Shwachman-Diamond syndrome : Clinical, genetic and radiological study

Shwachman-Diamond syndrome (SDS) is a rare autosomal recessive disorder in which the cardinal symptoms arise from exocrine pancreatic insufficiency and bone marrow dysfunction. Previous studies have suggested increased risk of fatal complications among Finnish SDS infants. The genetic defect responsible for the disease was recently identified; the SBDS gene is located at chromosome 7q11 and encodes a protein that is involved in ribosome biosynthesis. The discovery of the SBDS gene has opened new insights into the pathogenesis of this multi-organ disease. This study aimed to assess phenotypic and genotypic features of Finnish patients with SDS. Seventeen Finnish patients with a clinical diagnosis of SDS were included in the study cohort. Extensive clinical, biochemical and imaging assessments were performed to elucidate the phenotypic features, and the findings were correlated with the SBDS genotype. Imaging studies included abdominal magnetic reso-nance imaging (MRI), brain MRI, cardiac echocardiography including tissue Doppler examination, and cardiac MRI. The skeletal phenotype was assessed by dual-energy X-ray absorptiometry and bone histomorphometry. Twelve patients had mutations in the SBDS gene. In MRI, a characteristic pattern of fat-replaced pancreas with occasional enhancement of scattered parenchymal foci and of pancreatic duct was noted in the SBDS mutation-positive patients while the mutation-negative patients did not have pancreatic fat accumulation. The patients with SBDS mutations had significantly reduced bone mineral density associated with low-energy peripheral fractures and vertebral compression fractures. Bone histomorphometry confirmed low-turnover osteoporosis. The patients with SBDS mutations had learning difficulties and smaller head size and brain volume than control subjects. Corpus callosum, cerebellar vermis, and pos-terior fossa structures were significantly smaller in SDS patients than in controls. Patients with SDS did not have evidence of clinical heart disease or myocardial fibrosis. However, subtle diastolic changes in the right ventricle and exercise-induced changes in the left ventricle contractile reserve were observed. This study expanded the phenotypic features of SDS to include primary low-turnover osteoporosis and structural alterations in the brain. Pancreatic MRI showed characteristic changes in the SBDS mutation-positive patients while these were absent in the mutation-negative patients, suggesting that MRI can be used to differentiate patients harbouring SBDS mutations from those without mutations. No evidence for clinical cardiac manifestations was found, but imaging studies revealed slightly altered myocardial function that may have clinical implications. These findings confirm the pleiotropic nature of SDS and underscore the importance of careful multidisciplinary follow-up of the affected individuals.

The impact of the metabolic syndrome and parental risk factors in patients with type 1 diabetes

Background: One-third of patients with type 1 diabetes develop diabetic complications, such as diabetic nephropathy. The diabetic complications are related to a high mortality from cardiovascular disease, impose a great burden on the health care system, and reduce the health-related quality of life of patients. Aims: This thesis assessed, whether parental risk factors identify subjects at a greater risk of developing diabetic complications. Another aim was to evaluate the impact of a parental history of type 2 diabetes on patients with type 1 diabetes. A third aim was to assess the role of the metabolic syndrome in patients with type 1 diabetes, both its presence and its predictive value with respect to complications. Subjects and methods: This study is part of the ongoing nationwide Finnish Diabetic Nephropathy (FinnDiane) Study. The study was initiated in 1997, and, thus far, 4,800 adult patients with type 1 diabetes have been recruited. Since 2004, follow-up data have also been collected in parallel to the recruitment of new patients. Studies I to III have a cross-sectional design, whereas Study IV has a prospective design. Information on parents was obtained from the patients with type 1 diabetes by a questionnaire. Results: Clustering of parental hypertension, cardiovascular disease, and diabetes (type 1 and type 2) was associated with diabetic nephropathy in patients with type 1 diabetes, as was paternal mortality. A parental history of type 2 diabetes was associated with a later onset of type 1 diabetes, a higher prevalence of the metabolic syndrome, and a metabolic profile related to insulin resistance, despite no difference in the distribution of human leukocyte antigen genotypes or the presence of diabetic complications. A maternal history of type 2 diabetes, seemed to contribute to a worse metabolic profile in the patients with type 1 diabetes than a paternal history. The metabolic syndrome was a frequent finding in patients with type 1 diabetes, observed in 38% of males and 40% of females. The prevalence increased with worsening of the glycemic control and more severe renal disease. The metabolic syndrome was associated with a 3.75-fold odds ratio for diabetic nephropathy, and all of the components of the syndrome were independently associated with diabetic nephropathy. The metabolic syndrome, independent of diabetic nephropathy, increased the risk of cardiovascular events and cardiovascular and diabetes-related mortality over a 5.5-year follow-up. With respect to progression of diabetic nephropathy, the role of the metabolic syndrome was less clear, playing a strong role only in the progression from macroalbuminuria to end-stage renal disease. Conclusions: Familial factors and the metabolic syndrome play an important role in patients with type 1 diabetes. Assessment of these factors is an easily applicable tool in clinical practice to identify patients at a greater risk of developing diabetic complications.

Cyclins in Breast Cancer

Breast cancer is the most common malignancy in women in Western countries. It is a heterogeneous disease with varying biological characteristics and aggressiveness. Family history is one of the strongest predisposing factors for breast cancer. The known susceptibility genes explain only around 25% of all familial breast cancers. At least part of the unknown familial aggregation may be caused by several low-penetrance variants that occur commonly in the general population. Cyclins are cell cycle-regulating proteins. Cyclin expression oscillates during the cell cycle and is under strict control. In cancer cells, cyclin expression often becomes deregulated, leading to uncontrolled cell division and proliferation, one of the hallmarks of cancer. In this study, we investigated the role of cyclins in breast cancer predisposition, pathogenesis, and tumor behavior. Cyclin A immunohistochemistry was evaluated both on traditional large sections and on tissue microarrays (TMA). The concordance of the results was good, indicating that TMA is a reliable method for studying cyclin expression in breast cancer. The expression of cyclins D1, E, and B1 was studied among 1348 invasive breast cancers on TMA. Familial BRCA1/2-mutation negative tumors had significantly more often low cyclin E and high cyclin D1 expression than BRCA1/2 related or sporadic tumors. Unique cyclin E and D1 expression patterns among familial non-BRCA1/2 breast cancers may reflect different predisposition and pathogenesis in these groups and help to differentiate mutation-positive from mutation-negative familial cancers. High cyclin E expression was associated with an aggressive breast cancer phenotype and was an independent marker of poor metastasis-free survival. High cyclin D1 was associated with high grade and high proliferation among estrogen receptor (ER)-positive but with low grade and low proliferation among ER-negative breast cancers. Among ER-positive cancers not treated with chemotherapy, high cyclin D1 showed a trend towards shorter metastasis-free survival. These results suggest that different mechanisms may drive proliferation in ER-negative and -positive breast cancers and that cyclin D1 has a particularly important role in tumorigenesis of hormone receptor-positive breast cancer. High cyclin B1 expression was associated with aggressive breast cancer features and had an independent impact on survival. The results suggest that cyclin B1 immunohistochemistry is a method that could easily be adapted for routine use and is an independent prognostic factor, adding specificity to prognostic evaluation conducted with traditional markers. A commonly occurring cyclin D1 gene polymorphism A870G was associated with increased breast cancer risk in a large material of Finnish and Canadian breast cancer patients. The interaction of the high-activity alleles of cyclin D1 gene and estrogen metabolism gene COMT conferred an even higher risk. These results show that cyclin D1 and COMT act synergistically to contribute to breast cancer progression and that individual risk for breast cancer can be altered by the combined effect of polymorphisms with low-penetrance alleles. By investigating critical cell cycle regulator protein cyclins, we revealed new aspects of breast cancer predisposition, pathogenesis, and clinical course.

Cancer incidence, mortality, and pregnancy outcome among women treated for cervical intraepithelial neoplasia

Cervical cancer develops through precursor lesions, i.e. cervical intraepithelialneoplasms (CIN). These can be detected and treated before progression to invasive cancer. The major risk factor for developing cervical cancer or CIN is persistent or recurrent infection with high-risk human papilloma virus (hrHPV). Other associated risk factors include low socioeconomic status, smoking, sexually transmitted infections, and high number of sexual partners, and these risk factors can predispose to some other cancers, excess mortality, and reproductive health complications as well. The aim was to study long-term cancer incidence, mortality, and reproductive health outcomes among women treated for CIN. Based on the results, we could evaluate the efficacy and safety of CIN treatment practices and estimate the role of the risk factors of CIN patients for cancer incidence, mortality, and reproductive health. We collected a cohort of 7 599 women treated for CIN at Helsinki University Central Hospital from 1974 to 2001. Information about their cancer incidence, cause of death, birth of children and other reproductive endpoints, and socio-economic status were gathered through registerlinkages to the Finnish Cancer Registry, Finnish Population Registry, and Statistics Finland. Depending on the endpoints in question, the women treated were compared to the general population, to themselves, or to an age- and municipality-matched reference cohort. Cervical cancer incidence was increased after treatment of CIN for at least 20 years, regardless of the grade of histology at treatment. Compared to all of the colposcopically guided methods, cold knife conization (CKC) was the least effective method of treatment in terms of later CIN 3 or cervical cancer incidence. In addition to cervical cancer, incidence of other HPV-related anogenital cancers was increased among those treated, as was the incidence of lung cancer and other smoking-related cancers. Mortality from cervical cancer among the women treated was not statistically significantly elevated, and after adjustment for socio-economic status, the hazard ratio (HR) was 1.0. In fact, the excess mortality among those treated was mainly due to increased mortality from other cancers, especially from lung cancer. In terms of post-treatment fertility, the CIN treatments seem to be safe: The women had more deliveries, and their incidence of pregnancy was similar before and after treatment. Incidence of extra-uterine pregnancies and induced abortions was elevated among the treated both before and after treatment. Thus this elevation did not occur because they were treated rather to a great extent was due to the other known risk factors these women had in excess, i.e. sexually transmitted infections. The purpose of any cancer preventive activity is to reduce cancer incidence and mortality. In Finland, cervical cancer is a rare disease and death from it even rarer, mostly due to the effective screening program. Despite this, the women treated are at increased risk for cancer; not just for cervical cancer. They must be followed up carefully and for a long period of time; general health education, especially cessation of smoking, is crucial in the management process, as well as interventions towards proper use of birth control such as condoms.

Genetic Variation and Clinical Manifestations in Inflammatory Bowel Disease

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

Individualized immunosuppression after renal transplantation in childhood

Pediatric renal transplantation (TX) has evolved greatly during the past few decades, and today TX is considered the standard care for children with end-stage renal disease. In Finland, 191 children had received renal transplants by October 2007, and 42% of them have already reached adulthood. Improvements in treatment of end-stage renal disease, surgical techniques, intensive care medicine, and in immunosuppressive therapy have paved the way to the current highly successful outcomes of pediatric transplantation. In children, the transplanted graft should last for decades, and normal growth and development should be guaranteed. These objectives set considerable requirements in optimizing and fine-tuning the post-operative therapy. Careful optimization of immunosuppressive therapy is crucial in protecting the graft against rejection, but also in protecting the patient against adverse effects of the medication. In the present study, the results of a retrospective investigation into individualized dosing of immunosuppresive medication, based on pharmacokinetic profiles, therapeutic drug monitoring, graft function and histology studies, and glucocorticoid biological activity determinations, are reported. Subgroups of a total of 178 patients, who received renal transplants in 1988 2006 were included in the study. The mean age at TX was 6.5 years, and approximately 26% of the patients were <2 years of age. The most common diagnosis leading to renal TX was congenital nephrosis of the Finnish type (NPHS1). Pediatric patients in Finland receive standard triple immunosuppression consisting of cyclosporine A (CsA), methylprednisolone (MP) and azathioprine (AZA) after renal TX. Optimal dosing of these agents is important to prevent rejections and preserve graft function in one hand, and to avoid the potentially serious adverse effects on the other hand. CsA has a narrow therapeutic window and individually variable pharmacokinetics. Therapeutic monitoring of CsA is, therefore, mandatory. Traditionally, CsA monitoring has been based on pre-dose trough levels (C0), but recent pharmacokinetic and clinical studies have revealed that the immunosuppressive effect may be related to diurnal CsA exposure and blood CsA concentration 0-4 hours after dosing. The two-hour post-dose concentration (C2) has proved a reliable surrogate marker of CsA exposure. Individual starting doses of CsA were analyzed in 65 patients. A recommended dose based on a pre-TX pharmacokinetic study was calculated for each patient by the pre-TX protocol. The predicted dose was clearly higher in the youngest children than in the older ones (22.9±10.4 and 10.5±5.1 mg/kg/d in patients <2 and >8 years of age, respectively). The actually administered oral doses of CsA were collected for three weeks after TX and compared to the pharmacokinetically predicted dose. After the TX, dosing of CsA was adjusted according to clinical parameters and blood CsA trough concentration. The pharmacokinetically predicted dose and patient age were the two significant parameters explaining post-TX doses of CsA. Accordingly, young children received significantly higher oral doses of CsA than the older ones. The correlation to the actually administered doses after TX was best in those patients, who had a predicted dose clearly higher or lower (> ±25%) than the average in their age-group. Due to the great individual variation in pharmacokinetics standardized dosing of CsA (based on body mass or surface area) may not be adequate. Pre-Tx profiles are helpful in determining suitable initial CsA doses. CsA monitoring based on trough and C2 concentrations was analyzed in 47 patients, who received renal transplants in 2001 2006. C0, C2 and experienced acute rejections were collected during the post-TX hospitalization, and also three months after TX when the first protocol core biopsy was obtained. The patients who remained rejection free had slightly higher C2 concentrations, especially very early after TX. However, after the first two weeks also the trough level was higher in the rejection-free patients than in those with acute rejections. Three months after TX the trough level was higher in patients with normal histology than in those with rejection changes in the routine biopsy. Monitoring of both the trough level and C2 may thus be warranted to guarantee sufficient peak concentration and baseline immunosuppression on one hand and to avoid over-exposure on the other hand. Controlling of rejection in the early months after transplantation is crucial as it may contribute to the development of long-term allograft nephropathy. Recently, it has become evident that immunoactivation fulfilling the histological criteria of acute rejection is possible in a well functioning graft with no clinical sings or laboratory perturbations. The influence of treatment of subclinical rejection, diagnosed in 3-month protocol biopsy, to graft function and histology 18 months after TX was analyzed in 22 patients and compared to 35 historical control patients. The incidence of subclinical rejection at three months was 43%, and the patients received a standard rejection treatment (a course of increased MP) and/or increased baseline immunosuppression, depending on the severity of rejection and graft function. Glomerular filtration rate (GFR) at 18 months was significantly better in the patients who were screened and treated for subclinical rejection in comparison to the historical patients (86.7±22.5 vs. 67.9±31.9 ml/min/1.73m2, respectively). The improvement was most remarkable in the youngest (<2 years) age group (94.1±11.0 vs. 67.9±26.8 ml/min/1.73m2). Histological findings of chronic allograft nephropathy were also more common in the historical patients in the 18-month protocol biopsy. All pediatric renal TX patients receive MP as a part of the baseline immunosuppression. Although the maintenance dose of MP is very low in the majority of the patients, the well-known steroid-related adverse affects are not uncommon. It has been shown in a previous study in Finnish pediatric TX patients that steroid exposure, measured as area under concentration-time curve (AUC), rather than the dose correlates with the adverse effects. In the present study, MP AUC was measured in sixteen stable maintenance patients, and a correlation with excess weight gain during 12 months after TX as well as with height deficit was found. A novel bioassay measuring the activation of glucocorticoid receptor dependent transcription cascade was also employed to assess the biological effect of MP. Glucocorticoid bioactivity was found to be related to the adverse effects, although the relationship was not as apparent as that with serum MP concentration. The findings in this study support individualized monitoring and adjustment of immunosuppression based on pharmacokinetics, graft function and histology. Pharmacokinetic profiles are helpful in estimating drug exposure and thus identifying the patients who might be at risk for excessive or insufficient immunosuppression. Individualized doses and monitoring of blood concentrations should definitely be employed with CsA, but possibly also with steroids. As an alternative to complete steroid withdrawal, individualized dosing based on drug exposure monitoring might help in avoiding the adverse effects. Early screening and treatment of subclinical immunoactivation is beneficial as it improves the prospects of good long-term graft function.

Migraine Comorbidities : A Clinical and Molecular Genetic Study

Migraine is a highly prevalent disease, and despite several important breakthroughs there are still a many questions unanswered in the clinical, genetic and pathophysiological aspects of migraine research. Migraine has been linked to several other diseases such as epilepsy and stroke, but there are still unsolved issues concerning the true nature of these associations. Three genes predisposing to hemiplegic migraine and several loci associated to migraine have been identified, but so far no genes responsible for common forms of migraine have been recognized. Triptans have provided an important step in migraine treatment, but their usefulness in rare forms of migraine have been controversial. The Finnish Migraine Gene Project (FMGP) includes more than 1600 families and 7500 individuals. We evaluated comorbidity from 1000 consecutive subjects in the FMGP. To search for novel loci, we performed a genome-wide linkage scan in 36 families with high prevalences of migraine with visual aura. We collected 76 subjects from the FMGP who suffer from hemiplegic migraine and have used triptans. Finally, to study possible links between stroke and migraine we evaluated the prevalence of migraine in subjects with cervical artery dissection (CAD) and healthy controls. Migraine was associated with increased prevalence of allergy, hypotension and psychiatric diseases. Additionally, men suffering from migraine with aura had increased prevalence of epilepsy and stroke. Further evidence of association between migraine and epilepsy was found in our linkage study. The parametric two-point linkage analysis showed significant evidence of linkage between migraine aura and a locus on 9q21-q22. Interestingly, the same locus has been associated with occipitotemporal epilepsy. CAD seems to be a migraine risk factor, and therefore a link between stroke and migraine. Notably, CAD seems to alleviate migraine activity further indicating the association between these two conditions. Despite the contraindications of triptans, it seems that they are safe and effective in the abortive treatment of hemiplegic migraine.

New approaches to improve the cord blood unit hematopoietic progenitor cell content

Cord blood is a well-established alternative to bone marrow and peripheral blood stem cell transplantation. To this day, over 400 000 unrelated donor cord blood units have been stored in cord blood banks worldwide. To enable successful cord blood transplantation, recent efforts have been focused on finding ways to increase the hematopoietic progenitor cell content of cord blood units. In this study, factors that may improve the selection and quality of cord blood collections for banking were identified. In 167 consecutive cord blood units collected from healthy full-term neonates and processed at a national cord blood bank, mean platelet volume (MPV) correlated with the numbers of cord blood unit hematopoietic progenitors (CD34+ cells and colony-forming units); this is a novel finding. Mean platelet volume can be thought to represent general hematopoietic activity, as newly formed platelets have been reported to be large. Stress during delivery is hypothesized to lead to the mobilization of hematopoietic progenitor cells through cytokine stimulation. Accordingly, low-normal umbilical arterial pH, thought to be associated with perinatal stress, correlated with high cord blood unit CD34+ cell and colony-forming unit numbers. The associations were closer in vaginal deliveries than in Cesarean sections. Vaginal delivery entails specific physiological changes, which may also affect the hematopoietic system. Thus, different factors may predict cord blood hematopoietic progenitor cell numbers in the two modes of delivery. Theoretical models were created to enable the use of platelet characteristics (mean platelet volume) and perinatal factors (umbilical arterial pH and placental weight) in the selection of cord blood collections with high hematopoietic progenitor cell counts. These observations could thus be implemented as a part of the evaluation of cord blood collections for banking. The quality of cord blood units has been the focus of several recent studies. However, hemostasis activation during cord blood collection is scarcely evaluated in cord blood banks. In this study, hemostasis activation was assessed with prothrombin activation fragment 1+2 (F1+2), a direct indicator of thrombin generation, and platelet factor 4 (PF4), indicating platelet activation. Altogether three sample series were collected during the set-up of the cord blood bank as well as after changes in personnel and collection equipment. The activation decreased from the first to the subsequent series, which were collected with the bank fully in operation and following international standards, and was at a level similar to that previously reported for healthy neonates. As hemostasis activation may have unwanted effects on cord blood cell contents, it should be minimized. The assessment of hemostasis activation could be implemented as a part of process control in cord blood banks. Culture assays provide information about the hematopoietic potential of the cord blood unit. In processed cord blood units prior to freezing, megakaryocytic colony growth was evaluated in semisolid cultures with a novel scoring system. Three investigators analyzed the colony assays, and the scores were highly concordant. With such scoring systems, the growth potential of various cord blood cell lineages can be assessed. In addition, erythroid cells were observed in liquid cultures of cryostored and thawed, unseparated cord blood units without exogenous erythropoietin. This was hypothesized to be due to the erythropoietic effect of thrombopoietin, endogenous erythropoietin production, and diverse cell-cell interactions in the culture. This observation underscores the complex interactions of cytokines and supporting cells in the heterogeneous cell population of the thawed cord blood unit.

Molecular Genetics of Age-related Macular Degeneration

Age-related macular degeneration (AMD; OMIM # 603075) is an eye disease of the elderly, signs of which appear after the age of 50. In the Western world it is a leading cause of permanent visual loss with a prevalence of 8.5% in persons under 54 years of age and of 37% in persons over 75 years of age. Early forms of AMD may be asymptomatic, but in the late forms usually a central scotoma in the visual field follows severely complicating daily tasks. Smoking, age, and genetic predisposition are known risk factors for AMD. Until recently no true susceptibility genes had been identified though the composition of drusen deposits, the hallmarks of AMD, has suggested that the complement system might play a role in the pathogenesis of AMD. When four groups reported in March 2005, that, on chromosome 1q32, a Y402H variant in the complement factor H (CFH) gene confers risk for AMD in independent Caucasian samples, a new period in the field of genetic research of AMD started. CFH is a key regulator of the complement system. Thus, it is logical to speculate, that it plays a role in the pathogenesis of AMD. We performed a case-control association study to analyse whether the CFH Y402H variant contain a risk for AMD in the Finnish population. Although the population of Finland represents a genetic isolate, the CFH Y402H polymorphism was associated with AMD also in our patient sample with similar risk allele frequencies as in the other Caucasian populations. We further evaluated the effects of this variant, but no association between lesion subtype (predominantly classic, minimally classic or occult lesion) or lesion size of neovascular AMD and the CFH Y402H variant was detected. Neither did the variant have an effect on the photodynamic therapy (PDT) outcome. The patients that respond to PDT carried the risk genotype as frequently as those who did not respond, and no difference was found in the number of PDT sessions needed in patients with or without the risk genotypes of CFH Y402H. Functional analyses, however, showed that the binding of C-reactive protein (CRP) to CFH was significantly reduced in patients with the risk genotype of Y402H. In the past two years, the LOC387715/ high-temperature requirement factor A1 (HTRA1) locus on 10q26 has also been repeatedly associated with AMD in several populations. The recent discovery of the LOC387715 protein on the mitochondrial outer membrane suggests that the LOC387715 gene, not HTRA1, is the true predisposing gene in this region, although its biological function is still unknown. In our Finnish patient material, patients with AMD carried the A69S risk genotype of LOC387715 more frequently than the controls. Also, for the first time, an interaction between the CFH Y402H and the LOC387715 A69S variants was found. The most recently detected susceptibilty gene of AMD, the complement component 3 (C3) gene, encodes the central component of the complement system, C3. In our Finnish sample, an additive gene effect for the C3 locus was detected, though weaker than the effects for the two main loci, CFH and LOC387715. Instead, the hemicentin-1 or the elongation of very long chain fatty acids-like 4 genes that have also been suggested as candidate genes for AMD did not carry a risk for AMD in the Finnish population. This was the first series of molecular genetic study of AMD in Finland. We showed that two common risk variants, CFH Y402H and LOC387715 A69S, represent a high risk of AMD also in the isolated Finnish population, and furthermore, that they had a statistical interaction. It was demonstrated that the CFH Y402H risk genotype affects the binding of CFH to CRP thus suggesting that complement indeed plays an important role in the pathogenesis of AMD.

Molecular characterization of mitochondrial ataxias : with emphasis on MIRAS and IOSCA

Uusi hermoston rappeumasairaus MIRAS: Suomessa kantajia joka 125. väestöstä Tässä väitöskirjatyössä on kuvattu uusi peittyvästi periytyvä hermoston rappeumasairaus, MIRAS (mitochondrial recessive ataxia syndrome), ja sen geenitausta. Tauti osoittautui tutkimuksessamme Suomen yleisimmäksi perinnölliseksi ataksiasairaudeksi. Tutkimuksessa on tutkittu perinnöllisiä aivosairauksia, joissa yhtenä oireena on ataksia (kävelyn epävarmuus, tasapainovaikeus ja liikkeiden haparointi), sekä lukuisia muita aivojen toimintahäiriöstä johtuvia oireita. Seuloessamme suomalaisilta ataksiapotilailta MIRAS-geenivirhettä, 27 potilasta sai diagnoosin aikaisemmin tuntemattomalle, etenevälle ataksiasairaudelleen. Tutkimuksen tuloksena kyseisen geenivirheen DNA-diagnostiikka on otettu käyttöön suomalaisissa ja eurooppalaisissa laboratorioissa, ja toista sataa potilasta ympäri maailman on saanut diagnoosin. Suomen väestössä joka 125. kantaa MIRAS geenivirhettä, mutta taudin saa vain, jos perii geenivirheen molemmilta vanhemmiltaan. MIRAS on taudinkuvaltaan vaihteleva, mutta vaikea etenevä neurologinen sairaus. Useilla potilailla esiintyvät oireet ovat ataksia, puheen puuromaisuus (dysartria), ääreishermorappeuma (neuropatia), pakkoliikkeet, psykiatriset oireet sekä vaikea epilepsia. Erityisen tärkeää MIRAS-taudin tunnistaminen on siihen liittyvän epilepsian hoitopäätöksessä: valproaatti-lääkitys voi aiheuttaa MIRAS-potilaille vaikean maksavaurion. Väitöskirjatyön tuloksena selvisi, että kaikki suomalaiset, norjalaiset, belgialaiset, englantilaiset, australialaiset ja uusi-seelantilaiset MIRAS potilaat olivat kaukaista sukua samalle, tuhansia vuosia sitten eläneelle eurooppalaiselle esivanhemmalle. Ataksiasairauksien tautimekanismeja selvitimme tutkimalla MIRAS-ataksiaa ja sitä muistuttavaa IOSCA sairautta (infantile onset spinocerebellar ataxia), jonka aiheuttaa peittyvästi periytyvä geenivirhe Twinkle-geenissä. Tutkimuksessa löydettiin myös uusi, Twinkle-geenin geenivirheestä johtuva taudinkuva: vaikea-asteinen, varhaisella iällä alkava aivosairaus, jossa on lisäksi viitteitä maksasairaudesta. Löysimme potilaiden aivoista muutoksia mitokondrioiden eli solun voimalaitosten perimän määrässä. Nämä tulokset antavat arvokasta lisätietoa ataksiasairauksien taustalla olevista muutoksista, joiden ymmärtäminen on välttämätön edellytys hoitomahdollisuuksien tutkimiselle tulevaisuudessa.

Psychiatric disturbances in children with intellectual disability : Prevalence, risk factors and assessment

Children with intellectual disability are at increased risk for emotional and behavioural problems, but many of these disturbances fail to be diagnosed. Structured checklists have been used to supplement the psychiatric assessment of children without intellectual disability, but for children with intellectual disability, only a few checklists are available. The aim of the study was to investigate psychiatric disturbances among children with intellectual disability: the prevalence, types and risk factors of psychiatric disturbances as well as the applicability of the Finnish translations of the Developmental Behaviour Checklist (DBC-P) and the Child Behavior Checklist (CBCL) in the assessment of psychopathology. The subjects comprised 155 children with intellectual disability, and data were obtained from case records and five questionnaires completed by the parents or other carers of the child. According to case records, a psychiatric disorder had previously been diagnosed in 11% of the children. Upon careful re-examination of case records, the total proportion of children with a psychiatric disorder increased to 33%. According to checklists, the frequency of probable psychiatric disorder was 34% by the DBC-P, and 43% by the CBCL. The most common diagnoses were pervasive developmental disorders and hyperkinetic disorders. The results support previous findings that compared with children without intellectual disability, the risk of psychiatric disturbances is 2-3-fold in children with intellectual disability. The risk of psychopathology was most significantly increased by moderate intellectual disability and low socio-economic status, and decreased by adaptive behaviour, language development, and socialisation as well as living with both biological parents. The results of the study suggest that both the DBC-P and the CBCL can be used to discriminate between children with intellectual disability with and without emotional or psychiatric disturbance. The DBC-P is suitable for children with any degree of intellectual disability, and the CBCL is suitable at least for children with mild intellectual disability. Because the problems of children with intellectual disability differ somewhat from those of children without intellectual disability, checklists designed specifically for children with intellectual disability are needed.

CHEK2 in Breast and Colorectal Cancer

Breast and colorectal cancers, are common types of cancer, with over two million newly diagnosed cases annually worldwide. Cancer is a genetic disease and defects in DNA integrity restoring functions make a significant contribution to cancer risk. CHEK2 is a checkpoint kinase functioning as a regulator of cell cycle checkpoints, apoptosis, and DNA repair in response to DNA double-strand breaks. The aim of this study was to evaluate the role of CHEK2 in breast cancer predisposition in Finnish breast cancer families and in breast cancer risk at the population level. We were interested in the clinical and biological characteristics of the breast tumors associated with the CHEK2 germline mutations or aberrant CHEK2 protein expression and the effect on survival of patients with these CHEK2 defects. We also assessed the role of CHEK2 mutations, namely 1100delC and I157T, in colorectal cancer susceptibility in Finland. CHEK2 I157T was found to be a low-penetrance breast cancer susceptibility allele, conferring a 1.4-fold risk for carriers. Reduced or absent CHEK2 protein expression was observed in one-fifth of breast tumors from patients unselected for family history, implying that defective CHEK2 signaling contributes to tumorigenesis. Reduction in CHEK2 expression was more common in tumors with larger diameter and ER expression, but with regard to other tumor characteristics and prognosis of a patient no association was observed. Results from comparison of CHEK2 1100delC carrier tumors with noncarrier tumors were in line with the findings from the CHEK2 expression study. Tumors from CHEK2 1100delC carriers were more often of higher grade than tumors from noncarriers, and they also tended to be ER-positive more often, although generally 1100delC status does not seem to radically affect the tumor characteristics. Our results suggest that CHEK2 1100delC may not be a susceptibility allele for CRC, although a very small effect cannot be excluded. Furthermore, CHEK2 1100delC is equally frequent in HBCC (hereditary breast and colorectal cancer) phenotype families and in breast cancer families. Over 1000 CRC cases were screened for CHEK2 I157T, and a significantly higher frequency of I157T was observed among both familial and sporadic CRC cases. The relation of CHEK2 I157T with familial CRC has not been studied previously. CHEK2 I157T seems to be a susceptibility allele for both familial and sporadic CRC, conferring a 1.5-fold risk for carriers of this variant. CHEK2 I157T has been proposed to have a role as a multiple cancer susceptibility allele, which is supported by our results since we observed a trend towards higher frequency of the variant among cases with multiple primary tumors or those with a family history of cancer. During the last five years CHEK2 has established its role as an important cancer susceptibility gene. It has become apparent that CHEK2 is a low-penetrance susceptibility gene for several cancer types, significantly contributing to familial cancer risk as well as to cancer risk at the population level.

Molecular Genetic Studies of Melanocortin Receptors in Morbid Obesity

The prevalence of obesity is increasing at an alarming rate in all age groups worldwide. Obesity is a serious health problem due to increased risk of morbidity and mortality. Although environmental factors play a major role in the development of obesity, the identification of rare monogenic defects in human genes have confirmed that obesity has a strong genetic component. Mutations have been identified in genes encoding proteins of the leptin-melanocortin signaling system, which has an important role in the regulation of appetite and energy balance. The present study aimed at identifying mutations and genetic variations in the melanocortin receptors 2-5 and other genes active on the same signaling pathway accounting for severe early-onset obesity in children and morbid obesity in adults. The main achievement of this thesis was the identification of melanocortin-4 receptor (MC4R) mutations in Finnish patients. Six pathogenic MC4R mutations (308delT, P299H, two S127L and two -439delGC mutations) were identified, corresponding to a prevalence of 3% in severe early-onset obesity. No obesity causing MC4R mutations were found among patients with adult-onset morbid obesity. The MC4R 308delT deletion is predicted to result in a grossly truncated nonfunctional receptor of only 107 amino acids. The C-terminal residues, which are important in MC4R cell surface targeting, are totally absent from the mutant 308delT receptor. In vitro functional studies supported a pathogenic role for the S127L mutation since agonist induced signaling of the receptor was impaired. Cell membrane localization of the S127L receptor did not differ from that of the wild-type receptor, confirming that impaired function of the S127L receptor was due to reduced signaling properties. The P299H mutation leads to intracellular retention of the receptor. The -439delGC deletion is situated at a potential nescient helix-loop-helix 2 (NHLH2) -binding site in the MC4R promoter. It was demonstrated that the transcription factor NHLH2 binds to the consensus sequence at the -439delGC site in vitro, possibly resulting in altered promoter activity. Several genetic variants were identified in the melanocortin-3 receptor (MC3R) and pro-opiomelanocortin (POMC) genes. These polymorphisms do not explain morbid obesity, but the results indicate that some of these genetic variations may be modifying factors in obesity, resulting in subtle changes in obesity-related traits. A risk haplotype for obesity was identified in the ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1) gene through a candidate gene single nucleotide polymorphism (SNP) genotyping approach. An ENPP1 haplotype, composed of SNPs rs1800949 and rs943003, was shown to be significantly associated with morbid obesity in adults. Accordingly, the MC3R, POMC and ENPP1 genes represent examples of susceptibility genes in which genetic variants predispose to obesity. In conclusion, pathogenic mutations in the MC4R gene were shown to account for 3% of cases with severe early-onset obesity in Finland. This is in line with results from other populations demonstrating that mutations in the MC4R gene underlie 1-6% of morbid obesity worldwide. MC4R deficiency thus represents the most common monogenic defect causing human obesity reported so far. The severity of the MC4-receptor defect appears to be associated with time of onset and the degree of obesity. Classification of MC4R mutations may provide a useful tool when predicting the outcome of the disease. In addition, several other genetic variants conferring susceptibility to obesity were detected in the MC3R, MC4R, POMC and ENPP1 genes.

Occupational Rhinitis : Diagnosis and Health-related Quality of Life

Occupational rhinitis is mainly caused by work environment and not by stimuli encountered outside the workplace. It differs from rhinitis that is worsened by, but not mainly caused by, workplace exposures. Occupational rhinitis can develop in response to allergens, inhaled irritants, or corrosive gases. The thesis evaluated the use of challenge tests in occupational rhinitis diagnostics, studied the long-term health-related quality of life among allergic occupational rhinitis patients, and the allergens of wheat grain among occupational respiratory allergy patients. The diagnosed occupational rhinitis was mainly allergic rhinitis, which was caused by occupational agents, most commonly flours and animal allergens. The non-IgE-mediated rhinitis reactions were less frequent and caused more often asthma than rhinitis. Both nasal challenges and inhalation challenges were found to be safe tests. The inhalation challenge tests had considerably resource-intensive methodology. However, the evaluation of nasal symptoms and signs together with bronchial reactions saved time and expense compared with the organization of multiple individual challenges. The scoring criteria used matched well with the weighted amount of discharge ≥ 0.2 g and in most cases gave comparable results. The challenge tests are valuable tools when there is uncertainty whether the patient's exposure should be reduced or discontinued. It was found that continuing exposure decreases health-related quality of life among patients with allergic occupational rhinitis despite of rhinitis medications, still approximately ten years after the diagnosis. Health-related quality of life among occupational rhinitis patients without any longer occupational exposure was mainly similar than that of the healthy controls. This highlights the importance of the reduction and cessation of occupational exposure. To achieve this, 17% of occupational rhinitis patients had been re-educated. Alpha-amylase inhibitors, lipid transfer protein 2G, thaumatin -like protein, and peroxidase I were found to be relevant allergens in Finnish patients with occupational respiratory wheat allergy. Of these allergens, thaumatin-like protein and lipid transfer protein 2G were found as new allergens associated with baker's rhinitis and asthma. The knowledge of the new clinically relevant proteins can be used in the future in the development of better standardized diagnostic preparations.

Distal anterior cerebral artery aneurysms

Objective: Distal anterior cerebral artery (DACA) aneurysms represent about 6% of all intracranial aneurysms. So far, only small series on treatment of these aneurysms have been published. Our aim is to evaluate the anatomic features, microneurosurgical techniques, treatment results, and long-term outcome in patients treated for DACA aneurysms. Patients and methods: We analyzed the clinical and radiological data on 517 consecutive patients diagnosed with DACA aneurysm at two neurosurgical centers serving solely the Southern (Helsinki) and Eastern (Kuopio) Finland in 1936–2007, and used a defined subgroup of the whole study population in each part of the study. Detailed anatomic analysis was performed in 101 consecutive patients from 1998 to 2007. Treatment results were analyzed in 427 patients treated between 1980 to 2005, the era of CT imaging and microneurosurgery. Long-term treatment outcome of ruptured DACA aneurysm(s) was evaluated in 280 patients with a median follow-up of 10 years; no patients were lost to follow-up. Results: DACA aneurysms, found most often (83%) at the A3 segment of the anterior cerebral artery (ACA), were smaller (median 6 mm vs. 8 mm), more frequently associated with multiple aneurysms (35% vs. 18%), and presented more often with intracerebral hematomas (ICHs) (53% vs. 26%) than ruptured aneurysms in general. They were associated with anomalies of the ACA in 23% of the patients. Microsurgical treatment showed similar complication rates (treatment morbidity 15%, treatment mortality 0.4%) as for other ruptured aneurysms. At one year after subarachnoid hemorrhage (SAH), DACA aneurysms had equally favorable outcome (GOS≥4) as other ruptured aneurysms (74% vs. 69%) but their mortality was lower (13% vs. 24%). Factors predicting unfavorable outcome for ruptured DACA aneurysms were advanced age, Hunt&Hess≥3, rebleeding before treatment, ICH, intraventricular hemorrhage, and severe preoperative hydrocephalus. The cumulative relative survival ratio showed 16% excess mortality in patients with ruptured DACA aneurysm during the first three years after SAH compared to the matched general population. From the fourth year onwards, there was no excess mortality during the follow-up. There were four episodes of recurrent SAH, only one due to treated DACA aneurysm, with a 10-year cumulative risk of 1.4%. Conclusions: The special neurovascular features and frequent association with anterior cerebral artery anomalies must be taken into account when planning occlusive treatment of DACA aneurysms. Clipping of DACA aneurysms provides a long-lasting result, with very small rates of rebleeding. After surviving three years from rupture of DACA aneurysm, the long-term survival of these patients becomes similar to that of the matched general population.