Low-Frequency and Rare-Coding Variation Contributes to Multiple Sclerosis Risk / Erratum

Erratum in Low-Frequency and Rare-Coding Variation Contributes to Multiple Sclerosis Risk. International Multiple Sclerosis Genetics Consortium. Electronic address: [email protected]; International Multiple Sclerosis Genetics Consortium. Cell. 2020 Jan 23;180(2):403. doi: 10.1016/j.cell.2020.01.002. PMID: 31978348 Free PMC article

High resolution melting curve analysis for high-throughput SNP genotyping in IL23R gene and association of IL23R with Slovenian inflammatory bowel diseases patients

Single nucleotide polymorphism (SNP) analysis is important tool in the studies of genetic factors associated with complex diseases and with genetically influenced response to drug therapy (pharmacogenetics). Recently, a new generation of generic dsDNA binding dyes (LCGreen$^{TM}$) contributed to the development of fast and low-cost method for SNP detection and/or genotyping based on high resolution melting (HRM) analysis. The aim of our study was to develop HRM assay for IL23R gene (rs7517847) and to perform association study in Slovenian inflammatory bowel diseases (IBD) patients. We genotyped 345 Slovenian healthy controls and 295 IBD patients including 159 with Crohn\u27s disease (CD) and 136 with ulcerative colitis (UC) for rs7517847 polymorphism in IL23R gene using standard RFLP and optimized HRM methods. In this study, we showed, that HRM is a simple, fast and reliable method for genotyping of clinical samples where homozygotes (GG and TT) were determined by Tm calling method and difference between homozygotes and heterozygotes was determined by different melting curve shape using gene scanning method. With combination of results from Tm calling and gene scanning methods, we achieved 98,6% concordance between PCR-RFLP and PCRHRM results, based on the analysis of 640 samples. We found statistically significant association of IL23R polymorphism with Slovenian Crohn\u27s disease patients when comparing genotype and allele frequencies between CD patients and controls. Allele frequency of minor allele G was 0,46 in controls and was reduced to 0,33 in CD patients (p < 0,001, OR = 0,588). The frequency of T/T genotype carriers was higher in CD patients (50,3%) than in controls (26,7%, p = 0,002, OR = 2,558). We found weak association between IL23R polymorphism and Slovenian UC patients. Carriers of T/T genotype have higher risk for UC (p = 0,035, OR = 1,599). These results suggest IL23R plays important role in CD and UC development in Slovenian patients.Analiza polimorfizmov posameznega nukleotida (ang. SNP za single nucleotide polymorphism) in mutacij je ključnega pomena pri ugotavljanju genetskih dejavnikov tveganja za nastanka kompleksnih bolezni in genetsko pogojenega odziva posameznikov na zdravila (farmakogenetika). Razvoj nove generacije fluorescentnih označevalcev (LCGreen), ki se z večjo afiniteto vežejo na dvojno vijačnico DNK, je omogočil razvoj nove metode za hitro in cenovno ugodno analizo polimorfizmov SNP in mutacij na osnovi analize talilne krivulje visoke ločljivosti (ang. HRM za Highresolution melting). Namen naše študije je bil razvoj metode HRM za gensko tipizacijo polimorfizma rs7517847 v genu IL23R in izvedba asociacijske študije za omenjeni polimorfizem pri slovenskih bolnikih s kronično vnetno črevesno boleznijo (KVČB). V asociacijski študiji smo z uporabo standardne metode PCR-RFLP in optimizirane metode PCRHRM določili genotipe za polimorfizem gena IL23R (rs7517847) za 345 zdravih posameznikov in za 295 bolnikov s KVČB, med katerimi je bilo 159 bolnikov s Crohnovo boleznijo (CB) in 136 bolnikov z ulceroznim kolitisom (UK). Ugotovili smo, da je HRM enostavna, hitra in zanesljiva metoda za določanje genotipov v kliničnih vzorcih. Razlike med skupinama homozigotov (GG in TT) smo ugotavljali z algoritmom "Tm calling", heterozigote in homozigote pa smo razlikovali na osnovi različnih oblik talilnih krivulj z algoritmom "gene scanning". S kombinacijo obeh algoritmov za analizo HRM podatkov smo določili genotipe za 640 vzorcev in ugotovili 98,6% skladnost z genotipi, ki so bili določeni z referenčno RFLP metoda na istih vzorcih. V asociacijski študiji smo s primerjavo alelnih in genotipskih frekvenc med zdravimi posamezniki in bolniki s CB odkrili statistično značilno povezavo med polimorfizmom gena IL23R in skupino bolnikov s CB. Pri zdravih posameznikih je bila alelna frekvenca alela G 0,46, pri bolnikih s CB pa 0,33 (p < 0,001, OR = 0,588). Frekvenca posameznikov z genotipom T/T je bila pri bolnikih s CB (50,3%) višja kot pri skupini zdravih posameznikov (26,7%, p = 0,002, OR = 2,558). Odkrili smo tudi šibko povezavo med polimorfizmom gena IL23R in bolniki z UC, kjer so imeli nosilci genotipa T/T višje tveganje za UC (p = 0,035, OR = 1,599). Ti rezultati kažejo, da igra gen IL23R pomembno vlogo v patogenezi pri slovenskih bolnikih s CB in UC

High-resolution melting curve analysis for high-throughput genotyping of NOD2/CARD15 mutations and distribution of these mutations in Slovenian inflammatory bowel diseases patients

Inflammatory bowel diseases (IBD) are usually classified into Crohn\u27s disease (CD) and ulcerative colitis (UC). NOD2/CARD15 was the first identified CD-susceptibility gene and was confirmed as the most potent disease gene in CD pathogenesis. Three NOD2/CARD15 variants, namely two missense polymorphisms R702W (rs2066844) and G908R (rs2066845), and a frame shift polymorphism L1007fs (rs2066847), were associated with CD in Caucasian populations. High resolution melting analysis (HRMA) with saturation LCGreen dyes was previously reported as a simple, inexpensive, accurate and sensitive method for genotyping and/or scanning of rare variants. For this reasons we used qPCR-HRMA for genotyping NOD2/CARD15 variants in 588 Slovenian IBD patients and 256 healthy controls. PCR-RFLP was used as a reference method for genotyping of clinical samples. The optimization of an HRM experiment required careful design and adjustment of main parameters, such as primer concentration, MgCl_{2} concentration, probe design and template DNA concentration. Different HRMA approaches were tested and used to develop a reliable and low-cost SNP genotyping assays for polymorphisms in NOD2/CARD15 gene. Direct HRMA was the fastest and cheapest HRMA approach for L1007fs and R702W polymorphisms, yet for G908R polymorphism sufficient reliability was achieved after introduction of unlabeled probe. In association analysis, we found statistically significant association of L1007fs (p =0.001, OR=3.011, CI95%=1.494-6.071) and G908R (p=2.62 * 10^{-4}, OR=14.117, CI95%= 1.884-105.799) polymorphisms with CD patients. At least one of NOD2/CARD15 polymorphisms was found in 78/354 (22.03% (12.69%) in UC patients and in 26/256 (10.15%) in healthy controls. We have successfully implemented NOD2/CARD15 HRMA assays, which may contribute to the development of genetic profiles for risk prediction of developing CD and for differential diagnosis of CD vs. UC

Genome-wide association analysis of Slovenian inflammatory bowel disease patients

V preteklih desetletjih je bil v raziskave in razumevanje patogeneze KVČB vložen precejšen napor, vendar natančni vzroki nastanka bolezni še niso povsem pojasnjeni. Po zdaj najbolj uveljavljeni hipotezi se bolezen pojavi zaradi pretiranega imunskega odziva na normalno črevesno mikrofloro pri gensko dovzetnih posameznikih. Bolniki s KVČB zbolijo za eno od dveh precej podobnih oblik kroničnega vnetja črevesja: ulceroznim kolitisom (UK) ali Crohnovo boleznijo (CB). V 10−15 odstotkih primerov pa bolniki ne kažejo jasnih kliničnih, endoskopskih in histoloških značilnosti CB ali UK, in govorimo o intermediarnem kolitisu (IK). Največjo pogostost bolezni so ugotovili v Kanadi in Severni Evropi, najmanjšo pa v deželah v razvoju in v Aziji, pri čemer je UK pogostejši kot CB. Za slovensko populacijo sicer ni konkretnih epidemioloških podatkov, vendar je bilo ob koncu 90. let v Sloveniji 1150 bolnikov s KVČB, kar nas uvršča na dno evropske lestvice. Na nastanek bolezni vplivajo različni dejavniki okolja, iz epidemioloških raziskav družin in dvojčkov pa nedvomno izhaja, da k tveganju za KVČB prispevajo tudi genski dejavniki. Kljub številnim dokazom o genskem ozadju bolezni se je natančno določanje genskih determinant začelo šele v zadnjih petnajstih letih. Temeljilo je na številnih raziskovalnih pristopih. Asociacijske študije na celotnem genomu (GWAS) so z odkritjem povezav med KVČB in 99 lokusi največ prispevale k razumevanju genskega ozadja bolezni. Med pomembnejše izzive v obdobju po GWAS spadata odkrivanje vzročnih variant in analiza njihovih funkcionalnih posledic, s tem pa posledično tudi rešitev problema manjkajočega dednostnega deleža pri kompleksnih boleznih. Zaradi tega so bili predlagani številni pristopi, kot so npr. fino kartiranje, analiza eQTL-ov in GWAS pri družinah obolelih, vključno s preučevanjem učinkov starševskega izvora (angl. parent of origin − POO) na izražanje genov. Ob tem pa so številne raziskovalne skupine, ki se ukvarjajo z genetiko KVČB, pod okriljem konzorcija International Inflammatory Bowel Disease Consortium (IIBDGC) združile moči v projektu ImmunoChip (iCHIP), ki predstavlja tarčno mikromrežo DNK za analiziranje imunsko posredovanih bolezni. V raziskavi smo izvedli z iCHIP-om prvo tarčno študijo GWAS pri 227 bolnikih s KVČB in 210 zdravih posameznikih. Zaradi razmeroma omejenega števila preiskovancev sicer nismo imeli zadostne statistične moči za odkrivanje statistično značilnih povezav, vendar smo nakazali na nominalne povezave z boleznijo na kromosomih 1, 5, 8, 13 in 16. V sodelovanju z drugimi člani projekta iCHIP, ki je v metaanalizi vključeval približno 75.000 bolnikov s KVČB in zdravih posameznikov, smo prispevali k odkritju 71 novih statistično značilnih povezav z boleznijo, tako da se je skupno število bolezenskih lokusov z 99 povzpelo na 163. Glede odkrivanja skupnih mehanizmov kompleksnih bolezni je pomembna tudi ugotovitev, da je več kot dve tretjini lokusov KVČB impliciranih v patogenezi drugih imunsko posredovanih bolezni, kot so ankilizirajoči spondilitis, psoriaza in mikobakterijske okužbe. V raziskavi smo ločeno izvedli tudi asociacijsko analizo SNP-jev genov NOD2 in IL23R ter prvič povezali omenjena gena s patogenezo KVČB pri slovenskih bolnikih. Genotipske podatke smo pridobili z optimizacijo metode analize DNK s talilnimi krivuljami visoke ločljivosti (HRMA), ki se je izkazala kot hitra, preprosta, natančna in cenovno ugodna metoda za odkrivanje redkih mutacij in za gensko tipizacijo. V študiji smo s kartiranjem cis-eQTL-ov v regiji s tremi kandidatnimi geni ( ) ugotovili, da polimorfizma rs10178214, rs1041973 in redka varianta ccc-2-102248784-A-G statistično značilno vplivajo na izražanje gena IL1RL1 v periferni venski krvi. Nismo pa opazili statistično značilnega izražanja tarčnih genov v črevesnih biopsijah bolnikov s CB. V raziskavi smo pri nizozemskih bolnikih s KVČB odkrili omejen učinek POO genov IL12B, PRDM1 in NOD2, ki pa nam ga v večji neodvisni kohorti ni uspelo replicirati.Crohn’s disease (CD) and ulcerative colitis (UC) are the two main forms of chronic relapsing inflammatory bowel diseases (IBD). The etiology of IBD has been extensively studied in the past few decades however, disease pathogenesis is not fully understood. It is a chronic disease characterized by recurring inflammation of the gut, and is thought to arise in response to the commensal microflora in a genetically susceptible host. IBD is believed to be associated with industrialization of nations, with the highest incidence rates and prevalence of IBD in North America and Northern Europe. There are no precise epidemiological data available for Slovenians, yet in the late 90. there were 1.190 individuals affected with IBD. The abundance of epidemiological data is suggesting influence of different environmental and genetic determinants in the disease pathogenesis. Complex disease genetics has been revolutionised in recent years by the advent of genome-wide association (GWA) studies. The chronic inflammatory bowel diseases (IBDs), Crohn\u27s disease and ulcerative colitis have seen notable successes culminating in the discovery of 99 published susceptibility loci/genes to date. One of the future challenges in this post-GWAS era is to identify potential sources of the remaining heritability. Such sources may include common variants with limited effect size, rare variants with higher effect sizes, or even more complicated mechanisms such as epistatic and epigenetic interactions. Many approaches to meet these challenges have been suggested, including fine-mapping, eQTL mapping and family studies, focused on parent of origin (POO) analysis. To fully unravel the hidden heritability of IBD, collaborations between genome research centers are crucial, since the solutions to identify the hidden heritability are either costly or require a huge number of cases and controls. The IIBDGC is a good example of what can be achieved by performing large meta­analyses, and it is currently joining forces in the ImmunoChip project (iCHIP), a custom made array encompasing loci from GWAS of 12 immune-mediated diseases. We conducted iCHIP based association analysis on 227 IBD patients and 210 healthy controls. Unfortunately, due to small sample size we did not have sufficient power to detect significant associations. However we show a trend towards association on chromosomes 1, 5, 8, 13 and 16. In collaboration with other IIBDGC members we expanded on the knowledge of relevant pathways by undertaking a meta-analysis of Crohn’s disease and ulcerative colitis genome-wide association scans, followed by extensive validation of significant findings, with a combined total of more than 75,000 cases and controls. We identify 71 new associations, for a total of 163 IBD loci, that meet genome-wide significance thresholds. Many IBD loci are also implicated in other immune-mediated disorders, most notably with ankylosing spondylitis and psoriasis. We also observe considerable overlap between susceptibility loci for IBD and mycobacterial infection. We also conducted a separate association analysis for SNPs in genes NOD2 and IL23R, which were associated with Slovenian IBD patients for the first time. Genotypes were obtained by high-resolution melting analysis (HRMA). The results of this study suggest that HRMA yields significant savings on analysis time and costs and has proven as a simple high-throughput technique for screening for polymorphisms. In this study, we also conducted eQTL mapping of IL18RAP locus, which contains 3 candidate genes IL18R1, IL18RAP in IL1RL1. Polymorphisms rs10178214, rs1041973 and a rare variant ccc- 2-102248784-A-G, were influencing expression of IL1RL1 in whole-blood samples. We did not observe any changes of gene expression of the candidate genes when intestinal biopsies were taken into consideration. We identified POO effects for NOD2 for Dutch CD triosthese results could not be replicated in an independent cohort. A POO effect in IBD was observed for IL12B and PRDM1. In the Indian trios the IL10 locus showed a nominal POO effect

Limited evidence for parent-of-origin effects in inflammatory bowel disease associated loci

Background Genome-wide association studies of two main forms of inflammatory bowel diseases (IBD), Crohnʼs disease (CD) and ulcerative colitis (UC), have identified 99 susceptibility loci, but these explain only ~23% of the genetic risk. Part of the Žhidden heritabilityʼ could be in transmissible genetic effects in which mRNA expression in the offspring depends on the parental origin of the allele (genomic imprinting), since children whose mothers have CD are more often affected than children with affected fathers. We analyzed parent-of-origin (POO) effects in Dutch and Indian cohorts of IBD patients. Methods We selected 28 genetic loci associated with both CD and UC, and testedthem for POO effects in 181 Dutch IBD case-parent trios. Three susceptibility variants in NOD2 were tested in 111 CD trios and a significant finding was re-evaluated in 598 German trios. The UC-associated gene, BTNL2, reportedly imprinted, was tested in 70 Dutch UC trios. Finally, we used 62 independent Indian UC trios to test POO effects of five established Indian UC risk loci. Results We identified POO effects for NOD2 (L1007fsOR = 21.0, P-value = 0.013) for CDthese results could not be replicated in an independent cohort (OR = 0.97, P-value = 0.95). A POO effect in IBD was observed for IL12B (OR = 3.2, P-value = 0.019) and PRDM1 (OR = 5.6, P-value = 0.04). In the Indian trios the IL10 locus showed a POO effect (OR = 0.2, P-value = 0.03). Conclusions Little is known about the effect of genomic imprinting in complex diseases such as IBD. We present limited evidence for POO effects for the tested IBD loci. POO effects explain part of the hidden heritability for complex genetic diseases but need to be investigated further

Low-Frequency and Rare-Coding Variation Contributes to Multiple Sclerosis Risk / Erratum

Erratum in Low-Frequency and Rare-Coding Variation Contributes to Multiple Sclerosis Risk. International Multiple Sclerosis Genetics Consortium. Electronic address: [email protected]; International Multiple Sclerosis Genetics Consortium. Cell. 2020 Jan 23;180(2):403. doi: 10.1016/j.cell.2020.01.002. PMID: 31978348 Free PMC article

Low-Frequency and Rare-Coding Variation Contributes to Multiple Sclerosis Risk

Multiple sclerosis is a complex neurological disease, with ∼20% of risk heritability attributable to common genetic variants, including >230 identified by genome-wide association studies. Multiple strands of evidence suggest that much of the remaining heritability is also due to additive effects of common variants rather than epistasis between these variants or mutations exclusive to individual families. Here, we show in 68,379 cases and controls that up to 5% of this heritability is explained by low-frequency variation in gene coding sequence. We identify four novel genes driving MS risk independently of common-variant signals, highlighting key pathogenic roles for regulatory T cell homeostasis and regulation, IFNγ biology, and NFκB signaling. As low-frequency variants do not show substantial linkage disequilibrium with other variants, and as coding variants are more interpretable and experimentally tractable than non-coding variation, our discoveries constitute a rich resource for dissecting the pathobiology of MS

Erratum: Low-Frequency and Rare-Coding Variation Contributes to Multiple Sclerosis Risk (Cell (2019) 178(1) (262), (S0092867419306798), (10.1016/j.cell.2019.06.016))

(Cell 175, 1679–1687.e1–e7; November 29, 2018) It has come to our attention that in preparing the final version of this article, the authors inadvertently misspelled the last name of author Charlotte E. Teunissen as “Charlotte E. Theunissen.” This error has been corrected in the article online. In the Editorial Note (Cell 178, 262, June 27, 2019), the editors refer to the original version of the published manuscript. That version contained a misspelled name, and as that has now been corrected, we are updating the Editorial Note as well