Leveraging pQTL-based Mendelian randomization to identify new treatment prospects for primary biliary cholangitis and primary sclerosing cholangitis

Results

Figure 1 demonstrates the whole design and procedure of this study.

Figure 1. Overall flow chart. Abbreviations: PBC: Primary biliary cholangitis; PSC: Primary sclerosing cholangitis; SMR: Summary-data-based Mendelian randomization.

MR analysis of druggable proteins

We selected 1046 proteins encoded by druggable genes from 4907 proteins in the deCODE database (Supplementary Table 3) and performed two-sample Mendelian randomization analysis with PBC and PSC. The p-values of the analysis results were adjusted using the stringent Bonferroni method (p-value < 0.05/1046). From the results in Table 1, it could be observed that there were 8 proteins with causal relationships with PBC. Among them, HSPB1, LEFTY2, FGF2, TNFAIP6, ITIH5, and PBC had a positive correlation, while EPHA1, ERAP1, and PBC had a negative correlation (all results were shown in Supplementary Table 4). From the results in Table 2, it could be observed that there are another 8 proteins with causal relationships with PSC. Among them, GSTO1, RNASET2, FCGR2A, FCGR2B, RNASE6, and CTSB had a negative correlation with PSC, while FCGR3A, FCGR3B, and PSC had a positive correlation (all results were shown in Supplementary Table 5).

Table 1. The result of Mendelian randomization between protein and primary biliary cholangitis.

Exposure	Gene	UniProt	Outcome	Method	Nsnp	b	se	p-val
11103_24_HSPB1_HSP_27.txt.gz	HSPB1	P04792	PBC	IVW	55	0.485995906	0.090246638	7.24E-08
15503_15_LEFTY2_Lefty_A.txt.gz	LEFTY2	O00292	PBC	IVW	16	1.03573495	0.222256859	3.16E-06
15503_20_LEFTY2_Lefty_A.txt.gz	LEFTY2	O00292	PBC	IVW	115	0.279742942	0.045272049	6.44E-10
3025_50_FGF2_bFGF.txt.gz	FGF2	P09038	PBC	IVW	81	0.310168146	0.074496891	3.13E-05
3431_54_EPHA1_EphA1.txt.gz	EPHA1	P21709	PBC	IVW	64	−0.216252104	0.047195482	4.60E-06
4964_67_ERAP1_ARTS1.txt.gz	ERAP1	Q9NZ08	PBC	IVW	138	−0.114925345	0.027587082	3.10E-05
5036_50_TNFAIP6_TSG_6.txt.gz	TNFAIP6	P98066	PBC	IVW	79	0.258985304	0.06156953	2.59E-05
8233_2_ITIH5_ITIH5.txt.gz	ITIH5	Q86UX2	PBC	IVW	94	0.312619443	0.069467875	6.79E-06
Abbreviation: PBC: primary biliary cholangitis.

Table 2. The result of Mendelian randomization between protein and primary sclerosing cholangitis.

Exposure	Gene	UniProt	Outcome	Method	Nsnp	b	se	p-val
12436_84_GSTO1_GST_omega_1.txt.gz	GSTO1	P78417	PSC	IVW	112	−0.155230396	0.028185457	3.64E-08
15388_24_FCGR3A_FcRIIIa.txt.gz	FCGR3A	P08637	PSC	IVW	176	0.108085588	0.019235973	1.92E-08
16913_8_RNASET2_RNT2.txt.gz	RNASET2	O00584	PSC	IVW	54	−0.225056815	0.050975082	1.01E-05
3309_2_FCGR2A_FCG2A.txt.gz	FCGR2A	P12318	PSC	IVW	158	−0.09643699	0.018820999	2.99E-07
3310_62_FCGR2B_FCG2B.txt.gz	FCGR2B	P31994	PSC	IVW	164	−0.091293154	0.019245418	2.10E-06
3311_27_FCGR3B_FCG3B.txt.gz	FCGR3B	O75015	PSC	IVW	45	0.271596135	0.064005135	2.20E-05
5646_20_RNASE6_RNAS6.txt.gz	RNASE6	Q93091	PSC	IVW	150	−0.116694162	0.025940446	6.84E-06
8007_19_CTSB_Cathepsin_B.txt.gz	CTSB	P07858	PSC	IVW	87	−0.167020198	0.032992719	4.14E-07
Abbreviation: PSC: primary sclerosing cholangitis.

Sensitivity analysis on the proteins and PBC

First, we conducted heterogeneity tests on HSPB1, LEFTY2, LEFTY2, FGF2, TNFAIP6, ITIH5, EPHA1, ERAP1, and PBC (Table 3). The q-values of the heterogeneity tests for the 8 proteins were all greater than 0.05, regardless of whether the MR Egger method or IVW method was used. This indicates that there was no significant heterogeneity among the instrumental variables we used, providing reliable results. The funnel plot in Figure 2 demonstrated an even distribution of instrumental variables for the 8 proteins without obvious heterogeneity, consistent with the calculation results.

Table 3. The result of heterogeneity test between protein and primary biliary cholangitis.

Exposure	Gene	UniProt	Outcome	Method	Q	Q_df	Q_p-val
11103_24_HSPB1_HSP_27.txt.gz	HSPB1	P04792	PBC	MR Egger	33.57942018	53	0.982844971
11103_24_HSPB1_HSP_27.txt.gz	HSPB1	P04792	PBC	IVW	34.28677871	54	0.983326124
15503_15_LEFTY2_Lefty_A.txt.gz	LEFTY2	O00292	PBC	MR Egger	15.34154428	14	0.355212901
15503_15_LEFTY2_Lefty_A.txt.gz	LEFTY2	O00292	PBC	IVW	15.63659144	15	0.406606267
15503_20_LEFTY2_Lefty_A.txt.gz	LEFTY2	O00292	PBC	MR Egger	127.1386199	113	0.171611091
15503_20_LEFTY2_Lefty_A.txt.gz	LEFTY2	O00292	PBC	IVW	127.1858557	114	0.187957209
3025_50_FGF2_bFGF.txt.gz	FGF2	P09038	PBC	MR Egger	67.26242789	79	0.824010298
3025_50_FGF2_bFGF.txt.gz	FGF2	P09038	PBC	IVW	69.3766221	80	0.795841665
3431_54_EPHA1_EphA1.txt.gz	EPHA1	P21709	PBC	MR Egger	36.30233909	62	0.996253655
3431_54_EPHA1_EphA1.txt.gz	EPHA1	P21709	PBC	IVW	38.80564268	63	0.992956538
4964_67_ERAP1_ARTS1.txt.gz	ERAP1	Q9NZ08	PBC	MR Egger	124.9118521	136	0.742409772
4964_67_ERAP1_ARTS1.txt.gz	ERAP1	Q9NZ08	PBC	IVW	124.9186857	137	0.761735543
5036_50_TNFAIP6_TSG_6.txt.gz	TNFAIP6	P98066	PBC	MR Egger	67.40211869	77	0.774509578
5036_50_TNFAIP6_TSG_6.txt.gz	TNFAIP6	P98066	PBC	IVW	69.59190645	78	0.740574298
8233_2_ITIH5_ITIH5.txt.gz	ITIH5	Q86UX2	PBC	MR Egger	101.1836469	92	0.24055945
8233_2_ITIH5_ITIH5.txt.gz	ITIH5	Q86UX2	PBC	IVW	104.0331413	93	0.203980833
Abbreviation: PBC: primary biliary cholangitis.

Figure 2. Funnel plots of Mendelian randomization (MR) analysis on PBC. (A) MR funnel plot for HSPB1 and PBC; (B) MR funnel plot for LEFTY2 with primer 15503_15 and PBC; (C) MR funnel plot for LEFTY2 with primer 15503_20 and PBC; (D) MR funnel plot for FGF2 and PBC; (E) MR funnel plot for EPHA1 and PBC; (F) MR funnel plot for ERAP1 and PBC; (G) MR funnel plot for TNFAIP6 and PBC; (H) MR funnel plot for ITIH5 and PBC. Abbreviations: MR: Mendelian randomization; PBC: primary biliary cholangitis.

Next, a pleiotropy test was performed on HSPB1, LEFTY2, LEFTY2, FGF2, TNFAIP6, ITIH5, EPHA1, ERAP1, and PBC (Table 4). The q-values of the pleiotropy tests for the 8 proteins were all greater than 0.05, suggesting that the selected SNPs do not act on PBC through other pathways.

Table 4. The result of pleiotropy test between protein and primary biliary cholangitis.

Exposure	Gene	UniProt	Outcome	Egger_intercept	se	p-val
11103_24_HSPB1_HSP_27.txt.gz	HSPB1	P04792	PBC	−0.022169912	0.026359926	0.404100774
15503_15_LEFTY2_Lefty_A.txt.gz	LEFTY2	O00292	PBC	0.036260473	0.069880865	0.611942028
15503_20_LEFTY2_Lefty_A.txt.gz	LEFTY2	O00292	PBC	0.003522901	0.017193505	0.83802105
3025_50_FGF2_bFGF.txt.gz	FGF2	P09038	PBC	0.035640536	0.024511607	0.149900303
3431_54_EPHA1_EphA1.txt.gz	EPHA1	P21709	PBC	−0.028371055	0.017931587	0.118696115
4964_67_ERAP1_ARTS1.txt.gz	ERAP1	Q9NZ08	PBC	−0.001297117	0.015691191	0.934239228
5036_50_TNFAIP6_TSG_6.txt.gz	TNFAIP6	P98066	PBC	0.032322808	0.021842788	0.14300839
8233_2_ITIH5_ITIH5.txt.gz	ITIH5	Q86UX2	PBC	−0.034397758	0.021370146	0.110907565
Abbreviation: PBC: primary biliary cholangitis.

Furthermore, leave-one-out tests were conducted on the SNPs of the 8 proteins. Supplementary Figure 1 showed that the beta values of all the results lay on one side of 0, indicating that individual SNPs had minimal impact on the results of Mendelian randomization, and the results remained stable. Additional analysis using the Steiger direction test revealed that, except for the direction between the protein encoded by ERAP1 and PBC being uncertain, the p-values of the Steiger direction tests for HSPB1, LEFTY2, LEFTY2, FGF2, TNFAIP6, ITIH5, EPHA1, and PBC were all much smaller than 0.05. This indicated that the direction was correct and that the protein products of these 7 genes potentially contributed to the occurrence of PBC (Table 5). To further verify the direction between the protein encoded by ERAP1 and PBC, a reverse Mendelian randomization analysis was conducted, resulting in a p-value of 0.18, indicating no reverse causal relationship existed (Supplementary Table 6).

Table 5. The result of Steiger direction test between protein and primary biliary cholangitis.

Exposure	Gene	UniProt	Outcome	Snp_r2. exposure	Snp_r2. outcome	Correct_causal_direction	Steiger_p-val
11103_24_HSPB1_HSP_27.txt.gz	HSPB1	P04792	PBC	0.179613954	0.000256323	TRUE	0
15503_15_LEFTY2_Lefty_A.txt.gz	LEFTY2	O00292	PBC	0.037895293	0.000135877	TRUE	4.08E-237
15503_20_LEFTY2_Lefty_A.txt.gz	LEFTY2	O00292	PBC	0.545890549	0.000617874	TRUE	0
3025_50_FGF2_bFGF.txt.gz	FGF2	P09038	PBC	0.30229971	0.000317004	TRUE	0
3431_54_EPHA1_EphA1.txt.gz	EPHA1	P21709	PBC	0.318174206	0.000212982	TRUE	0
4964_67_ERAP1_ARTS1.txt.gz	ERAP1	Q9NZ08	PBC	1.036182549	0.000535196	TRUE	NA
5036_50_TNFAIP6_TSG_6.txt.gz	TNFAIP6	P98066	PBC	0.290984971	0.000323357	TRUE	0
8233_2_ITIH5_ITIH5.txt.gz	ITIH5	Q86UX2	PBC	0.328213373	0.000496922	TRUE	0
Abbreviation: PBC: primary biliary cholangitis.

Finally, the scatter plot of the Mendelian randomization results for HSPB1, LEFTY2, LEFTY2, FGF2, TNFAIP6, ITIH5, EPHA1, ERAP1, and PBC was plotted. As shown in Figure 3, HSPB1, LEFTY2, FGF2, TNFAIP6, ITIH5, and PBC exhibited positive correlation, while EPHA1, ERAP1, and PBC showed negative correlation, consistent with the results of the Mendelian randomization.

Figure 3. Scatter plots of Mendelian randomization (MR) results on PBC. (A) Scatter plot of MR results for HSPB1 and PBC; (B) Scatter plot of MR results for LEFTY2 with primer 15503_15 and PBC; (C) Scatter plot of MR results for LEFTY2 with primer 15503_20 and PBC; (D) Scatter plot of MR results for FGF2 and PBC; (E) Scatter plot of MR results for EPHA1 and PBC; (F) Scatter plot of MR results for ERAP1 and PBC; (G) Scatter plot of MR results for TNFAIP6 and PBC; (H) Scatter plot of MR results for ITIH5 and PBC. Abbreviations: MR: Mendelian randomization; PBC: primary biliary cholangitis.

Sensitivity analysis on the proteins and PSC

Firstly, heterogeneity tests were conducted on GSTO1, FCGR3A, RNASET2, FCGR2A, FCGR2B, FCGR3B, RNASE6, CTSB, and PSC (Table 6). The results from both the MR Egger method and the IVW method indicated that all genes, except FCGR2B, demonstrated q-values greater than 0.05. This implies that there was no significant heterogeneity among the instrumental variables of GSTO1, FCGR3A, RNASET2, FCGR2A, FCGR3B, RNASE6, and CTSB, ensuring the reliability of the outcomes. The uniform distribution of instrumental variables without significant heterogeneity can be observed in the funnel plot displayed in Figure 4. Therefore, for the Mendelian randomization analysis between FCGR2B and PSC, we rely on the random-effects model results obtained using the Inverse Variance Weighted method, which revealed a p-value of 2.10e-06, suggesting a causal relationship between the two.

Table 6. The result of heterogeneity test between protein and primary sclerosing cholangitis.

Exposure	Gene	UniProt	Outcome	Method	Q	Q_df	Q_p-val
12436_84_GSTO1_GST_omega_1.txt.gz	GSTO1	P78417	PSC	MR Egger	130.5352222	110	0.088355553
12436_84_GSTO1_GST_omega_1.txt.gz	GSTO1	P78417	PSC	IVW	131.409545	111	0.090455909
15388_24_FCGR3A_FcRIIIa.txt.gz	FCGR3A	P08637	PSC	MR Egger	164.2225705	174	0.690884965
15388_24_FCGR3A_FcRIIIa.txt.gz	FCGR3A	P08637	PSC	IVW	165.1510566	175	0.691760694
16913_8_RNASET2_RNT2.txt.gz	RNASET2	O00584	PSC	MR Egger	51.66881974	52	0.486868401
16913_8_RNASET2_RNT2.txt.gz	RNASET2	O00584	PSC	IVW	53.89309321	53	0.439994777
3309_2_FCGR2A_FCG2A.txt.gz	FCGR2A	P12318	PSC	MR Egger	161.4126697	156	0.366700706
3309_2_FCGR2A_FCG2A.txt.gz	FCGR2A	P12318	PSC	IVW	161.6123403	157	0.383872503
3310_62_FCGR2B_FCG2B.txt.gz	FCGR2B	P31994	PSC	MR Egger	195.8890975	162	0.035781113
3310_62_FCGR2B_FCG2B.txt.gz	FCGR2B	P31994	PSC	IVW	195.8895765	163	0.040224913
3311_27_FCGR3B_FCG3B.txt.gz	FCGR3B	O75015	PSC	MR Egger	55.14172947	43	0.101425523
3311_27_FCGR3B_FCG3B.txt.gz	FCGR3B	O75015	PSC	IVW	57.33132758	44	0.085637624
5646_20_RNASE6_RNAS6.txt.gz	RNASE6	Q93091	PSC	MR Egger	148.4066695	148	0.475134707
5646_20_RNASE6_RNAS6.txt.gz	RNASE6	Q93091	PSC	IVW	150.3312716	149	0.454027288
8007_19_CTSB_Cathepsin_B.txt.gz	CTSB	P07858	PSC	MR Egger	82.79503252	85	0.547491994
8007_19_CTSB_Cathepsin_B.txt.gz	CTSB	P07858	PSC	IVW	89.71617707	86	0.370679142
Abbreviation: PSC: primary sclerosing cholangitis.

Figure 4. Funnel plots of Mendelian randomization (MR) analysis on PSC. (A) MR funnel plot for GSTO1 and PSC; (B) MR funnel plot for FCGR3A and PSC; (C) MR funnel plot for RNASET2 and PSC; (D) MR funnel plot for FCGR2A and PSC; (E) MR funnel plot for FCGR2B and PSC; (F) MR funnel plot for FCGR3B and PSC; (G) MR funnel plot for RNASE6 and PSC; (H) MR funnel plot for CTSB and PSC. Abbreviations: MR: Mendelian randomization; PSC: primary sclerosing cholangitis.

Next, a pleiotropy test was conducted on GSTO1, FCGR3A, RNASET2, FCGR2A, FCGR2B, FCGR3B, RNASE6, CTSB, and PSC (Table 7). It was noticeable that all genes, except CTSB, exhibited q-values greater than 0.05 in the pleiotropy tests, indicating that the selected SNPs do not exert their effects on PSC through other pathways. However, CTSB’s SNP demonstrates horizontal pleiotropy, rendering the results unreliable, and consequently it is excluded from subsequent analyses.

Table 7. The result of pleiotropy test between protein and primary sclerosing cholangitis.

Exposure	Gene	UniProt	Outcome	Egger_intercept	se	p-val
12436_84_GSTO1_GST_omega_1.txt.gz	GSTO1	P78417	PSC	−0.010567731	0.012311568	0.392561392
15388_24_FCGR3A_FcRIIIa.txt.gz	FCGR3A	P08637	PSC	−0.008928035	0.009265486	0.336593845
16913_8_RNASET2_RNT2.txt.gz	RNASET2	O00584	PSC	0.019043534	0.012768899	0.141898527
3309_2_FCGR2A_FCG2A.txt.gz	FCGR2A	P12318	PSC	−0.004008726	0.009125482	0.661059868
3310_62_FCGR2B_FCG2B.txt.gz	FCGR2B	P31994	PSC	−0.000175424	0.008813695	0.98414481
3311_27_FCGR3B_FCG3B.txt.gz	FCGR3B	O75015	PSC	0.023774026	0.018193929	0.198259968
5646_20_RNASE6_RNAS6.txt.gz	RNASE6	Q93091	PSC	0.010788007	0.007786936	0.168015118
8007_19_CTSB_Cathepsin_B.txt.gz	CTSB	P07858	PSC	−0.028469396	0.010821546	0.01011224
Abbreviation: PSC: primary sclerosing cholangitis.

Furthermore, leave-one-out tests were performed on the SNPs of the 8 proteins, as depicted in Supplementary Figure 2. The outcomes revealed that the beta values for individual SNPs in the MR analysis lay on one side of zero, suggesting that individual SNPs had negligible impact on the analysis results and ensuring their stability. Steiger direction tests were additionally conducted, indicating that the p-values for the Steiger direction tests between these 8 proteins and PSC are significantly less than 0.05, implying the correct directionality, namely, the protein products of these 8 genes influence the occurrence of PSC (Table 8).

Table 8. The result of Steiger direction test between protein and primary sclerosing cholangitis.

Exposure	Gene	UniProt	Outcome	Snp_r2. exposure	Snp_r2. outcome	Correct_causal_direction	Steiger_p-val
12436_84_GSTO1_GST_omega_1.txt.gz	GSTO1	P78417	PSC	0.592299575	0.000537659	TRUE	0
15388_24_FCGR3A_FcRIIIa.txt.gz	FCGR3A	P08637	PSC	0.934075888	0.000699525	TRUE	0
16913_8_RNASET2_RNT2.txt.gz	RNASET2	O00584	PSC	0.178739732	0.000236886	TRUE	0
3309_2_FCGR2A_FCG2A.txt.gz	FCGR2A	P12318	PSC	0.855566158	0.000697265	TRUE	0
3310_62_FCGR2B_FCG2B.txt.gz	FCGR2B	P31994	PSC	0.77594143	0.000765493	TRUE	0
3311_27_FCGR3B_FCG3B.txt.gz	FCGR3B	O75015	PSC	0.122381344	0.000278588	TRUE	0
5646_20_RNASE6_RNAS6.txt.gz	RNASE6	Q93091	PSC	0.505588478	0.000564529	TRUE	0
8007_19_CTSB_Cathepsin_B.txt.gz	CTSB	P07858	PSC	0.500938131	0.000420383	TRUE	0
Abbreviation: PSC: primary sclerosing cholangitis.

Lastly, scatter plots illustrating the MR results for GSTO1, FCGR3A, RNASET2, FCGR2A, FCGR2B, FCGR3B, RNASE6, CTSB, and PSC were generated in Figure 5. From the figure, a positive correlation among FCGR3A, FCGR3B, and PSC could be observed, while GSTO1, RNASET2, FCGR2A, FCGR2B, RNASE6, CTSB, and PSC exhibited negative correlations, consistent with the MR results.

Figure 5. Scatter plots of Mendelian randomization (MR) results on PSC. (A) Scatter plot of MR results for GSTO1 and PSC; (B) Scatter plot of MR results for FCGR3A and PSC; (C) Scatter plot of MR results for RNASET2 and PSC; (D) Scatter plot of MR results for FCGR2A and PSC; (E) Scatter plot of MR results for FCGR2B and PSC; (F) Scatter plot of MR results for FCGR3B and PSC; (G) Scatter plot of MR results for RNASE6 and PSC; (H) Scatter plot of MR results for CTSB and PSC. Abbreviations: MR: Mendelian randomization; PSC: primary sclerosing cholangitis.

SMR and colocalization analysis

The presence of pleiotropy was further confirmed through the HEIDI test in the SMR analysis. The results showed that except for the protein encoded by LEFTY2 with primer 15503_15 and the protein encoded by FCGR2A, all other HEIDI test p-values were greater than 0.05 (Table 9). Therefore, the SNPs of the protein encoded by LEFTY2 with primer 15503_15 and the protein encoded by FCGR2A demonstrated pleiotropy.

Table 9. The result of summary-data-based Mendelian randomization.

Exposure	Gene	Topsnp	Outcome	p_HEIDI	nsnp_HEIDI
15503_15_LEFTY2_Lefty_A.txt.gz	LEFTY2	rs360058	PBC	0.01569885	20
15503_20_LEFTY2_Lefty_A.txt.gz	LEFTY2	rs10915893	PBC	0.0537142	20
5036_50_TNFAIP6_TSG_6.txt.gz	TNFAIP6	rs2278089	PBC	0.890924	20
3025_50_FGF2_bFGF.txt.gz	FGF2	rs308403	PBC	0.9544156	20
4964_67_ERAP1_ARTS1.txt.gz	ERAP1	rs30185	PBC	0.9706626	20
11103_24_HSPB1_HSP_27.txt.gz	HSPB1	rs2868371	PBC	0.8863009	20
3431_54_EPHA1_EphA1.txt.gz	EPHA1	rs4725617	PBC	0.1184055	20
8233_2_ITIH5_ITIH5.txt.gz	ITIH5	rs10795561	PBC	0.1364776	20
3309_2_FCGR2A_FCG2A.txt.gz	FCGR2A	rs1801274	PSC	0.01835392	20
15388_24_FCGR3A_FcRIIIa.txt.gz	FCGR3A	rs10919543	PSC	0.2464716	20
3311_27_FCGR3B_FCG3B.txt.gz	FCGR3B	rs2099684	PSC	0.1935976	20
3310_62_FCGR2B_FCG2B.txt.gz	FCGR2B	rs6665610	PSC	0.1977813	20
16913_8_RNASET2_RNT2.txt.gz	RNASET2	rs35614625	PSC	0.2032446	20
8007_19_CTSB_Cathepsin_B.txt.gz	CTSB	rs2645425	PSC	0.8469198	20
12436_84_GSTO1_GST_omega_1.txt.gz	GSTO1	rs45596840	PSC	0.6104043	20
5646_20_RNASE6_RNAS6.txt.gz	RNASE6	rs986193	PSC	0.2111333	20
Abbreviations: PSC: primary sclerosing cholangitis; PBC: primary biliary cholangitis.

The coloc analysis results (Table 10) indicated a strong colocalization between the protein encoded by LEFTY2 with primer 15503_20 and PBC, and between FCGR3B and PSC (PP.H4 > 0.8). There was a moderate colocalization between HSPB1 and PBC, and between FCGR2A and PSC (0.5 < PP.H4 < 0.8).

Table 10. The result of colocalization.

Exposure	Gene	UniProt	Outcome	Nsnps	PP.H0.abf	PP.H1.abf	PP.H2.abf	PP.H3.abf	PP.H4.abf
11103_24_HSPB1_HSP_27.txt.gz	HSPB1	P04792	PBC	7604	1.02E-293	0.244948657	6.43E-294	0.154662126	0.600389217
15503_15_LEFTY2_Lefty_A.txt.gz	LEFTY2	O00292	PBC	7702	9.72E-96	0.359608508	1.37E-95	0.505349337	0.135042155
15503_20_LEFTY2_Lefty_A.txt.gz	LEFTY2	O00292	PBC	7702	9.30E-303	0.044440725	1.31E-302	0.06189221	0.893667065
3025_50_FGF2_bFGF.txt.gz	FGF2	P09038	PBC	8401	3.86E-302	0.612355464	2.12E-302	0.336159162	0.051485374
3431_54_EPHA1_EphA1.txt.gz	EPHA1	P21709	PBC	5680	2.38E-295	0.617372726	1.03E-295	0.266127574	0.1164997
4964_67_ERAP1_ARTS1.txt.gz	ERAP1	Q9NZ08	PBC	9300	4.37E-306	0.589286656	2.39E-306	0.321659709	0.089053635
5036_50_TNFAIP6_TSG_6.txt.gz	TNFAIP6	P98066	PBC	8110	1.25E-303	0.630683591	6.43E-304	0.325367544	0.043948865
8233_2_ITIH5_ITIH5.txt.gz	ITIH5	Q86UX2	PBC	11520	4.55E-294	0.473506481	3.39E-294	0.352917859	0.17357566
12436_84_GSTO1_GST_omega_1.txt.gz	GSTO1	P78417	PSC	7830	6.39E-305	0.580905661	3.24E-305	0.294613073	0.124481266
15388_24_FCGR3A_FcRIIIa.txt.gz	FCGR3A	P08637	PSC	8778	7.19E-306	0.270997462	6.89E-306	0.259484414	0.469518124
16913_8_RNASET2_RNT2.txt.gz	RNASET2	O00584	PSC	10388	3.76E-216	0.415100579	1.93E-216	0.213417558	0.371481863
3309_2_FCGR2A_FCG2A.txt.gz	FCGR2A	P12318	PSC	8928	1.48E-306	0.240712985	1.41E-306	0.230091232	0.529195782
3310_62_FCGR2B_FCG2B.txt.gz	FCGR2B	P31994	PSC	8634	2.06E-306	0.296974467	1.96E-306	0.282948095	0.420077438
3311_27_FCGR3B_FCG3B.txt.gz	FCGR3B	O75015	PSC	8619	4.07E-304	0.051060059	3.91E-304	0.04855231	0.900387631
5646_20_RNASE6_RNAS6.txt.gz	RNASE6	Q93091	PSC	10149	2.00E-305	0.518012159	1.09E-305	0.282150919	0.199836922
8007_19_CTSB_Cathepsin_B.txt.gz	CTSB	P07858	PSC	9602	5.30E-302	0.586984533	2.95E-302	0.326537269	0.086478198
Abbreviations: PSC: primary sclerosing cholangitis; PBC: primary biliary cholangitis.

Validation of external datasets

For further verification of the results, we validated using pQTL data from “Phenome-wide Mendelian randomization: mapping the influence of the plasma proteome on complex diseases”. The protein dataset in this data differs from the deCODE dataset. We used this new protein data to validate five genes (ITIH5, HSPB1, FGF2, TNFAIP6, ERAP1) encoding proteins in PBC, where HSPB1 had a p-value of < 0.05. Furthermore, in PSC, we validated five genes (FCGR3B, FCGR2B, GSTO1, CTSB, RNASE6) encoding proteins, where FCGR3B, FCGR2B, and RNASE6 had a p-value of < 0.05 (Table 11). Nearly half of the genes encoding proteins were able to reproduce positive results. However, due to the smaller number of SNPs and sample size in the validation dataset compared to the deCODE dataset, we relied on the data from the deCODE database.

Table 11. The result of Mendelian randomization between protein and primary cholangitis (validation).

Gene	UniProt	Outcome	Method	Nsnp	b	se	p-val
ITIH5	G5E9D8; Q86UX2; Q96JW9; C9J2H1; A0A096LP62	PBC	Wald ratio	1	0.281800435	0.21906797	0.198317196
HSPB1	P04792; V9HW43	PBC	Wald ratio	1	−0.465030556	0.204754444	0.02313716
FGF2	P09038	PBC	Wald ratio	1	0.229518318	0.120573948	0.056969056
TNFAIP6	P98066	PBC	Wald ratio	1	0.170681511	0.113453002	0.132472021
ERAP1	Q9NZ08	PBC	Wald ratio	1	−0.112176807	0.078958294	0.155401199
FCGR3B	O75015; M9MML6; A0A087WZR4; A0A087WU90	PSC	Wald ratio	1	0.323937514	0.087694693	0.00022082
FCGR2B	P31994; P31995	PSC	Wald ratio	1	−0.074429666	0.03395003	0.028355693
GSTO1	P78417; V9HWG9	PSC	Wald ratio	1	−0.062942688	0.039904699	0.114720716
CTSB	Q5HYG5; A0A024R374; P07858; B4DMY4	PSC	Wald ratio	1	−0.143471974	0.101695949	0.158305542
RNASE6	Q6IB39; Q93091	PSC	Wald ratio	1	−0.08948982	0.039917461	0.024969673
Abbreviations: PSC: primary sclerosing cholangitis; PBC: primary biliary cholangitis.

Pharmaceutical targets prediction

To identify potential drug targets, we categorized potential target proteins into three groups based on the results of our analysis. Group one included proteins with a p-value < 0.05 after Mendelian randomization correction, a HEIDI test p-value > 0.05, and a posterior probability of H4 (PP.H4) for colocalization analysis of ≥ 0.8. The second group consisted of proteins with a p-value < 0.05 after Mendelian randomization correction, a HEIDI test p-value > 0.05, and a PP.H4 for colocalization analysis between 0.5 and < 0.8. The third group included proteins with a p-value < 0.05 after Mendelian randomization correction, a HEIDI test p-value > 0.05, and a PP.H4 for colocalization analysis < 0.5. Using these criteria, we identified LEFTY2-encoded protein (targeted by primer 15503_20) as a potential drug target in group one, HSPB1 as a potential drug target in group two, and LEFTY2-encoded protein (targeted by primer 15503_15), TNFAIP6, FGF2, ERAP1, EPHA1, and ITIH5 as potential drug targets in group three for PBC. For PSC, we identified FCGR3B as a potential drug target in group one, and FCGR3A, FCGR2B, RNASET2, CTSB, GSTO1, and RNASE6 as potential drug targets in group three. FCGR2A was excluded from the analysis due to a HEIDI test p-value < 0.05, while CTSB was not considered for further analysis due to its SNP’s horizontal pleiotropy.

We analyzed the potential pharmaceuticals for group one and two druggable targets in DURGBANK database and found that LEFTY2 did not have corresponding drugs in PBC, while HSPB1 had three potential drugs: Apatorsen, Phenethyl Isothiocyanate, and Artenimol. In PSC, FCGR3B corresponded to 12 potential drugs: Alefacept, Alemtuzumab, Catumaxomab, Cetuximab, Daclizumab, Etanercept, Gemtuzumab ozogamicin, Human immunoglobulin G, Muromonab, Natalizumab, Palivizumab, and Sarilumab (Table 12).

Table 12. Protein corresponding drug in DRUGBANK.

Disease	Tier	Target	Drugbank id	Name	Durg group	Pharmacological action	Actions
PBC	1	LEFTY2	NA
	2	HSPB1	DB06094	Apatorsen	Investigational	Unknown
		DB12695	Phenethyl Isothiocyanate	Investigational	Unknown
		DB11638	Artenimol	Approved, experimental, investigational	Unknown	Ligand
PSC	1	FCGR3B	DB00092	Alefacept	Approved, investigational, withdrawn	Unknown
			DB00002	Cetuximab	Approved	Unknown	Binder
			DB00005	Etanercept	Approved, investigational	Unknown	Ligand
			DB00028	Human immunoglobulin G	Approved, investigational	Yes	Antagonist
			DB00056	Gemtuzumab ozogamicin	Approved, investigational	Unknown
			DB00075	Muromonab	Approved, investigational	Unknown
			DB00087	Alemtuzumab	Approved, investigational	Unknown	Binder
			DB00108	Natalizumab	Approved, investigational	Unknown
			DB00110	Palivizumab	Approved, investigational	Unknown
			DB00111	Daclizumab	Investigational, withdrawn	Unknown
			DB11767	Sarilumab	Approved, investigational	Unknown	Unknown
			DB06607	Catumaxomab	Approved, investigational, withdrawn
Abbreviations: PSC: primary sclerosing cholangitis; PBC: primary biliary cholangitis.

Differential expression verification

Following the data normalization procedure, Top DEGs in PBC and PSC, respectively, were selected using a threshold of |log2 fold change (FC)| ≥ 1 and a p-value < 0.05. By intersecting with the genes identified in previous MR analysis, we found two differentially expressed genes with drug targets associated with PBC and three differentially expressed genes with drug targets associated with PSC. The expression levels of ITIH5 (p-value < 0.001) were increased and HSPB1 (p-value < 0.01) were decreased in the PBC group compared to the control group (Figure 6A). Regarding PSC, all of the expression levels of FCGR2A (p-value < 0.001), FCGR3B (p-value < 0.001), and RNASET2 (p-value < 0.05) were higher compared to the control group (Figure 6B).

Figure 6. Validation of external dataset and IHC experiment. (A) Expression levels of HSPB1 and ITIH5 between PBC and control groups in GSE119600 dataset; (B) Expression levels of FCGR2A, FCGR3B, and RNASET2 between PSC and control groups in GSE119600 dataset; (C) IHC staining of HSPB1 encoding proteins in liver samples of PBC patients and normal individuals; (D) IHC staining of LEFTY2 encoding proteins in liver samples of PBC patients and normal individuals (E) IHC staining of FCGR3B encoding proteins in liver samples of PSC patients and normal individuals. Abbreviations: IHC: immunohistochemistry; PBC: primary biliary cholangitis; PSC: primary sclerosing cholangitis. ^*p-value < 0.05, ^**p-value < 0.01, ^***p-value < 0.001.

Immunohistochemistry (IHC) was also utilized for examining the protein expression of HSPB1 and LEFTY2 in liver tissues from patients with PBC, as well as FCGR3B in liver tissues from patients with PSC. Based on the staining intensity in the cytoplasm and nuclei, we clearly observed an increased expression of HSPB1 and LEFTY2 in PBC tissues compared to normal tissues (p-value < 0.05) (Figure 6C, 6D). Likewise, FCGR3B exhibited higher expression in the PSC group compared to the normal group (p-value < 0.05) (Figure 6E).

Author Contributions

Conception and writing-original draft preparation, L.D.; charting and methodology, J.M.; data analysis and visualization, L.D.; reference acquisition, Y.Y.Y.; comments and suggestions, J.H. and Z.T.H.; manuscript revision and funding acquisition, C.J.L. All the authors approved the final manuscript.

Conflicts of Interest

The authors declare no conflicts of interest related to this study.

Ethical Statement and Consent

The research procedures involving human subjects were approved by the research ethics committee at Lihuili Hospital, affiliated with Ningbo University (Approval No. KY2023ML052). Written informed consent was obtained from all patients, following the principles outlined in the Helsinki Declaration of 1964 and its subsequent revisions, as well as relevant ethical standards, prior to their enrollment. All the data acquisition complied with the accessibility statement of the database.

Funding

This study was supported by Ningbo Health Branding Subject Fund (PPXK2018-03), Municipal Key Technical Research and Development Program of Ningbo (2023Z160) and Natural Science Foundation of Ningbo (2023J221).

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