Jenetik Osteopontin nan pasyan ki gen maladi ren kwonik: etid Alman sou maladi ren kwonik Ⅳ

Varyab debaz ak mezi

Yo te mezire yon seri biomakè estanda nan yon laboratwa santral sètifye lè l sèvi avèk pwotokòl estanda [81]. Pami lòt moun, yo te quantifye kreyatinin ak albumin ki soti nan serom ak pipi lè l sèvi avèk yon metodoloji IDMS trasable (Kreatinin plis, Roche, Almay) ak yon metòd turbidimetrik (Tina-quant, Roche, Almay) Roche/Hitachi MODULAR P, respektivman. Yo te estime pousantaj filtraj glomerulèr (GFR) lè l sèvi avèk CKD-EPI ki baze sou kreyatinin pou mula (inite: mL/min/1.73m2, [82]). UACR te kalkile kòm mezire albumin urin / kreyatinin urin (mg / g, [83]). Yo te rapòte laj ak sèks nan vizit debaz la. Nan 2015, OPN te mezire nan echantiyon serik debaz nan kowòt etid konplè GCKD la lè l sèvi avèk yon teknik imunoassay anzim sandwich quantitative (ELISA solid-phase; Quantikine Human OPN Immunoassay DOST00 nan R&D Systems (R&D Systems Europe, Abingdon, UK). )). Kantifikasyon te pote soti nan Enstiti a nan Chimi klinik ak Medsin Oratory Laboratwa, Greifswald, Almay. Koefisyan varyasyon (antra-essai) yo te 4.5%, 5.3% ak 3.5% pou nivo ba, medyàn ak wo, respektivman. Koyefisyan varyasyon ant tès la te 6.4%. Reyaktif ak estanda segondè yo te itilize jan manifakti a rekòmande.

ZÈB TRADITIONNEL ÒGANIC POU SANTE REN

Genotype, kontwòl kalite ak impitasyon

Enfòmasyon detaye sou jenotip ak netwayaj done nan etid GCKD yo te dekri deja [18]. Yon ti tan, ADN te izole nan san antye ak jenotip nan 2,612,357 varyant foumi pou 5,123 patisipan GCKD lè l sèvi avèk etalaj Illumina HumanOmni2.5 Exome BeadChip (Illumina, GenomeStudio, Genotyping Modil Version 1.9.4) nan Helmholtz Center Minik. Netwayaj done yo te fèt separeman pou kontni an Omni2.5 ak kontni an chip exome nan etalaj la. Ki baze sou pwotokòl ofisyèl [84], yo te itilize scripts ekri koutim (R, Perl) ak lojisyèl Plink1.9 [85] pou kontwòl kalite (QC) nan kontni Omni2.5 la. Etap QC ki baze sou echantiyon yo enkli chèk pousantaj apèl, sèks, eterozigozite, zansèt jenetik ak relasyon, ki mennen ale nan esklizyon 89 echantiyon. Nan nivo Variant, polimorfism sèl nukleotid (SNP) yo te eskli si to apèl la te<0.96, and whenever the assumption of the Hardy-Weinberg equilibrium was violated (p-value <1.0E-05). After removing SNPs on duplicate positions, the cleaned dataset contained 5,034 individuals and 2,337,794 SNPs (S1 Fig). Genotypes were then imputed using minimac3 v2.0.1 at the Michigan Imputation Server [86]. The Haplotype Reference Consortium (HRC) haplotypes version r1.1 were used as the reference panel, and Eagle 2.3 was used for phasing. The final dataset contains data of 5,034 participants and 7,750,367 high-quality autosomal bi-allelic variants (imputation quality of R2 �0.3, MAF �1%). For the exome chip content, QC was similarly conducted [18]. In addition, checks specific for exome variants were added [87]. In brief, 96 individuals and 3,818 SNPs were removed, the latter of which had a call rate <0.95 and a Hardy-Weinberg equilibrium p-value <1.0E−05. The final exome chip dataset contains 5,027 participants with 226,233 variants (S1 Fig). For the exome chip association analysis, the genotypes were post-processed using zCall with a zscore threshold of six [88]. Genomic positions base on human genome build GRCh37.

Etid asosyasyon nan tout genòm nan varyant komen

Kòm te rapòte deja [17,18], GWAS te fèt pou patisipan GCKD ak genotype konplè (Omni2.5), eGFR, UACR ak mezi log2(OPN) (N=4,897) done lè l sèvi avèk regression zòrèy lin nan log2. (OPN) sou SNPs (modèl jenetik aditif) ak yon MAF �1%, ajiste pou laj, sèks, log (eGFR), ak log (UACR) (S1 Fig). Analiz asosyasyon yo te fèt lè l sèvi avèk SNPTEST v2.5 [89]. Rezime estatistik yo te tcheke pou bon jan kalite lè l sèvi avèk GWAtoolbox [90] ak pou enflasyon lè l sèvi avèk kontwòl jenomik [91]. Yon koreksyon kontwòl jenomik, sepandan, pa te mande (λ=1.01). Asosyasyon ki gen yon p-valè<5.0E-08 were considered significant. Per chromosome, an index SNP was defined as the SNP with the lowest genome-wide p-value with a 1-Mb interval centered around this SNP. This approach was repeated until no further SNP outside the interval(s) was available passing the genome-wide significance threshold. In order to discover further independent signals, we repeated GWAS analysis for chromosomes with significant results by conditioning on the genotype of the SNP with the lowest association pvalue of the respective chromosome. This procedure was repeated until no further genomewide signal was observed.

Te fè anotasyon fonksyonèl nan variants lè l sèvi avèk ANNOVAR [92], SNiPA [93], Open Targets Genetics [94], FAVOR [95], ak RegulomeDB [96]. Yo te trase trase asosyasyon rejyonal yo lè l sèvi avèk LocusZoom v1.3 [97].

Amann-kat jeyografik

Statistical fine-mapping [21] was carried out as previously described [17] for the two replicated SNPs within a region ±500kb. Approximate Bayes factors (ABFs) were then derived from the original GWAS statistics estimates. The SD prior was chosen as 0.61 because 95% of the effect size estimates fell within the −1.2 to 1.2 interval [21]. The ABF of the SNPs were used to calculate the posterior probability for each variant driving the association signal (PPA, 'causal variant'). Credible sets were determined by summing up PPA-ranked variants until the cumulative PPA was >99%.

Analiz kolokalizasyon

Pou nou ka konprann pi lwen mekanis molekilè yo ak fenotip ki asosye ki kache nan asosyasyon yo, nou te fè analiz kolokalizasyon nan estatistik rezime OPN GWAS ki gen rapò ak de repwodiksyon OPN yo ak estatistik rezime GWAS ki soti nan twa lòt sous jan sa endike anba a. Pou tout analiz colocalisation, nou te itilize fonksyon 'coloc.fast' ki soti nan R package gtx ak paramèt default ak definisyon anvan (https://github.com/tobyjohnson/gtx), yon aplikasyon yon vèsyon adapte metòd colocalisation prezante. pa Giambartolomei et al. [98]. Nou konsidere yon kolokalizasyon pozitif lè pwobabilite apre yon varyant kozatif pataje nan pozisyon asosyasyon pou tou de karakteristik (H4, p12) te > 0.8.

Ekspresyon jèn.

Premyèman, nou te itilize estatistik rezime GWAS sou done ekspresyon jèn ki soti nan pwojè GTEx [99] ak etid NEPTUNE [100]. Done eQTL ki soti nan GTEx V8 (49 tisi) ak etid NEPTUNE (NephQTL ki soti nan pòsyon glomerulus ak tubulointerstitial ren) yo te telechaje nan Portal GTEx (https://www.gtexportal.org/home/) ak sit entènèt NephQTL (http://www.gtexportal.org/home/). ://nephqtl.org/), respektivman. Etap analiz kolokalizasyon yo te dekri an detay yon lòt kote [17]. Premyerman, rezime GWAS nan GTEx ak NephQTL nan rejyon jenomik ± 100kb nan de SNP OPN yo te ekstrè. Jèn yo nan GWAS ekstrè yo idantifye epi pou chak jèn, yo defini yon fennèt cis 500kb ki antoure kòmansman ak fen jèn nan. Lè sa a, pou chak fenèt jèn cis sa yo, ak omwen yon SNP ki gen yon asosyasyon p-valè <0.001, rezime GWAS nan tisi GTEx ak NephQTL ak OPN GWAS yo te ekstrè epi itilize kòm opinyon pou analiz kolokizasyon.