. Genetic variability together with food neophobic tendency partially explained these differences. Food neophobia and genetic differences may form a barrier through which individual flavor preferences are generated.
  • Knaapila, Antti (Helsingin yliopisto, 2008)
    Olfaction, the sense of smell, has many important functions in humans. Human responses to odors show substantial individual variation. Olfactory receptor genes have been identified and other genes may also influence olfaction. However, the proportion of phenotypic variation in odor response due to genetic variation remains largely unknown. Little is also known about which genes modify specific responses to odors. This study aimed to elucidate genetic and environmental influences on human responses to odors. Individuals from Finnish families (n=146) and Australian (n=413), British (n=163), Danish (n=336), and Finnish (n=399) twins rated intensity and pleasantness of a set of 12 (families) or 6 (twins) odors and tried to identify the odors. In addition, the participants rated their own sense of smell and annoyance experienced with different environmental odors. The odor stimuli of a commercial smell test (The Brief Smell Identification Test; banana, chocolate, cinnamon, gasoline, lemon, onion, paint thinner, pineapple, rose, smoke, soap, and turpentine) were presented in the family study. Based on the results of the family study and a literature survey, a new set of odor stimuli (androstenone, chocolate, cinnamon, isovaleric acid, lemon, and turpentine) was designed for the twin studies. In the family sample, heritabilities of the traits were estimated and underlying genomic regions were searched using a genome-wide linkage scan. In the pooled twin sample, variation in the measured traits was decomposed into genetic and environmental components using quantitative genetic modeling. In addition, associations between nongenetic factors (e.g., sex, age, and smoking) and olfactory-related traits were explored. Suggestive evidence for a genetic linkage for pleasantness of cinnamon at a locus on chromosome 4q32.3 emerged from the family sample. High heritability for the pleasantness of cinnamon was found in the family but not the twin study. Heritability of perceived intensity of androstenone odor was determined to be ~30% in the twin sample. A strong genetic correlation between perceived intensity and pleasantness of androstenone, in the absence of any environmental correlation, indicated that only the genetic correlation explained the phenotypic correlation between the traits (r=-0.27) and that the traits were influenced by an overlapping set of genes. Self-rated olfactory function appeared to reflect the odor annoyance experienced rather than actual olfactory acuity or genetic involvement. Results from nongenetic analyses supported the speculated superiority of females' olfactory abilities, the age-related diminishing of olfactory acuity, and the influences of experience-dependent factors on odor responses. This was the first study to estimate heritabilities and perform linkage screens for individual odors. A genetic effect was detected for only a few responses to specific odors, suggesting the predominance of environmental effects in odor perceptions.
  • Kuismanen, Shannon (Helsingin yliopisto, 2006)
    Colorectal cancer is one of the three most common cancers today, for both men and women. Approximately 90% of the cases are sporadic while the remaining 10% is hereditary. Among this 10% is hereditary nonpolyposis colorectal cancer (HNPCC), an autosomal dominant disease, accounting for up to 13% of these cases. HNPCC is associated with germline mutations in four mismatch repair (MMR) genes, MLH1, MSH2, MSH6, and PMS2, and is characterized by a familial accumulation of endometrial, gastric, urological, and ovarian tumors, in addition to colorectal cancer. An important etiological characteristic of HNPCC is the presence of microsatellite instability (MSI), caused by mutations of the MMR genes. Approximately 15% of sporadic cases share the MSI+ trait. Colon cancer is believed to be a consequence of an accumulation of mutations in tumor suppressor genes and oncogenes, eventually resulting in tumor development. This phenomena is accelerated in HNPCC due the presence of an inherited mutation in the MMR genes, accounting for one of the two hits proposed to be needed by Knudson (1971) in order for the manifestation of the MSI phenotype. MMR alterations alone, however, do not occur in the majority of sporadic colon cancers, prompting searches for other mechanisms. One such mechanism found to play a role in colon cancer development was DNA methylation, which is known to play a role in MLH1 inactivation. Our objective was clarification of mechanisms associated with tumor development in both HNPCC and sporadic colorectal cancer in relation to tumorigenic mechanisms. Of particular interest were underlying mechanisms of MSI in sporadic colorectal cancers, with attention to DNA methylation changes and their correlation to MSI. Of additional interest were the genetic and epigenetic events leading to the HNPCC tumor spectrum, chiefly colon and endometrial cancers, in regards to what extent the somatic changes in target tissue explained this phenomenon. We made a number of important findings pertaining to these questions. First, MSI tumor development differs epigenetically from stable tumor development, possibly underlying developmental pathway differences. Additionally, while epigenetic modification, principally DNA methylation, is a major mechanism in sporadic MSI colorectal cancer MLH1 inactivation it does not play a significant role in HNPCC tumors with germline MLH1 mutations. This is possibly an explanation for tumorigenic pathways and clinicopathological characteristic differences between sporadic and hereditary MSI colorectal cancers. Finally, despite indistinguishable genetic predisposition for endometrial and colorectal cancers, instability profiles highlighting organ-specific differences, may be important HNPCC tumor spectrum determinants.
  • Nieminen, Taina (Helsingin yliopisto, 2011)
    Individuals with inherited deficiency in DNA mismatch repair(MMR) (Lynch syndrome) LS are predisposed to different cancers in a non-random fashion. Endometrial cancer (EC) is the most common extracolonic malignancy in LS. LS represents the best characterized form of hereditary nonpolyposis colorectal carcinoma (HNPCC). Other forms of familial non-polyposis colon cancer exist, including familial colorectal cancer type X (FCCX). This syndrome resembles LS, but MMR gene defects are excluded and the predisposition genes are unknown so far. To address why different organs are differently susceptible to cancer development, we examined molecular similarities and differences in selected cancers whose frequency varies in LS individuals. Tumors that are common (colorectal, endometrial, gastric) and less common (brain, urological) in LS were characterized for MMR protein expression, microsatellite instability (MSI), and by altered DNA methylation. We also studied samples of histologically normal endometrium, endometrial hyperplasia,and cancer for molecular alterations to identify potential markers that could predict malignant transformation in LS and sporadic cases. Our results suggest that brain and kidney tumors follow a different pathway for cancer development than the most common LS related cancers.Our results suggest also that MMR defects are detectable in endometrial tissues from a proportion of LS mutation carriers prior to endometrial cancer development. Traditionally (complex) atypical hyperplasia has been considered critical for progression to malignancy. Our results suggest that complex hyperplasia without atypia is equally important as a precursor lesion of malignancy. Tumor profiles from Egypt were compared with colorectal tumors from Finland to evaluate if there are differences specific to the ethnic origin (East vs.West). Results showed for the first time a distinct genetic and epigenetic signature in the Egyptian CRC marked by high methylation of microsatellite stable tumors associated with advanced stage, and low frequency of Wnt signaling activation, suggesting a novel pathway. DNA samples from FCCX families were studied with genome wide linkage analysis using microsatellite markers. Selected genes from the linked areas were tested for possible mutations that could explain predisposition to a large number of colon adenomas and carcinomas seen in these families. Based on the results from the linkage analysis, a number of areas with tentative linkage were identified in family 20. We narrowed down these areas by additional microsatellite markers to found a mutation in the BMPR1A gene. Sequencing of an additional 17 FCCX families resulted in a BMPR1A mutation frequency of 2/18 families (11%). Clarification of the mechanisms of the differential tumor susceptibility in LS increases the understanding of gene and organ specific targets of MMR deficiency. While it is generally accepted that widespread MMR deficiency and consequent microsatellite instability (MSI) drives tumorigenesis in LS, the timing of molecular alterations is controversial. In particular, it is important to know that alterations may occur several years before cancer formation, at stages that are still histologically regarded as normal. Identification of molecular markers that could predict the risk of malignant transformation may be used to improve surveillance and cancer prevention in genetically predisposed individuals. Significant fractions of families with colorectal and/or endometrial cancer presently lack molecular definition altogether. Our findings expand the phenotypic spectrum of BMPR1A mutations and, for the first time, link FCCX families to the germline mutation of a specific gene. In particular, our observations encourage screening of additional families with FCCX for BMPR1A mutation, which is necessary in obtaining a reliable estimate of the share of BMPR1A-associated cases among all FCCX families worldwide. Clinically, the identification of predisposing mutations enables targeted cancer prevention in proven mutation carriers and thereby reduces cancer morbidity and mortality in the respective families.
  • Fodstad, Heidi (Helsingin yliopisto, 2005)
  • Penttinen, Petri (Helsingin yliopisto, 2014)
    Nitrogen is an essential nutrient for plants. Biological and industrial nitrogen fixation are the major sources of soil nitrogen. This study focuses on rhizobia that are the bacterial partners in the biologically nitrogen fixing symbiosis between leguminous plants and bacteria. In symbiosis, the plant and the rhizobia form a specified organ called nodule. In the nodule rhizobia differentiate into nitrogen fixing forms. Phenolic molecules secreted by the plant roots induce the rhizobia to produce lipochitooligosaccharide signalling molecules (LCOs). LCOs have an amino sugar backbone consisting of N-acetylglucosamine residues (GlcNAc), an acyl group on the non-reducing terminal GlcNAc residue and commonly other side-groups. When growing legumes in the field, inoculating the plants with compatible rhizobia usually results in better growth. The soil may not contain rhizobial strains that nodulate selective legumes. The promiscuous legumes are easily nodulated, yet most of the nodulating strains may be ineffective in nitrogen fixation. Environmental conditions, e.g. soil salinity, affect nodulation. The general aim of this study was to gain knowledge on the features of potential inoculant rhizobia strains. The specific aims were to assess host specific characteristics and the effect of salt stress on rhizobial signalling molecules, LCOs; to find potential inoculant strains for a promiscuous legume tree; and to characterize potential inoculant strains isolated from promiscuous legume trees and forage legumes to assess genetic features behind the characteristics of the strains. To reach the aims, LCOs of rhizobia nodulating Prosopis chilensis and Acacia senegal were analyzed using mass spectrometry; rhizobial strains were isolated from Leucaena leucocephala growing in China, and characterized with phenotypic and genotypic methods; and the genetic features behind the desirable characteristics of S. arboris and wild-type S. meliloti strains were analyzed by comparative genomic hybridization (CGH) using a model rhizobium S. meliloti Rm1021 microarray. The LCOs from prosopis- and acacia-nodulating strains were similar to those of other Acacia-nodulating rhizobia. The L. leucocephala nodulating Ensifer strains, representing at least three species, shared a common nodC, implying that their LCOs might be similar. Similarly, no divergence was detected between the S. meliloti model strain and the S. meliloti wild type strains nodulation genes. Even though rhizobia having a shared host range produced similar LCOs and carried similar nod genes, comparison of major LCOs between rhizobia with different host ranges suggested that LCOs have only a limited role in host specificity. L. leucocephala hosted eleven rhizobial strains that were efficient in nitrogen fixation making them good candidates as inoculants. Ten isolates had a growth slowing effect on the host. The variation in effectiveness between the isolates implied that the ability to compete with other strains is an important characteristic for the inoculant of a promiscuous host; the inoculant is of no use if nodules are mainly occupied by less efficient strains. The species distribution was different from those isolated from L. leucocephala in other locations, suggesting that strains adapted to local soil conditions would be preferred when choosing inoculants. The genotypic and phenotypic characteristics of Leucaena isolates did not distinguish the efficient from the inefficient and parasitic isolates. The results of the CGH analysis suggested that the osmotic shock response of the S. arboris and wild-type S. meliloti strains is considerably different to that of S. meliloti Rm1021, and that the stress tolerance of S. arboris strain might be partially due to a more efficient protein biosynthesis. The genes related to the induction of LCO synthesis and LCO secretion were duplicated in wild-type S. meliloti strains, which is possibly connected to the efficient nodulation capabilities of the strains. The LCO production of a salt-stressed S. arboris HAMBI 2361 was approximately one tenth of that of the non-stressed one, partially explaining the decrease in nodulation under salt stress. The Leucaena isolates were phenotypically diverse, and the S. arboris and wild-type S. meliloti strains diverged from the model rhizobium S. meliloti Rm1021 mainly in the accessory genome implying that the divergence was important in shaping the adaptability of the strains. However, it is impossible to conclude if the diversity and divergence gave the strains any competitive advantage.
  • Häggman, Johanna (Helsingin yliopisto, 2014)
    Growing public interest in production animal welfare and the considerable costs (e.g. milk loss and involuntary culling) associated with claw disorders and lameness have resulted in a need to study how claw health can be improved in practice. Good claw health is essential for cows in modern dairy farming since herd sizes are increasing and almost all new herds are loose housed, with the cows walking to the milking parlour or to the automatic milking system and to feed. The overall aim of this thesis was to identify the best means to improve claw health through genetic selection and housing and management practices on Finnish dairy farms. The study was divided into three parts. The aim of the first part was to evaluate genetic parameters for claw traits and to determine whether feet and leg conformation traits could be used as indicator traits when selecting for better claw health. In the second part, cow-level and herd-level risk factors affecting infectious and non-infectious claw disorders in tie stalls and loose house herds were evaluated. The aim of the third part was to investigate the effect of lameness on feeding behaviour, feed consumption, and milk yield of dairy cows and to determine whether feeding behaviour of a cow can be used as a reliable indicator for lameness. The heritabilities for different claw disorders from logistic models vary from 0.01 to 0.20 for the Ayrshire breed and from 0.02 to 0.13 for the Holstein breed. The heritabilities for feet and leg conformation traits were higher than for claw disorders, varying from 0.07 to 0.39 for Ayrshire and from 0.09 to 0.19 for Holstein cows. The genetic correlation between overall claw health and individual feet and leg conformation traits varied from -0.40 to 0.42 for the Ayrshire breed. For Holstein cows, the corresponding figure ranged from -0.51 to 0.45. Most cow-level factors (breed, parity, season, year, stage of lactation) were similar for infectious and non-infectious claw disorders in tie stall and loose house herds. By contrast, most herd-level factors (e.g. feeding system, bedding material, bed surface, outdoor access) differed between claw disorder groups and herd types. Lameness seemed to have an effect on feeding behaviour; daily feeding time decreased and feeding rate increased with lameness scores, especially with severely lame primiparous cows. Selection index calculations were performed to illustrate the approximate gain in accuracy of selection when using direct selection for claw traits and/or indirect selection of feet and leg conformation traits. According to the results, the genetic evaluation method currently used by Nordic cattle genetic evaluation for claw traits seems to be suitable for Finnish Ayrshire and Holstein breeds; thus there is no need to include feet and leg conformation traits as indicator traits in genetic evaluations. These findings highlight the importance of claw trimming data collection. However, the inter-claw trimmer variances in the national data were quite high, especially for infectious claw disorders. To enable more reliable national data collection, the training of claw trimmers should be standardized. Because of differences found in herd-level factors between different herd types and disorder groups, the recommendations for housing and management options for individual farms should be based on the previous disease status of the herd and the current housing type. Changes in cows´ feeding behaviour can be used when developing automated lameness detection systems.
  • Väinölä, Anu (Helsingin yliopisto, 2000)
  • Alakulppi, Noora (Helsingin yliopisto, 2008)
    Kidney transplantation (Tx) is the treatment of choice for end stage renal disease. Immunosuppressive medications are given to prevent an immunological rejection of the transplant. However, immunosuppressive drugs increase e.g. the risk of infection, cancer or nephrotoxicity. A major genetic contributors to immunological acceptance of the graft are human leukocyte antigen (HLA) genes. Also other non-HLA gene polymorphisms may predict the future risk of complications before Tx, possibly enabling individualised immunotherapy. Graft function after Tx is monitored using non-specific clinical symptoms and laboratory markers. The definitive diagnosis of graft rejection however relies on a biopsy of the graft. In the acute rejection (AR) diagnostics there is a need for an alternative to biopsy that would be an easily repeatable and simple method for regular use. Frequent surveillance of acute or subclinical rejection (SCR) may improve long-term function. In this thesis, associations between cytokine and thrombosis associated candidate genes and the outcome of kidney Tx were studied. Cytotoxic and co-stimulatory T lymphocyte molecule gene expression biomarkers for the diagnosis of the AR and the SCR were also investigated. We found that polymorphisms in the cytokine genes tumor necrosis factor and interleukin 10 (IL10) of the recipients were associated with AR. In addition, certain IL10 gene polymorphisms of the donors were associated with the incidence of cytomegalovirus infection and occurrence of later infection in a subpopulation of recipients. Further, polymorphisms in genes related to the risk of thrombosis and those of certain cytokines were not associated with the occurrence of thrombosis, infarction, AR or graft survival. In the study of biomarkers for AR, whole blood samples were prospectively collected from adult kidney Tx patients. With real-time quantitative PCR (RT-QPCR) gene expression quantities of CD154 and ICOS differentiated the patients with AR from those without, but not from the patients with other causes of graft dysfunction. Biomarkers for SCR were studied in paediatric kidney Tx patients. We used RT-QPCR to quantify the gene expression of immunological candidate genes in a low-density array format. In addition, we used RT-QPCR to validate the results of the microarray analysis. No gene marker differentiated patients with SCR from those without SCR. This research demonstrates the lack of robust markers among polymorphisms or biomarkers in investigated genes that could be included in routine analysis in a clinical laboratory. In genetic studies, kidney Tx can be regarded as a complex trait, i.e. several environmental and genetic factors may determine its outcome. A number of currently unknown genetic factors probably influence the results of Tx.
  • Palo, Outi M (Helsingin yliopisto, 2010)
    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.
  • Saarinen, Silva (Helsingin yliopisto, 2013)
    Lymphomas are classified into Hodgkin and non-Hodgkin lymphomas (HL and NHL), and they are further categorized into tens of different subtypes. This thesis has focused on two rare lymphoma subtypes: nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) and primary mediastinal large B-cell lymphoma (PMBCL). Although the majority of lymphomas are sporadic, a familial component has been observed in both HL and NHL. The aim of this thesis was to study the genetic background of familial NLPHL and PMBCL predisposition. HL is classified into NLPHL and classical Hodgkin lymphoma (cHL). Germline mutations of the KLHDC8B gene have been observed in some families with cHL. In this study, the KLHDC8B gene was analyzed in four families with NLPHL, but mutations were not detected. The results suggest that germline mutations of KLHDC8B do not underlie familial NLPHL susceptibility. To clarify the genetic basis of NLPHL predisposition, a Finnish family with NLPHL susceptibility was studied with new genome-wide methods, SNP microarray and exome sequencing. Data from these two efforts were integrated, and a germline deletion of two nucleotides (c.2437-2438delAG) was observed in the NPAT gene. Subsequently, NPAT was screened in a number of familial and sporadic HL cases, and an in-frame deletion of one serine residue was found to be more frequent in the cases versus healthy controls (odds ratio 4.11, p = 0.018). Thus, NPAT was identified as a candidate gene for NLPHL predisposition. The familial risk in NLPHL was studied by using the Finnish Cancer Registry (FCR) and the Finnish Population Registries. Altogether 693 patients with NLPHL were identified, and a family member cohort comprising of the first-degree relatives of 692 patients was collected. Cancer data for the relatives was derived from the FCR. We calculated the standardized incidence ratios (SIRs) for all cancers, NHL, cHL and NLPHL in the family member cohort. In addition, the primary tumor samples from 20 first-degree relatives with HL diagnosis were histopathologically reviewed. In the family member cohort, the SIR for NLPHL was 19. Thus, a high familial risk in NLPHL was observed, suggesting that familial factors contribute to NLPHL susceptibility. An increased relative risk for NHL and cHL was also observed. We studied a family of three siblings with PMBCL and a cousin affected by diffuse large B-cell lymphoma (DLBCL). Re-examination of tumor samples revealed that all four tumors resembled each other histopathologically, proposing a common background. Genome-wide SNP array, linkage analysis and exome sequencing were performed to identify the genetic factor underlying PMBCL/DLBCL in this family. A missense change c.5533C>A (His1845Asn) was detected in the MLL gene and was the only non-database variation residing in linked regions and segregating in lymphoma affected family members. To our knowledge, this is the first description of familial clustering of PMBCL. Our findings suggest that sometimes PMBCL may arise in the context of familial predisposition, and MLL was identified as a candidate gene for PMBCL susceptibility. However, our observations are preliminary, and need to be confirmed in future studies.
  • Renkonen, Elise (Helsingin yliopisto, 2006)
    Hereditary nonpolyposis colorectal cancer (HNPCC) and familial adenomatous polyposis (FAP) are characterized by a high risk and early onset of colorectal cancer (CRC). HNPCC is due to a germline mutation in one of the following MMR genes: MLH1, MSH2, MSH6 and PMS2. A majority of FAP and attenuated FAP (AFAP) cases are due to germline mutations of APC, causing the development of multiple colorectal polyps. To date, over 450 MMR gene mutations and over 800 APC mutations have been identified. Most of these mutations lead to a truncated protein, easily detected by conventional mutation detection methods. However, in about 30% of HNPCC and FAP, and about 90% of AFAP families, mutations remain unknown. We aimed to clarify the genetic basis and genotype-phenotype correlation of mutation negative HNPCC and FAP/AFAP families by advanced mutation detection methods designed to detect large genomic rearrangements, mRNA and protein expression alterations, promoter mutations, phenotype linked haplotypes, and tumoral loss of heterozygosity. We also aimed to estimate the frequency of HNPCC in Uruguayan CRC patients. Our expression based analysis of mutation negative HNPCC divided these families into two categories: 1) 42% of families linked to the MMR genes with a phenotype resembling that of mutation positive, and 2) 58% of families likely to be associated with other susceptibility genes. Unbalanced mRNA expression of MLH1 was observed in two families. Further studies revealed that a MLH1 nonsense mutation, R100X was associated with aberrant splicing of exons not related to the mutation and an MLH1 deletion (AGAA) at nucleotide 210 was associated with multiple exon skipping, without an overall increase in the frequency of splice events. APC mutation negative FAP/AFAP families were divided into four groups according to the genetic basis of their predisposition. Four (14%) families displayed a constitutional deletion of APC with profuse polyposis, early age of onset and frequent extracolonic manifestations. Aberrant mRNA expression of one allele was observed in seven (24%) families with later onset and less frequent extracolonic manifestations. In 15 (52%) families the involvement of APC could neither be confirmed nor excluded. In three (10%) of the families a germline mutation was detected in genes other than APC: AXIN2 in one family, and MYH in two families. The families with undefined genetic basis and especially those with AXIN2 or MYH mutations frequently displayed AFAP or atypical polyposis. Of the Uruguayan CRC patients, 2.6% (12/461) fulfilled the diagnostic criteria for HNPCC and 5.6% (26/461) were associated with increased risk of cancer. Unexpectedly low frequency of molecularly defined HNPCC cases may suggest a different genetic profile in the Uruguayan population and the involvement of novel susceptibility genes. Accurate genetic and clinical characterization of families with hereditary colorectal cancers, and the definition of the genetic basis of "mutation negative" families in particular, facilitate proper clinical management of such families.
  • Georgitsi, Marianthi (Helsingin yliopisto, 2008)
    Much of the global cancer research is focused on the most prevalent tumors; yet, less common tumor types warrant investigation, since A rare disorder is not necessarily an unimportant one . The present work discusses a rare tumor type, the benign adenomas of the pituitary gland, and presents the advances which, during the course of this thesis work, contributed to the elucidation of a fraction of their genetic background. Pituitary adenomas are benign neoplasms of the anterior pituitary lobe, accounting for approximately 15% of all intracranial tumors. Pituitary adenoma cells hypersecrete the hormones normally produced by the anterior pituitary tissue, such as growth hormone (GH) and prolactin (PRL). Despite their non-metastasizing nature, these adenomas can cause significant morbidity and have to be adequately treated; otherwise, they can compromise the patient s quality of life, due to conditions provoked by hormonal hypersecretion, such as acromegaly in the case of GH-secreting adenomas, or due to compressive effects to surrounding tissues. The vast majority of pituitary adenomas arise sporadically, whereas a small subset occur as component of familial endocrine-related tumor syndromes, such as Multiple Endocrine Neoplasia type 1 (MEN1) and Carney complex (CNC). MEN1 is caused by germline mutations in the MEN1 tumor suppressor gene (11q13), whereas the majority of CNC cases carry germline mutations in the PRKAR1A gene (17q24). Pituitary adenomas are also encountered in familial settings outside the context of MEN1 and CNC, but unlike in the latter syndromes, their genetic background until recently remained elusive. Evidence in previous literature supported the notion that a tumor suppressor gene on 11q13, residing very close to but still distinct from MEN1, causes genetic susceptibility to pituitary tumors. The aim of the study was to identify the genetic cause of a low penetrance form of Pituitary Adenoma Predisposition (PAP) in families from Northern Finland. The present work describes the methodological approach that led to the identification of aryl hydrocarbon receptor interacting protein (AIP) as the gene causing PAP. Combining chip-based technologies (SNP and gene expression arrays) with traditional gene mapping methods and genealogy data, we showed that germline AIP mutations cause PAP in familial and sporadic settings. PAP patients were diagnosed with mostly adenomas of the GH/PRL-secreting cell lineage. In Finland, two AIP mutations accounted for 16% of all patients diagnosed with GH-secreting adenomas, and for 40% of patients being younger than 35 years of age at diagnosis. AIP is suggested to act as a tumor suppressor gene, a notion supported by the nature of the identified mutations (most are truncating) and the biallelic inactivation of AIP in the tumors studied. AIP has been best characterized as a cytoplasmic interaction partner of aryl hydrocarbon receptor (AHR), also known as dioxin receptor, but it has other partners as well. The mechanisms that underlie AIP-mediated pituitary tumorigenesis are to date largely unknown and warrant further investigation. Because AIP was identified in the genetically homogeneous Finnish population, it was relevant to examine its contribution to PAP in other, more heterogeneous, populations. Analysis of pituitary adenoma patient series of various ethnic origins and differing clinical settings revealed germline AIP mutations in all cohorts studied, albeit with low frequencies (range 0.8-7.4%). Overall, PAP patients were typically diagnosed at a young age (range 8-41 years), mainly with GH-secreting adenomas, without strong family history of endocrine disease. Because many PAP patients did not display family history of pituitary adenomas, detection of the condition appeared challenging. AIP immunohistochemistry was tested as a molecular pre-screening tool on mutation-positive versus mutation-negative tumors, and proved to be a potentially useful predictor of PAP. Mutation screening of a large cohort of colorectal, breast, and prostate tumors did not reveal somatic AIP mutations. These tumors, apart from being the most prevalent among men and women worldwide, have been associated with acromegaly, particularly colorectal neoplasia. In this material, AIP did not appear to contribute to the pathogenesis of these common tumor types and other genes seem likely to play a role in such tumorigenesis. Finally, the contribution of AIP in pediatric onset pituitary adenomas was examined in a unique population-based cohort of sporadic pituitary adenoma patients from Italy. Germline AIP mutations may account for a subset of pediatric onset GH-secreting adenomas (in this study one of seven GH-secreting adenoma cases or 14.3%), and appear to be enriched among young (≤25 years old) patients. In summary, this work reveals a novel tumor susceptibility gene, namely AIP, which causes genetic predisposition to pituitary adenomas, in particular GH-secreting adenomas. Moreover, it provides molecular tools for identification of individuals predisposed for PAP. Further elaborate studies addressing the functional role of AIP in normal and tumor cells will hopefully expand our knowledge on endocrine neoplasia and reveal novel cellular mechanisms of pituitary tumorigenesis, including potential drug targets.
  • Wan, Xing (2012)
    Class IIa (pediocin-like) bacteriocins are a major group of bacteriocins produced by lactic acid bacteria (LAB) characterised by their antilisterial activity. As a protective LAB strain for meat products, Leuconostoc carnosum 4010 kills Listeria by producing two class IIa bacteriocins, the well characterised leucocin A (LeuA) and the less studied leucocin C (LecC). Although the amino acid sequence of the secreted LecC has been published, the genes required for its production remain unknown. The aims of this study were to characterise the genes needed for LecC production and to express the lecC gene in Lactococcus lactis. The lecC gene was localised by Southern blot in a large plasmid different from the one harbouring LeuA genes in Ln. carnosum 4010 genome. Five genes in two operons were identified mainly by PCR-based methods and sequencing, namely, the structural gene (lecC) with a 72-bp signal sequence, the immunity gene (lecI) encoding a 97-aa immunity protein, two genes lecTS for an ABC transporter and the gene lecX for an accessory protein. The immunity function of LecI was demonstrated by expressing the lecI gene in LecC sensitive Listeria monocytogenes. Compared to the wild type, LecI-producing Listeria was more tolerant to LecC, thus corroborating the immunity function of LecI. For heterologous expression of LecC, the lecC gene was fused to the lactococcal usp45 signal sequence in the nisin-selectable and nisin-inducible food-grade secretion vector pLEB690. Consequently, bioactive LecC was secreted efficiently by the recombinant Lc. lactis. In conclusion, novel genes for the production of LecC in Ln. carnosum 4010 were identified. The findings indicate that LecC is produced by a dedicated system independent of LeuA. The successful production of functional LecC in Lc. lactis offers an attractive approach for the future application of bacteriocins in food production.
  • Metlin, Artem (Helsinki University Printing House, 2008)
    Rabies is a fatal disease that affects the central nervous system of all warmblooded mammals. The rabies virus belongs to the order Mononegavirales, family Rhabdoviridae, genus Lyssavirus. This virus has a negative single-stranded RNA genome and the virions are bullet-shaped. Rabies is reported in many countries throughout the world and has been registered in all continents except Australia, where only the bat Lyssaviruses have been found, and in Antarctica where the main vectors of rabies are absent. Russia and most of the bordering countries are affected by rabies. Finland was a rabies-free country from 1959 to 1988, when a sylvatic rabies epidemic appeared with raccoon dogs as the main host and vector of infection. That epidemic was eradicated by the oral vaccination of wild carnivores and the parenteral immunization of dogs and cats; and Finland has been rabies-free since 1991. However, this status is constantly under threat because rabies is endemic in Russia and Estonia. In June 2003, a horse imported to Finland from Estonia was clinically and laboratory diagnosed as rabies positive. The close relationship of the isolated equine virus strain with the current Estonian strains was verified during subsequent molecular epidemiological studies. Because the case was imported, it did not affect Finland’s rabies-free status. Also in 2007 another 2 imported cases of rabies were recorded: one in a human being from Philippines and the other in a dog from India. Five different antigenic variants of the rabies virus were identified among rabies positive field samples from Russia, Finland, and Estonia by using antinucleocapsid monoclonal antibodies. Two rabies virus field isolates showed a different reaction pattern that was similar to that of the vaccine strains of the SAD group, which might suggest a new antigen variant or reverted vaccine strain. Nevertheless, the sequence analysis showed that the vaccine strains RV-97 and SAD B19 included in the oral anti-rabies vaccine “Sinrab” (Russia) and “Fuchsoral” (Germany), respectively, differ considerably from all the field strains. Field rabies viruses collected in recent years from different regions of the Russian Federation were chosen on the basis of mAb studies and geographical origin for molecular epidemiological studies to characterize their genetic heterogeneity and to study their molecular epidemiology. In addition to the Russian viruses, archival samples from Estonia and Finland and Russian vaccine strains were also included in this study. Among the field viruses studied, two main phylogenetic groups were found, and designated as the Pan-Eurasian and Caucasian based on their geographical origin. The Pan-Eurasian 7 group including some reference viruses from Europe was further divided into four subgroups. All the vaccine strains were clearly different from the field strains. No recombination between the field and vaccine virus strains was observed. The critical roles of geographical isolation, the limitation of the genetic clustering, and the evolution of the rabies virus were shown during this study. The rabies virus vaccine strain RV-97 is widely used in Russia as a component of the oral anti-rabies vaccine “Sinrab”. To characterize the molecular properties of this strain, entire genome sequencing was conducted. A simple technique was developed to obtain this sequence, including the 3’- and 5’- ends. The entire genome sequence and deduced amino-acid sequences of the major viral proteins were compared with the sequences of other known fixed rabies viruses. The strain RV-97 formed a separate phylogenetic branch and seems to be more related to the group of Japanese strains. The field strains from the Caucasian group seem to be phylogenetically the nearest group to the RV-97 strain. The data shown herein makes it possible to develop molecular methods for distinguishing between the field rabies viruses from the vaccine strains for the rapid recognition of the vaccine strains that are unstable or have reverted back to their pathogenic form. The wide genetic heterogeneity verified in this study indicates that it is important to remain on permanent alert for the appearance of rabies.
  • Hytönen, Marjo (Helsingin yliopisto, 2013)
    Since the sequencing of the Canis lupus familiaris genome the dog has become a powerful tool for scientists. Selective breeding has created more than 400 different breeds each representing genetic isolates with breed-specific morphological and behavioral characteristics. Unique population history, available genealogical records, veterinary diagnostics and novel genomic tools greatly facilitate gene mapping studies in dogs. Given that over 600 genetic disorders have been described in dogs and that most of them are similar to human conditions, dogs have emerged as a clinically relevant model for human inherited disorders. This study explores the genetics of three different inherited developmental defects in dogs, caudal dysplasia, ectodermal dysplasia, and mucopolysaccharidosis VII, which all have counterparts in human. In this study, various clinical and pathological techniques have been used to characterize the phenotypes, and genetic methods such as genome-wide association studies and next-generation sequencing to resolve the genetics of the diseases. Moreover, functional studies in mice have been performed to explore the molecular role during embryonic development. The discoveries made here have established the affected breeds as models to further explore disease mechanisms and therapeutic methods, identified new disease pathways, and offered novel approaches for further developmental studies. Furthermore, this work has enabled the development of genetic tests for breeding purposes. Three different phenotypes have been investigated in this study. First, we studied genetics of caudal dysplasia, which in its mildest form is presenting as short-tail phenotype in dogs. A mutation in T (brachyury homolog) was earlier identified to cause this phenotype in Pembroke Welsh Corgis. Homozygous mutations of T in mouse result in severe caudal dysplasia and embryonic lethality suggesting an essential role for the T gene during mammalian development. The presence of the documented T mutation, c.189C>G, was investigated in 23 other breeds demonstrating that short-tailed dogs from 17 breeds were heterozygous for the mutation that associated completely with the phenotype. The homozygous mutation was suggested to be lethal, as no dogs homozygous for the mutation were found and an approximately 30% decrease was seen in the size of Swedish Vallhund litters when both parents were short-tailed. However, short-tailed dogs were found from six breeds that did not carry the known substitution or any other mutations in the T coding regions and therefore other genetic factors are yet to be discovered that affect the development of the posterior mesodermal region. The short-tailed dogs which do not have T mutation will serve as models in future studies to identify possible novel genetic factors for caudal dysplasia and related medical conditions. Second, a new gene was identified for a hairless phenotype and some of its upstream regulators were characterized. Hairless dog breeds show a breed characteristic which is in clinical terms an ectodermal dysplasia. In this study, the causative mutation for canine ectodermal dysplasia (CED) was sought and subsequently the function of this novel gene in the ectodermal organ development was explored. Accordingly, a genome-wide association study was performed and CED was mapped to dog chromosome 17. Haplotype association testing revealed a 160-kb haplotype, which was fine-mapped using three different breeds. The causative genetic mutation for CED was identified as a 7-bp duplication producing a frameshift and premature stop codon in a previously uncharacterized canine gene forkhead box protein I3 (FOXI3). The study provided a novel gene focus to aid research into ectodermal development. Therefore, a detailed expression pattern of murine Foxi3 during the development of the ectodermal organs was constructed and a series of tissue culture experiments and expression analyses with mouse embryos were performed to assess the function of Foxi3 in mammalian embryogenesis. The results suggest that Foxi3 regulates hair follicle and tooth formation as well as the development of mammary and salivary gland, nail, and eye. Ectodysplasin and activin A were identified as upstream regulators of Foxi3. Third, Brazilian Terriers with severe skeletal defects at early puppyhood were identified through information provided by breeders. Subsequently, a major aim of this work was to describe the clinical and pathological features of the syndrome and to identify its genetic cause. Clinicopathological examinations and pedigree analysis demonstrated that the affected puppies had a recessive spondyloepihyseal dysplasia. The disease locus was mapped to chromosome 6 and a mutation leading to pathogenic p.P289L change in a conserved functional domain of β-glucuronidase (GUSB) was identified. Elevated glycosaminoglycans were detected in urine and only a residual β-glucuronidase activity was observed in the serum of the affected dogs, which confirmed the pathogenity of the mutation. GUSB defects result in mucopolysaccharidosis VII (MPS VII) in several species and thus the mutation defined the syndrome as MPS VII in Brazilian Terriers. Overall, this study illustrates how unique morphological diversity and enriched genetic alterations in closed populations can be efficiently harnessed to gain new insights into developmental biology across species. For example, the identification of the CED mutation in FOXI3 revealed a completely novel gene with a previously unknown essential function in ectodermal development. This work has established several novel large animal models to further explore disease mechanisms and to develop therapeutic methods. Moreover, several new DNA tests have been developed for different breeds of dogs to eradicate or, to control better, the conditions through improved breeding plans. This will improve the welfare of our beloved pets.