Browsing by discipline "Genetics"
Now showing items 21-40 of 48
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(2013)Intellectual disability (ID) is a clinically diverse and genetically heterogeneous disorder characterized by central nervous system defects of varying severity resulting in substantial impairment of intellectual and adaptive functioning as expressed in conceptual (IQ<70), social and practical adaptive skills diagnosed before 18 years of age. The condition is referred to as non-syndromic when ID is the only clinical feature and syndromic when ID is accompanied by specific other features, for example, Down syndrome. Intellectual disability is one of the largest unsolved problems of health care with a prevalence of 2-3% in the population. There is a 30-40% excess of male versus female patients in ID which refers to over-representation of X chromosomal defects causing ID. In this study, exome sequencing of the X chromosome was applied in order to identify genes and their mutations in two Finnish families with intellectual disability of unknown cause. The mutations were identified using Agilent Sure select array that covers almost 93% of the coding region of the chromosome. Exome sequencing resulted in 11 variations in total. Segregation of these variants was studied using PCR, ExoSAP-IT purification protocol and BigDye® Terminator v3.1 Cycle Sequencing Kit. Eventually, two novel mutations were identified: one for each family. Both mutations reside in genes that have previously been shown to cause X-linked intellectual disability. Both of the mutations were absent in over 120 control DNA samples. In one family with three affected males, a novel splice mutation was identified in discs large homolog 3 (DLG3), which encodes synapse-associated protein 102 (SAP102). The mutation is located at the splice site in intron 1 (500+1 G>C) and its effect on protein function needs to be analyzed at the RNA-level using cDNA-sequencing. The clinical phenotype of the three affected brothers is mild to moderate intellectual disability. In the other family with three severely affected male patients, a novel mutation in exon 12 was identified on glutamate receptor, ionotropic, AMPA 3 (GRIA3) resulting in amino acid glycine (GGG) changing to arginine (CGG) at codon 630 (G630R). GRIA3 belongs to AMPA receptors implicated in the regulation of several biological processes. Our findings elucidate the power of exome sequencing in the diagnosis of rare, genetically heterogeneous disorders like intellectual disability. The results obtained will help in assessing the prognosis of the disease, in estimating the risk of the disorder to other family members, and in facilitating the development of future therapies for these devastating disorders. The results also further confirm the role of DLG3 and GRIA3 in human cognitive development.
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(2020)The Clustered Regularly-Interspaced Short Palindromic Repeats (CRISPR) and CRISPR associated protein (Cas9) (CRISPR-Cas9) system is a widely used gene editing technology due to its potential to alter the genome precisely in desired locations. Due to the potential of the CRISPR-Cas9 system, the objective of the thesis is to improve the precise editing of genes by modifying the CRISPR-Cas9 platform. Ultimately, the aim is to develop a platform that can edit any mutation and repair it to a normal, functional gene in patient cells. In general, CRISPR-Cas9 provides opportunities in treating monogenic diseases, for example by modifying long-term hematopoietic stem cells in immunodeficiencies. CRISPR-Cas9 can target disease-causing mutation sites and introduce double-strand breaks. Afterwards, the native DNA repair machinery of a cell repairs the cut site either by more efficient, error-prone non-homologous end joining (NHEJ) or precise homology-directed recombination (HDR). In most clinically oriented genome editing studies, the desired repair outcome is the latter because it allows precise repair of the mutation according to the exogenous repair template. Despite all its positive features, the optimization of CRISPR-based editing system is crucial before medical use; CRISPR-Cas9 induces a p53-mediated DNA damage response, which leads to a transient G1 cell cycle arrest and hampers HDR-based precision genome editing. Other problems include the repair pathway depending on the cell cycle phase, repair template proximity, and off-target activity. This thesis demonstrates that Cas9 fusions allow addressing the problems mentioned above. Cas9 fusions with DNA repair proteins ensure improved editing efficiency at the close proximity to the target site in HEK293T, BJ5-ta and RPE reporter cell lines. In addition, Cas9 coupled with the engineered cell cycle timer, AcrⅡA2-cdt1, favors the editing at the S/G2 cell cycle phases avoiding the p53-mediated response. AcrⅡA2-cdt1 is a reversible, phage-derived CRISPR inhibitor that selectively inhibit CRISPR-Cas9 at the G1 cell cycle phase and releasing it at the S phase. This thesis provides extensive look on the CRISPR-Cas9 editing and its challenges in immortalized cell lines and primary cells. In the thesis, the generation of reporter cell lines is prior to the validation of the novel Cas9-fusions. Furthermore, the optimization of primary T cell and CD34+ hematopoietic stem cell electroporation with different electroporation systems brings the study closer to clinical applications. The thesis provides insights about the effect of the target site and the cell type for genome editing outcomes. The editing efficiencies depend on the Cas9 fusion protein, cell type and its proliferation rate. The editing efficiency in primary T cells and CD34+ hematopoietic stem cells can significantly improve by optimizing transfection and culturing conditions, such as concentration of the CRISPR-Cas9 complex, cell culturing time and electroporation program. Cas9 fusions improve the safety and efficiency of the CRISPR-Cas9 system depending the cell type and the proliferation rate of the cell. Timing the induction of double-strand breaks also improves the editing efficiency. Overall, the methods used in the thesis give useful tools for eventual translational applications.
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(2020)Isolating mechanisms of the species usually prevent interspecific hybridisation. At times, these mechanisms might break down temporarily and lead to the birth of interspecific hybrids. Introgression is a term related to a set of consecutive backcrossings in which the hybrids reproduce with one of their parental species. It is characterised as a long process associated with alleles which are transferred from a population of one of the parental species to a population of the other parental species. Introgression is adaptive if phenotypic variation is increased in the recipient population by the genetic variants of the donor population and maintained by natural selection. The Baltic grey seal (Halichoerus grypus) and the Baltic ringed seal (Pusa hispida botnica) have interbred when they were kept in captivity in a shared pond. According to the findings from a previous study, interbreeding could have happened in the wild as well. The purpose of this study is to examine the proportion of introgression between the Baltic grey and ringed seals. The genomewide introgression is analysed using Patterson’s D-statistic, F4-ratio test and specific introgression intervals defined from the seals of analysed data. Introgression is assumed to have contributed intraspecific morphological variation detected in phocine teeth. Therefore, it is also examined whether the genes involved in tooth development express signs of introgression in the grey and ringed seals and whether the introgression intervals include potential variants. The results of Patterson D-statistic and F4-ratio test show both hybridisation and introgression between the Baltic grey and ringed seals. Based on the introgression intervals, a longer period has passed since the species interbred. Similar proportions of introgressed DNA as those defined from the genomes of the ringed seals have been detected in brown bears, bovines and modern humans. Furthermore, several genes affecting the shape of a developing mammalian tooth show signs of introgression in the seals. The individuals also carry variants in their introgression intervals. Introgression and the variants can account for the intraspecific morphological variation in the phocine dentitions. Potential introgressed genome intervals in the regulatory sequences of the tooth genes might also affect phocine tooth shape, which should be examined more in the future.
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(2018)Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) is a plant pathogen which causes bacterial speck in the host plants, especially tomato. The genome sequence of Pst DC3000 was resolved in 2003 by Buell et al. and since then it has served as an important model organism in plant pathology. Pst DC3000 secretes proteins through so-called type III pathway (T3SS). The secreted proteins, called Hrp (Hypersensitive response and pathogenicity) or Hop (Hrp outer protein), are considered as main virulence determinants of the bacterium. Aim of the study was to find out the suitability of the phage Mu DNA transposition complex to generate a transposon insertion mutant library for Gram-negative phytopathogen. Then further screen the mutant library for Hop effector protein knock-out mutants and evaluate their virulence in Lycopersicon esculentum cv. Moneymaker tomato plants. The thesis was part of a research project that characterized virulence determinants of Gram-negative phytopathogenic bacteria, including their gene regulation and effects on the plant-bacterium interaction. Pst DC3000 transposon insertion mutant library was generated by electroporation of phage Mu DNA transposition complex and a nested PCR method was used for screening the library. Three hop transposon insertion mutants were found, and those strains were extracted from the library. The three mutant strains where then used to infect tomato plants. Virulence was evaluated through phenotypic lesions and bacterial endophytic growth. Some of the results were different from the previous studies. The ability of HopM1 mutant to form lesions was weakened and it´s endophytic growth was slowed down. In previous studies the ability to form lesions was also weakened, but the endophytic growth was not affected until avrE gene was also deleted. HopM1 deletion alone did not have an effect to bacterial growth. In HopAO1 mutant the virulence was not affected. In other studies, HopAO1 deletion weakened bacterial growth in Arabidopsis and tobacco plants, but in tomato it has not been tested. HopD1 mutant was avirulent in this study. In previous studies HopD1 was essential for Pst DC3000 bacteria in defeating plants effector-triggered immunity (ETI), but hopD1 deletion alone did not make the bacteria avirulent. Pst DC3000 has 28 different effector proteins so usually one effector protein mutation does not affect its virulence. Hence the next step of the study was to make double mutants for Hop proteins, and this included deletion of the antibiotic marker by Cre-lox mechanism, which was started in this study.
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(2018)Recently, our group reported that mutations in KCNQ1, a potassium channel gene usually linked to long QT syndrome, cause growth hormone deficiency and maternally inherited gingival fibromatosis. Expression of the mutated KCNQ1 with KCNE2 subunit was shown to reduce pituitary hormone secretion in functional experiments in the original study. The aim of this thesis was to investigate if germline mutations in KCNQ1 and KCNE2, a gene encoding an auxiliary potassium channel subunit, could also play a role in the opposite phenomenon, growth hormone excess. Growth hormone (GH) excess causes acromegaly, a condition that is typically due to a GH secreting pituitary adenoma. I screened KCNQ1 and KCNE2 for germline mutations in 45 acromegaly patients by Sanger sequencing and predicted effects of the mutations on protein function by in silico tools. Only deep intronic and synonymous polymorphisms were detected in KCNQ1. These findings were likely insignificant based on the in silico predictions and the variants’ frequencies in the general population. In KCNE2, a heterozygous c.22A>G, p.(Thr8Ala) mutation with an unknown significance was found in two patients. It was present in the general population with a frequency of 0.0038. In conclusion, no evidence of KCNQ1 or KCNE2 mutations being associated with growth hormone excess was found. Mutation screenings of larger patient series and additional functional experiments are needed to shed more light on the roles of KCNQ1 related genes in growth hormone secretion.
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(2015)Darwinian selection can be measured and investigated from gene sequences. A certain gene form favored by positive selection will become more common in the population. Detecting strong positive selection is rare, but it has been found to affect genes involved in immune defense and perception of odorants. Genes under positive selection have a possible role in speciation or adaptation. This is why chemical communication, being based on the sense of smell, is an interesting topic for measuring natural selection and positive selection in particular. Social insects, such as ants, are model organisms for chemical communication. They use chemical communication not only for finding nutrition and detecting intruders, but also in coordinating the activities of several thousands of colony members. Insects perceive odorant signal molecules with their antennae. Odorant binding proteins (OBP) and chemosensory proteins (CSP) bind and transport odorant molecules through the sensillar lymph. OBPs and CSPs are also suspected to have a role in selecting odorants. This work focuses on two OBP genes and two CSP genes to study natural selection. All of these genes are conserved among all ant species. Three of these genes, OBP1, CSP1 and CSP7, are strongly expressed in the antennae suggesting that they function in chemical communication. CSP7 also has a known function in nest mate recognition in ants and OBP1 is known to bind a queen pheromone in the honeybee. The data includes gene sequences from 7 Formica ant species (270 sequences in total). The main goals of the research were to find out 1) the extent of variation between and within closely related ant species, 2) which evolutionary forces, natural selection or random drift, are behind the variation and 3) are there systematic differences between the two social forms of ants suggesting that these genes would affect the social structure of an ant colony. The variation in the sequence data was visualized by phylogenetic, principal coordinate and fixed differences analyses. Differences between populations were studied by FST values. The evolutionary forces shaping chemical communication genes within the species and populations, were studied by McDonald-Kreitman test, Tajima s D, Fu and Li test and MFDM test. The data shows that two of the species, F. cinerea and F. exsecta, significantly differ from the other five ant species, the F. rufa group species, and that the F. rufa group species don t significantly differ from each other based on these genes and this data. This could be due to their recent speciation or crossing between the species leading to hybrids in the data. The results of the evolutionary analyses are inconsistent. However, CSP7 has the strongest indication of selection based on all of the tests. Possible selection and sequence variation was detected at a predicted transcription factor binding site in F. cinerea. This indicates that selection might affect the regulation of CSP7. In the future it would be interesting to check the true transcription factor binding sites experimentally.
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(University of HelsinkiHelsingin yliopistoHelsingfors universitet, 2017)Hammasresorptio on kissojen yleisin hammassairaus. Sairauden aiheuttavat odontoklastit eli hampaan luunsyöjäsolut, joiden aikaansaama hammaskudoksen tuhoutuminen johtaa lopulta hampaan menetykseen. Hammasresorptio voi alkaa mistä tahansa hampaan pinnalta, joten hampaiden röntgenkuvaus on olennainen osa diagnostiikkaa. Pitkälle edetessään muutokset ovat kivuliaita ja niiden ainoa hoito on tapauksesta riippuen hampaan poisto tai kruunuamputaatio. Hammasresorptioiden esiintyvyys on korkea, aikaisemmissa tutkimuksissa 29-67 % välillä. Sairauden etiologia on pitkälti epäselvä, vaikka lukuisia teorioita on esitetty. Kirjallisuudessa hammasresorptioiden on epäilty liittyvän mm. korkeaan ikään, rotuun, muihin suu- ja hammassairauksiin, virusinfektioihin sekä kissan ruokavalioon ja elinympäristöön liittyviin tekijöihin. Tämän tutkimuksen kirjallisuuskatsauksessa kartoitettiin hammasresorptioihin liitettyjä erilaisia etiologisia teorioita ja erilaisille populaatioille tehtyjä esiintyvyystutkimuksia. Sairauden patogeneesiä, diagnosointia ja hoitoa käsiteltiin lyhyesti. Tutkielma sisältää alkuperäistutkimuksen, joka on poikittaistutkimusaineistosta tehty tapaus-verrokkianalyysi. Tutkimuksessa käytettiin internetkyselynä toteutettua kissojen terveyskyselyaineistoa. Tähän analyysiin otettiin mukaan vastaukset 8115 kissasta. Tutkimuksen ensimmäisenä tavoitteena oli selvittää hammasresorption esiintyvyyttä suomalaisilla maatiais- ja rotukissoilla. Hypoteesina oli, että hammasresorption esiintyvyys olisi korkeampi puhdasrotuisilla kissoilla ja tietyissä roduissa. Toisena tavoitteena oli arvioida hammasresorption riskitekijöitä tarkastelemalla sairautta eri ikäryhmissä sekä suhteessa sukupuoleen, ruokavalioon, elinympäristöön, muihin suu- ja hammassairauksiin ja kissojen muihin sairauksiin. Hypoteesina oli, että hammasresorption riskitekijät olisivat kirjallisuudessa aikaisemmin kuvattujen kaltaisia. Monimuuttujaisen logistisen regressiomallin avulla laskettiin ennusteita hammasresorption esiintymisen todennäköisyydelle erilaisilla kissoilla. Kissojen hammasresorption esiintyvyys suomalaisille kissanomistajille suunnatussa internetkyselyssä oli 3,9 %. Esiintyvyys oli tässä tutkimuksessa alhaisempi kuin sellaisissa, joissa kissojen hampaita on tutkittu kliinisesti. Esiintyvyys oli tietyissä roduissa huomattavasti muita rotuja korkeampi. Hammasresorptioon tilastollisesti merkitsevästi assosioituvia ominaisuuksia olivat rotu, ruoan saatavuus, stomatiitti, parodontiitti ja interaktioina toistensa kanssa ikä, ientulehdus ja hammaskivi. Hammasresorptioon assosioituvat tekijät olivat pääasiassa aikaisemmin kirjallisuudessa kuvattujen kaltaisia. Tietyillä roduilla on lisääntynyt alttius, mikä voi viitata geneettiseen komponenttiin hammasresorption etiologiassa. Rodun yhteys hammasresorptioon vaatisi kuitenkin tarkempaa selvitystä kliinisillä jatkotutkimuksilla.
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(2015)Charcot-Marie-Tooth disease is a clinically and genetically heterogeneous group of disorders of the peripheral nervous system. It affects both peripheral motor and sensory nerves that lead to progressive degeneration of muscles in the lower limbs. Prevalence of CMT is 1:2500. It can be divided into two groups: CMT1 is the demyelinating type and CMT2 is the axonal type. There are 20 known genes associated with CMT2 but not all of them have been found yet. Hereditary spastic paraplegia (HSP) is a group of inherited neurological disorders and it is also genetically and clinically heterogeneous. HSP is caused by degeneration of the upper motor neurons in the brain and spinal cord. HSP can be divided into pure and complex forms. In pure forms the main symptoms are progressive spasticity and weakness in the lower limbs. In complex forms there are additional symptoms such as epilepsy and mental retardation. The prevalence of HSP is 2-10:100 000. There are over 70 loci associated with HSP and 55 genes identified so far but there is probably more to be found. With both of these diseases it has been difficult to diagnose patients at the genetic level because of the great amount of disease genes. The aim of this study was to identify potential disease causing mutations by using targeted next-generation sequencing (NGS) and also to do functional testing with the discovered mutations. There were 48 patients, 28 diagnosed with CMT and 20 with HSP. For the targeted sequencing there was a gene panel with 167 genes, including genes associated with neuropathy, paraplegia, ALS and mitochondrial functions. Analyzing of the gene variants was largely based on their frequencies in the population and several exome databases were used. All possibly pathogenic mutations were confirmed with Sanger-sequencing and also sequenced from the patients’ relatives, if DNA samples were available. Possible genetic diagnosis was found for five CMT patients and six HSP patients. Only one of the mutations was previously known. One of the probably pathogenic mutations was a deletion, others were missense mutations. Large portion of the patients were left without a genetic diagnosis probably because there are many more genes associated with these diseases to be found. De novo heterozygous missense mutations in the KIF1A gene were found to cause a novel phenotype. Symptoms of these patients include classical HSP symptoms and additionally intellectual disability and other symptoms, such as cerebellar atrophy, in some patients. In this study we found a deletion in the gene HSPB1 that causes premature STOP codon that leads to disappearance of nearly the entire C-terminus. In this study I built a HSPB1-Western blot assay that was used in further research. It was found out that the truncated protein was stabile, it forms dimers with the normal protein and impairs the cells response against stress caused by misfolded proteins.
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(2020)Cancer-associated fibroblasts (CAF) form a heterogenous stromal cell population of a solid tumor. They are known to promote tumor growth and survival through metabolic reprogramming and inflammation. It is unclear though whether CAF are crucial component of tumor initiation and whether CAFs are dispensable altogether from the fully developed neoplasm. Tumor suppressor LKB1 regulates AMPK and AMPK-related kinases (ARK), and its function is compromised in familial disorder Peutz-Jeghers syndrome (PJS). Fibroblast specific haploinsufficiency of LKB1 alone is sufficient of initiating gastrointestinal polyposis but the mechanism through which LKB1 mediates this is only partially understood. We provide evidence that LKB1 is downregulated in multiple human malignancies including high grave serous ovarian cancer (HGSOC). Human ovarian cancer is the most lethal gynecological disease, characterized by metastasis of omentum. Loss-of LKB1 in ovarian fibroblasts was accompanied with metabolic changes associated with CAF-transformation. We screened down critical LKB1 substrates through transcriptomic and functional assays revealing AMPKa1 and MARK3 as potential downstream effectors of oxidative phosphorylation. AMPKa1, MARK1 and SIK-family were the glycolytic counterparts. We also took an initiative of cataloguing published human cancer stroma data in order to gain more comprehensive look of tumor heterogeneity. Metabolic rewiring was also observable in published cancer-stroma datasets. Human cancer-stroma divided into metabolically active and highly inflamed subtypes. These results highlights LKB1’s role as a conserved metabolic caretaker in fibroblasts. Our data also support mechanistic model in which LKB1 and ARKs regulate mitochondrial metabolism, essential for CAF transformation.
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(2019)Retrotransposons are genetic elements with the ability to make a copy of themselves and insert the copy into a new location in a genome. Most of the retrotransposons in the human genome are not transposition competent and the remaining copies are prevented from moving by epigenetics. However, some tumors experience abnormal retrotransposon activity resulting in many copies of retrotransposons inserted into new locations. Retrotransposons can be detected from sequenced genome data by bioinformatic tools. One of them is TraFiC, a tool designed to detect somatic retrotransposon insertions from tumor samples. In this Master’s thesis, I test TraFiC with 201 colorectal cancer tumors and one colorectal adenoma and develop tools to further analyze retrotransposon insertions. These tools are TraID, a pipeline to detect transductions, insertions with flanking sequence from source elements, and InSeqR, a pipeline to recreate the inserted sequence from known insertion sites. TraFiC detected 4744 somatic insertions and TraID detected 346 somatic transductions from the tumor samples. 80 % of the detected insertions were identified as true somatic insertions based on visual examination of a subset of the calls. 87 % of insertions detected by TraFiC and 82 % of the insertions detected by TraID had their insertion breakpoints and the sequence flanking them recreated by InSeqR. The detected insertions with their sequence form a reliable and comprehensive call set that can be used to create new knowledge of somatic retrotransposon insertions in colorectal cancer.
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(2019)Ihmistoiminnan vaikutukset ovat muuttaneet voimakkaasti ekosysteemien rakennetta ja toimintaa maailmanlaajuisesti viimeisten vuosikymmenten aikana. Ekologisen tilan heikentyminen aiheuttaa vakavan uhan virtavesien eliöyhteisöjen monimuotoisuudelle. Tulevaisuudessa tarve kustannustehokkaille, luotettaville ja nopeille keinoille arvioida vesistöjen ekologista tilaa kasvaa. Biomonitorointi, eli eliöiden käyttö ympäristömuutosten seurantaan ja mittaamiseen, pyrkii tunnistamaan, seuraamaan ja arvioimaan ekosysteemien toimintaa uhkaavia ympäristön stressitekijöitä. Tulevaisuudessa yksi mahdollinen biomonitoroinnin työkalu ja ekologisen tilan mittari yhdessä muiden biologisten (esim. piilevät, pohjaeläimet, vesikasvit ja kalat) ja fysikaalis-kemiallisten mittarien kanssa voi olla mikrobit. Uuden polven sekvensointimenetelmien kehitys ja sekvensointikustannusten aleneminen viime vuosina on mahdollistanut yhä useamman ympäristömikrobiaineiston analysoinnin uuden polven sekvensointimenetelmillä. Tämän pro gradu -tutkielman tarkoitus on tuoda lisätietoa siitä, mitkä eri ympäristötekijät vaikuttavat virtaavien vesien biofilmibakteerien yhteisörakenteeseen, yhteisö-ympäristösuhteen voimakkuuteen, yhteisöjen koostumusta selittävien muuttujien identiteettiin ja miten harvinaisten bakteerisukujen poisto aineistosta vaikuttaa erilaisiin ympäristömuuttujiin. Tutkimusaineistoa analysoidaan redundanssianalyysin (redundancy analysis, RDA) ja monimuuttujaregressiopuiden (Multivariate regression trees, MRT) avulla. Tutkielman tavoitteena on myös kuvata länsisuomalaisten virtavesien koskipaikkojen kivien pinnalla elävien biofilmibakteerien ominaispiirteitä pääjakso-, luokka-, lahko-, heimo- ja sukutasoille tehtävien BLAST-analyysien (basic local alignment search tool) avulla. Pro gradu tutkielmani bakteeriaineiston näytteet on otettu 21 suomalaiselta kolmannen jakovaiheen jokivaluma-alueelta, joilta eristettiin erillisten purojen koskipaikoista bakteerinäytteitä yhteensä 84 kappaletta. Samaan aikaan bakteerinäytteiden ottamisen kanssa tutkimuskohteilla toteutettiin vesinäytteenotto, ja kohteista määritettiin myös piilevä- ja pohjaeläinlajisto. Näytteet sekvensoitiin Ion Torrent Semiconductor -sekvensaattoria käyttäen ja valmis sekvenssiaineisto analysoitiin QIIME (Quantitative Insights Into Microbial Ecology) –ohjelmalla. Sekvenssit klusteroitiin toiminnallisiksi taksonomisiksi yksiköiksi (OTUiksi) 97 % samankaltaisuusastetta käyttäen. OTUille haettiin BLASTia (basic local alignment search tool) käyttäen paras mahdollinen pääjakso-, luokka-, lahko-, heimo- ja sukutason vastaavuus. Yleisimmät havaitut luokat olivat Alphaproteobacteria (28.9%), Betaproteobacteria (27.7%), Gammaproteobacteria (7.5%) Cytophagia (7.3%), Saprospirae (6.5%) ja Flavobacteriia (5.1%). Tuloksista havaitaan, että yhteisön ympäristösuhteisiin vaikuttavat erityisesti yleiset lajit. Harvinaisten lajien poistolla ei tässä tutkimuksessa siten havaita olevan suurta merkitystä, vaan merkitsevät muuttujat pysyvät lähes samoina huolimatta siitä, poistetaanko aineistosta ne lajit, jotka esiintyvät vain yhdellä paikalla vai kaikki ne lajit, jotka esiintyvät vain ≤ 40 paikalla. Myös selitysasteet jäävät alhaisiksi ja satunnaisten eli stokastisten tekijöiden vaikutus yhteisön ympäristöolosuhteisiin luultavasti korostuu. Jatkossa eri bakteeritaksonien roolista muuttuvissa ympäristöolosuhteissa tarvitaan tarkempaa tietoa ennen kuin virtavesien biofilmibakteereja voidaan laajamittaisesti käyttää biomonitoroinnin täysimittaisena työkaluna ja ympäristön tilan kuvaajana. Tämän tutkielman redundanssianalyysin, vaihtelun osituksen ja monimuuttujaregressiopuiden avulla saatujen tulosten perusteella voidaan todeta virtaavien vesien mikrobiyhteisöjen yhteisö-ympäristösuhteiden olevan heikkoja. Ympäristön tilan seurannassa tulisikin jatkossa tulosten perusteella käyttää mikrobien osalta muita vastemuuttujia kuin yhteisörakennetta.
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(2020)MikroRNA:t ovat tärkeitä, transkription jälkeen geenien ilmenemiseen vaikuttavia säätelijöitä. MikroRNA:iden tuotantoa on löydetty konservoituneena useista eri eliöryhmistä ja kudoksista, mikä osaltaan kertoo niiden toiminnan tärkeydestä. Rakenteeltaan miRNA:t ovat lyhyitä, noin 22 nukleotidin mittaisia yksijuosteisia RNA:ita, jotka tuotetaan pidemmistä esi-RNA:sta entsymaattisesti leikkaamalla. Geenien säätelyssä miRNA:iden teho perustuu niiden kykyyn tunnistaa kohdegeenit miRNA:n ja lähetti-RNA:n sekvenssien komplementaarisuuden perusteella. MikroRNA:iden kohteiden tunnistuksen kannalta tärkeätä, 6-8 nukleotidin jaksoa kutsutaan miRNA:n ydinalueeksi (seed-region). Yhdellä miRNA:lla voi potentiaalisesti olla satoja kohdegeenejä, joita se säätelee, minkä vuoksi näitä miRNA-mRNAinteraktioita on pyritty bioinformatiivisesti ennakoimaan useiden ohjelmien ja algoritmien avulla. miRNA:iden toiminnan tutkimista mutkistavat isomiR:it, jotka ovat yleisesti tunnettujen, kanonisten, miRNA:iden vaihtoehtoisia muotoja. IsomiR:ejä tuottavat poikkeamat miRNA:iden esiasteiden prosessoinnissa ja mikroRNA:ihin kohdistuva RNA-editointi. Tuloksena saadaan mikroRNA:ita joiden pituus tai emäskompositio eroavat kanonisista sekvensseistä. Myös miRNA:iden toiminta voi muun muassa alkaa kohdistamaan säätelyä eri geeneihin, kuin mitä kanonisen sekvenssin perusteella voisi olettaa. Tässä pro gradu -projektissa analysoin suurtehosekvensoinnilla tuotettua dataa kahdesta aivoalueesta kuudelta eri laboratoriohiirikannalta. Normalisoin datan ja todettuani sen laadun hyväksi jatkoin miRNA:iden ilmenemisen tarkastelua eri analyyseillä: Selvitin mitkä miRNA:t ilmentyivät tutkituilla aivoalueille jamiten niiden ekspressio erosi eri aivoalueiden ja hiirikantojen välillä. Tämän jälkeen tarkastelin miRNA:sta tuotettujen vaihtoehtoisten sekvenssien, isomiR:ien ilmentymistä. Vertailimme lisäksi aineistossa havaitsemiemme isomiR:ien ekspressiossa eroja aivoalueiden ja hiirikantojen välillä. Lopuksi pyrimme osoittamaan havaittujen isomiR:ejä tuottavien RNAeditointitapahtumien olevan funktionaalisesti merkittäviä. Tätä tarkoitusta varten transfektoimme soluviljelmiä lusiferaasi-reportterivektorilla ja editoiduilla ja editoimattomilla miRNA:ta muistuttavilla sekvensseillä. Analyysien tuloksena havaitsimme lukuisia miRNA:ita, jotka olivat eritavoin ilmentyneitä joko aivoaluiden, hiirikantojen tai molempien välillä. Lisäksi havaitsimme lukuisia isomiR-sekvenssejä: Noin 90%:sta havaittuja miRNA:ita löydettiin vähintään yksi vaihtoehtoinen, ei-kanoninen sekvenssi. IsomiR:ien suuresta määrästä huolimatta,miRNA:iden kokonaisekspressiosta suurin osaoli yleensä vain muutaman isomiR:in aikaansaannosta. Aineistoista löysimme myös runsaasti merkkejä RNA-editoinnista, ja erityisesti ADAR-entsyymin toiminnasta. Funktionaaliset kokeemme antoivat myös vahvoja viitteitä siitä, että miRNA:iden ydinalueisiin kohdistuvat editoinnit ovat funktionaalisesti merkittäviä tapahtumia. Muutosten seurauksena havaitsimme muuttuneiden ydinaluiden tunnistavan eri kohdegeenejä kuin kanoninen muuntelematon miRNA-sekvenssi. Johtopäätöksenä miRNA:iden ja erityisesti niiden isomiR:ien muunteluun on jatkossa syytä kiinnittää nykyistä enemmän huomiota. Parempi ymmärrys isomiR:eistä ja niitä synnyttävistä mekanismeista voivat jatkossa mahdollistaa tehokkaamman koesuunnittelun ja helpottaa saatujen tulosten tulkintaa.
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(2018)nxiety disorders are the most common psychiatric disorders. Iiris Hovatta’s group (Neurogenomics lab) uses inbred mice to study effects of psychosocial stress. Mice can be divided to susceptible and resilient phenotypes, based on their behavioral response to psychosocial stress. Differences between phenotypes can be examined for example on brain structural or transcriptional levels. These studies have shown changes in brain myelination and expression levels of myelin-related genes between phenotypes. Transcriptomic studies have also revealed differences in microRNA (miRNA) expression profiles. Especially miR-219a, a known regulator of myelination, appears to be differentially expressed. Bioinformatic studies also revealed that many predicted target genes of miR-219a are connected to Wnt/β-catenin signaling pathway, which is also known to regulate myelination. This led to a hypothesis that miR-219a regulates myelination through Wnt/β-catenin signaling pathway in adult mouse brain. Prediction of miRNA target genes is based on algorithms using varying criteria, and their biological significance is uncertain. Assumptions about the biological role of specific miRNA require experimental validation of the silencing interaction between the miRNA and the target gene mRNA. The purpose of this study was to validate five Wnt-signaling related mouse genes (Gsk3b, Esr1, Tcf7l2, Fkbp5 and Acvr1b) as targets for miR-219a-5p or miR-219a-1-3p. For validation a luciferase-reporter assay was used, in which the target sequence of the gene’s mRNA is cloned to a reporter vector, then cotransfected to cultured cells (human HEK293 cells) with a miRNA-mimic. The light emitting luciferase activity is measured to examine the possible silencing effect of the miRNA. Results of this study showed that miR-219a mimics did silence the expression of the target genes. Although some variation was observed in the efficiency of silencing between the genes and based on the mimic concentration. The most efficient silencing was observed between miR-219a-5p and Gsk3b construct. These results implicate that miR-219a regulates several genes related to Wnt/β-catenin pathway, and that stress-related differences in its expression may be associated to differential myelination observed between phenotypes that react differentially to psychosocial stress.
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(2019)Myopatiat ovat laaja joukko erilaisia luurankolihasten heikkoutena ja rappeumana ilmeneviä sairauksia. Ne ovat geneettisesti ja ilmiasuiltaan heterogeenisiä: pelkän kliinisen taudinkuvan perusteella taudin alatyypin tarkka määrittely on hankalaa. Aiemmin näitä tauteja on tutkittu ja diagnosoitu lähinnä melko työläällä Sanger-sekvensointimenetelmällä. Sekvensointimenetelmien kehittyminen on mullistanut myopatioiden tutkimuksen ja diagnostiikan. Uusien massiivisten rinnakkaissekvensointimenetelmien (Massively parallel sequencing, MPS) suoritusteho on valtava ja esimerkiksi koko ihmisen genomi on mahdollista sekvensoida yhdessä sekvensointiajossa. Tampereen yliopiston Lihastautien tutkimuskeskus hyödyntää myopatioiden tutkimuksessa ja diagnostiikassa kohdennettua MPS-menetelmää. Tutkimus on nimeltään MYOcap ja se on kohdennettu myopatioita tunnetusti tai ennustetusti aiheuttavien geenien eksoneihin. MYOcap-geenipaneelia on asteittain laajennettu ja nykyään se kattaa 345 geenin eksonit. Intronaalisia alueita, joilta on kuvattu patogeenisia muutoksia, on sisällytetty paneelin viimeisimpään versioon. Onnistuneessakin MYOcap-geenipaneelitutkimuksessa havaitaan kuitenkin matalasti katettuja alueita (alle 20X-lukupeitto). Nämä alueet voivat pahimmillaan aiheuttaa patogeenisen muutoksen havaitsematta jäämisen. Näitä huonosti katettuja alueita ei aiemmin ollut systemaattisesti selvitetty. Tämän tutkielman tarkoituksena oli kartoittaa MYOcap-geenipaneelitutkimuksen matalan lukupeiton alueita geenipaneelin eri versioissa ja tunnistaa toistuvasti huonosti katettuja alueita. Lisäksi tämän tutkielman tarkoituksena oli selvittää, onko tietyn geenin koettimien suhteellisen määrän lisäämisestä (tehostuksesta) seurannut lukupeittoa parantavaa vaikutusta sekä, onko toistuvasti matalasti katetuilta alueilta kuvattu patogeenisia muutoksia. Tämän tutkielman käytössä oli koko Lihastautien tutkimuskeskuksen MYOcap-sekvensointiaineisto. Aineisto sisälsi 1898 näytteen sekvensointiaineistot. Sekvensointiaineistoista koottiin tietokanta, josta voitiin tunnistaa eri MYOcap-geenipaneeliversioilla toistuvasti matalasti katetut kohdealueet. Näillä kohdealueilla sijaitsevat tunnetut patogeeniset muutokset kerättiin Human Gene Mutation Database 2018.1 -tietokannasta. Sekvensointiaineistojen analysointi tehtiin R-ohjelmointikielellä. Keskeiset käytetyt paketit olivat tidyversen dplyr, tidyr, readr ja ggplot2. Kaikissa MYOcap-versioissa voitiin havaita toistuvasti matalasti katettuja kohdealueita. Näiden huonosti sekvensoituvien kohdealueiden määrä kuitenkin väheni merkittävästi uusien geenipaneeliversioiden myötä. Huonoa sekvensoituvuutta voitiin selittää kohdealueen korkealla GC-pitoisuudella ja kohdealueen sekvenssin epäuniikkiudella. Osa ongelmakohdealueista jäi selittämättömästä syystä toistuvasti matalasti katetuiksi. Tunnettuja patogeenisia muutoksia sijoittui vain osaan toistuvasti matalasti katetuista alueista. Lukupeiton parantamistoimenpiteet voisi keskittää näille alueille, mikäli tutkimuksesta halutaan ensisijaisesti tunnettujen muutosten osalta hyvin kattava. Toisaalta geenipaneeli sisältää laajasti myös ennustettuja tautigeenejä ja siten tutkimuksen ideologiaan sopisi kaikkien toistuvasti matalasti katettujen kohdealueiden lukupeiton parantaminen myös uusien, toistaiseksi tuntemattomien muutosten havaitsemiseksi. Tehostuksen vaikutusta kohdealueiden lukupeittoon oli vaikea arvioida menetelmän epäsystemaattisen käytön takia. Tämän tutkielman perusteella voitiinkin todeta, että tehostuksen kohdistaminen ilman järjestelmällistä lukupeiton kattavuuden arviointia on vaikeaa. Tulosten perusteella käytössä olevaa tehostusta voidaan haluttaessa purkaa ja puolestaan lisätä tehostusta osalle tutkielman perusteella ongelmallisille kohdealueille.
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(2019)Nucleosides are one of the fundamental building blocks of the cell and are precursors of DNA and RNA and serve as universal signalling molecules for cell surface receptors. These nucleosides play vital roles in myriad of physiological processes, such as cardioprotection, platelet aggregation and coronary vasodilation. In clinical settings, synthetic analogs of nucleosides are used as therapeutic against cancer and other viral diseases. In human, uptake and regulation of nucleosides and their analog drugs is facilitated by the two families solute carrier membrane transporter proteins (SLC); SLC28 and SLC29. SLC29 consists of four human equilibrative nucleoside transporter members (hENT1-4) with different sub-cellular localization, tissue distribution and substrate selectivities. Human equilibrative nucleoside transporter 1 (hENT1) is the major plasma membrane nucleoside transporter and has shown to play an important role in adenosine signalling and anticancer therapies. Despite the significant physiological and pathophysiological role, little is known about the structure and molecular function of ENT homologs. Structural studies of hENT1 and other nucleoside transporter is hampered by their low expression and hydrophobic nature. The aim of this thesis work is to find suitable nanobodies (Nbs) that may serve as crystallization chaperone to facilitate the structural studies of hENT1 transporter. Camelid heavy chain only antibodies (Nanobodies) raised against recombinant hENT1 were screened for their suitability for future structural investigation of hENT1. Selected nanobodies were expressed and purified from E.coli cell in milligram quantities. Affinity of nanobodies with hENT1 was assessed using co-elution on size exclusion chromatography. High affinity binders were further screened for their ability to conformationally stabilize hENT1. In future, high affinity nanobodies will be explored for x-ray crystallization and single particle cryo-electron microscopy of hENT1. For cryo-EM it's important to convert the selected Nbs into megabody which constitute large scaffold proteins. 3D structure determination of hENT1 will be significant in understanding its molecular function and to accelerate rational drug designing against cancer, HIV and other viral infections.
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(University of HelsinkiHelsingin yliopistoHelsingfors universitet, 2015)Tässä tutkimuksessa selvitettiin sairauksien ja ongelmien esiintyvyyttä norjalaisilla metsäkissoilla Suomessa. Ensisijaisena tavoitteena oli kerätä tietoa rodulla esiintyvistä sairauksista, mutta samalla selvitettiin myös kissojen taustatietoja (kissan ja vanhempien rekisteröintitiedot, sukupuoli, ikä, paino, synnyinmaa, vieroitus- ja omistajalle saapumisikä, elinympäristö, ravinto, veriryhmä), perinnöllisten sairauksien (glykogeenin kertymäsairaus tyyppi IV ja hypertrofinen kardiomyopatia) varalta tehtyjä tutkimuksia ja niiden tuloksia, kuolinsyitä ja jalostukseen liittyviä tekijöitä (kasvattajien osuus, pentujen ja pentueiden kissakohtainen määrä, kissan käyttö jalostukseen tulevaisuudessa). Rotukissoilla, kuten norjalaisilla metsäkissoilla, ei ole aiemmin Suomessa tehty terveystietojen kartoitusta tieteellisesti tai yhtä kattavasti kuin tässä tutkimuksessa. Tutkimusta tehdessä ei myöskään ollut tiedossa vastaavanlaisia tutkimuksia ulkomailta. Nollahypoteesina oli, että norjalaisilta metsäkissoilta ei löytyisi mitään sairautta epätavallisen paljon. Tutkimus toteutettiin sähköisen kyselylomakkeen avulla. Tavoitteena oli kerätä 400 kissan terveystiedot. Tietoja kerättiin kolmen kuukauden ajan vuonna 2012 ja niitä saatiin 604 kissalta. Tutkimustulosten perusteella todettiin, että yleisimmät eläinlääkärissä todetut sairaudet ja ongelmat kuuluivat suun ja hampaiden (11,4 %), munuaisten ja virtsateiden (9,6 %) ja sukupuolielinten (6,8 %) sairauksiin ja ongelmiin. Omistajien itse toteamat ongelmat liittyivät useimmin käytöshäiriöihin (35,3 %). Käytöshäiriöiden osuus oli odotettua korkeampi ja joitain sairauksia, kuten korvasairauksia, esiintyi odotettua vähemmän. Saavutettu otoskoko on tilastollisesti riittävä kuvaamaan norjalaisilla metsäkissoilla esiintyvien sairauksien ja ongelmien esiintyvyyttä Suomessa. Useimpien taustatietojen osalta saatiin myös riittävän kattavasti tietoa, jotta tuloksia voidaan yleistää koskemaan koko populaatiota. Kunkin elin- tai ongelmaryhmän osalta on kuvattu yksittäisten sairauksien ja ongelmien jakauma ryhmän sisäisesti, jotta todennäköisimmät ja harvinaisimmat ryhmänsisäiset sairaudet saataisiin selville. Nämä luvut kuvaavat luotettavasti niiden jakaumaa vain tutkimuksen kissoilla. Sama pätee kuolinsyihin. Tämän tutkimuksen tuloksista on apua etenkin norjalaisten metsäkissojen kasvattajille. Tiedostamalla rodun keskeisimmät ongelmat, voidaan niiden ehkäisemiseen kiinnittää huomiota jalostuksessa ja valistaa kissojen omistajia todennäköisimmistä terveysongelmista. Tutkimuksen tuloksia voidaan hyödyntää rodulle rakennettavassa terveystietorekisterissä ja jalostuksen tavoiteohjelmassa. Eläinlääkäreille tulokset antavat tärkeää tietoa mihin ongelmiin rodulla pitäisi erityisesti kiinnittää huomiota. Lisäksi tutkimus edesauttaa ja kannustaa kaikkien suomalaisten kissojen terveystietojen keräystä. Tutkimus on myös ollut osa laajempaa käynnissä olevaa kissojen geenitutkimushanketta Suomessa (Kissojen geenitutkimus 2015).
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(2020)Mammalian dentitions exhibit extraordinary diversity in morphology and function. Yet,the mechanisms governing dental development are considered highly conserved across Mammalia. The inhibitory cascade (IC) model is a developmental model explaining variation in molar size proportions observed in mammals. The IC model predicts a range of dental phenotypes based on a dynamic of cumulative inhibition and activation in the sequential development of molars. Whereas most mammals fit these predictions well, bears(Ursidae) are a known exception.Here I employ dental topographic analysis to examine the developmental basis of ursine molar dentitions defying the IC model.I quantified two aspects of tooth shape:molar complexity using orientation patch count (OPC), and tooth size as the area of occlusal surface. As the complexity and size of a tooth mainly emerge based on two different developmental processes –patterning and growth, respectively –these measurements were used to decompose the two phases of tooth development producing the final phenotype. To this end, also an estimate of feature density was calculated.As in previous studies, the molar size proportions of bears were highly incongruent with the IC model. However, complexity along the molar row followed a trend more closely matching the model. Feature density was highest in the third molar. Altogether, these observations suggest an early arrest in the growth of the third molar as the principal cause for bears falling outside the predictions of the IC model –consequently supporting the idea of the inhibitory cascade as a plesiomorphy of Mammalia.As an auxiliary part of this project, I assessed the functionality of Morphoviewer, a new piece of software for measuring complexity. Morphoviewer was successfully applied to infer diet from tooth complexity in a limited sample of carnivorans; and was thus used for all further OPC analyses.
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(2020)Endocytosis is the process responsible for internalising membrane components and as such plays a key role in the biology of this structure. Mammalian cells have evolved various endocytic strategies, but Clathrin-Mediated Endocytosis (CME) is the most common type. Since the discovery of CME, around 50 years ago, the field has built a remarkable wealth of knowledge on the core CME components. In stark contrast, our understanding on the relationship between CME and the actin cytoskeleton, which is present throughout the process, is still in its infancy. In this thesis, I show the production and characterisation of recombinant, SpyCatcher tagged transferrin (TF), a canonical CME ligand. TF was expressed in E. coli and using an optimised protocol, successfully solubilised and refolded from inclusion bodies. The protein was then labelled with a fluorophore and purified to a high level of purity. Tests in mammalian cells showed that home-made TF has the same endocytic behaviour as TF purified from human plasma. Moreover, I could show that the SpyCatcher moiety attached to our home-made TF is capable to mediate its covalent linkage to its counterpart SpyTag. The successful production, refolding and functional characterization of recombinant TF in this study is an important first step to examine the participation of the actin cytoskeleton during CME.
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(2012)In multicellular organisms, complex signalling mechanisms have evolved to guide the behaviour of individual cells. Growth factors are secreted proteins that can stimulate the proliferation and/or differentiation of cells. Vascular endothelial growth factor D (VEGF-D) is a ligand for VEGF receptor 2 (VEGFR-2) and for VEGFR-3, which are predominantly expressed on blood vascular endothelial cells and on lymphatic endothelial cells, respectively. Thus VEGF-D can contribute to growth of both blood vessels (angiogenesis) and lymphatic vessels (lymphangiogenesis). Although there have been many reports showing the angiogenic and lymphangiogenic effects of VEGF-D, its physiological role is still largely unknown. Most of these reports are severely hampered by incomplete characterization of the specific form of VEGF-D that was used. During or after secretion, VEGF-D undergoes complicated proteolytic processing. Alternative Nterminal cleavage results in two different fully processed forms, VEGF-D major and VEGF-D minor. Processing significantly increases the activity of VEGF-D towards its receptors. Surprisingly, it is still unknown whether the differential N-terminal cleavage of VEGF-D has any effect on receptor binding activity or on receptor activation. The goal of this study was to produce and purify high quality biologically active VEGF-D which is needed for studying the physiological role of this growth factor. Several different forms of recombinant human VEGF-D were produced using the Drosophila Schneider 2 insect cell system. A bioassay utilizing the Ba/F3 cells expressing chimeric VEGFR/EpoR receptors was used to determine the receptor binding activities of recombinant VEGF-Ds. Two constructs producing biologically active VEGF-Ds were chosen for chromatographic purification (untagged major and his-tagged major forms). During purification, the activity of both VEGF-D forms towards their receptors decreased significantly. In case of the untagged form, this was presumably due to some residual proteolytic activity during purifications. The results might indicate that only the major form is responsible for the activation of VEGFR-3. The fact that no activity of the minor forms was detected when screening the cell supernatants with Ba/F3-VEGFR-3-EpoR-bioassay, supports this explanation. If this explanation can be verified, the role of the alternative N-terminal cleavage becomes obvious: By proteolysis the activity of VEGF-D can be redirected from the lymphatics towards the blood vessels.
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(2020)Tiivistelmä – Referat – Abstract Celiac disease (CD) is a serious lifelong condition, in which the immune system attacks an individual’s own tissue when eating gluten. This leads to inflammation and damage to the small intestine. Celiac disease often goes undiagnosed because many of its symptoms are nonspecific. The prevalence of combined undiagnosed and diagnosed CD is estimated to affect 1 in 100 people throughout Europe and USA. CD is a polygenic disease, it is known that the human leukocyte antigen (HLA) system plays a crucial role. HLA-DQ2/DQ8 risk allele genotyping screening test from a whole blood sample (B -HLAKeli) is routinely used to estimate the genetic risk of a patient having CD. HLA genotyping test result is routinely used to rule out celiac disease rather than confirming it; if an individual does not have celiac disease related risk alleles, it is very unlikely that he or she has celiac disease. The Celiac disease diagnosis decision making process is based on the classic triple combination of serological antibody tests, the HLA-DQ2/DQ8 genotyping test and duodenal biopsies. The aim of this master’s thesis was to study evaluate how the two different risk classification praxis for HLA-DQx.5 allele used for celiac disease diagnostics in SYNLAB Finland and Estonia central laboratory and in SYNLAB Suomi central laboratory might influence the clinical process and final diagnosis. In SYNLAB Suomi central laboratory HLA-DQx.5 is classified and interpreted as a risk allele predisposing to celiac disease. In SYNLAB Finland and Estonia central laboratory this allele is classified as CD-non-risk-allele based on recommendations in international guideline. In addition, the aim was to get a general understanding of celiac disease prevalence and risk allele distributions among the study population. From the study population of 196 celiac disease suspect patients, 9% had a celiac disease positive laboratory result and the HLA risk genotype distribution among positive cases was well aligned with the expected values described in the literature. Study results indicated that there’s no additional clinical value if HLA-DQx.5 is classified as a celiac disease predisposing risk allele; the study data implies that it is very unlikely to find celiac disease positive cases from laboratory test perspective among HLA-DQx.5 carriers. Based on the study, approximately 7% of the celiac disease suspects carry the allele HLA-DQx.5 and therefore probably go through additional celiac disease related laboratory testing if this allele is interpreted as a risk allele. According to the study findings and general recommendations based on international guideline of celiac disease diagnosis, it seems that there is no clear clinical benefit if HLA-DQx.5 is classified as a CD risk allele.
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