Cutaneous porphyrias : Clinical and histopathological study

The prevalence of variegate porphyria (VP) (2.1:100 000, in 2006 n=108) was higher in Finland than elsewhere in European countries due to a founder effect (R152C). The incidence of VP was estimated at 0.2:1 000 000 based on the number of new symptomatic patients yearly. The prevalence of porphyria cutanea tarda (PCT) was 1.2:100 000 (in 2006 n=63), which is only one fourth of the numbers reported from other European countries. The estimated incidence of PCT was 0.5:1 000 000. Based on measurements of the uroporphyrinogen decarboxylase activity in erythrocytes, the proportion of familial PCT was 49% of the cases. The prevalence of erythropoietic protoporphyria (EPP) was at 0.8:100 000 (in 2006 n=39) including asymptomatic carriers of a mutation in the ferrochelatase (FECH) gene. The incidence of EPP was estimated at 0.1:1 000 000. After 1980 the penetrance was 37% among patients with VP. Of the mutation carriers (n=57) 30% manifested with skin symptoms. Frequency of skin symptom as only clinical sign was stable before or after 1980 (22% vs. 21%), but acute attacks became infrequent (29% vs. 7%). Of the symptomatic patients 30% had both acute attacks and skin symptoms and 80% had skin symptoms. Fragility (95%) and blistering (46%) of the skin in the backs of the hands were the most common skin symptoms. Transient correction of porphyrin metabolism using eight haem arginate infusions within five weeks had no effect on the skin symptoms in three of four patients with VP. In one case skin symptoms disappeared transiently. One patient with homozygous VP had severe photosensitivity since birth. Sensory polyneuropathy, glaucoma and renal failure developed during the 25-year follow-up without the presence of acute attacks. The I12T mutation was detected in both of his alleles in the protoporphyrinogen oxidase gene. Lack of skin symptoms and infrequency of acute attacks (1/9) in the patients with I12T mutation at the heterozygous stage indicate a mild phenotype (the penetrance 11%). Four mutations (751delGAGAA, 1122delT, C286T, C343T) in the FECH gene were characterised in four of 15 families with EPP. Burning pain (96%) and swelling (92%) of the sun-exposed skin were the major skin symptoms. Hepatopathy appeared in one of 25 symptomatic patients (4%). Clinical manifestations and associated factors of PCT were similar in the sporadic and familial types of PCT. The majority of the patients with PCT had one to three precipitating factors: alcohol intake (78%), mutations in hemochromatosis associated gene (50%), use of oestrogen (25% of women) and hepatitis B or C infections (25 %). Fatty liver disease (67%) and siderosis (67%) were commonly found in their liver biopsies. The major histopathological change of the sun-exposed skin in the patients with VP (n=20), EPP (n=8) and PCT (n=5) was thickening of the vessel walls of the upper dermis suggesting that the vessel wall is the primary site of the phototoxic reaction in each type of porphyria. The fine structure of the vessel walls was similar in VP, EPP and PCT consisting of the multilayered basement membrane and excess of finely granular substance between the layers which were surrounded by the band of homogenous material. EPP was characterised by amorphous perivascular deposits extending also to the extravascular space. In direct immunofluorescence study homogenous IgG deposits in the vessel walls of the upper dermis of the sun-exposed skin were demonstrated in each type of porphyria. In EPP the excess material around vessel walls consisted of other proteins such as serum amyloid protein, and kappa and lambda light chains in addition to the basement membrane constituents such as collagen IV and laminin. These results suggest that the alterations of the vessel walls are a consequence of the repeated damage and the repairing process in the vessel wall. The microscopic alterations could be demonstrated even in the normal looking but sun-exposed skin of the patients with EPP during the symptom-free phase suggesting that vascular change can be chronic. The stability of vascular changes in the patients with PCT after treatment indicates that circulating porphyrins are not important for the maintenance of the changes.

Tumour necrosis factor receptor-associated periodic syndrome (TRAPS) : Identification of novel TNFRSF1A mutations and intracellular signalling defects

The systemic autoinflammatory disorders are a group of rare diseases characterized by periodically recurring episodes of acute inflammation and a rise in serum acute phase proteins, but with no signs of autoimmunity. At present eight hereditary syndromes are categorized as autoinflammatory, although the definition has also occasionally been extended to other inflammatory disorders, such as Crohn s disease. One of the autoinflammatory disorders is the autosomally dominantly inherited tumour necrosis factor receptor-associated periodic syndrome (TRAPS), which is caused by mutations in the gene encoding the tumour necrosis factor type 1 receptor (TNFRSF1A). In patients of Nordic descent, cases of TRAPS and of three other hereditary fevers, hyperimmunoglobulinemia D with periodic fever syndrome (HIDS), chronic infantile neurologic, cutaneous and articular syndrome (CINCA) and familial cold autoinflammatory syndrome (FCAS), have been reported, TRAPS being the most common of the four. Clinical characteristics of TRAPS are recurrent attacks of high spiking fever, associated with inflammation of serosal membranes and joints, myalgia, migratory rash and conjunctivitis or periorbital cellulitis. Systemic AA amyloidosis may occur as a sequel of the systemic inflammation. The aim of this study was to investigate the genetic background of hereditary periodically occurring fever syndromes in Finnish patients, to explore the reliability of determining serum concentrations of soluble TNFRSF1A and metalloproteinase-induced TNFRSF1A shedding as helpful tools in differential diagnostics, as well as to study intracellular NF-κB signalling in an attempt to widen the knowledge of the pathomechanisms underlying TRAPS. Genomic sequencing revealed two novel TNFRSF1A mutations, F112I and C73R, in two Finnish families. F112I was the first TNFRSF1A mutation to be reported in the third extracellular cysteine-rich domain of the gene and C73R was the third novel mutation to be reported in a Finnish family, with only one other TNFRSF1A mutation having been reported in the Nordic countries. We also presented a differential diagnostic problem in a TRAPS patient, emphasizing for the clinician the importance of differential diagnostic vigiliance in dealing with rare hereditary disorders. The underlying genetic disease of the patient both served as a misleading factor, which possibly postponed arrival at the correct diagnosis, but may also have predisposed to the pathologic condition, which led to a critical state of the patient. Using a method of flow cytometric analysis modified for the use on fresh whole blood, we studied intracellular signalling pathways in three Finnish TRAPS families with the F112I, C73R and the previously reported C88Y mutations. Evaluation of TNF-induced phosphorylation of NF-κB and p38, revealed low phosphorylation profiles in nine out of ten TRAPS patients in comparison to healthy control subjects. This study shows that TRAPS is a diagnostic possibility in patients of Nordic descent, with symptoms of periodically recurring fever and inflammation of the serosa and joints. In particular in the case of a family history of febrile episodes, the possibility of TRAPS should be considered, if an etiology of autoimmune or infectious nature is excluded. The discovery of three different mutations in a population as small as the Finnish, reinforces the notion that the extracellular domain of TNFRSF1A is prone to be mutated at the entire stretch of its cysteine-rich domains and not only at a limited number of sites, suggesting the absence of a founder effect in TRAPS. This study also demonstrates the challenges of clinical work in differentiating the symptoms of rare genetic disorders from those of other pathologic conditions and presents the possibility of an autoinflammatory disorder as being the underlying cause of severe clinical complications. Furthermore, functional studies of fresh blood leukocytes show that TRAPS is often associated with a low NF-κB and p38 phosphorylation profile, although low phosphorylation levels are not a requirement for the development of TRAPS. The aberrant signalling would suggest that the hyperinflammatory phenotype of TRAPS is the result of compensatory NF-κB-mediated regulatory mechanisms triggered by a deficiency of the innate immune response.

Dissection of the genetic background of childhood onset progressive myoclonic epilepsies

The progressive myoclonic epilepsies (PMEs) are a clinically and etiologically heterogeneous group of symptomatic epilepsies characterized by myoclonus, tonic-clonic seizures, psychomotor regression and ataxia. Different disorders have been classified as PMEs. Of these, the group of neuronal ceroid lipofuscinoses (NCLs) comprise an entity that has onset in childhood, being the most common cause of neurodegeneration in children. The primary aim of this thesis was to dissect the molecular genetic background of patients with childhood onset PME by studying candidate genes and attempting to identify novel PME-associated genes. Another specific aim was to study the primary protein properties of the most recently identified member of the NCL-causing proteins, MFSD8. To dissect the genetic background of a cohort of Turkish patients with childhood onset PME, a screen of the NCL-associated genes PPT1, TPP1, CLN3, CLN5, CLN6, MFSD8, CLN8 and CTSD was performed. Altogether 49 novel mutations were identified, which together with 56 mutations found by collaborators raised the total number of known NCL mutations to 364. Fourteen of the novel mutations affect the recently identified MFSD8 gene, which had originally been identified in a subset of mainly Turkish patients as the underlying cause of CLN7 disease. To investigate the distribution of MFSD8 defects, a total of 211 patients of different ethnic origins were evaluated for mutations in the gene. Altogether 45 patients from nine different countries were provided with a CLN7 molecular diagnosis, denoting the wide geographical occurrence of MFSD8 defects. The mutations are private with only one having been established by a founder-effect in the Roma population from the former Czechoslovakia. All mutations identified except one are associated with the typical clinical picture of variant late-infantile NCL. To address the trafficking properties of MFSD8, lysosomal targeting of the protein was confirmed in both neuronal and non-neuronal cells. The major determinant for this lysosomal sorting was identified to be an N-terminal dileucine based signal (9-EQEPLL-14), recognized by heterotetrameric AP-1 adaptor proteins, suggesting that MFSD8 takes the direct trafficking pathway en route to the lysosomes. Expression studies revealed the neurons as the primary cell-type and the hippocampus and cerebellar granular cell layer as the predominant regions in which MFSD8 is expressed. To identify novel genes associated with childhood onset PME, a single nucleotide polymorphism (SNP) genomewide scan was performed in three small families and 18 sporadic patients followed by homozygosity mapping to determine the candidate loci. One of the families and a sporadic patient were positive for mutations in PLA2G6, a gene that had previously been shown to cause infantile neuroaxonal dystrophy. Application of next-generation sequencing of candidate regions in the remaining two families led to identification of a homozygous missense mutation in USP19 for the first and TXNDC6 for the second family. Analysis of the 18 sporadic cases mapped the best candidate interval in a 1.5 Mb region on chromosome 7q21. Screening of the positional candidate KCTD7 revealed six mutations in seven unrelated families. All patients with mutations in KCTD7 were reported to have early onset PME, rapid disease progression leading to dementia and no pathologic hallmarks. The identification of KCTD7 mutations in nine patients and the clinical delineation of their phenotype establish KCTD7 as a gene for early onset PME. The findings presented in this thesis denote MFSD8 and KCTD7 as genes commonly associated with childhood onset symptomatic epilepsy. The disease-associated role of TXNDC6 awaits verification through identification of additional mutations in patients with similar phenotypes. Completion of the genetic spectrum underlying childhood onset PMEs and understanding of the gene products functions will comprise important steps towards understanding the underlying pathogenetic mechanisms, and will possibly shed light on the general processes of neurodegeneration and nervous system regulation, facilitating the diagnosis, classification and possibly treatment of the affected cases.

The effect of interrogative function on intonation in spontaneous and read Finnish

Goals This study aims to map the effect of interrogative function on the intonation of spontaneous and read Finnish. Earlier research shows that the most prominent feature in Finnish question intonation is an appeal to the listener. Question word questions typically start with a high peak which is followed by falling intonation. In yes/no questions, F0 remains on a high level until the word carrying sentence stress and then falls. Final rises are mainly found in intonation clichés such as "Ai mitä?" ("What?") These earlier results are based on read speech and enacted dialogues. In this study, questions and statements found in spontaneous dialogues were compared. These utterances were also compared with read versions of the same utterances. Fundamental frequency values were compared using a mixed model. Contours were also grouped using auditory and visual inspection. Thus it was possible to compare frequencies of contour types according to utterance type and speech style. The position of questions in the F0 distribution of the whole material was also investigated in this study. Method The material consisted of four spontaneous dialogues and their read versions. The speakers were young adults from the Helsinki metropolitan area, four females and four males. The whole material was first divided into broad dialogue function categories arising from the material and F0 curves were calculated for each category. After this, 277 questions and 244 statements were selected for closer inspection. Values reflecting F0 distribution and contour shape were measured from the F0 contours of these utterances. A mixed model was used to analyse the differences. Utterance type, question type, speech style and speaker gender were used as fixed effects. The frequencies of F0 contour types were compared using a Chi square test. Additional material in this study came from eight young female speakers in central Finland. Results and conclusions In the mixed model analysis, significant differences were found both between questions and statements and between spontaneous and read speech. Generally, utterance type affected the variables reflecting contour type while speech style affected the variables reflecting F0 distribution. The effect of question type was not clearly visible. In read speech the contours resembled earlier results more closely. Speakers had different strategies in differentiating between questions and statements. In the whole material, F0 was slightly higher in questions than in statements. The effect of dialectal background could be seen in the contour types. The results show that interrogative function affects intonation in both spontaneous and read Finnish.

Living on the edge : Population genetics of Finno-Ugric-speaking humans in North Eurasia

The major aim of this thesis was to examine the origins and distribution of uniparental and autosomal genetic variation among the Finno-Ugric-speaking human populations living in Boreal and Arctic regions of North Eurasia. In more detail, I aimed to disentangle the underlying molecular and population genetic factors which have produced the patterns of uniparental and autosomal genetic diversity in these populations. Among Finno-Ugrics the genetic amalgamation and clinal distribution of West and East Eurasian gene pools were observed within uniparental markers. This admixture indicates that North Eurasia was colonized through Central Asia/ South Siberia by human groups already carrying both West and East Eurasian lineages. The complex combination of founder effects, gene flow and genetic drift underlying the genetic diversity of the Finno-Ugric- speaking populations were emphasized by low haplotype diversity within and among uniparental and biparental markers. A high prevalence of lactase persistence allele among the North Eurasian Finno- Ugric agriculturalist populations was also shown indicating a local adaptation to subsistence change with lactose rich diet. Moreover, the haplotype background of lactase persistence allele among the Finno- Ugric-speakers strongly suggested that the lactase persistence T-13910 mutation was introduced independently more than once to the North Eurasian gene pool. A significant difference in genetic diversity, haplotype structure and LD distribution within the cytochrome P450 CYP2C and CYP2D regions revealed the unique gene pool of the Finno-Ugric Saami created mainly by population genetic processes compared to other Europeans and sub-Saharan Mandenka population. From all studied populations the Saami showed also significantly the highest allele frequency of a CYP2C19 gene mutation causing variable drug reactions. The diversity patterns observed within CYP2C and CYP2D regions emphasize the strong effect of demographic history shaping genetic diversity and LD especially among such small and constant size populations as the Finno-Ugric-speaking Saami. Moreover, the increased LD in Saami due to genetic drift and/or admixture was shown to offer an advantage for further attempts to identify alleles associated to common complex pharmacogenetic traits.

Effect of waiting time on health outcomes and service utilization : A prospective randomized study on patients admitted to hospital for hip or knee replacement

Although the principle of equal access to medically justified treatment has been promoted by official health policies in many Western health care systems, practices do not completely meet policy targets. Waiting times for elective surgery vary between patient groups and regions, and growing problems in the availability of services threaten equal access to treatment. Waiting times have come to the attention of decision-makers, and several policy initiatives have been introduced to ensure the availability of care within a reasonable time. In Finland, for example, the treatment guarantee came into force in 2005. However, no consensus exists on optimal waiting time for different patient groups. The purpose of this multi-centre randomized controlled trial was to analyse health-related quality of life, pain and physical function in total hip or knee replacement patients during the waiting time and to evaluate whether the waiting time is associated with patients health outcomes at admission. This study also assessed whether the length of waiting time is associated with social and health services utilization in patients awaiting total hip or knee replacement. In addition, patients health-related quality of life was compared with that of the general population. Consecutive patients with a need for a primary total hip or knee replacement due to osteoarthritis were placed on the waiting list between August 2002 and November 2003. Patients were randomly assigned to a short waiting time (maximum 3 months) or a non-fixed waiting time (waiting time not fixed in advance, instead the patient followed the hospitals routine practice). Patients health-related quality of life was measured upon being placed on the waiting list and again at hospital admission using the generic 15D instrument. Pain and physical function were evaluated using the self-report Harris Hip Score for hip patients and a scale modified from the Knee Society Clinical Rating System for knee patients. Utilization measures were the use of home health care, rehabilitation and social services, physician visits and inpatient care. Health and social services use was low in both waiting time groups. The most common services used while waiting were rehabilitation services and informal care, including unpaid care provided by relatives, neighbours and volunteers. Although patients suffered from clear restrictions in usual activities and physical functioning, they seemed primarily to lean on informal care and personal networks instead of professional care. While longer waiting time did not result in poorer health-related quality of life at admission and use of services during the waiting time was similar to that at the time of placement on the list, there is likely to be higher costs of waiting by people who wait longer simply because they are using services for a longer period. In economic terms, this would represent a negative impact of waiting. Only a few reports have been published of the health-related quality of life of patients awaiting total hip or knee replacement. These findings demonstrate that, in addition to physical dimensions of health, patients suffered from restrictions in psychological well-being such as depression, distress and reduced vitality. This raises the question of how to support patients who suffer from psychological distress during the waiting time and how to develop strategies to improve patients initiatives to reduce symptoms and the burden of waiting. Key words: waiting time, total hip replacement, total knee replacement, health-related quality of life, randomized controlled trial, outcome assessment, social service, utilization of health services

Molecular Genetics of Schizophrenia and Related Intermediate Phenotypes in a Founder Population

Schizophrenia, affecting about 1% of population worldwide, is a severe mental disorder characterized by positive and negative symptoms, such as psychosis and anhedonia, as well as cognitive deficits. At present, schizophrenia is considered a complex disorder of neurodevelopmental origin with both genetic and environmental factors contributing to its onset. Although a number of candidate genes for schizophrenia have been highlighted, only very few schizophrenia patients are likely to share identical genetic liability. This study is based on the nation-wide schizophrenia family sample of the National Institute for Health and Welfare, and represents one of the largest and most well-characterized familial series in the world. In the first part of this study, we investigated the roles of the DTNBP1, NRG1, and AKT1 genes in the background of schizophrenia in Finland. Although these genes are associated with schizophrenia liability in several populations, any significant association with clinical diagnostic information of schizophrenia remained absent in our sample of 441 schizophrenia families. In the second part of this study, we first replicated schizophrenia linkage on the long arm of chromosome 7 in 352 schizophrenia families. In the following association analysis, we utilized additional clinical disorder features and intermediate phenotypes – endophenotypes - in addition to diagnostic information from altogether 290 neuropsychologically assessed schizophrenia families. An intragenic short tandem repeat allele of the regional RELN gene, supposed to play a role in the background of several neurodevelopmental disorders, showed significant association with poorer cognitive functioning and more severe schizophrenia symptoms. Additionally, this risk allele was significantly more prevalent among the individuals affected with schizophrenia spectrum disorders. We have previously identified linkage of schizophrenia and its cognitive endophenotypes on the long arms of chromosomes 2, 4, and 5. In the last part of this study, we selected altogether 104 functionally relevant candidate genes from the linked regions. We detected several promising associations, of which especially interesting are the ERBB4 gene, showing association with the severity of schizophrenia symptoms and impairments in traits related to verbal abilities, and the GRIA1 gene, showing association with the severity of schizophrenia symptoms. Our results extend the previous evidence that the genetic risk for schizophrenia is at least partially mediated via the effects of the candidate genes and their combinations on relevant brain systems, resulting in alterations in different disorder domains, such as the cognitive deficits.

The effect of temperature on height growth of Scots pine in northern Finland

The effect of temperature on height growth of Scots pine in the northern boreal zone in Lapland was studied in two different time scales. Intra-annual growth was monitored in four stands in up to four growing seasons using an approximately biweekly measurement interval. Inter-annual growth was studied using growth records representing seven stands and five geographical locations. All the stands were growing on a dry to semi-dry heath that is a typical site type for pine stands in Finland. The applied methodology is based on applied time-series analysis and multilevel modelling. Intra-annual elongation of the leader shoot correlated with temperature sum accumulation. Height growth ceased when, on average, 41% of the relative temperature sum of the site was achieved (observed minimum and maximum were 38% and 43%). The relative temperature sum was calculated by dividing the actual temperature sum by the long-term mean of the total annual temperature sum for the site. Our results suggest that annual height growth ceases when a location-specific temperature sum threshold is attained. The positive effect of the mean July temperature of the previous year on annual height increment proved to be very strong at high latitudes. The mean November temperature of the year before the previous had a statistically significantly effect on height increment in the three northernmost stands. The effect of mean monthly precipitation on annual height growth was statistically insignificant. There was a non-linear dependence between length and needle density of annual shoots. Exceptionally low height growth results in high needle-density, but the effect is weaker in years of average or good height growth. Radial growth and next year s height growth are both largely controlled by current July temperature. Nevertheless, their growth variation in terms of minimum and maximum is not necessarily strongly correlated. This is partly because height growth is more sensitive to changes in temperature. In addition, the actual effective temperature period is not exactly the same for these two growth components. Yet, there is a long-term balance that was also statistically distinguishable; radial growth correlated significantly with height growth with a lag of 2 years. Temperature periods shorter than a month are more effective variables than mean monthly values, but the improvement is on the scale of modest to good when applying Julian days or growing-degree-days as pointers.

Effect of phenolic-rich plant materials on protein and lipid oxidation reactions

The antioxidant activity of natural plant materials rich in phenolic compounds is being widely investigated for protection of food products sensitive to oxidative reactions. In this thesis plant materials rich in phenolic compounds were studied as possible antioxidants to prevent protein and lipid oxidation reactions in different food matrixes such as pork meat patties and corn oil-in water emulsions. Loss of anthocyanins was also measured during oxidation in corn oil-in-water emulsions. In addition, the impact of plant phenolics on amino acid level was studied using tryptophan as a model compound to elucidate their role in preventing the formation of tryptophan oxidation products. A high-performance liquid chromatography (HPLC) method with ultraviolet and fluorescence detection (UV-FL) was developed that enabled fast investigation of formation of tryptophan derived oxidation products. Byproducts of oilseed processes such as rapeseed (Brassica rapa L.), camelina (Camelina sativa) and soy meal (Glycine max L.) as well as Scots pine bark (Pinus sylvestris) and several reference compounds were shown to act as antioxidants toward both protein and lipid oxidation in cooked pork meat patties. In meat, the antioxidant activity of camelina, rapeseed and soy meal were more pronounced when used in combination with a commercial rosemary extract (Rosmarinus officinalis). Berry phenolics such as black currant (Ribes nigrum) anthocyanins and raspberry (Rubus idaeus) ellagitannins showed potent antioxidant activity in corn oil-in-water emulsions toward lipid oxidation with and without β-lactoglobulin. The antioxidant effect was more pronounced in the presence of β-lactoglobulin. The berry phenolics also inhibited the oxidation of tryptophan and cysteine side chains of β-lactoglobulin. The results show that the amino acid side chains were oxidized prior the propagation of lipid oxidation, thereby inhibiting fatty acid scission. In addition, the concentration and color of black currant anthocyanins decreased during the oxidation. Oxidation of tryptophan was investigated in two different oxidation models with hydrogen peroxide (H2O2) and hexanal/FeCl2. Oxidation of tryptophan in both models resulted in oxidation products such as 3a-hydroxypyrroloindole-2-carboxylic acid, dioxindolylalanine, 5-hydroxy-tryptophan, kynurenine, N-formylkynurenine and β-oxindolylalanine. However, formation of tryptamine was only observed in tryptophan oxidized in the presence of H2O2. Pine bark phenolics, black currant anthocyanins, camelina meal phenolics as well as cranberry proanthocyanidins (Vaccinium oxycoccus) provided the best antioxidant effect toward tryptophan and its oxidation products when oxidized with H2O2. The tryptophan modifications formed upon hexanal/FeCl2 treatment were efficiently inhibited by camelina meal followed by rapeseed and soy meal. In contrast, phenolics from raspberry, black currant, and rowanberry (Sorbus aucuparia) acted as weak prooxidants. This thesis contributes to elucidating the effects of natural phenolic compounds as potential antioxidants in order to control and prevent protein and lipid oxidation reactions. Understanding the relationship between phenolic compounds and proteins as well as lipids could lead to the development of new, effective, and multifunctional antioxidant strategies that could be used in food, cosmetic and pharmaceutical applications.