Whole-genome sequencing identifies EN1 as a determinant of bone density and fracture

The extent to which low-frequency (minor allele frequency (MAF) between 1-5%) and rare (MAF Bone mineral density (BMD) is highly heritable, a major predictor of osteoporotic fractures, and has been previously associated with common genetic variants, as well as rare, population-specific, coding variants. Here we identify novel non-coding genetic variants with large effects on BMD (ntotal = 53,236) and fracture (ntotal = 508,253) in individuals of European ancestry from the general population. Associations for BMD were derived from whole-genome sequencing (n = 2,882 from UK10K (ref. 10); a population-based genome sequencing consortium), whole-exome sequencing (n = 3,549), deep imputation of genotyped samples using a combined UK10K/1000 Genomes reference panel (n = 26,534), and de novo replication genotyping (n = 20,271). We identified a low-frequency non-coding variant near a novel locus, EN1, with an effect size fourfold larger than the mean of previously reported common variants for lumbar spine BMD (rs11692564(T), MAF = 1.6%, replication effect size = +0.20 s.d., Pmeta = 2 x 10(-14)), which was also associated with a decreased risk of fracture (odds ratio = 0.85; P = 2 x 10(-11); ncases = 98,742 and ncontrols = 409,511). Using an En1(cre/flox) mouse model, we observed that conditional loss of En1 results in low bone mass, probably as a consequence of high bone turnover. We also identified a novel low-frequency non-coding variant with large effects on BMD near WNT16 (rs148771817(T), MAF = 1.2%, replication effect size = +0.41 s.d., Pmeta = 1 x 10(-11)). In general, there was an excess of association signals arising from deleterious coding and conserved non-coding variants. These findings provide evidence that low-frequency non-coding variants have large effects on BMD and fracture, thereby providing rationale for whole-genome sequencing and improved imputation reference panels to study the genetic architecture of complex traits and disease in the general population.

Nephrin-associated molecules in human glomerular diseases

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

Mutations as molecular tools : The metabolic-rate dependent molecular clock and DNA barcoding of allied species

Mutation and recombination are the fundamental processes leading to genetic variation in natural populations. This variation forms the raw material for evolution through natural selection and drift. Therefore, studying mutation rates may reveal information about evolutionary histories as well as phylogenetic interrelationships of organisms. In this thesis two molecular tools, DNA barcoding and the molecular clock were examined. In the first part, the efficiency of mutations to delineate closely related species was tested and the implications for conservation practices were assessed. The second part investigated the proposition that a constant mutation rate exists within invertebrates, in form of a metabolic-rate dependent molecular clock, which can be applied to accurately date speciation events. DNA barcoding aspires to be an efficient technique to not only distinguish between species but also reveal population-level variation solely relying on mutations found on a short stretch of a single gene. In this thesis barcoding was applied to discriminate between Hylochares populations from Russian Karelia and new Hylochares findings from the greater Helsinki region in Finland. Although barcoding failed to delineate the two reproductively isolated groups, their distinct morphological features and differing life-history traits led to their classification as two closely related, although separate species. The lack of genetic differentiation appears to be due to a recent divergence event not yet reflected in the beetles molecular make-up. Thus, the Russian Hylochares was described as a new species. The Finnish species, previously considered as locally extinct, was recognized as endangered. Even if, due to their identical genetic make-up, the populations had been regarded as conspecific, conservation strategies based on prior knowledge from Russia would not have guaranteed the survival of the Finnish beetle. Therefore, new conservation actions based on detailed studies of the biology and life-history of the Finnish Hylochares were conducted to protect this endemic rarity in Finland. The idea behind the strict molecular clock is that mutation rates are constant over evolutionary time and may thus be used to infer species divergence dates. However, one of the most recent theories argues that a strict clock does not tick per unit of time but that it has a constant substitution rate per unit of mass-specific metabolic energy. Therefore, according to this hypothesis, molecular clocks have to be recalibrated taking body size and temperature into account. This thesis tested the temperature effect on mutation rates in equally sized invertebrates. For the first dataset (family Eucnemidae, Coleoptera) the phylogenetic interrelationships and evolutionary history of the genus Arrhipis had to be inferred before the influence of temperature on substitution rates could be studied. Further, a second, larger invertebrate dataset (family Syrphidae, Diptera) was employed. Several methodological approaches, a number of genes and multiple molecular clock models revealed that there was no consistent relationship between temperature and mutation rate for the taxa under study. Thus, the body size effect, observed in vertebrates but controversial for invertebrates, rather than temperature may be the underlying driving force behind the metabolic-rate dependent molecular clock. Therefore, the metabolic-rate dependent molecular clock does not hold for the here studied invertebrate groups. This thesis emphasizes that molecular techniques relying on mutation rates have to be applied with caution. Whereas they may work satisfactorily under certain conditions for specific taxa, they may fail for others. The molecular clock as well as DNA barcoding should incorporate all the information and data available to obtain comprehensive estimations of the existing biodiversity and its evolutionary history.

Applications of coralline hydroxyapatite with bioabsorbable containment and reinforcement as bone graft substitute : An experimental study

The aim of the present experimental study was to find out if the applications of coralline hydroxyapatite (HA) can be improved by using bioabsorbable containment or binding substance with particulate HA in mandibular contour augmentation and by using bioabsorbable fibre-reinforced HA blocks in filling bone defects and in anterior lumbar interbody fusion. The use of a separate curved polyglycolide (PGA) containment alone or together with a fast resorbing polyglycolide/polylactide (PGA/PLA) binding substance were compared to the conventional non-contained method in ridge augmentation in sheep. The contained methods decreased HA migration, but the augmentations did not differ significantly. The use of the containment caused a risk for wound dehiscence and infection. Histologically there was a rapid connective tissue ingrowth into the HA graft and it was more abundant with the PGA containment compared to the non-contained augmentation and even additionally rich when the HA particles were bound with PGA/PLA copolymer. However, the bone ingrowth was best in the non-contained augmentation exceeding 10-12 % of the total graft area at 24 weeks. Negligible or no bone ingrowth was seen in the cases where the polymer composite was added to the HA particles and, related to that, foreign-body type cells were seen at the interface between the HA and host bone. The PGA and poly-dl/l-lactide (PDLLA) fibre-reinforced coralline HA blocks were studied in the metaphyseal and in the diaphyseal defects in rabbits. A rapid bone ingrowth was seen inside the both types of implants. Both PGA and PDLLA fibres induced an inflammatory fibrous reaction around themselves but it did not hinder the bone ingrowth. The bone ingrowth pattern was directed according to the loading conditions so that the load-carrying cortical ends of the implants as well as the implants sited in the diaphyseal defects were the most ossified. The fibre-reinforced coralline HA implants were further studied as stand-alone grafts in the lumbar anterior interbody implantation in pigs. The strength of the HA implants proved not to be adequate, the implants fractured in six weeks and the disc space was gradually lost similarly to that of the discectomized spaces. Histologically, small quantities of bone ingrowth was seen in some of the PGA and PDLLA reinforced coralline implants while no bone formation was identified in any of the PDLLA reinforced synthetic porous HA implants. While fragmented, the inner structure of the implants was lost, the bone ingrowth was minimal, and the disc was replaced by the fibrous connective tissue. When evaluated radiologically the grade of ossification was assessed as better than histologically, and, when related to the histologic findings, CT was more dependable than the plain films to show ossification of the implanted disc space. Local kyphosis was a frequent finding along with anterior bone bridging and ligament ossification as a consequence of instability of the implanted segment.

Novel surgical and imaging methods of the middle ear and temporal bone

Välikorvaleikkauksiin usein liittyvän välikorvan ja kuuloluuketjun kirurgisen rekonstruktion tavoitteena on luoda olosuhteet, jotka mahdollistavat hyvän kuulon sekä välikorvan säilymisen tulehduksettomana ja ilmapitoisena. Välikorvan rekonstruktiossa on käytetty implanttimateriaaleina perinteisesti potilaan omia kudoksia sekä tarvittaessa erilaisia hajoamattomia biomateriaaleja, mm. titaania ja silikonia. Ongelmana biomateriaalien käytössä voi olla bakteerien adherenssi eli tarttuminen vieraan materiaalin pintaan, mikä saattaa johtaa biofilmin muodostumiseen. Tämä voi aiheuttaa kroonisen, huonosti antibiootteihin reagoivan infektion kudoksessa, mikä usein käytännössä johtaa uusintaleikkaukseen ja implantin poistoon. Maitohappo- ja glykolihappopohjaiset biologisesti hajoavat polymeerit ovat olleet kliinisessä käytössä jo vuosikymmeniä. Niitä on käytetty erityisesti tukimateriaaleina mm. ortopediassa sekä kasvo- ja leukakirurgiassa. Niitä ei ole toistaiseksi käytetty välikorvakirurgiassa. Korvan kuvantamiseen käytetään ensisijaisesti tietokonetomografiaa (TT). TT-tutkimuksen ongelmana on potilaan altistuminen suhteellisen korkealle sädeannokselle, joka kasvaa kumulatiivisesti, jos kuvaus joudutaan toistamaan. Väitöskirjatyö selvittää uuden, aiemmin kliinisessä työssä rutiinisti lähinnä hampaiston ja kasvojen alueen kuvantamiseen käytetyn rajoitetun kartiokeila-TT:n soveltuvuutta korvan alueen kuvantamiseen. Väitöskirjan kahdessa ensimmäisessä osatyössä tutkittiin ja verrattiin kahden kroonisia ja postoperatiivisia korvainfektioita aiheuttavan bakteerin, Staphylococcus aureuksen ja Pseudomonas aeruginosan, in vitro adherenssia titaanin, silikonin ja kahden eri biohajoavan polymeerin (PLGA) pintaan. Lisäksi tutkittiin materiaalien albumiinipinnoituksen vaikutusta adherenssiin. Kolmannessa osatyössä tutkittiin eläinmallissa PLGA:n biokompatibiliteettia eli kudosyhteensopivuutta kokeellisessa välikorvakirurgiassa. Chinchillojen välikorviin istutettiin PLGA-materiaalia, eläimiä seurattiin, ja ne lopetettiin 6 kk:n kuluttua operaatiosta. Biokompatibiliteetin arviointi perustui kliinisiin havaintoihin sekä kudosnäytteisiin. Neljännessä osatyössä tutkittiin kartiokeila-TT:n soveltuvuutta korvan alueen kuvantamiseen vertaamalla sen tarkkuutta perinteisen spiraali-TT:n tarkkuuteen. Molemmilla laitteilla kuvattiin ohimo- eli temporaaliluita korvan alueen kliinisesti ja kirurgisesti tärkeiden rakenteiden kuvantumisen tarkkuuden arvioimiseksi. Viidennessä osatyössä arvioitiin myös operoitujen temporaaliluiden kuvantumista kartiokeila-TT:ssa. Bakteeritutkimuksissa PLGA-materiaalin pintaan tarttui keskimäärin korkeintaan saman verran tai vähemmän bakteereita kuin silikonin tai titaanin. Albumiinipinnoitus vähensi bakteeriadherenssia merkitsevästi kaikilla materiaaleilla. Eläinkokeiden perusteella PLGA todettiin hyvin siedetyksi välikorvassa. Korvakäytävissä tai välikorvissa ei todettu infektioita, tärykalvon perforaatioita tai materiaalin esiin työntymistä. Kudosnäytteissä näkyi lievää tulehdusreaktiota ja fibroosia implantin ympärillä. Temporaaliluutöissä rajoitettu kartiokeila-TT todettiin vähintään yhtä tarkaksi menetelmäksi kuin spiraali-TT välikorvan ja sisäkorvan rakenteiden kuvantamisessa, ja sen aiheuttama kertasäderasitus todettiin spiraali-TT:n vastaavaa huomattavasti vähäisemmäksi. Kartiokeila-TT soveltui hyvin välikorvaimplanttien ja postoperatiivisen korvan kuvantamiseen. Tulokset osoittavat, että PLGA on välikorvakirurgiaan soveltuva, turvallinen ja kudosyhteensopiva biomateriaali. Biomateriaalien pinnoittaminen albumiinilla vähentää merkittävästi bakteeriadherenssia niihin, mikä puoltaa pinnoituksen soveltamista implanttikirurgiassa. Kartiokeila-TT soveltuu korvan alueen kuvantamiseen. Sen tarkkuus kliinisesti tärkeiden rakenteiden osoittamisessa on vähintään yhtä hyvä ja sen potilaalle aiheuttama sädeannos pienempi kuin nykyisen korva-spiraali-TT:n. Tämä tekee menetelmästä spiraali-TT:aa potilasturvallisemman vaihtoehdon erityisesti, jos potilaan tilanne vaatii seurantaa ja useampia kuvauksia, ja jos halutaan kuvata rajoitettuja alueita uni- tai bilateraalisesti.

Liver fat in the metabolic syndrome and type 2 diabetes

Introduction: The epidemic of obesity has been accompanied by an increase in the prevalence of the metabolic syndrome, type 2 diabetes, and non-alcoholic fatty liver disease (NAFLD). However, not all obese subjects develop these metabolic abnormalities. Hepatic fat accumulation is related to hepatic insulin resistance, which in turn leads to hyperglycemia, hypertriglyceridemia, and a low HDL cholesterol con-centration. The present studies aimed to investigate 1) how intrahepatic as compared to intramyocellular fat is related to insulin resistance in these tissues and to the metabolic syndrome (Study I); 2) the amount of liver fat in subjects with and without the metabolic syndrome, and which clinically available markers best reflect liver fat content (Study II); 3) the effect of liver fat on insulin clearance (Study III); 4) whether type 2 diabetic patients have more liver fat than age-, gender-, and BMI-matched non-diabetic subjects (Study IV); 5) how type 2 diabetic patients using exceptionally high doses of insulin respond to addition of a PPARγ agonist (Study V). Subjects and methods: The study groups consisted of 45 (Study I), 271 (Study II), and 80 (Study III) non-diabetic subjects, and of 70 type 2 diabetic patients and 70 matched control subjects (Study IV). In Study V, a total of 14 poorly controlled type 2 diabetic patients treated with high doses of insulin were studied before and after rosiglitazone treatment (8 mg/day) for 8 months. In all studies, liver fat content was measured by proton magnetic resonance spectroscopy, and sub-cutaneous and intra-abdominal fat content by MRI. In addition, circulating markers of insulin resistance and serum liver enzyme concentrations were determined. Hepatic (i.v. insulin infusion rate 0.3 mU/kg∙min combined with [3-3H]glucose, Studies I, III, and V) and muscle (1.0 mU/kg min, Study I) insulin sensitivities were measured by the euglycemic hyperinsulinemic clamp technique. Results: Fat accumulation in the liver rather than in skeletal muscle was associated with features of insulin resistance, i.e. increased fasting serum (fS) triglycerides and decreased fS-HDL cholesterol, and with hyperinsulinemia and low adiponectin concentrations (Study I). Liver fat content was 4-fold higher in subjects with as compared to those without the metabolic syndrome, independent of age, gender, and BMI. FS-C-peptide was the best correlate of liver fat (Study II). Increased liver fat was associated with both impaired insulin clearance and hepatic insulin resistance independent of age, gender, and BMI (Study III). Type 2 diabetic patients had 80% more liver fat than age-, weight-, and gender-matched non-diabetic subjects. At any given liver fat content, S-ALT underestimated liver fat in the type 2 diabetic patients as compared to the non-diabetic subjects (Study IV). In Study V, hepatic insulin sensitivity increased and glycemic control improved significantly during rosiglitazone treatment. This was associated with lowering of liver fat (on the average by 46%) and insulin requirements (40%). Conclusions: Liver fat is increased both in the metabolic syndrome and type 2 diabetes independent of age, gender, and BMI. A fatty liver is associated with both hepatic insulin resistance and impaired insulin clearance. Rosi-glitazone may be particularly effective in type 2 diabetic patients who are poorly controlled despite using high insulin doses.

Men´s health behaviour, health beliefs and need for health counselling : A study amongst 40-year-old males from one Helsinki City region

Tavoitteena oli tutkia 40-vuotiaiden miesten terveyskäyttäytymistä, terveysuskomuksia ja miesten saamaa terveysneuvontaa Helsingissä. 273 miestä vastasi kyselyyn ja osallistui terveystutkimuksiin. Terveydentilan perusteella miehet arvioitiin matalan (n=145) ja korkean (n=128) riskin ryhmiin. Khin neliö-testillä tutkittiin elämäntapa- ja riskitekijöitä koetun terveyden (hyvä, keskinkertainen/huono) luokissa ja verrattiin matalan ja korkean riskin ryhmiä em. tekijöiden osalta. Askeltavalla logistisella regressiomallilla analysoitiin tulosmuuttujia taustatekijöiden, terveyskäyttäytymisen, terveysuskomusten ja kliinisten riskitekijöiden avulla sekä arvioitiin oireiden ja vaivojen suhdetta koettuun terveydentilaan. Korkeassa riskissä olevien terveyttä seurattiin vuosina 2001–2004 analysoimalla mini-intervention vaikutusta terveysriskeihin ja elintapoihin varianssianalyysin avulla (ANOVA) (n=46). Matalasta vastausprosentista johtuen (39.6%), ei-vastanneiden aineistoa kerättiin käyttämällä syvähaastattelua (n=28) sekä puhelinkyselyä (n=40). Lopullinen aineisto koostui 341 miehestä. Tulokset osoittivat, että miehillä oli sydän- ja verisuonitautiriskejä. Kaksi kolmesta osallistuneista oli ylipainoisia tai lihavia, yli kolmanneksella vyötärönympärys oli ≥100 cm, ja yli 40%:llä oli diastolinen verenpaine ≥90 mmHg. Yli puolet tupakoi päivittäin ja 40% käytti alkoholia runsaasti. Ristiriitaisuutta ilmensi se, että huolimatta riskitekijöistä noin puolet miehistä koki terveydentilansa hyväksi. Sairauden tai vamman puute, hyvä suun terveydentila ja normaali vyötärönympärys olivat yhteydessä hyväksi koettuun terveydentilaan. Suora yhteys voitiin havaita omaisten tarjoaman neuvonnan ja vähäisen alkoholin käytön välillä. Masennus ja unettomuus olivat voimakkaasti yhteydessä loppuun palamiseen. Miehillä oli erilaisia fyysisiä ja psyykkisiä oireita, jotka korreloivat voimakkaasti masennuksen kanssa. Pieni määrä miehistä koki saaneensa terveysneuvontaa hoitohenkilökunnalta verrattuna perheenjäseniltä saatuun ohjaukseen. Korkeariskisten miesten (n=46) arvot parantuivat merkitsevästi lyhyellä aikavälillä. Kolesteroliarvoja lukuunottamatta ne palautuivat kolmen vuoden kuluttua alkumittausarvoja kohti. Laadullinen tutkimus osoitti, että “ei-vastanneet“ eivät osallistuneet projektiin, sillä he olivat oireettomia tai kiireisiä. Heillä todettiin samoja terveysriskejä kuin projektiin osallistuneilla. Syvähaastattelussa miehet toivat esille kokemuksiaan huolista, vihan tunteista, peloista ja yksinäisyydestä. Hoidonantajien on tärkeää ymmärtää ristiriidat miesten subjektiivisen ja objektiivisen terveydentilan välillä, mikä auttaa havaitsemaan esteitä terveyskäyttäytymiselle. Yhä enemmän tarvitaan yhteistyötä yksityisen ja julkisen terveydenhuollon välillä varmistamaan terveystottumusten jatkuminen miesten keskuudessa.

Impact of hysterectomy or levonorgestrel-releasing intrauterine system on ovarian function, bone, and sexual functioning in menorrhagic patients

The `VuoKKo` trial consisted of 236 women referred and randomised due to menorrhagia in the five university hospitals of Finland between November 1994 and November 1997. Of these women, 117 were randomised to hysterectomy and 119 to use levonorgestrel-releasing intrauterine system (LNG-IUS) to treat this complaint. Their follow-up visits took place six and twelve months after the treatment and five years after the randomisation. The first aim in the primary trial was quality-of-life and monetary aspects, and secondly in the present study to compare ovarian function, bone mineral density (BMD) and sexual functioning after these two treatment options. Ovarian function seemed to decrease after hysterectomy, demonstrated by increased hot flashes and serum follicle-stimulating hormone concentrations twelve months after the operation. Such an increase was not seen among LNG-IUS users. The pulsatility index of intraovarian arteries measured by two-dimensional ultrasound decreased in the hysterectomy group, but not in the LNG-IUS group. The decrease in serum inhibin B concentrations was similar in both groups, while ovarian artery circulation remained unchanged. BMD of the women measured by dual x-ray absorptiometry (DXA) at the lumbar spine and femoral neck at baseline and at five years after treatment showed BMD decrease at the lumbar spine among hysterectomised women, but not among LNG-IUS users. In both groups, BMD at the femoral neck had decreased. Differences between the groups were not, however, significant. Sexual functioning assessed by McCoy s sexual scale showed that sexual satisfaction as well as intercourse frequency had increased and sexual problems decreased among hysterectomised women six months after treatment. Among LNG-IUS users, sexual satisfaction and sexual problems remained unchanged. Although, the two groups did not differ in terms of sexual satisfaction or sexual problems at one-year and five-year follow-ups, LNG-IUS users were less satisfied with their partners than hysterectomised women.

Childhood-onset mitochondrial diseases : with emphasis on molecular diagnostics of neurological disorders

Childhood-onset mitochondrial diseases comprise a heterogeneous group of disorders, which may manifest with almost any symptom and affect any tissue or organ. Due to challenging diagnostics, most children still lack a specific aetiological diagnosis. The aim of this thesis was to find molecular causes for childhood-onset mitochondrial disorders in Finland. We identified the underlying cause for 25 children, and found three new diseases, which had not been diagnosed in Finland before. These diseases caused severe progressive infantile-onset encephalomyopathies, and were due to defects in mitochondrial DNA (mtDNA) maintenance. Furthermore, the thesis provides the molecular background of Finnish patients with ‘leukoencephalopathy with brain stem and spinal cord involvement and elevated brain lactate’ (LBSL). A new phenotype was identified to be due to mutations in Twinkle, resembling ‘infantile onset spinocerebellar ataxia’ (IOSCA). These mutations caused mtDNA depletion in the liver, thus confirming the essential role of Twinkle in mtDNA maintenance, and expanding the molecular background of mtDNA depletion syndromes. The major aetiology for infantile mitochondrial myopathy in Finland was discovered to be due to mutations in thymidine kinase 2 (TK2). A novel mutation with Finnish ancestry was identified, and a genotype-phenotype correlation with mutation-specific distribution of tissue involvement was found, thus proving that deficient TK2 may cause multi-tissue depletion and impair neuronal function. This work established the molecular diagnosis and advanced the knowledge of phenotypes among paediatric patients with polymerase gamma (POLG) mutations. The patients showed severe early-onset encephalopathy with intractable epilepsy. POLG mutations are not a prevalent cause of children’s ataxias, although ataxia is a major presenting symptom among adults. Our findings indicate that POLG mutations should be investigated even if typical MRI, histochemical or biochemical abnormalities are lacking. LBSL patients showed considerable variation in phenotype despite identical mutations. A common, most likely European, ancestry, and a relative high carrier frequency of these mutations in Finland were discovered; suggesting that LBSL may be a quite common leukoencephalopathy in other populations as well. The results suggest that MRI findings are so unique that the diagnosis of LBSL is possible to make without genetic studies. This thesis work has resulted in identification of new mitochondrial disorders in Finland, enhancing the understanding of the clinical variability and the importance of tissue-specificity of these disorders. In addition to providing specific diagnosis to the patients, these findings give light to the underlying pathogenetic mechanisms of childhood-onset mitochondrial disorders.

Directional dominance on stature and cognition in diverse human populations

Homozygosity has long been associated with rare, often devastating, Mendelian disorders1, and Darwin was one of the first to recognize that inbreeding reduces evolutionary fitness2. However, the effect of the more distant parental relatedness that is common in modern human populations is less well understood. Genomic data now allow us to investigate the effects of homozygosity on traits of public health importance by observing contiguous homozygous segments (runs of homozygosity), which are inferred to be homozygous along their complete length. Given the low levels of genome-wide homozygosity prevalent in most human populations, information is required on very large numbers of people to provide sufficient power3, 4. Here we use runs of homozygosity to study 16 health-related quantitative traits in 354,224 individuals from 102 cohorts, and find statistically significant associations between summed runs of homozygosity and four complex traits: height, forced expiratory lung volume in one second, general cognitive ability and educational attainment (P < 1 × 10−300, 2.1 × 10−6, 2.5 × 10−10 and 1.8 × 10−10, respectively). In each case, increased homozygosity was associated with decreased trait value, equivalent to the offspring of first cousins being 1.2 cm shorter and having 10 months’ less education. Similar effect sizes were found across four continental groups and populations with different degrees of genome-wide homozygosity, providing evidence that homozygosity, rather than confounding, directly contributes to phenotypic variance. Contrary to earlier reports in substantially smaller samples5, 6, no evidence was seen of an influence of genome-wide homozygosity on blood pressure and low density lipoprotein cholesterol, or ten other cardio-metabolic traits. Since directional dominance is predicted for traits under directional evolutionary selection7, this study provides evidence that increased stature and cognitive function have been positively selected in human evolution, whereas many important risk factors for late-onset complex diseases may not have been.

Health-Related Quality of Life, Costs and Treatment of Inflammatory Rheumatic Diseases in Routine Practice : With special emphasis on biological drugs

Rheumatoid arthritis (RA) and other chronic inflammatory joint diseases already begin to affect patients health-related quality of life (HRQoL) in the earliest phases of these diseases. In treatment of inflammatory joint diseases, the last two decades have seen new strategies and treatment options introduced. Treatment is started at an earlier phase; combinations of disease-modifying anti-rheumatic drugs (DMARDs) and corticosteroids are used; and in refractory cases new drugs such as tumour necrosis factor (TNF) inhibitors or other biologicals can be started. In patients with new referrals to the Department of Rheumatology of the Helsinki University Central Hospital, we evaluated the 15D and the Stanford Health Assessment Questionnaire (HAQ) results at baseline and approximately 8 months after their first visit. Altogether the analysis included 295 patients with various rheumatic diseases. The mean baseline 15D score (0.822, SD 0.114) was significantly lower than for the age-matched general population (0.903, SD 0.098). Patients with osteoarthritis (OA) and spondyloarthropathies (SPA) reported the poorest HRQoL. In patients with RA and reactive arthritis (ReA) the HRQoL improved in a statistically significant manner during the 8-month follow-up. In addition, a clinically important change appeared in patients with systemic rheumatic diseases. HAQ score improved significantly in patients with RA, arthralgia and fibromyalgia, and ReA. In a study of 97 RA patients treated either with etanercept or adalimumab, we assessed their HRQoL with the RAND 36-Item Health Survey 1.0 (RAND-36) questionnaire. We also analysed changes in clinical parameters and the HAQ. With etanercept and adalimumab, the values of all domains in the RAND-36 questionnaire increased during the first 3 months. The efficacy of each in improving HRQoL was statistically significant, and the drug effects were comparable. Compared to Finnish age- and sex-matched general population values, the HRQoL of the RA patients was significantly lower at baseline and, despite the improvement, remained lower also at follow-up. Our RA patients had long-standing and severe disease that can explain the low HRQoL also at follow-up. In a pharmacoeconomic study of patients treated with infliximab we evaluated medical and work disability costs for patients with chronic inflammatory joint disease during one year before and one year after institution of infliximab treatment. Clinical and economic data for 96 patients with different arthritis diagnoses showed, in all patients, significantly improved clinical and laboratory variables. However, the medical costs increased significantly during the second period by 12 015 (95% confidence interval, 6 496 to 18 076). Only a minimal decrease in work disability costs occurred mean decrease 130 (-1 268 to 1 072). In a study involving a switch from infliximab to etanercept, we investigated the clinical outcome in 49 patients with RA. Reasons for switching were in 42% failure to respond by American College of Rheumatology (ACR) 50% criteria; in 12% adverse event; and in 46% non-medical reasons although the patients had responded to infliximab. The Disease Activity Score with 28 joints examined (DAS28) allowed us to measure patients disease activity and compare outcome between groups based on the reason for switching. In the patients in whom infliximab was switched to etanercept for nonmedical reasons, etanercept continued to suppress disease activity effectively, and 1-year drug survival for etanercept was 77% (95% CI, 62 to 97). In patients in the infliximab failure and adverse event groups, DAS28 values improved significantly during etanercept therapy. However, the 1-year drug survival of etanercept was only 43% (95% CI, 26 to 70) and 50% (95% CI, 33 to 100), respectively. Although the HRQoL of patients with inflammatory joint diseases is significantly lower than that of the general population, use of early and aggressive treatment strategies including TNF-inhibitors can improve patients HRQoL effectively. Further research is needed in finding new treatment strategies for those patients who fail to respond or lose their response to TNF-inhibitors.

The effects of low-intensity ultrasound in bioabsorbable self-reinforced poly-L-lactide -fixed cancellous bone fracture

The purpose of the present study was to investigate the effects of low-intensity ultrasound on bioabsorbable self-reinforced poly-L-lactide (SR-PLLA) screws and on fracture healing after SR-PLLA device fixation in experimental and clinical cancellous bone fracture. In the first experimental study, the assessment of the mechanical strengths of the SR-PLLA screws was performed after 12 weeks of daily 20-minute ultrasound exposure in vitro. In the second experimental study, 32 male Wistar rats with an experimental distal femur osteotomy fixed with an SR-PLLA rod were exposed for daily low-intensity ultrasound treatment for 21 days. The effects on the healing bone were assessed. The clinical studies consist of three prospective, randomized, and placebo-controlled series of dislocated lateral malleolar fractures fixed with one SR-PLLA screw. The total number of the patients in these series was 52. Half of the patients were provided randomly with a sham ultrasound device. The patients underwent ultrasound therapy 20 minutes daily for six weeks. Radiological bone healing was assessed both by radiographs at two, six, nine, and 12 weeks and by multidetector computed tomography (MDCT) scans at two weeks, nine weeks, and 18 months. Bone mineral density was assessed by dual-energy X-ray absorptiometry (DXA). The clinical outcome was assessed by both Olerud-Molander scoring and clinical examination of the ankle. Low-intensity ultrasound had no effects on the mechanical properties and degradation behaviour of the SR-PLLA screws in vitro. There were no obvious signs of low-intensity ultrasound-induced enhancement in the bone healing in SR-PLLA-rod-fixed metaphyseal distal femur osteotomy in rats. The biocompatibility of low-intensity ultrasound treatment and SR-PLLA was found to be good. In the clinical series low-intensity ultrasound was observed to have no obvious effects on the bone mineral density of the fractured lateral malleolus. There were no obvious differences in the radiological bone healing times of the SR-PLLA-screw-fixed lateral malleolar fractures after low-intensity ultrasound treatment. Low-intensity ultrasound did not have any effects on radiological bone morphology, bone mineral density or clinical outcome 18 months after the injury. There were no obvious findings in the present study to support the hypothesis that low-intensity pulsed ultrasound enhances bone healing in SR-PLLA-rod-fixed experimental metaphyseal distal femur osteotomy in rats or in clinical SR-PLLA-screw-fixed lateral malleolar fractures. It is important to limit the conclusions of the present set of studies only to lateral malleolar fractures fixed with an SR-PLLA screw.