Genetic association and transcriptome integration identify contributing genes and tissues at cystic fibrosis modifier loci
[['Greater than 348 CF transmembrane conductance regulator ( CFTR ) gene variants are known to cause CF , where epithelial function is impaired in several organs including the lung , pancreas , and intestine , amongst others ( see CFTR2 ( Web Resources in Material and Methods ) and [1] ) .', 'Individuals with the same causal CFTR genotype display a wide range of disease burden within and across organs and exhibit variable response to costly CFTR mutation-directed therapies .', 'Modifier gene relationships with CFTR may involve tissue-specific facets that explain some of this variability , and need to be understood to improve therapeutic strategies [2] .', 'Significant morbidity and mortality in CF is a consequence of progressive lung disease , for which the estimated heritability is approximately 50% [3] and correlates poorly with CFTR genotype [4] .', 'Meconium ileus , intestinal obstruction due to failure to pass the meconium , occurs in 13–21% [5] of CF newborns , with equal frequency in males and females .', 'Meconium ileus displays heritability estimates of ~88% [6] and can be discordant for CF siblings with identical CFTR mutations , further highlighting the importance of genetic background beyond CFTR .', 'Meconium , or first stool of a newborn , is typically comprised of intestinal mucins , proteins , bile salts , and cellular debris that are shed from the intestinal mucosa during the fetal period [7] .', 'The meconium exhibits increased thickness and adherent properties in CF , with blockage .', 'Evidence for blockage or meconium ileus can be seen on ultrasound as early as the second trimester , and although there can be resolution in utero , persistence to beyond birth is fatal without enema or surgical intervention [7] .', 'Given the early occurrence , unambiguous presentation and high heritability , genes that contribute to meconium ileus may be more amenable to gene mapping .', 'Moreover , genetic contributors to meconium ileus have been shown to inform disease severity in other CF affected organs .', 'Genome-wide association studies ( GWAS ) have identified modifier genes that contribute to morbidity across multiple CF-affected organs [8] , with the associated variants frequently annotated to putative regulatory regions of nearby genes .', 'For example , allelic variation near the transcription start site ( TSS ) of SLC26A9 , which encodes an anion transporter in epithelial cells that interacts with CFTR to enhance its functional expression [9–11] , has been shown to associate with aspects of CF lung disease [2] , meconium ileus [12] , CF-related diabetes ( CFRD ) [13] , as well as exocrine pancreatic damage at birth [14 , 15] .', 'Although the same modifier can show association with multiple CF co-morbidities , it is unclear whether these are independent effects as their timing of impact and expression levels vary .', 'Exemplary is how SLC26A9 appears to contribute to CFRD through exocrine pancreatic damage in utero [15] , an example of vertical pleiotropy [16] .', 'Here , we are interested in understanding the relation between the intestine , where meconium develops , and the pancreas .', 'The presence of meconium ileus is correlated with disease in other CF affected organs , in particular pancreatic disease .', 'Direct pancreatic function studies have shown that individuals with CF have reduced digestive enzyme secretory fluid with low pH and high protein concentrations [17 , 18] .', 'Meconium ileus occurs almost exclusively in individuals with severe CFTR mutations known to confer pancreatic exocrine insufficiency [5 , 19] ( ~90% of the CF population [20] ) , therefore modifier gene studies of meconium ileus have been restricted to this CF sub-population .', 'It has been noted that those with meconium ileus on average have relatively less elevated immunoreactive trypsinogen ( IRT ) levels at birth compared to patients without meconium ileus ( [15 , 21] ) ; elevated IRT is the newborn screening biomarker for CF and is also reflective of the extent of prenatal pancreatic injury [19 , 22] .', 'The phenotypic correlation between meconium ileus and CF pancreatic disease , including CFRD [13] , leads one to consider the involvement of the pancreas in the development of meconium ileus .', 'Evidence that gene expression in the pancreas is mediating the association between meconium ileus and the GWAS variants would support this hypothesis .', 'Using the largest study population of individuals with CF to date from the International CF Gene Modifier Consortium ( GMC ) , we ( 1 ) identify genetic loci contributing to meconium ileus using a combination of whole genome genotype arrays and next generation sequencing; ( 2 ) integrate publicly available cross-tissue gene expression [23] ( and epigenomic data [24–27] ) with genotypes ( Material and Methods and S1 Appendix ) to test the hypothesis that the variants associated with meconium ileus are colocalizing with variants that impact gene expression and ( 3 ) determine the most probable originating tissue for the meconium ileus modifiers .', 'We also ( 4 ) integrate RNA-sequencing of nasal epithelia from individuals with CF to determine cross-phenotypic mechanisms and tissue-specific GWAS signals .'], ['Genome-wide association analysis used generalized estimating equations with an exchangeable correlation structure ( to account for siblings present in the data ) , and included adjustment for consortium site , genotype platform and population stratification ( S1 Fig for the first three principal components ) .', 'Three genome-wide significant loci with nearest genes SLC6A14 , SLC26A9 and ATP12A ( p-values<5x10-8 based on the conventional family-wise error control [30] ) , and three additional loci with nearest genes PRSS1 , TARS , CEBPB ( q-values<0 . 05 based on the false discovery rate control [31] ) were evident ( Fig 1 ) .', 'The chromosome X ( SLC6A14; panel', '( a ) of Fig 2 ) and chromosome 1 ( SLC26A9; panel', '( b ) of Fig 2 ) loci were identified as genome-wide significant in an earlier meconium ileus study by the GMC [12] using a subset of the current study sample ( S3 Table ) ; this earlier study also demonstrated that a set of 157 genes that code for constituents of the apical plasma membrane where CFTR resides ( see Supplementary Table 3 in [12] ) were significantly enriched for variants associated with meconium ileus , and this gene-set association was replicated in the French Gene Modifier Study ( FGMS ) cohort [12 , 32] .', 'The expanded sample ( Table 1 and S1 Table ) analyzed here provides further support for the same variants at the SLC6A14 and SLC26A9 loci ( Table 2 , minimum p<2 . 2x10-16 and p = 2 . 81x10-11 in column ‘Mega-all’ , and S3 Table ) .', 'Support for CFTR ( min p = 2 . 34x10-5 at rs213972; gene-based permutation p = 0 . 0001; S2 Fig ) and the association between the apical plasma membrane gene-set and meconium ileus was also observed in this study ( permutation p<1x10-4 in a subset of 5 , 869 unrelated individuals from the full consortium data and p = 0 . 017 in the North American Phase II subgroup alone ) .', 'The confirmation of multiple contributing genes supports the existence of genetic heterogeneity , indicating that meconium ileus is a phenotype of complex genetic etiology within a mendelian disease .', 'The SLC6A14 association region is subject to X-inactivation based on the H3K27me3 histone methylation pattern provided by the Roadmap Epigenomics Mapping Consortium ( REMC ( Web Resources in Material and Methods ) ; experiments ENCSR204NFO and ENCSR727VOB [25] in female lung tissue where strong SLC6A14 expression occurs , GTEx [23] ) .', 'Therefore , the observation that the risk alleles for the genome-wide significant SNPs display lower odds ratios ( OR ) in females ( with two versus zero copies of the risk alleles ) than the ORs in males ( one versus zero copies of the risk alleles ) , p = 0 . 074 , 0 . 046 , and 0 . 058 , respectively , for rs3768766 A , rs5905177 T , and rs12710568 C risk alleles ( S4 Table ) , suggests the possibility of sex-specific differences beyond what is expected under random X-inactivation .', 'A new genome-wide significant locus was identified on chromosome 13 with minimum p-value = 3 . 83x10-10 at rs61948108 ( Table 2 and S3 Table ) , and located in intron 17 of the ATPase H+/K+ Transporting Non-Gastric Alpha2 Subunit ( ATP12A ) ( panel', '( c ) in Fig 2 ) .', 'Restricting the association analysis to the 4 , 343 individuals homozygous for Phe508del resulted in a larger effect size ( OR = 1 . 7 vs . 1 . 55 , Table 2 , S3 Fig ) .', 'Although this did not result in statistically significant GxG interaction ( interaction p = 0 . 057 ) , the dependence on CFTR genotype of the ATP12A-meconium ileus relationship requires further investigation .', 'Modifier-CFTR-mutation relationships have previously been reported for CF lung disease [2 , 33] , and ATP12A was previously implicated as a modifier of CF lung disease using candidate gene analysis in pig models [34] .', 'Conditional analysis at each of the three genome-wide significant loci displayed no evidence for allelic heterogeneity ( S4 Fig ) .', 'Top variants from the three genome-wide significant loci explain 7% of the phenotypic variability for meconium ileus .', 'Of the three FDR significant loci ( Table 2 ) , the chromosome 7 locus ( ‘Mega-all’ p = 2 . 55x10-7 at rs3757377 located upstream of PRSS1; S5 Fig ) was suggestive in the previous consortium study ( Fig 1 of [12] ) , and is replicated in the additional North American cohort ( ‘Phase II-NA’ p = 8 . 43x10-3 , Table 2 ) .', 'We note that the physical landscape encompassing this locus has been revised in recent genome map versions , such that it remains unclear if variation at this locus has been fully appreciated .', 'The new chromosomes 5 and 20 loci were not identified in the previous study [12] and will need to be investigated further .', 'The identified associated variants and those nearby in high linkage disequilibrium ( LD ) do not occur in gene coding regions; however , they include variants near the transcription start sites ( TSS ) of the nearest genes suggesting roles in gene regulation .', 'PRSS1 , or cationic trypsinogen , is used as a measure of early pancreatic disease in CF [35 , 36] .', 'At the PRSS1 locus , the meconium ileus associated SNP , rs3757377 ( Table 2 , Chr7:142455538 , 1 . 8kb 5’ of the TSS , OR = 1 . 29 , p = 2 . 55x10-7; S5 Fig ) is in high LD ( r2 = 0 . 71 and D’ = -0 . 99 ) with rs10273639 , the variant that was identified in a GWAS of alcoholic-related chronic pancreatitis in Europeans [37] .', 'A follow-up study [38] further showed that rs4726576 ( chr7:142457132 , 186bp upstream of PRSS1; S3 Table ) , a variant in high LD with both GWAS study-associated variants ( rs3757377 and rs10273639 ) , is contributing to the pancreatitis relationship by directly influencing gene expression levels based on reporter gene assays in acinar pancreatic cells .', 'These assays and consideration that the encoded digestive enzyme function originates from the pancreas argue that variants associated with meconium ileus at the PRSS1 locus impact gene expression in this organ .', 'The pancreatitis protective T allele of rs10273639 that is associated with less PRSS1 expression is a risk allele for meconium ileus .', 'The meconium of individuals with CF contain high levels of protein and less trypsinogen may adversely retain consistency [39] .', 'We did note that no eQTLs for PRSS1 are reported in the pancreas in the genotype tissue expression project ( GTEx ) .', 'This may be reflecting its extremely high expression ( the PRSS1 transcript is the most highly expressed gene in the pancreas with median TPM of 9 . 91 x 104 [23] ) , or that eQTL determination is affected by the local genome landscape that has been altered in the most recent genome assembly ( GRCh38/hg38 ) with added genes as well as indication of alternative assemblies .', 'To determine whether gene expression variation at the three genome-wide significant loci ( SLC6A14 , SLC26A9 and ATP12A ) influence meconium ileus risk and in which tissue ( s ) , we used data from the GTEx v7 ( accessed on Oct 1 , 2018 ) [23] and considered the enhancer and promoter states from the Regulatory Regions Map ( Reg2map; see Web Resources in Material and Methods ) [24 , 25] .', 'We first display the association findings ( Fig 2 , colored circles as in the LocusZoom plots [40] ) at the three loci in the context of the available eQTLs of gastro-intestinal tissues ( Fig 2 , colored lines indicating different tissues; eQTLs for SLC6A14 in the pancreas and ATP12A in the transverse colon were included in the GTEx v6 but not v7 . They were calculated using v7 data and included here; Material and Methods and sex-stratified analysis S6 Fig ) , with the locations of tissue-specific enhancers and promoters noted when available to complement the comparisons .', 'Notably , the pattern of eQTL evidence for each of SLC6A14 , SLC26A9 and ATP12A in the pancreas ( black solid line ) mirrors the genetic association pattern ( colored circles ) , with the enhancer and promoter states overlapping with pancreas-specific regulatory elements for at least SLC26A9 and ATP12A .', 'In contrast , eQTLs in the small intestine ( blue dashed line ) do not meet expression threshold criteria by either GTEx v6 or v7 ( due to a very low level of expression ) for SLC26A9 and ATP12A ( panels', '( b ) and', '( c ) in Fig 2 ) or do not overlap ( SLC6A14 , panel', '( a ) in Fig 2 ) with the GWAS findings .', 'Based on GTEx data , the meconium ileus risk alleles are associated with decreased SLC26A9 expression , but increased ATP12A and SLC6A14 expression in the pancreas .', 'The chromosome X locus associated with meconium ileus also displayed association with CF lung function severity [41] , with similar effects in males and females .', 'However , the previously identified broad peak of association for lung function appeared shifted and was notably closer to the angiotensin II receptor type 2 ( AGTR2 ) gene ( panel', '( a ) in Fig 3 , blue/green dot palette for lung function association [41] , and red/yellow dot palette for meconium ileus association ) .', 'The SLC6A14 eQTLs from the GTEx lung samples ( panel', '( a ) in Fig 3 , brown dash-dotted line ) did not appear to colocalize with the lung GWAS association evidence .', 'Given that the GTEx protocol for lung tissue collection largely avoids the respiratory epithelia ( which include the most relevant cell types in CF ) , we confirmed the lung eQTLs by RNA sequencing of nasal epithelia ( a surrogate airway model in CF [42] ) from 63 Canadians with CF included in the GWAS ( panel', '( a ) in Fig 3 , blue dash line ) .', 'The SLC6A14 eQTLS from the nasal epithelia ( blue dashed line ) and pancreas ( black solid line ) tissues appeared to coincide , respectively , with lung disease and meconium ileus associated variants .', 'eQTLs for AGTR2 in lung from GTEx also colocalize with the lung association signals in this region ( S7 Fig ) , however AGTR2 is not expressed in the nasal epithelia .', 'To formally evaluate the colocalization patterns observed in Figs 2 and 3 , we developed a frequentist regulatory integration analytic framework , referred to as the Simple Sum ( SS , Material and Methods and S1 Appendix ) , to directly test whether eQTLs for a given gene and in a given tissue colocalize with meconium ileus-associated GWAS variants , and can also test whether one tissue ( or expression for one gene ) shows greater colocalization over another .', 'Only summary statistics for the respective eQTL and association evidence are required , and the colocalization p-values are simple to calculate analytically .', 'We developed the SS because detecting colocalization in the presence of high LD and/or allelic heterogeneity is challenging for several commonly used colocalization methods [43–48] , the majority of which are Bayesian approaches , such as COLOC [43] and eCAVIAR [44] , that estimate the posterior probability of co-localization under specific causal variation assumptions .', 'COLOC and eCAVIAR can be used with summary statistics , but COLOC assumes a single causal variant in both eQTL and GWAS , and the presence of allelic heterogeneity reduces the co-localization posterior probability .', 'eCAVIAR can accommodate allelic heterogeneity , but the posterior probability of co-localization will be averaged across several variants in high LD .', 'In contrast , the goal of the SS approach is not to estimate which variant is causal , but rather to assess the evidence for a regulatory mechanism at the associated locus .', 'The performance of the SS approach , including in the presence of high LD and allelic heterogeneity , was confirmed by extensive simulation studies ( Material and Methods , S1 Appendix , and S5–S7 Tables ) , demonstrating correct type 1 error control across most settings ( S8–S13 Figs , and S8 and S9 Tables ) and good power performance ( S11–S16 Tables ) , as well as agreement with permutation-based results in applications ( Table 3 and S10 Table ) .', 'Results of method comparison with the alternative approaches COLOC [43] and eCAVIAR[44] are also provided ( Table 3 , and S8 , S11–S16 Tables ) .', 'Consistent with the patterns seen in Fig 2 , the eQTL evidence for SLC6A14 , SLC26A9 and ATP12A in the pancreas ( as measured by −log10 ( eQTL p ) are significantly correlated with the GWAS evidence ( as measured by meconium ileus-SNP Wald χ12 association statistic ) at each of these three loci , with SS p-values = 1 . 12x10-10 , 4 . 48x10-5 and 3 . 35x10-8 , respectively for the three loci ( Table 3 , analytical results , confirmed by the permutation-based results also provided in Table 3 ) .', 'Posterior probabilities of colocalization using the alternative COLOC [43] and eCAVIAR [44] methods provide supportive although less conclusive findings ( Table 3 ) , particularly for the ATP12A locus .', 'In addition , eCAVIAR , which calculates SNP-level colocalization statistics and can be challenged by substantial LD in regions of interest , did not provide a regional colocalization probability that exceeded 0 . 50 across any of the three regions or tissues; the eCAVIAR maximum occurred at rs3788766 at chromosome X for the pancreas .', 'At the chromosome X lung GWAS locus ( Fig 3 and S7 Fig ) , application of the SS colocalization analysis indicates that eQTLs from GTEx for AGTR2 in the lungs show evidence of colocalization with the lung-associated variants but not for SLC6A14 ( colocalization p-values = 1 and 2 . 82x10-7 for SLC6A14 and AGTR2 , respectively; S17 Table ) .', 'Given sub-optimal content of epithelia in the GTEx lung tissue samples by design , we calculated SLC6A14 eQTLs from the CF nasal epithelia which provided colocalization evidence with the lung disease associated variants ( p = 2 . 4x10-4 ) ; AGTR2 was not expressed in the nasal epithelia .', 'Individually , the lung and meconium ileus GWAS association findings could be interpreted as simply indicating distinct genes for the two CF phenotypes .', 'However , the integration of the tissue-specific eQTL patterns for SLC6A14 ( Fig 3 ) support a role for SLC6A14 in both CF lung and pancreas .', 'Further , as regulatory elements can exert effects over long distances , the differential positioning of the GWAS peaks with the co-alignment of eQTL peaks could accordingly reflect alternate modes of cis-regulation of SLC6A14 .', 'Significant colocalization can occur in more than one tissue ( Table 3 , S10 Table and S14 Fig ) or for expression of more than one gene .', 'Therefore , it is also of interest to directly contrast the colocalization evidence between tissues or genes ( Material and Methods , Table 3 , S10 and S17 Tables ) , suggesting for example , the most probable tissue ( s ) of origin or target gene for an association locus identified via GWAS .', 'Contrast colocalization analysis for the meconium ileus GWAS loci showed that eQTLs for SLC6A14 , SLC26A9 and ATP12A in the pancreas colocalize with meconium ileus-associated variants significantly more than eQTLs for these genes in the other meconium ileus-relevant tissues tested including the intestine ( Table 3 , SSC colocalization p-value results ) .', 'The maximum posterior probability of colocalization from COLOC and eCAVIAR also occurred with the pancreas eQTLs versus the other tissues evaluated ( Table 3 ) .', 'These findings are consistent with the pancreas-specific PRSS1 locus in that variation in SLC6A14 , SLC26A9 and ATP12A gene expression in the pancreas appears to mediate the association between meconium ileus and the GWAS variants .', 'We previously used the SLC26A9 variant rs7512462 ( S3 Table ) as an instrumental variable in a Mendelian Randomization ( MR ) study to assess whether damage to the exocrine pancreas is a causal contributor to CF-related diabetes [15] .', 'To determine whether variation in exocrine pancreatic injury in utero ( measured by immunoreactive trypsinogen ( IRT ) at birth ) is a causal contributor to meconium ileus , we now likewise applied a two-sample MR procedure [50 , 51] using the same instrumental variable ( Material and Methods ) .', 'The two-sample method first assessed the effect of rs7512462 on meconium ileus in the Canadian Gene Modifier Study ( CGMS ) Phase I sample ( n = 1 , 661 , OR = 1 . 39 , 95% CI = [1 . 14 , 1 . 70] ) .', 'Then a sub-sample of n = 126 was used , for whom IRT measurements were available , to estimate the effect of rs7512462 on log ( IRT ) levels estimated at birth ( mean change in log ( IRT ) per additional rs7512462 risk allele ( T ) = -0 . 60 , 95% CI = [-1 . 06 , -0 . 14] ) .', 'These lead to the estimate of the causal effect of pancreatic damage on meconium ileus risk ( OR = e[log ( 1 . 39 ) /-0 . 60] = 0 . 58 , 95% CI = [0 . 34 , 0 . 99] ) .', 'This significant causal estimate is similar to that obtained from a logistic regression of estimated log ( IRT ) on meconium ileus status ( OR = 0 . 54 , 95% CI = [0 . 40 , 0 . 70] ) , suggesting this direct association is largely free of confounding .', 'Together these data contribute to evidence that variation in gene expression in the exocrine pancreas may be a causal contributor to meconium ileus , although the MR assumption of no horizontal pleiotropy [16] is difficult to confirm for single-instrument analyses .', 'In support of this assumption , we note that expression of SLC26A9 is reported in the pancreas ( median TPM 1 . 60 ) , with essentially none in the relevant terminal ileum tissue ( i . e . small intestine; median TPM 0 . 02 ) in human ( GTEx , [23] ) .', 'Similarly , Slc26a9 was noted in the pancreas but was undetectable in the distal segments of the small intestine by RT-PCR analysis of murine tissues at adult stages ( see below and [52] ) .'], ['Together with the identification of PRSS1 as a meconium ileus modifier , the integration of GTEx eQTL evidence and meconium ileus susceptibility associations through colocalization analysis support that variation at the identified modifier loci may reflect SLC6A14 , SLC26A9 and ATP12A gene expression , and in the CF pancreas rather than in the intestine or any other meconium ileus-relevant tissues tested .', 'The underlying mechanism of this complex intestinal obstruction phenotype that requires loss of CFTR in the intestine [53] must then include the critical roles played by the transporters in early development and/or the early tissue damage that arises with loss of CFTR in the pancreas .', 'SLC6A14 encodes a neutral and cationic amino acid transporter [54] that based on GTEx information is most notably expressed in human lung .', 'Various cancers of epithelial origin , including breast [55] , colon [56] and pancreas [57] also exhibit robust expression that is relatively elevated from their source tissues .', 'The SLC6A14 rs3788766 was the most significant SNP in this ( Table 2 , panel', '( a ) in Fig 2 ) and our previous meconium ileus GWAS [12] ( S3 Table ) , and its risk allele T corresponds to increased transcript level based on the GTEx eQTL data of adult pancreas tissue ( panel', '( a ) in Fig 2 ) .', 'Although human data from early stages of development would be preferable , we did examine pancreatic and lung tissue from the mouse spanning late embryonic to adult stages of development ( panel', '( b ) and', '( c ) in Fig 3 ) to find maintained , albeit relatively low , Slc6a14 transcript levels in the pancreas , with decreasing levels in lung over time .', 'The rs3788766 SNP is in perfect LD ( r2 = 0 . 8 and D’ = 1 ) with rs12710568 , which is just 714bp 5’ of the SLC6A14 TSS .', "ChIP-seq evidence [58] demonstrates the binding of primed AR ( androgen receptor ) , which has been shown in seven independent studies ( GSE58428 , GSE84432 , GSE56288 , GSE28950 , GSE56086 , GSE65478 , GSE32892 ) and this SNP falls within the androgen receptor binding motif ( 5'-GGA/TACA[N]NNTGTTCT-3'—SNP position marked in brackets [59] ) and could potentially contribute to the sex-specific risk differences observed ( S4 Table , S6 Fig ) .", 'In contrast to the chromosome 13 and X loci , there are additional genes and tissues that show evidence of colocalization with the GWAS variants ( S14 Fig ) within 1Mbp of the chromosome 1 locus .', 'As a consequence it is more challenging to pinpoint the responsible gene and tissue from the colocalization evidence alone .', 'However , published experimental evidence for SLC26A9 complements the colocalization results .', 'SLC26A9 has been characterized as an anion channel in epithelial cells [60 , 61] .', 'Slc26a9-/- /Cftr-/- double knock out models have a lower rate of survival post-birth than either Cftr-/- or Slc26a9-/- mice [52] .', 'SLC26A9 is expressed in several tissues including the lung , stomach , upper intestinal tract , kidney , and pancreas [25 , 52 , 62 , 63] .', 'Several studies indicate that CFTR and SLC26A9 interact in lung tissue , with biochemical and electrophysiological studies indicating that interaction enhances functional expression of CFTR [9–11] .', 'Interestingly , this genome-wide study , as well as previous studies of early-onset pancreatic phenotypes that identified SLC26A9 as a CF modifier [12–14] , highlight that SLC26A9 and CFTR may not require interaction in the pancreas in the same way apparent for the lung phenotype .', 'The meconium ileus association is seen in patient cohorts composed of many different CFTR genotypes , and the effect size is similar in individuals who are Phe508del/Phe508del ( OR = 1 . 41 , se = 0 . 058 ) versus other CFTR genotypes ( OR = 1 . 32 , se = 0 . 083 ) .', 'Indeed , notable differences in murine mRNA levels between late gestation and adult stages for Slc26a9 in the pancreas and the lung , in opposing directions , suggests the function of SLC26A9 may be different across time and tissue ( panel', '( c ) in Fig 4 of [2] ) .', 'Detailed analysis of murine Slc26a9 expression in the intestine by others have also indicated changes in expression with young versus older ages [52] .', 'Taken together with previous reports of SLC26A9-associated pancreatic phenotypes [13–15] we purport that SLC26A9 may provide alternative chloride transport at early developmental stages , and that its contribution to meconium ileus , is at least partly , from the pancreas .', 'ATP12A encodes the α-subunit of the non-gastric H+/K+ transporter .', 'In a recent study , its expression in human and pig versus the negligible expression in the murine airway was argued to explain differences in airway surface liquid pH , viscosity and host defense in human and pig versus murine CF airways [34] .', 'Although we were not able to detect Atp12a mRNA in total mouse pancreas using RT-PCR , ATP12A expression has been seen in human pancreatic ductal lines [64] and in isolated rat pancreatic ducts with localization toward the lumen [65] .', 'The essential contributions of H+/HCO3- movements and pH maintenance for fluid secretion in the pancreatic duct has been studied extensively , and any disturbance with decrease in pH would increase concern for effective digestive enzyme transport and risk of auto-activation .', 'With the eQTL findings pointing to the pancreas , the common allele variants in ATP12A that associated with meconium ileus risk are also associated with increased ATP12A expression , where even modest increases in proton secretion , may be critical in the absence of CFTR [66] .', 'Meconium ileus has complex etiology .', 'The pig and murine models have highlighted the engagement of the gastrointestinal tract , where confined restoration of CFTR is sufficient to ameliorate both meconium ileus [53] and the intestinal obstruction phenotypes to substantial degrees [67 , 68] .', 'Although the genes identified in this GWAS are candidates for contributions to CF given their roles in epithelial transport and function , a working model in which their contribution to intestinal obstruction at birth is through common variation of their expression in the CF pancreas was not immediately obvious , beyond PRSS1 .', 'Understanding the organ of origin for any modifier gene is highly relevant as we embark on personalized medicine , first , toward designing the most optimal functional studies to test therapies and second , toward development of the complex models and signatures of prediction for both prognosis and therapeutic responses .', 'Access to tissue-specific expression data for colocalization analysis here , beyond the nasal epithelia data , was limited to that available in the GTEx consortium project , and therefore our conclusions must be viewed in that context .', 'Consideration of expression of modifier genes in early pancreatic stages rather than just adult tissue would be helpful for confirmation .', 'There is substantial variability in response to the recently developed CFTR-modulatory therapies between [69 , 70] and within [71] individuals , and modifier studies highlight that individual level genetic background beyond CFTR genotype contributes to these differences .', 'For example , we have shown that variation in lung response to modulator therapy can be explained in part by the SLC26A9 modifier gene , possibly via interaction with CFTR [2] .', 'Consideration of temporal and spatial expression of CFTR and modifier genes is needed when assessing CFTR function and therapeutic response for the purpose of choosing therapeutic strategies .', 'This is specifically relevant where current testing paradigms use primary CF-tissue models such as nasal and lung epithelia , or rectal and lung organoid models [72–74] .', 'Maximum benefit will be achieved with consideration of genetic background , as these models are being considered to decipher how to implement therapeutic decision-making and achieve personalized medicine [73] .', 'Predictive genetic signatures that include organ specific aspects hold great promise to direct and complement therapeutic decision making in CF and other inherited diseases .', 'They have additional potential in assisting clinical monitoring regimes , and toward stratification of patients for clinical trials to optimize outcome .'], ['Studies were approved by institutional review boards at participating sites and include Johns Hopkins School of Medicine eIRB2 ( Committee: IRB-3 ) ; Research Ethics Board of the Hospital for Sick Children; Biomedical Institutional Review Board , Office of Human Research , University of North Carolina at Chapel Hill; University Hospitals Case Medical Center , Institutional Review Board for Human Investigation; and the French ethical committee ( CPP n°2004/15 ) and the information collection was approved by CNIL ( n°04 . 404 ) .', 'Written informed consent was obtained from adults , and for patients<18 years old consent was obtained from parents or guardians for participation in the study .', 'The research ethics approval number for this cystic fibrosis gene modifier study was obtained from the Hospital for Sick Children Research Ethics Board , number 1000016662 .', 'The International CF GMC consists of individuals from four consortium sites , Johns Hopkins University ( JHU ) , the University of North Carolina/Case Western Reserve study ( UNC/Case ) , the Canadian Gene Modifier Study ( CGMS ) and the French Gene Modifier Study ( FGMS ) .', 'The North American ( NA ) data were genotyped in two phases , Phase I-NA with 5 , 132 individuals and Phase II-NA with 2 , 830 individuals ( before quality control; see below ) .', 'Data from the FGMS were genotyped separately and include 1 , 300 individuals with CF .', 'The Phase I-NA data were the basis of 
['Introduction', 'Results', 'Discussion', 'Material and methods']
['Cystic Fibrosis ( CF ) exhibits morbidity in several organs , including progressive lung disease in all patients and intestinal obstruction at birth ( meconium ileus ) in ~15% .', 'Individuals with the same causal CFTR mutations show variable disease presentation which is partly attributed to modifier genes .', 'With >6 , 500 participants from the International CF Gene Modifier Consortium , genome-wide association investigation identified a new modifier locus for meconium ileus encompassing ATP12A on chromosome 13 ( min p = 3 . 83x10-10 ) ; replicated loci encompassing SLC6A14 on chromosome X and SLC26A9 on chromosome 1 , ( min p<2 . 2x10-16 , 2 . 81x10−11 , respectively ) ; and replicated a suggestive locus on chromosome 7 near PRSS1 ( min p = 2 . 55x10-7 ) .', 'PRSS1 is exclusively expressed in the exocrine pancreas and was previously associated with non-CF pancreatitis with functional characterization demonstrating impact on PRSS1 gene expression .', 'We thus asked whether the other meconium ileus modifier loci impact gene expression and in which organ .', 'We developed and applied a colocalization framework called the Simple Sum ( SS ) that integrates regulatory and genetic association information , and also contrasts colocalization evidence across tissues or genes .', 'The associated modifier loci colocalized with expression quantitative trait loci ( eQTLs ) for ATP12A ( p = 3 . 35x10-8 ) , SLC6A14 ( p = 1 . 12x10-10 ) and SLC26A9 ( p = 4 . 48x10-5 ) in the pancreas , even though meconium ileus manifests in the intestine .', 'The meconium ileus susceptibility locus on chromosome X appeared shifted in location from a previously identified locus for CF lung disease severity .', 'Using the SS we integrated the lung disease association locus with eQTLs from nasal epithelia of 63 CF participants and demonstrated evidence of colocalization with airway-specific regulation of SLC6A14 ( p = 2 . 3x10-4 ) .', 'Cystic Fibrosis is realizing the promise of personalized medicine , and identification of the contributing organ and understanding of tissue specificity for a gene modifier is essential for the next phase of personalizing therapeutic strategies .']
['Cystic Fibrosis ( CF ) impacts the normal functioning of several organs including the pancreas , intestines and lungs .', 'CF is caused by mutations in the CF transmembrane conductance regulator , but individuals with the same mutations have different disease severity .', 'For example , only ~15% of individuals with CF are born with intestinal obstruction at birth ( meconium ileus ) while all have progressive lung disease with varying severity .', 'Modifier genes contribute to variation across individuals with CF .', 'In this study we identified modifiers of meconium ileus susceptibility , encompassing the previously identified SLC26A9 and SLC6A14 , and two new loci ATP12A and PRSS1 .', 'To understand the mechanism by which these modifier genes impact CF severity we developed a new statistical method that assesses colocalization with regulatory information .', 'Using this methodology we show , surprisingly , that all the meconium ileus association signals colocalize with gene expression in the pancreas rather than in intestinal tissues .', 'A distinct and neighbouring locus near SLC6A14 also contributes to CF lung disease .', 'Aided by transcriptomics of nasal epithelia from CF patients we found that each locus impacts variation in gene expression of SLC6A14 with tissue specificity .', 'Understanding the contributing tissue and responsible gene are necessary to prioritize modifiers as alternative therapeutic targets .']
['genome-wide association studies', 'medicine and health sciences', 'sociology', 'genetic diseases', 'fibrosis', 'social sciences', 'pulmonology', 'simulation and modeling', 'developmental biology', 'cystic fibrosis', 'genome analysis', 'research and analysis methods', 'autosomal recessive diseases', 'gene expression', 'exocrine glands', 'consortia', 'research assessment', 'genetic loci', 'clinical genetics', 'anatomy', 'pancreas', 'genetics', 'endocrine system', 'biology and life sciences', 'genomics', 'research errors', 'computational biology', 'human genetics']