Microbial Seromarkers in Coeliac Disease

Keliakia on krooninen immuunivälitteinen tauti, jossa ravinnon gluteeni aiheuttaa ohutsuolen limakalvovaurion geneettisesti alttiilla henkilöillä. Taudinkuvaan liittyy usein myös vasta-ainemuodostus gluteenia sekä elimistön omia antigeenejä, kuten tyypin 2 transglutaminaasia ja endomysiumia, kohtaan. Keliakia on asteittain kehittyvä sairaus, jonka varhaiseen vaiheeseen liittyy vain suolen limakalvon tulehdus ja / tai vasta-ainemuodostus, joita seuraa kryptahyperplasian sekä villusatrofian kehittyminen. Nykyään keliakian ainoa tiedeyhteisön hyväksymä hoitomuoto on elinikäinen gluteeniton ruokavalio. Vaikka gluteenin poistaminen ruokavaliosta yleensä johtaa ohutsuolen limakalvon parantumiseen, osalla potilaista vaurio ei korjaudu, ja keliakiaan liittyvät oireet jatkuvat, kuten hoitoon reagoimattomassa keliakiassa. Keliakialla ajatellaan olevan monitekijäinen tausta, jossa sekä geneettiset että ympäristötekijät vaikuttavat taudin puhkeamiseen. Lähes kaikilla keliakiapotilailla on HLA-DQ2- ja/tai HLA-DQ8-haplotyypit, ja lisäksi tunnetaan yli 40 HLA-alueen ulkopuolista kohonneeseen sairastumisriskiin liitettyä lokusta. Kuitenkin vain pieni osa geneettisessä riskissä olevista sairastuu keliakiaan, mikä viittaa ympäristötekijöiden merkitykseen. Suolistomikrobeilla tiedetään olevan keskeinen rooli suolen immuunijärjestelmän kehityksessä ja toiminnassa. Keliakiapotilailla on kuvattu muutoksia suolistomikrobistossa verrattuna ei keliakiaa sairastaviin kontrolleihin. Aktiivisessa keliakiassa on myös kuvattu lisääntynyttä vasta- ainemuodostusta suolistossa esiintyviä mikrobeja, kuten Saccharomyces cerevisiae -hiivaa sekä Pseudomonas fluorescens -ja Bacteroides caccae -bakteereja kohtaan. Vasta-ainetasot laskevat gluteenittoman dieetin ja suolen limakalvon parantumisen myötä. On hyvin vähän tutkimustietoa siitä, missä vaiheessa tautiprosessia kyseisiä mikrobivasta- aineita alkaa muodostua, ja onko vasta-ainepositiivisuudella yhteyttä keliakian taudinkuvaan. Kyseisiä vasta-aineita ei juurikaan ole tutkittu keliakiapotilaiden ensimmäisen asteen sukulaisilla, joilla on kohonnut riski sairastua keliakiaan sairastuneen perheenjäsenen kanssa jaettujen geneettisten ja usein myös ympäristön riskitekijöiden kanssa. Tämän väitöskirjan tavoitteena oli tutkia seerumin vasta-aineita Saccharomyces cerevisiae -hiivaa sekä Pseudomonas fluorescens -ja Bacteroides caccae -bakteereja kohtaan keliakiapotilailla taudin eri vaiheissa ja erilaisissa taudinkuvissa, keliakiapotilaiden ensimmäisen asteen sukulaisilla sekä verenluovuttajilla, jotka eivät sairasta keliakiaa. Lisäksi tutkittiin vasta-ainepositiivisuuden yhteyttä keliakialle altistaviin HLA-DQ2- ja HLA-DQ8-haplotyyppeihin sekä vasta-ainepositiivisuuden jakautumista perheiden sisällä ja perheiden välillä. Väitöskirja koostuu kolmesta eri osatyöstä. Osatyössä I mikrobivasta-aineita tutkittiin 44 potilaalla, joilla oli alkava keliakia. Määritykset toistettiin 16:lla potilaalla, kun heillä oli tähystyksessä todettu ohutsuolen limakalvovaurio, ja 33:lla vuosi gluteenittoman dieetin aloittamisen jälkeen. Viisi kuudesta keliakiavasta-ainenegatiivisesta alkavaa keliakiaa sairastavasta potilaasta oli jo seropositiivinen vähintään yhdelle mikrobivasta-aineelle. ASCA-positiivisten osuus laski merkitsevästi verrattaessa alkavan keliakian ja gluteenittoman dieetin ajanhetkiä. Sekä ASCA– että anti-OmpW-vasta-ainetasot myös laskivat merkitsevästi, kun taas anti-I2-vasta-ainepitoisuudet olivat korkeammat ainoastaan 16 potilaalla limakalvovaurion kehittymisen aikaan verrattuna ajankohtaan vuosi gluteenittoman dieetin aloittamisen jälkeen. Osatyössä II mikrobivasta-aineita mitattiin 20:lta ruokavaliohoitoon reagoimattomalta keliakiapotilaalta, jotka olivat olleet tarkalla gluteenittomalla dieetillä keskimäärin 3.5 vuotta. Tuloksia verrattiin 58 keliakiapotilaaseen diagnoosihetkellä ennen ruokavaliohoidon aloittamista sekä 55:een heistä vuosi tautia lieventäneen ruokavaliohoidon aloittamisen jälkeen sekä 80:een ei keliakiaa sairastavaan verenluovuttajaan. ASCA-positiivisuus oli kaikista yleisintä hoitoon reagoimattomilla keliakiapotilailla, ja sekä IgA- että IgG-luokan vasta-ainetasot olivat korkeammat kuin ruokavaliohoitoon suotuisasti reagoineilla potilailla ja kontrolleilla. Anti-I2- ja anti-OmpW-vasta-aineet eivät erottaneet hoitoon reagoimattomia keliakiapotilaita muista keliaakikoista, vaikka anti-I2-vasta-ainetasot olivat korkeammat kuin kontrolleilla. Osatyössä III mikrobivasta-aineita mitattiin 463:lta keliakiapotilaiden ensimmäisen asteen sukulaiselta, joista 49 oli keliakiavasta-ainepositiivisia. Tuloksia verrattiin osatyön II 58 hoitoon reagoineen keliakiapotilaan sekä 80 kontrollin vasta- ainepitoisuuksiin. Mikrobivasta-ainetasot olivat korkeammat keliakiavasta- ainepositiivisilla kuin vasta-ainenegatiivisilla. Mikrobivasta-ainepositiivisuus oli yleisempää myös seronegatiivisilla sukulaisilla kuin kontrolleilla, mutta harvinaisempaa kuin keliakiapotilailla. ASCA- ja anti-I2-vasta-ainetasot olivat kaikilla sukulaisilla korkeammat kuin kontrolleilla, mutta anti-OmpW-tasot olivat korkeammat ainoastaan keliakiavasta-ainepositiivisilla sukulaisilla. Seropositiivisuus mikrobivasta-aineille ei ollut yhteydessä tiettyyn HLA-haplotyyppiin. Tämä väitöskirjatutkimus osoitti, että mikrobivasta-ainemuodostusta voi esiintyä jo alkavassa keliakiassa ennen suolen limakalvovaurion syntymistä, osalla jo ennen keliakiavasta-aineita. Myös keliakiavasta-ainenegatiivisilla sukulaisilla esiintyi lisääntynyttä mikrobivasta-ainemuodostusta. Mikrobivasta-aineet voisivat siten mahdollisesti auttaa tunnistamaan ne geneettisesti alttiit henkilöt, joilla on erityisen korkea riski sairastua keliakiaan, mahdollisesti jo ennen keliakiavasta-aineiden muodostumista. Eri mikrobivasta-aineiden yhdistäminen lisää riskissä olevien tunnistamista. ASCA-positiivisuus oli kaikista yleisintä hoitoon reagoimattomassa keliakiassa, kun taas vasta-ainetasot laskevat suolen limakalvon paranemisen myöstä. ASCA voisi siten olla mahdollinen ei-invasiivinen markkeri keliakian hoitovasteen seurannassa. Se, onko mikrobivasta-aineilla tai niiden kohdemikrobeilla rooli keliakian patogeneesissä vaatii lisätutkimuksia.Coeliac disease is an enteropathy caused by an immune response to ingested gluten in genetically predisposed individuals. It is characterized by the presence of histological damage in the small-bowel mucosa and serum antibodies targeted against transglutaminase 2 (TG2-abs) and endomysium (EmA). The disease develops gradually from an early stage involving only mucosal inflammation and/or coeliac disease-associated autoantibodies to crypt hyperplasia and villous atrophy. Currently the only scientifically accepted treatment is a lifelong gluten-free diet. The normal mucosal architecture is usually restored upon treatment, but a subgroup of patients continues to have persistent clinical symptoms and histological damage; i.e., nonresponsive coeliac disease (NRCD). Coeliac disease is considered to have a multifactorial background. Nearly all patients carry the human leukocyte antigen (HLA) haplotypes DQ2 and/or DQ8, and over 40 loci outside the HLA region have also been associated with the disease. However, only a fraction of predisposed individuals develops the disease, indicating the presence of environmental modifiers. Intestinal microbiota has an essential role in the development and function of the immune system, and alterations in the microbial composition compared with non-coeliac controls have been associated with coeliac disease. Furthermore, increased serological responses towards commensal microbiota, including yeast Saccharomyces cerevisiae and Pseudomonas fluorescens and Bacteroides caccae bacteria have been reported in patients with active coeliac disease, as well as a decrease in the antibody levels during gluten-free diet. However, the time of the appearance of these microbial antibodies and whether they are associated with the clinical picture of coeliac disease remains unclear. There are also very limited data on the prevalence of microbial antibodies in the at-risk relatives of coeliac disease patients. The aim of this dissertation was to assess seroreactivity to IgA and IgG class anti- Saccharomyces cerevisiae antibodies (ASCA) and IgA class antibodies against the I2 protein of Pseudomonas fluorescens (anti-I2 antibodies) and the TonB-linked outer membrane protein of Bacteroides caccae (anti-OmpW) in patients with different types and stages of coeliac disease, the first-degree relatives of coeliac disease patients and non-coeliac controls. Further, association of microbial antibody positivity with HLA and distribution of seropositivity among families were evaluated. The dissertation consists of three individual studies. In Study I, the microbial antibodies were measured in 44 coeliac disease patients with early-stage disease and normal mucosal morphology, at the time when they developed diagnostic atrophy (n=16), and after one year on a gluten-free diet (n=33). It was found that five out of six patients seronegative to coeliac autoantibodies at an early stage already showed seropositivity to at least one of the microbial antibodies, and that the frequency of ASCA positivity decreased significantly on a gluten-free diet. Moreover, ASCA and anti-OmpW antibody levels were higher at the early stage than on a gluten-free diet, whereas the anti-I2 antibody levels were elevated at the time of coeliac disease diagnosis. In Study II, the microbial antibodies of 20 NRCD patients on a gluten-free diet for a median of 3.5 years were compared with those of 58 newly diagnosed coeliac patients before the initiation of dietary treatment, 55 of whom also gave serum samples after one year showing a beneficial response to gluten-free diet, and with those of 80 non-coeliac blood donors. The results showed that seropositivity to ASCA was most frequent in the NRCD group, and that the ASCA IgA and IgG levels were significantly higher in NRCD patients than in the treated diet-responsive patients and controls. Neither seropositivity rate to anti-I2 or anti-OmpW antibodies nor antibody levels distinguished NRCD from diet-responsive coeliac disease, but the anti-I2 levels were higher in NRCD patients than in the controls. In Study III, ASCA, anti-I2 and anti-OmpW antibodies were measured in 463 first-degree relatives of coeliac disease patients, 49 of whom were seropositive to TG2-ab and/or EmA. The results were compared with the 58 diet-responsive coeliac disease patients and the 80 controls also included in Study II. The microbial antibody levels were observed to be higher in the TG2-ab/EmA positive than the autoantibody negative relatives. Additionally, seropositivity was more common among the autoantibody negative relatives than among the controls but less common than among the coeliac disease patients. ASCA and anti-I2 antibody levels were also higher in all relatives than in the controls, whereas with anti-OmpW this was seen only in autoantibody positive relatives. Seropositivity to microbial antibodies was not significantly associated with HLA DQ haplotype. This dissertation demonstrates that adaptive immune response to microbial antibodies may already be present at the early stage of coeliac disease before the development of small-bowel mucosal atrophy, and, in some patients, even before the emergence of disease-associated autoantibodies. The autoantibody negative first- degree relatives also had increased seroreactivity to microbial antibodies. Therefore, measuring these markers might help to identify those at the highest risk among genetically predisposed individuals, in some cases prior to the emergence of coeliac disease-associated autoantibodies. Combining different microbial antibodies increases the detection rate of at-risk individuals. Elevated ASCA levels are associated with NRCD, while the antibody levels decrease along with the mucosal healing. ASCA could thus serve as an additional noninvasive marker in the follow- up of coeliac disease. Whether the antibody response and the targeted microbes have a causal role in the coeliac disease pathogenesis remains a subject for further research

A novel partial de novo duplication of JARID2 gene causing a neurodevelopmental phenotype

Publisher Copyright: © 2022 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals LLC.Background: Deletions covering the entire or partial JARID2 gene as well as pathogenic single nucleotide variants leading to haploinsufficiency of JARID2 have recently been shown to cause a clinically distinct neurodevelopmental syndrome. Here, we present a previously undescribed partial de novo duplication of the JARID2 gene in a patient displaying features similar to those of patients with JARID2 loss-of-function variants. Case report: The index patient presents with abnormalities in gross motor skills and speech development as well as neuropsychiatric disorders. The patient has markedly dark infraorbital circles and slightly prominent supraorbital ridges.Whole-genome sequencing and array comparative genomic hybridization revealed a novel disease-causing variant type, a partial tandem duplication of JARID2, covering the exons 1–7. Furthermore, RNA sequencing validated the increased expression of these exons. Expression alterations were also detected in target genes of the PRC2 complex, in which JARID2 acts as an essential member. Conclusion: Our data add to the variety of different pathogenic variants associated with JARID2 neurodevelopmental syndrome.Peer reviewe

First-degree Relatives of Celiac Disease Patients Have Increased Seroreactivity to Serum Microbial Markers

Risk of celiac disease (CD) is increased in relatives of CD patients due to genetic and possible environmental factors. We recently reported increased seropositivity to anti-Saccharomyces cerevisiae (ASCA), Pseudomonas fluorescens-associated sequence (anti-I2) and Bacteroides caccae TonB-linked outer membrane protein (anti-OmpW) antibodies in CD. We hypothesized these markers also to be overrepresented in relatives. Seropositivity and levels of ASCA, anti-I2 and anti-OmpW were compared between 463 first-degree relatives, 58 untreated and 55 treated CD patients, and 80 controls. CD-associated human leukocyte antigen (HLA)-haplotypes and transglutaminase (tTGab) and endomysium (EmA) antibodies were determined. One or more of the microbial antibodies was present in 75% of relatives, 97% of untreated and 87% of treated CD patients and 44% of the controls. The relatives had higher median ASCA IgA (9.13 vs. 4.50 U/mL, p < 0.001), ASCA IgG (8.91 vs. 5.75 U/mL, p < 0.001) and anti-I2 (absorbance 0.74 vs. 0.32, p < 0.001) levels than controls. There was a weak, positive correlation between tTGab and ASCA (r = 0.31, p < 0.001). Seropositivity was not significantly associated with HLA. To conclude, seropositivity to microbial markers was more common and ASCA and anti-I2 levels higher in relatives of CD patients than controls. These findings were not associated with HLA, suggesting the role of other genetic and environmental factors

First-degree Relatives of Celiac Disease Patients Have Increased Seroreactivity to Serum Microbial Markers

Risk of celiac disease (CD) is increased in relatives of CD patients due to genetic and possible environmental factors. We recently reported increased seropositivity to anti-Saccharomyces cerevisiae (ASCA), Pseudomonas fluorescens-associated sequence (anti-I2) and Bacteroides caccae TonB-linked outer membrane protein (anti-OmpW) antibodies in CD. We hypothesized these markers also to be overrepresented in relatives. Seropositivity and levels of ASCA, anti-I2 and anti-OmpW were compared between 463 first-degree relatives, 58 untreated and 55 treated CD patients, and 80 controls. CD-associated human leukocyte antigen (HLA)-haplotypes and transglutaminase (tTGab) and endomysium (EmA) antibodies were determined. One or more of the microbial antibodies was present in 75% of relatives, 97% of untreated and 87% of treated CD patients and 44% of the controls. The relatives had higher median ASCA IgA (9.13 vs. 4.50 U/mL, p < 0.001), ASCA IgG (8.91 vs. 5.75 U/mL, p < 0.001) and anti-I2 (absorbance 0.74 vs. 0.32, p < 0.001) levels than controls. There was a weak, positive correlation between tTGab and ASCA (r = 0.31, p < 0.001). Seropositivity was not significantly associated with HLA. To conclude, seropositivity to microbial markers was more common and ASCA and anti-I2 levels higher in relatives of CD patients than controls. These findings were not associated with HLA, suggesting the role of other genetic and environmental factors

Suomen merentutkimuksen ydinkysymykset - Merentutkimuslaitos suomalaisessa yhteiskunnassa

Julkaisu sisältää myös toisen artikkelin: Kimmo K. Kahma: Scientific impact of the Finnish Institute of Marine Research: a citation analysi

Microbial seromarkers of early celiac disease

Tutkielmaan liittyvä artikkeli / Article related to the thesis: Early Microbial Markers of Celiac Disease. Published in final edited form as: Journal of Clinical Gastroenterology, 2014 August ; 48(7): 620–624. doi:10.1097/MCG.000000000000008

Microbial Biomarkers in Patients with Nonresponsive Celiac Disease

Background and AimsIn nonresponsive celiac disease (NRCD), the symptoms and duodenal damage persist despite a gluten-free diet. Celiac disease patients with persistent symptoms are found to have a dysbiotic microbiota. We thus hypothesized that increased seroreactivity to the serum gluten-sensitive microbial antibodies Saccharomyces cerevisiae (ASCA), Pseudomonas fluorescens-associated sequence (I2), and Bacteroides caccae TonB-linked outer membrane protein (OmpW) is associated with NRCD.MethodsASCA, I2 and OmpW were measured in 20 seronegative CD patients with persistent villous damage despite strict dietary treatment (NRCD group). Fifty-eight responsive patients served as CD controls (55 on gluten-free treatment) and 80 blood donors as non-CD controls.ResultsAt least one microbial marker was positive in 80% of NRCD patients, in 97% of untreated CD and 87% of treated CD patients, and in 44% of controls. NRCD patients had the highest frequency of ASCA positivity (65% vs 52, 20, and 0%, respectively) and also significantly higher ASCA IgA (median 14.5 U/ml) and IgG (32.5 U/ml) titers than treated CD patients (7.0 U/ml, 13.0 U/ml) and non-CD controls (4.5 U/ml, 5.8 U/ml). The frequencies of I2 and OmpW were lower in NRCD than in untreated CD (65% and 45% vs 86% and 59%, respectively), and I2 titers were higher in NRCD (median absorbance 0.76) and untreated (1.0) and treated (0.83) CD than controls (0.32). OmpW was elevated in untreated (1.1) and treated (0.94) CD patients compared with controls (0.79).ConclusionsSeropositivity and high titers of ASCA are associated with NRCD and might serve as an additional follow-up tool in CD.Peer reviewe