3.1 Barrett's Esophagus
By extracting the outcome data of GWAS ID ebi-a-GCST90000515 in 1,531 exposed SNPs, 896 related outcome SNPs were obtained. By merging these with the previously obtained related SNPs, a total of 898 related SNPs were obtained. After excluding 26 palindromic SNPs, 118 bacterial species were retained. No SNPs with a P < 5×10-8 were found. No reverse causality SNPs were detected through the Steiger filter. After removing confounding factors such as diabetes[21](rs12636310),smoking[22](rs10167839, rs4506202, rs61771805), waist circumference[23](rs11109097, rs12288512, rs2590913, rs3761728, rs3800154, rs8066522), and hip circumference[24](rs17708276, rs2016057, rs2074881, rs2715439, rs35866622, rs9428102), 856 related SNPs and 118 bacterial species remained. Two bacterial species with less than 3 SNPs were removed, resulting in 116 bacterial species and 852 related SNPs. The outlier values of SNPs with more than 3 bacterial species were tested by MR-PRESSO (P = 0.188), and no abnormal values were found. Pleiotropic testing was performed (Tab.2), and two bacterial species with Pleiotropic were found. After removal,114 bacterial species and 835 SNPs remained(Fig.3).heterogeneity testing (Tab.2) was performed and random effect model MR analysis was selected for bacterial species with P <0.05,otherwise fixed effect model MR analysis was used. Data visualization showed that the results were robust with leave-one-out sensitivity analysis, scatter plots, forest plots, and funnel plots. Nine bacterial species with P <0.05 in the IVW method were identified, and a visual forest plot summary was created (Fig.1). The results showed that three bacterial species (Eubacterium eligens group, Actinomyces, Clostridium sensu stricto 1) had a protective effect on BE, and six bacterial species (Oxalobacter, Allisonella, Ruminococcaceae UCG009, Haemophilus, Sutterella, Anaerostipes) were risk factors for BE. The MR Egger, Weighted mode, and Simple mode results of these nine bacterial species all showed that P >0.05 (Fig.2). The weighted median results showed that three bacterial species had statistical significance (Allisonella (P=0.034,OR =1.152, ORCI=1.011-1.312), Anaerostipes (P=0.018,OR=1.420,ORCI=1.063-1.897), Eubacterium eligens group (P =0.013, OR =0.654, ORCI = 0.469-0.914)), while the remaining bacterial species had no statistical significance (P >0.05) (Fig.2).
Tab.2 Pleiotropic and Heterogeneity Test Results of IVW Positive Bacterial Groups
|
exposure
|
egger_intercept
|
pval
|
Q
|
Q_pval
|
Haemophilus
|
0.036
|
0.244
|
12.292
|
0.139
|
Allisonella
|
-0.029
|
0.561
|
2.581
|
0.764
|
Oxalobacter
|
-0.025
|
0.584
|
2.857
|
0.582
|
Sutterella
|
0.062
|
0.406
|
4.343
|
0.740
|
Anaerostipes
|
0.006
|
0.844
|
7.107
|
0.418
|
Clostridiumsensustricto1
|
-0.001
|
0.971
|
4.572
|
0.470
|
RuminococcaceaeUCG009
|
-0.067
|
0.133
|
5.663
|
0.580
|
Eubacteriumeligensgroup
|
-0.046
|
0.366
|
3.857
|
0.426
|
Actinomyces
|
-0.001
|
0.989
|
1.232
|
0.745
|
3.2 Gastroesophageal reflux disease
The GWAS ID for ebi-a-GCST90000514 extracted 897 related outcome SNPs in the exposure data. After merging and removing 25 reverse SNPs, 118 types of bacteria remained. No SNPs with P <5×10-8 as the outcome were found, and no reverse causality SNPs were detected through Steiger filtering. After excluding confounding factors such as body weight[25](rs12151423, rs60081663, rs9349693, rs17708276, rs2590913, rs2715439, rs3734633, rs3800154, rs7832116), body mass index[26](rs8130320, rs12288512, rs11109097, rs11990279, rs12636310, rs12894272, rs2016057, rs2057922, rs2074881, rs6761463), body fat rate[27](rs1689282, rs113054641, rs2930225, rs3761728, rs9900242), smoking[28](rs10167839, rs4506202, rs61771805), cholesterol[29](rs16940167, rs2548459, rs281379, rs35866622, rs4738679), and diabetes[26](rs6494306, rs59414781), 840 related SNPs and 118 types of bacteria remained. Among the 118 types of bacteria, two bacteria with fewer than 3 SNPs were removed, resulting in 116 bacteria and 836 SNPs (Fig.6). The MR-PRESSO test for outliers (P =0.204) showed no abnormal values for SNPs. The polyposis test (Tab.3) did not find any bacteria with pleiotropy. The heterogeneity test (Tab.3) was performed on the 116 bacteria and the random effect model MR analysis was chosen for P < 0.05, otherwise, the fixed effect model MR analysis was used. Data visualization analysis was conducted to observe the sensitivity analysis diagram, scatter plot, forest plot, and funnel plot of the leave-one-out method, all showing robust results. The summary of the IVW method showed that there were a total of 8 bacteria with P <0.05. The visualized forest plot showed(Fig.4) that 5 bacteria (Roseburia, Christensenellaceae R.7 group (not significantly associated), Lachnospiraceae UCG004, Methanobrevibacter, Erysipelatoclostridium (not significantly associated)) had a protective effect on GERD, and 3 bacteria (Prevotella 9, Allisonella, Anaerostipes) were risk factors for GERD. The MR Egger, Simple mode, and Weighted mode results of these 8 bacteria showed that all Pvalues were greater than 0.05 (Fig.5), and the Weighted median showed that one bacteria had statistical significance (Prevotella 9 (P = 0.006, OR = 1.082, ORCI = 1.023-1.450)), while the remaining bacteria showed no statistical significance (P > 0.05) (Fig.5).
Tab.3 Pleiotropic and Heterogeneity Test Results of IVW Positive Bacterial Groups
|
exposure
|
egger_intercept
|
pval
|
Q
|
Q_pval
|
Prevotella9
|
-0.004
|
0.530
|
7.021
|
0.797
|
Erysipelatoclostridium
|
-0.016
|
0.069
|
8.515
|
0.289
|
Allisonella
|
0.042
|
0.091
|
14.499
|
0.013
|
Roseburia
|
-0.011
|
0.435
|
33.486
|
0.001
|
Methanobrevibacter
|
-0.002
|
0.879
|
8.896
|
0.113
|
Anaerostipes
|
-0.011
|
0.234
|
6.080
|
0.530
|
ChristensenellaceaeR.7group
|
-0.011
|
0.379
|
2.098
|
0.718
|
LachnospiraceaeUCG004
|
0.005
|
0.646
|
12.434
|
0.087
|
3.3 Esophageal cancer
Among the 1531 exposed SNPs, 1521 relevant outcome SNPs were extracted. These were merged and 47 reciprocal SNPs were removed, leaving 119 species of bacteria. No SNPs with a Pvalue less than 5×10-8 were found. Steiger filtering did not identify any SNPs with reverse causality. Exclusions were made for confounding factors such as body mass index[30, 31](rs6761463, rs2016057, rs182549, rs1530559, rs12894272, rs12288512, rs11990279, rs114244418, rs11109097), body weight[32](rs9349693, rs60081663, rs3734633, rs2074881, rs12151423), body fat percentage[33](rs9900242, rs72814525, rs2930225, rs2590913, rs2387977, rs113054641), hip circumference[34](rs9428102, rs3761728, rs2715439, rs17708276), waist circumference[30](rs59068084, rs8066522), waist-to-hip ratio[30](rs6875660), total body fat[30](rs7832116), trunk fat[30](rs8039785, rs68093214, rs4936098, rs3800154, rs1689282, rs12925026), smoking[35, 36](rs61771805, rs4506202), alcohol consumption[36](rs646327, rs35866622, rs2548459), diabetes[37, 38](rs9276029, rs6494306, rs12636310), hypertension[39](rs6489992, rs1901739, rs1304512, rs112767262, rs10167839), plasma lipid levels[40](rs4738679, rs281379, rs16940167, rs10842464) and other confounding factors. This left 1428 relevant SNPs and 119 species of bacteria. No bacterial species with less than 3 SNPs were found. The MR-PRESSO test for outliers (P = 0.11) was performed on the bacterial species with more than 3 SNPs and no abnormal values were found. Pleiotropic testing was conducted (Tab.4) and 7 species with Pleiotropic were found. After removal, 112 species remained along with 1336 SNPs (Fig.9).The 112 species of bacteria were tested for heterogeneity (Tab.4) and random effect model MR analysis was chosen for those with P <0.05, while fixed effect model MR analysis was used for the opposite. The data were visualized and the leave-one-out sensitivity analysis diagram, scatter plot, forest plot, and funnel plot were observed, all showing robust results. A total of 9 species with IVW method P <0.05 were summarized (Fig.7), and the results showed that 3 species (Coprococcus1, Bilophila, CandidatusSoleaferrea) had a protective effect on EC, while 6 species (Coprobacter, Catenibacterium, Eubacteriumcoprostanoligenesgroup, Marvinbryantia, Sutterella, RuminococcaceaeUCG010) were risk factors for EC. The MR Egger, Simple mode, and Weighted mode results of these 9 species showed P > 0.05 (Fig.8), and the Weighted median showed that 1 species had statistical significance (CandidatusSoleaferrea, P = 0.013, OR = 0.697, ORCI = 0.525-0.925). The remaining species had no statistical significance (P >0.05) (Fig.8).
Tab.4 Pleiotropic and Heterogeneity Test Results of IVW Positive Bacterial Groups
|
exposure
|
egger_intercept
|
pval
|
Q
|
Q_pval
|
Catenibacterium
|
-0.017
|
0.925
|
1.795
|
0.773
|
Sutterella
|
0.108
|
0.116
|
6.919
|
0.806
|
RuminococcaceaeUCG010
|
-0.015
|
0.814
|
3.531
|
0.832
|
Marvinbryantia
|
-0.015
|
0.813
|
3.315
|
0.950
|
Coprobacter
|
0.019
|
0.656
|
12.339
|
0.500
|
Eubacteriumcoprostanoligenesgroup
|
0.020
|
0.655
|
9.653
|
0.787
|
CandidatusSoleaferrea
|
0.028
|
0.504
|
12.090
|
0.279
|
Bilophila
|
-0.025
|
0.682
|
23.519
|
0.074
|
Coprococcus1
|
-0.045
|
0.234
|
15.101
|
0.236
|
3.4 Esophritis
The outcome data of GWAS ID ukb-b-19354 in 1531 exposed SNPs were extracted, yielding a total of 1304 relevant SNPs. These were merged and 37 reciprocal SNPs were removed, leaving 119 species of bacteria. No SNPs with a P <5×10-8 were found. Steiger filtering did not identify any SNPs with reverse causality. Smoking-related SNPs[41](rs12288512, rs12996055, rs10167839, rs61771805), alcohol consumption-related SNPs[42](rs1901739, rs281379, rs35866622, rs2548459, rs646327), allergy-related SNPs[43](rs13157098, rs1492620, rs11597285, rs71481756, rs11736617), and celiac disease-related SNPs[44](rs9393920) were excluded as confounding factors, leaving 1256 relevant SNPs and 119 species of bacteria. No bacterial species with less than 3 SNPs were found. The MR-PRESSO test for outliers (P = 0.214) was performed on the bacterial species with more than 3 SNPs and no abnormal values were found. Pleiotropic testing was conducted (Tab.5), and one species with Pleiotropic was removed, leaving 1242 SNPs (Fig.12).Conducted heterogeneity testing on 118 species of bacteria (Tab.5) and chose random effect model MR analysis for those with P <0.05, otherwise, fixed effect model MR analysis was used. The data were visually analyzed, and the leave-one-out sensitivity analysis diagram, scatter plot, forest plot, and funnel plot all showed robust results. A total of 7 species of bacteria with IVW method P <0.05 were summarized (Fig.10), and the results showed that 4 species (Parasutterella, Barnesiella (not significantly associated), Howardella, Bilophila (not significantly associated)) had a protective effect on OE, while 3 species (CandidatusSoleaferrea (not significantly associated), Adlercreutzia (not significantly associated), Escherichia.Shigella (not significantly associated)) were risk factors for OE. The MR Egger, Simple mode, and Weighted mode results of these 7 species showed P >0.05 (Fig.11), and the Weighted median showed that 3 species had statistical significance (Howardella (P = 0.042, OR = 0.999, ORCI = 0.9967-0.9999), CandidatusSoleaferrea (P = 0.042, OR = 1.002, ORCI = 1.00007-1.00440), Parasutterella (P = 0.010, OR = 0.997, ORCI = 0.9948-0.9993)), while the remaining species had no statistical significance (P > 0.05) (Fig.11).
Tab.5 Pleiotropic and Heterogeneity Test Results of IVW Positive Bacterial Groups
|
exposure
|
egger_intercept
|
pval
|
Q
|
Q_pval
|
Escherichia.Shigella
|
-0.001
|
0.921
|
9.791
|
0.549
|
Barnesiella
|
-0.001
|
0.460
|
13.885
|
0.382
|
CandidatusSoleaferrea
|
-0.001
|
0.864
|
11.853
|
0.295
|
Parasutterella
|
-0.001
|
0.678
|
8.495
|
0.862
|
Bilophila
|
0.001
|
0.320
|
16.969
|
0.201
|
Adlercreutzia
|
-0.001
|
0.650
|
15.567
|
0.158
|
Howardella
|
0.001
|
0.900
|
3.485
|
0.900
|