Podosyyttiproteiinien rooli lapsuusiän nefroottisessa syndroomassa

The nephrotic syndrome (NS) is characterized by massive proteinuria, edema and hypertriglyceridemia. It can appear as a primary or a secondary disease, idiopathic or familial, monogenic or complex, responsive to medication or progressing inevitably towards end-stage renal disease. As the manifestations of the disease are varied so is the etiologies behind it and still much remains to be discovered. However, structural compromise can be observed in the glomeruli of the NS patients, especially in podocytes, specialized epithelial cells of the glomerular filtration barrier. When causative genetic variants are found they are predominantly in the genes coding proteins involved in the structure and function of the podocytes. To date, over thirty podocyte protein-coding genes have been implicated. In this study we looked into genetic and cellular mechanisms in the podocyte underlying different forms of NS: congenital nephrotic syndrome of the Finnish type (CNF), steroid sensitive nephrotic syndrome (SSNS) and steroid resistant nephrotic syndrome (SRNS). Specifically, we used CNF kidney samples to study how the different compartments of the podocyte are affected by the lack of nephrin, characteristic structural component of the unique cellular junction of the podocytes (slit diaphragm (SDs)). We also looked into genetic variation in the podocyte protein coding genes in the Finnish SRNS patients to map out the variant spectrum and to see if this population shows difference to other populations. In addition, we aimed to find genetic and clinical markers predicting the course and severity of SSNS, which would have significant clinical value. The analyses were carried out using an array of molecular biological methods. Protein expression was studied using immunohistochemistry and light microscopy, genetic variants with PCR and sequence analysis. In addition, immunoprecipitation and western blot analyses were carried out to study the functionality of a particular variant. Genome wide sequencing analysis was carried out to gain wider perspective on the variant spectrum in NS patients. Our results showed that the lack of nephrin leads to widespread effects in the podocyte on protein level, especially on the expression of other proteins of the SD. In other compartments of the cell (basal membrane, actin cytoskeleton, apical membrane) the effect was clear but considering the profound structural damage seen in CNF the orders of magnitude of the observed changes were modest. We also showed that the analyzed clinical features or variants in these key genes coding podocyte proteins cannot reliably predict severity of SSNS. Children with difficult disease (multiple relapses, dependence on steroids) are more likely to suffer from NS in adulthood but other correlations between early patient features and prognosis could not be made. Single nucleotide polymorphisms (SNP) analysis of children with idiopathic NS revealed that variants in some genes (e.g. MDR1) may be useful in predicting responsiveness to steroids but the correlation is not sufficiently strong to warrant routine clinical testing. We found few causative variants in patients with idiopathic SRNS but did uncover a de novo NPHS2 variant co-segregating with familial dominant SRNS and unusual course of the disease. Healthy function of the podocyte relies on interaction and communication between multitudes of protein components in the different compartments of the cell. If any of these components are faulty it may cascade into widespread podocyte damage and proteinuria. The precise nature of the defect may play a significant role in the disease phenotype and not only the function of the damaged gene but also the nature of the variant ought to be considered when carrying out genetic analysis. Not in all cases causative variants or precise faulty structures can be identified. The genetic factors behind complex traits remain elusive, and even though some pieces of the puzzle have been found, in small part by this study, much is still to be discovered.Nefroottiseen syndroomaan (NS) kuuluu väkevä valkuaisvirtsaisuus, turvotukset ja hypertriglyseridemia. Sen ilmentymät vaihtelevat primaarisairauksista sekundaarisiin, ja idiopaattisesta periytyvään. Osa tautimuodoista on monogeenisiä, toiset luonteeltaan monitekijäisiä. Osaan on löydetty tehokas lääkitys, kun taas osa johtaa vääjäämättä munuaisten vajaatoimintaan. Myös eri NS:n muotojen taustatekijät ovat moninaiset, eikä niitä kaikkia vielä tunneta. Usein kuitenkin NS potilaiden munuaiskerästen ja erityisesti niiden suodatuskerroksen epiteelisolujen, podosyyttien, rakenne on vaurioitunut. Löydetyt NS:n kanssa assosioituvat geneettiset muutokset ovat usein geeneissä, jotka koodaavat podosyyttien rakenteeseen ja toimintaan vaikuttavia proteiineja. Tähän mennessä variantteja on löytynyt yli kolmestakymmenestä podosyyttiproteiinia koodavasta geenistä. Tässä tutkimuksessa selvitimme podosyyttien erilaisia geneettisiä ja solubiologisia mekanismeja NS:n eri muodoissa: suomalaistyypin synnynnäisessä nefroosissa (CNF), steroidiresistentissä NS:ssä (SRNS) ja steroidisensitiivisessä NS:ssä (SSNS). CNF-munuaisnäytteistä tutkimme nefriini-proteiinin puutoksen aiheuttamia rakennemuutoksia podosyytin eri osissa. Etsimme myös variantteja yleisimmistä podosyyttiproteiineja koodaavista geeneistä suomalaisista SRNS potilaista nähdäksemme erottuuko tämä populaatio muista varianttien määrässä tai laadussa. Lisäksi etsimme geneettisiä ja kliinisiä tekijöitä joiden avulla voisi ennustaa SSNS:n taudinkuvaa ja kulkua. Tutkimuksessa käytettiin lukuisia molekyylibiologisia menetelmiä. Immunohistokemiaa ja valomikroskopiaa käytettiin proteiiniekspression tutkimiseen, PCR:ää ja sekvessianalyysiä geneettisten varianttien etsintään. Lisäksi immunopresipitaatiota ja Weatern blot menetelmää käytettiin tiettyjen varianttien toiminnallisten vaikutusten selvittämiseen. Koko genomin sekvensoinnilla haettiin laajempaa perspektiiviä NS-potilaiden geneettisten varianttien kirjoon. Osoitimme että proteiinitasolla nefriinin puutteen vaikutukset ilmenevät kaikissa podosyytin osissa, mutta erityisesti slit diaphragm rakenteen ympärillä. Muissa osissa (basaali- ja apikaalikalvot, aktiiniranka) vaikutus oli selvä, mutta huomioiden CNF:ssä havaitun histologisen vaurion laajuuden, mittaluokaltaan varsin hillitty. Näytimme myös etteivät kliiniset muuttujat tai geneettiset variantit keskeisiä podosyyttiproteiineja koodaavissa geeneissä ennusta taudin vakavuutta. Lapsuusiän vakava sairaus (mm. toistuvat relapsit, jatkuvaa steroidilääkitystä vaativa) korreloi aikuisena jatkuvan NS:n kanssa mutta muita yhteyksiä aikaisten kliinisten piirteiden ja taudin etenemisen kanssa ei voi vetää. Nukleotidimuutokset tietyissä geeneissä, kuten MDR1:ssä, voivat ennustaa sairauden steroidivastetta, mutta yhteys ei ole riittävän voimakas oikeuttaakseen rutiininomaisen kliinisen testauksen. Löysimme harvoja tautia aiheuttavia variantteja suomalaisista idiopaattista NS:ää sairastavista potilaista mutta paikansimme de novo variantin NPHS2 geenissä joka kulkeutuu yhdessä periytyvän, dominantin NS:n kanssa, ja assosioituu epätavallisen hitaasti kehittyvään tautimuotoon. Terveiden podosyyttien toiminta nojaa lukuisien proteiinien väliseen interaktioon ja kommunikaatioon solun eri osissa, ja jos jokin näistä komponenteista on vioittunut, se saattaa johtaa laajamittaiseen podosyyttivaurioon ja valkuiasvirtsaisuuteen. Saman geenin sisällä erilaiset muutokset voivat vaikuttaa kehittyvään taudinkuvaan eri tavoilla, joten geneettisissä analyyseissä tulee huomioida vaurioituneen geenin toiminnan lisäksi myös spesifin variantin laatu

SNPs in lncRNA regions and breast cancer risk

Peer reviewe

Pathogenic Variant Spectrum in Breast Cancer Risk Genes in Finnish Patients

Recurrent pathogenic variants have been detected in several breast and ovarian cancer (BC/OC) risk genes in the Finnish population. We conducted a gene-panel sequencing and copy number variant (CNV) analysis to define a more comprehensive spectrum of pathogenic variants in BRCA1, BRCA2, PALB2, CHEK2, ATM, BARD1, RAD51C, RAD51D, BRIP1, and FANCM genes in Finnish BC patients. The combined frequency of pathogenic variants in the BRCA1/2 genes was 1.8% in 1356 unselected patients, whereas variants in the other genes were detected altogether in 8.3% of 1356 unselected patients and in 12.9% of 699 familial patients. CNVs were detected in 0.3% of both 1137 unselected and 612 familial patients. A few variants covered most of the pathogenic burden in the studied genes. Of the BRCA1/2 carriers, 70.8% had 1 of 10 recurrent variants. In the other genes combined, 92.1% of the carrier patients had at least 1 of 11 recurrent variants. In particular, PALB2 c.1592delT and CHEK2 c.1100delC accounted for 88.9% and 82.9%, respectively, of the pathogenic variation in each gene. Our results highlight the importance of founder variants in the BC risk genes in the Finnish population and could be used in the designing of population screening for the risk variants

How self-efficacy beliefs are related to assessment practices : A study of experienced university teachers

Based on social cognitive theory, this study focused on how self-efficacy beliefs were related to assessment practices among experienced university teachers. The data consisted of 16 thematic interviews of university teachers from various faculties who had received awards of excellence in teaching. The thematic interviews were content analyzed both inductively and deductively. Academics' self-efficacy experiences were related in four assessment modes: assessment in general, diagnostic assessment, formative assessment and summative assessment. Sources of self-efficacy experience were identified by mastery experience, vicarious experience, social persuasions and physiological and affective states. The mastery experience was the most common source of self-efficacy in assessment practice, and for experienced teachers, fairness was the prominent feature in assessment.Peer reviewe

Breast cancer risks associated with missense variants in breast cancer susceptibility genes

Background Protein truncating variants in ATM, BRCA1, BRCA2, CHEK2, and PALB2 are associated with increased breast cancer risk, but risks associated with missense variants in these genes are uncertain. Methods We analyzed data on 59,639 breast cancer cases and 53,165 controls from studies participating in the Breast Cancer Association Consortium BRIDGES project. We sampled training (80%) and validation (20%) sets to analyze rare missense variants in ATM (1146 training variants), BRCA1 (644), BRCA2 (1425), CHEK2 (325), and PALB2 (472). We evaluated breast cancer risks according to five in silico prediction-of-deleteriousness algorithms, functional protein domain, and frequency, using logistic regression models and also mixture models in which a subset of variants was assumed to be risk-associated. Results The most predictive in silico algorithms were Helix (BRCA1, BRCA2 and CHEK2) and CADD (ATM). Increased risks appeared restricted to functional protein domains for ATM (FAT and PIK domains) and BRCA1 (RING and BRCT domains). For ATM, BRCA1, and BRCA2, data were compatible with small subsets (approximately 7%, 2%, and 0.6%, respectively) of rare missense variants giving similar risk to those of protein truncating variants in the same gene. For CHEK2, data were more consistent with a large fraction (approximately 60%) of rare missense variants giving a lower risk (OR 1.75, 95% CI (1.47-2.08)) than CHEK2 protein truncating variants. There was little evidence for an association with risk for missense variants in PALB2. The best fitting models were well calibrated in the validation set. Conclusions These results will inform risk prediction models and the selection of candidate variants for functional assays and could contribute to the clinical reporting of gene panel testing for breast cancer susceptibility.Peer reviewe

Sindbis Virus Strains of Divergent Origin Isolated from Humans and Mosquitoes During a Recent Outbreak in Finland

Sindbis virus (SINV) is a mosquito-borne avian hosted virus that is widely distributed in Europe, Africa, Asia, and Oceania. Disease in humans is documented mainly from Northern Europe and South Africa and associated with genotype I. In 2018 under extremely warm climatic conditions, a small outbreak of 71 diagnosed SINV infections was recorded in Finland. We screened 52 mosquito pools (570 mosquitoes) and 223 human sera for SINV with real-time RT-PCR and the positive samples with virus isolation. One SINV strain was isolated from a pool (n = 13) of genusOchlerotatusmosquitoes and three strains from patient serum samples. Complete genome analysis suggested all the isolates to be divergent from one another and related to previous Finnish, Swedish, and German strains. The study provides evidence of SINV strain transfer within Europe across regions with different epidemiological characteristics. Whether these are influenced by different mosquito genera involved in the transmission remains to be studied.Peer reviewe

Experienced academics' emotions related to assessment

The aim of this study was to examine the emotions higher education teachers associate with assessment and the factors in their teaching environment that triggered these emotions. As a starting point, Frenzel's model of teacher emotions and Pekrun's control-value theory of achievement emotions were used. The sample consisted of 16 experienced and pedagogically advanced teachers who participated in semi-structured interviews. After abductive content analyses, both positive and negative emotions were detected corresponding to Frenzel's and Pekrun's models. The main sources of emotions were validity of assessment, assessment methods, pedagogical development and assessment culture. This preliminary study indicates that assessment evokes both positive and negative emotions, and that validity of assessment is a prominent issue in evoking these emotions. Pedagogical training should deal with emotions and their regulation in assessment to help teachers in higher education to cope with negative emotions.Peer reviewe

A novel negevirus isolated from Aedes vexans mosquitoes in Finland

Negeviruses are insect-specific enveloped RNA viruses that have been detected in mosquitoes and sandflies from various geographical locations. Here, we describe a new negevirus from Northern Europe, isolated from pool ofAedes vexansmosquitoes collected in Finland, designated as Mekrijarvi negevirus (MEJNV). MEJNV had a typical negevirus genome organization, is 9,740 nucleotides in length, and has a GC content of 47.53%. The MEJNV genome contains three ORFs, each containing the following identified conserved domains: ORF1 (7,068 nt) encodes a viral methyltransferase, an FtsJ-like methyltransferase, a viral RNA helicase, and an RNA-dependent RNA polymerase, ORF2 (1,242 nt) encodes a putative virion glycoprotein, and ORF3 (660 nt) encodes a putative virion membrane protein. A distinctive feature relative to other currently known negeviruses is a 7-nucleotide-long overlap between ORF1 and ORF2. MEJNV shares the highest sequence identity with Ying Kou virus from China, with 67.71% nucleotide and 75.19% and 59.00% amino acid sequence identity in ORF 1 and ORF 2, respectively. ORF3 had the highest amino acid sequence similarity to Daeseongdong virus 1 and negevirus Nona 1, both with 77.61% identity, and to Ying Kou virus, with 71.22% identity. MEJNV is currently the northernmost negevirus described. Our report supports the view that negeviruses are a globally distributed, diverse group of viruses that can be found from mosquitoes in a wide range of terrestrial biomes from tropical to boreal forests.Peer reviewe

Novel NPHS2 variant in patients with familial steroid-resistant nephrotic syndrome with early onset, slow progression and dominant inheritance pattern

Background Steroid-resistant nephrotic syndrome (SRNS) is a common cause of end-stage renal disease in children but also occurs as an adult-onset condition. In a subset of SRNS patients, pathogenic variants are found in genes coding for podocyte foot process proteins. The aim of this study was to define the role of pathogenic variants in Finnish patients with familial and sporadic SRNS. Methods We analyzed SRNS-related genes NPHS1, NPHS2, NEPH1, ACTN4, TRPC6, INF2, WT1, CD2AP, LAMB2, and PLCE1 for disease-causing variants using direct sequencing of exons and intron/exon boundaries in all members of a family with dominant SRNS with early onset and slow progression to end-stage renal disease. We carried out a whole genome sequencing in two affected and two healthy family members. The function of found podocin variant was studied using co-immunoprecipitation and immunohistochemistry. Podocyte gene sequences were analyzed in a cohort of Finnish non-familial SRNS patients. Results A heterozygous de novo deletion, c. 988_989delCT in NPHS2, was found in all affected family members and in none of their healthy relatives, non-familial patients or controls. No other SRNS-related gene variant, coding or non-coding co-segregated with the disease phenotype in the family. While the truncated podocin remained able to bind nephrin, the expression of nephrin was fragmented and podocin expression reduced. The gene analysis of the non-familial SRNS patients revealed few variants. Conclusion The role of podocin variants in nephrotic syndrome may be more varied than previously thought.Peer reviewe

Characterisation of the RNA Virome of Nine Ochlerotatus Species in Finland

RNA viromes of nine commonly encountered Ochlerotatus mosquito species collected around Finland in 2015 and 2017 were studied using next-generation sequencing. Mosquito homogenates were sequenced from 91 pools comprising 16–60 morphologically identified adult females of Oc. cantans, Oc. caspius, Oc. communis, Oc. diantaeus, Oc. excrucians, Oc. hexodontus, Oc. intrudens, Oc. pullatus and Oc. punctor/punctodes. In total 514 viral Reverse dependent RNA polymerase (RdRp) sequences of 159 virus species were recovered, belonging to 25 families or equivalent rank, as follows: Aliusviridae, Aspiviridae, Botybirnavirus, Chrysoviridae, Chuviridae, Endornaviridae, Flaviviridae, Iflaviridae, Negevirus, Partitiviridae, Permutotetraviridae, Phasmaviridae, Phenuiviridae, Picornaviridae, Qinviridae, Quenyavirus, Rhabdoviridae, Sedoreoviridae, Solemoviridae, Spinareoviridae, Togaviridae, Totiviridae, Virgaviridae, Xinmoviridae and Yueviridae. Of these, 147 are tentatively novel viruses. One sequence of Sindbis virus, which causes Pogosta disease in humans, was detected from Oc. communis from Pohjois-Karjala. This study greatly increases the number of mosquito-associated viruses known from Finland and presents the northern-most mosquito-associated viruses in Europe to date