Relationship between mucin gene polymorphisms and different types of gallbladder stones.

The Clinical characteristics about the stone group and control group was displayed in Table  1 . The mean age of the stone group was 48.51 ± 13.96 years, while the mean age of the control group was 44.87 ± 14.19 years. The mean age of the stone group was slightly higher than that of the control group, but there was no statistically significant difference ( P  > 0.05). In the stone group, there were 48 males (39.67%) and 73 females (60.33%), while in the control group, there were 70 males (65.42%) and 37 females (34.58%). The percentage of females in the stone group was higher compared with the control group, and the gender difference between the two groups was statistically significant ( P  < 0.05). The mean BMI was 24.22 ± 3.5 kg/m2 in the stone group and 23.97 ± 4.34 kg/m2 in the control group, with no statistically significant difference in BMI between the two groups ( P  < 0.05). There were 35 cases with family history of gallbladder stones in the stone group, accounting for 28.93%, and 10 cases with family history of gallbladder stones in the control group, accounting for 9.35%, which was significantly higher than that of the control group, and the difference was statistically significant ( P  < 0.05). The number of smokers in the stone group was 17 (14.05%) and the number of smokers in the control group was 21 (19.63%), and there was no statistically significant difference in smoking between the two groups ( P  < 0.05). The number of alcohol drinkers in the stone group was 6, or 4.96%; the number of alcohol drinkers in the control group was 9, or 8.41%, and there was no statistically significant difference in the comparison of the number of alcohol drinkers in the two groups ( P  < 0.05). The number of people who could do regular exercise in the stone group was 24, accounting for 19.84%; the number of people who could do regular exercise in the control group was 40, accounting for 37.38%. The proportion of people who could adhere to regular exercise in the control group was significantly higher than that in the stone group, and the difference between the two groups was statistically different (P < 0.05). In the stone group, 88 people, or 72.73%, ate breakfast regularly; in the control group, 90 people, or 84.11%, ate breakfast regularly. The percentage of those who ate breakfast regularly in the stone group was lower than that in the control group, and the difference was statistically significant ( P  < 0.05). In the stone group, 9.917% had a history of diabetes mellitus and 21.49% had a history of hypertension; in the control group, 3.74% had a history of diabetes mellitus and 19.63% had a history of hypertension. There was no statistically significant difference in comparison between the two groups ( P  < 0.05). The number of people using lipid-lowering drugs in the stone group was 7.44%, which was higher than the 0.93% in the control group, and the difference was statistically significant ( P  < 0.05). In the experimental group, the mean LDL was 2.62 ± 0.72 mmol/L, the mean HDL was 1.19 ± 0.34 mmol/L, the mean total cholesterol was 4.31 ± 0.82 mmol/L, the mean triglyceride was 1.39 ± 0.67 mmol/L, the mean direct bilirubin was 5.18 ± 6.42 umol/L, the mean indirect bilirubin The mean direct bilirubin was 5.18 ± 6.42umol/L, the mean indirect bilirubin was 8.49 ± 11.17umol/L, and the mean total bilirubin was 13.72 ± 13.92umol/L. In the control group, the mean low-density lipoprotein (LDL) was 2.62 ± 0.72mmol/L, the mean high-density lipoprotein (HDL) was 1.25 ± 0.31mmol/L, the mean total cholesterol was 4.35 ± 0.85mmol/L, the mean triglyceride was 1.4 ± 0.85mmol/L, and the mean cholesterol was 1.3 ± 0.5mmol/L. The mean total cholesterol was 4.35 ± 0.85 mmol/L, the mean triglycerides were 1.4 ± 0.85 mmol/L, the mean direct bilirubin was 3.85 ± 1.58 umol/L, the mean indirect bilirubin was 7.86 ± 3.77 umol/L, and the mean total bilirubin was 11.7 ± 5.19 umol/L. There were no statistically significant differences in the laboratory parameters between the two groups (Table  1 ). The genotype frequencies of all control SNP loci were subjected to Hardy–Weinberg equilibrium test, and the results are shown in Table  2 . All SNP loci were in accordance with Hardy–Weinberg genetic equilibrium ( P  > 0.05). The genotypic distributions of this SNP locus in the control group were all in accordance with Hardy–Weinberg genetic equilibrium ( P  > 0.05), indicating that the population was genetically balanced, and the control group was representative of the population, which could be used for the next association study (Table  2 ). Table 2 Hardy–Weinberg genetic balance test SNP Genotypes MAF Control HWE Stone HWE MUC1 rs4072037 C/C 0.20 4(3.74%) 0.58 2(1.65%) 0.52 T/C 40(37.38%) 38(31.41%) T/T 63(58.88%) 81(66.94%) MUC2 rs41349846 A/A 0.35 8(7.48%) 0.13 19(15.70%) 0.44 G/A 55(51.40%) 52(42.98%) G/G 44(41.12%) 50(41.32%) rs41532344 T/T 0.10 0(0.00%) 1.00 1(0.83%) 1.00 C/T 17(15.89%) 26(21.49%) C/C 90(84.11%) 94(77.68%) rs2856111 C/C 0.49 31(28.97%) 0.56 22(18.18%) 0.27 T/C 50(46.73%) 67(55.37%) T/T 26(24.30%) 32(26.45%) MUC4 rs712005 G/G 0.22 5(4.67%) 0.33 12(9.92%) 0.10 A/G 29(27.10%) 39(32.23%) A/A 73(68.22%) 70(57.85%) rs2246980 C/C 0.28 7(6.54%) 1.00 11(9.09%) 0.83 G/C 43(40.19%) 49(40.50%) G/G 57(53.27%) 61(50.41%) rs2258447 T/T 0.21 2(1.87%) 0.73 7(5.78%) 0.81 C/T 33(30.84%) 43(35.54%) C/C 72(67.29%) 71(58.68%) rs2259292 C/C 0.28 6(5.61%) 0.80 13(10.74%) 0.52 T/C 43(40.19%) 48(39.67%) T/T 58(54.21%) 60(49.59%) MUC5AC rs28415193 G/G 0.27 6(5.66%) 0.57 14(11.57%) 0.40 C/G 34(32.08%) 48(39.67%) C/C 66(62.26%) 59(48.76%) rs56047977 A/A 0.20 5(4.67%) 0.53 3(2.48%) 0.40 G/A 31(28.97%) 42(34.71%) G/G 71(66.36%) 76(62.81%) MUC5B rs2037089 C/C 0.06 0(0.00%) 1.00 0(0.00%) 1.00 T/C 12(11.21%) 16(13.22%) T/T 95(88.79%) 105(86.78%) rs2075854 A/A 0.48 23(21.50%) 0.70 28(23.14%) 0.86 G/A 56(52.34%) 62(51.24%) G/G 28(26.17%) 31(25.62%) rs3829224 A/A 0.28 6(5.61%) 0.62 9(7.44%) 0.83 G/A 45(42.06%) 51(42.15%) G/G 56(52.34%) 61(50.41%) rs2672785 G/G 0.41 17(15.89%) 0.41 24(19.83%) 1.00 A/G 46(42.99%) 59(48.76%) A/A 44(41.12%) 38(31.41%) rs2735709 G/G 0.34 13(12.15%) 0.83 9(7.44%) 0.10 C/G 51(47.66%) 62(51.24%) C/C 43(40.19%) 50(41.32%) MUC6 rs10902268 A/A 0.17 1(0.93%) 0.30 6(4.96%) 0.09 G/A 34(31.78%) 28(23.14%) G/G 72(67.29%) 87(71.90%) rs573849895 T/T 0 0(0.00%) 1.00 0(0.00%) 1.00 G/T 0(0.00%) 0(0.00%) G/G 107(100.00%) 121(100.00%) rs59257210 T/T 0.28 9(8.41%) 0.62 8(6.61%) 0.65 C/T 40(37.38%) 52(42.98%) C/C 58(54.21%) 61(50.41%) rs61869016 C/C 0.38 14(13.08%) 1.00 22(18.18%) 0.12 T/C 50(46.73%) 49(40.50%) T/T 43(40.19%) 50(41.32%) rs7396383 T/T 0.42 18(16.82%) 0.69 20(16.53%) 0.36 A/T 49(45.79%) 65(53.72%) A/A 40(37.38%) 36(29.75%) rs74644072 T/T 0.31 15(14.02%) 0.28 11(9.09%) 0.50 C/T 43(40.19%) 46(38.02%) C/C 49(45.79%) 64(52.89%) rs7481521 T/T 0.27 5(4.67%) 0.32 10(8.26%) 1.00 C/T 46(42.99%) 49(40.50%) C/C 56(52.34%) 62(51.24%) rs9704308 C/C 0.14 2(1.87%) 0.67 3(2.48%) 1.00 T/C 23(21.50%) 31(25.62%) T/T 82(76.64%) 87(71.90%) MUC17 rs10229731 C/C 0.23 4(3.74%) 0.42 9(7.44%) 0.20 A/C 42(39.25%) 38(31.40%) A/A 61(57.01%) 74(61.16%) rs4729655 T/T 0.32 12(11.21%) 0.50 17(14.05%) 0.16 C/T 43(40.19%) 47(38.84%) C/C 52(48.60%) 57(47.11%) rs55903219 C/C 0.45 24(22.43%) 0.56 29(23.97%) 0.06 T/C 50(46.73%) 49(40.50%) T/T 33(30.84%) 43(35.53%) rs73168398 A/A 0.13 3(2.80%) 0.46 2(1.65%) 0.64 G/A 25(23.36%) 23(19.01%) G/G 79(73.83%) 96(79.34%) rs74974199 C/C 0.11 2(1.87%) 0.63 1(0.83%) 1.00 G/C 21(19.63%) 21(17.35%) G/G 84(78.50%) 99(81.82%) Hardy–Weinberg genetic balance test We conducted haplotype analyses of the gallbladder stones patients and control subjects (Fig.  1 and Table  3 ). In the two alleles’ haplotype ( MUC4 rs712005/rs2258447 ), the G-T (OR, 1.662855; 95% CI, 1.02403–2.7002; p  = 0.03978208) showed significantly increased stones group risk. In the two alleles’ haplotype ( MUC5AC rs28415193/rs56047977 ), the G-G (OR,1.569832; 95% CI, 1.031566–2.388962; p  = 0.03528528) was associated with increased stones group risk. Fig. 1 LD plot of MUC2, 4, 5, 6, 17 SNPs. a The linkage disequilibrium (LD) block structure consisted of the three SNPs located in MUC2. b The linkage disequilibrium (LD) block structure consisted of the four SNPs located in MUC4. c The linkage disequilibrium (LD) block structure consisted of the five SNPs located in MUC5. d The linkage disequilibrium (LD) block structure consisted of the seven SNPs located in MUC6. e The linkage disequilibrium (LD) block structure consisted of the five SNPs located in MUC17 Table 3 The haplotype analysis of the MUC4, 5, 6, 17 polymorphisms with an increased odds ratio in the stones and control participants Haplotypes Stones( n  = 242) Control( n  = 214) OR (95% CI) p * value MUC4(rs712005/rs2258447)  A-C 179(73.97%) 172(80.37%) - -  G-C 6(2.48%) 5(2.34%) 1.111111(0.32882–3.754541) 0.8653243  G-T 57(23.55%) 34(15.89%) 1.662855(1.02403–2.7002) 0.03978208 MUC4(rs2258447/rs2246980/rs2259292)  C–C-C 15(6.20%) 20(9.35%) 0.6902534(0.3466879–1.37429) 0.2913879  C-G-T 167(69.01%) 156(72.90%) - -  T-C–C 55(22.73%) 34(15.89%) 1.539945(0.9544595–2.484581) 0.07689022 MUC5AC(rs28415193/rs56047977)  C-A 48(19.83%) 41(19.34%) 1.127476(0.6966765–1.824665) 0.6252076  C-G 118(48.76%) 125(58.96%) - -  G-G 76(31.40%) 46(21.70%) 1.569832(1.031566–2.388962) 0.03528528 MUC5B(rs2075854/rs2735709)  A-C 117(48.35%) 98(45.79%) - -  A-G 1(0.41%) 4(1.87%) 0.2106742(0.023047–1.925787) 0.1677321  G-C 45(18.60%) 39(18.22%) 0.9572462(0.5803682–1.57886) 0.8641084  G-G 79(32.64%) 73(34.11%) 0.947188(0.613853–1.461531) 0.8063169 MUC5B(rs2735709/rs2037089/rs3829224)  C–C-G 16(6.61%) 12(5.61%) 1.138889(0.5094841–2.545846) 0.7513321  C-T-G 146(60.33%) 125(58.41%) - -  G-T-A 69(28.51%) 57(26.64%) 1.043457(0.67997–1.601251) 0.8456326  G-T-G 11(4.55%) 20(9.35%) 0.4710254(0.2171538–1.021695) 0.05669287 MUC6(rs74644072/rs10902268/rs7396383/rs9704308/rs7481521)  C-A-A-T-T 40(16.53%) 36(16.82%) 1.062572(0.623231–1.811624) 0.823564  C-G-A-T-T 28(11.57%) 19(8.88%) 1.439032(0.7401137–2.797965) 0.2833245  C-G-T-C–C 37(15.29%) 27(12.62%) 1.375254(0.7748834–2.440783) 0.2763135  C-G-T-T-C 68(28.10%) 58(27.10%) 1.247846(0.7844546–1.984971) 0.3498244  T-G-A-T-C 67(27.69%) 72(33.64%) - - MUC6(rs61869016/rs59257210)  C–C 93(38.43%) 77(35.98%) - -  T-C 81(33.47%) 79(36.92%) 0.8290935(0.559797–1.227938) 0.3496328  T-T 68(28.10%) 57(26.64%) 1.03915(0.66504–1.623712) 0.8660719 MUC17(rs10229731/rs74974199)  A-C 23(9.50%) 25(11.68%) 0.8306344(0.457141–1.509279) 0.5425077  A-G 163(67.36%) 139(64.95%) - -  C-G 56(23.14%) 50(23.36%) 0.9742359(0.6315192–1.50294) 0.9060612 MUC17(rs74974199/rs73168398/rs4729655/rs55903219)  C-G-C–C 23(9.50%) 25(11.68%) 0.8451384(0.4499853–1.587294) 0.6008195  G-A-T-T 26(10.74%) 31(14.49%) 0.648901(0.3625667–1.161365) 0.1453189  G-G-C–C 84(34.71%) 73(34.11%) - -  G-G-C-T 53(21.90%) 49(22.90%) 0.9453892(0.5985587–1.493188) 0.809697  G-G-T-T 55(22.73%) 36(16.82%) 1.324239(0.8192187–2.140586) 0.2517222 * p  < .05 indicates statistical significance LD plot of MUC2, 4, 5, 6, 17 SNPs. a The linkage disequilibrium (LD) block structure consisted of the three SNPs located in MUC2. b The linkage disequilibrium (LD) block structure consisted of the four SNPs located in MUC4. c The linkage disequilibrium (LD) block structure consisted of the five SNPs located in MUC5. d The linkage disequilibrium (LD) block structure consisted of the seven SNPs located in MUC6. e The linkage disequilibrium (LD) block structure consisted of the five SNPs located in MUC17 The haplotype analysis of the MUC4, 5, 6, 17 polymorphisms with an increased odds ratio in the stones and control participants * p  < .05 indicates statistical significance Statistical analysis of the distribution of mucin gene polymorphism loci in different types of gallbladder stones after correcting for sex and age, BMI, family history of gallbladder stone, regular sports, regular breakfast, antilipemic agents (Table  4 ). Table 4 Distribution of SNPs in each type of gallbladder stone group and control group Type of stone Genes SNPs Allele/ Genotype Stone Group Control OR(95%CI) P * value Cholesterol stone ( n  = 33) MUC4 rs712005 G 18(27.27%) 39(18.22%) 2.51(1.20–5.22) 0.014 A 48(72.73%) 175(81.78%) MUC5B rs2037089 C 7(10.61%) 12(5.61%) 3.54(1.14–11.01) 0.029 T 59(89.39%) 202(94.39%) MUC2 rs2856111 T/T 9(27.27%) 26(24.30%) 3.02(0.73–12.39) 0.126 C/T 20(60.61%) 50(46.73%) 3.81(1.06–13.68) 0.040 C/C 4(12.12%) 31(28.97%) - - MUC17 rs73168398 A/A 1(3.03%) 3(2.80%) 0.66(0.06–7.86) 0.741 G/A 4(12.12%) 25(23.36%) 0.26 (0.07–0.98) 0.046 G/G 28(84.85%) 79(73.83%) - - rs10229731 C/C 3(9.09%) 4(3.74%) 1.07(0.17–6.75) 0.944 A/C 8(24.24%) 42(39.25%) 0.33 (0.12–0.95) 0.039 A/A 22(66.67%) 61(57.01%) - - Pigment gallstone ( n  = 11) MUC6 rs10902268 A 1(4.55%) 36(16.82%) 0.00 (0.00–0.27) 0.011 G 21(95.45%) 178(83.18%) rs61869016 C 5(22.73%) 78(36.45%) 0.07 (0.01–0.63) 0.017 T 17(77.27%) 136(63.55%) Mixed stone ( n  = 77) MUC2 rs41532344 T 16(11.76%) 17(7.94%) 2.55(1.06–6.13) 0.036 C 120(88.24%) 197(92.06%) MUC5 AC rs28415193 G 45(33.09%) 46(21.70%) 1.77(1.02–3.07) 0.041 C 91(66.91%) 166(78.30%) MUC6 rs10902268 A/A 4(5.88%) 1(0.93%) 3.49(0.32–38.02) 0.305 G/A 16(23.53%) 34(31.78%) 0.40 (0.17–0.95) 0.038 G/G 48(70.59%) 72(67.29%) - - * P values were adjusted by sex and age, BMI, family history of gallbladder stone, regular sports, regular breakfast, antilipemic agents Distribution of SNPs in each type of gallbladder stone group and control group MUC5 AC * P values were adjusted by sex and age, BMI, family history of gallbladder stone, regular sports, regular breakfast, antilipemic agents Cholesterol stone group: allele frequencies or genotype frequency distributions of five SNP loci were statistically different in the cholesterol stone subgroup compared with the control group. The frequencies of MUC2 rs2856111 TT, CT and CC genotypes were 27.27%, 60.61% and 12.12%, respectively, in the cholesterol stone subgroup and 24.30%, 46.73% and 28.97%, respectively, in the control group, and the risk of disease in carriers of the genotype CT was 3.81-fold higher than that in carriers of the CC genotype (OR = 3.81, 95% CI: 1.06–13.68). The frequencies of the MUC17 rs10229731 CC, AC and AA genotypes were 9.09%, 24.24% and 66.67% in the cholesterol stone subgroup and 3.74%, 39.25% and 57.01% in the control group, respectively, and the prevalence risk of carrying the AC genotype was 0.33 times higher than that of carriers of the AA genotype (OR = 0.33, 95% CI: 0.12–0.95). The frequencies of MUC17 rs73168398 AA, GA and GG genotypes were 3.03%, 12.12% and 84.85% in the cholesterol stone subgroup and 2.80%, 23.36% and 73.83% in the control group, respectively, and the risk of prevalence in carriers of GA genotypes was 0.26 times higher than that in carriers of GG genotypes (OR = 0.26, 95% CI: 0.07–0.98). The MUC4 rs712005 G allele frequency and A allele frequency were 27.27% and 72.73% in the cholesterol stone subgroup, compared with 18.22% and 81.78% in the control group, respectively, and the risk of prevalence of carrying the G allele was 2.51 times higher than that of carriers of the A allele (OR = 2.51, 95% CI: 1.20–5.22). The MUC5B rs2037089 C allele frequency and T allele frequency in the cholesterol stone subgroup were 10.61% and 89.39%, respectively, compared with 5.61% and 94.39%, respectively, in the control group, and the risk of prevalence of carrying the C allele was 3.54 times higher than that of carriers of the T allele (OR = 3.54, 95% CI: 1.14–11.01). There were no significant differences in the distribution of allele frequencies or genotype frequencies for the remaining SNPs. Pigment gallstone group: allele frequency distributions for 2 SNP loci were statistically different in the cholestones subgroup compared with the control group. The frequencies of the A and G alleles of MUC6 rs10902268 were 4.55% and 95.45%, respectively, in the cholestasis subgroup and 16.82% and 83.18%, respectively, in the control group, and the risk of disease in carriers of the A allele was 0.004 times higher than that in carriers of the G allele (OR = 0.004, 95% CI: 0.00–0.27). The MUC6 rs61869016 C allele frequency and T allele frequency were 22.73% and 77.27% in the bile pigment stone subgroup, compared with 36.45% and 63.55% in the control group, respectively, and the risk of prevalence of carrying the C allele was 0.07 times higher than that of carriers of the T allele (OR = 0.07, 95% CI: 0.01–0.63). Mixed stone group: allele frequencies or genotype frequency distributions for three SNP loci were statistically different in the mixed stone subgroup compared with the control group. The MUC2 rs41532344 T allele frequency and C allele frequency were 11.76% and 88.24%, respectively, in the mixed stone subgroup, compared with 7.94% and 92.06%, respectively, in the control group, and the risk of disease in carriers of the T allele was 2.55 times higher than that in carriers of the C allele (OR = 2.55, 95% CI: 1.06–6.13). The MUC5AC rs28415193 G allele frequency and C allele frequency in the mixed stone subgroup were 33.09% and 66.91%, respectively, compared with 21.70% and 78.30%, respectively, in the control group, and the risk of prevalence of carrying the G allele was 1.77 times higher than that of carriers of the C allele (OR = 1.77, 95% CI: 1.02–3.07). The frequencies of the MUC6 rs10902268 AA, GA and GG genotypes were 5.88%, 23.53% and 70.59%, respectively, in the mixed stone subgroup, compared with 0.93%, 31.78% and 67.29%, respectively, in the control group, and the risk of prevalence of the GA-carrying genotype was 0.40 times higher than that of carriers of the GG genotype (OR = 0.40, 95% CI: 0.17–0.95).

In this study, 121 patients with gallbladder stones admitted to the Department of Hepatobiliary and Pancreatic Surgery from October 2021 to February 2022 were collected as the stone group and 107 patients with non-gallbladder stones as the control group. Basic information (such as gender, age, BMI, etc.) for both groups was collected using the inpatient workstation system, along with the results of blood sampling tests for total bilirubin, direct bilirubin, indirect bilirubin, total cholesterol, triglycerides, low-density lipoproteins, and high-density lipoproteins. The collected gallbladder stones were cleaned using distilled water to wash away impurities such as bile, mucus and blood from the surface. The traits of the stones were observed and then the maximum diameter of the stones was measured using a graduated scale in each case and the number of stones was recorded. All stone specimens were dried and prepared for use. The diameter of the largest of the multiple stones was measured. Since all patients with gallbladder stones were analysed for composition, cases with sediment-like stones have been excluded (Table  1 ). Table 1 Clinical characteristics of the study subjects Characteristics Stone ( n  = 121) Control ( n  = 107) OR(95% CI) P* value Age (years ± SD) 48.51 ± 13.96 44.87 ± 14.19 1.0002(1–1.0004) 0.0535 BMI (kg/m2 ± SD) 24.22 ± 3.50 23.97 ± 4.34 1.0002(0.9995–1.0009) 0.6313 Gender, n (%) Male 48(39.67%) 70(65.42%) 1.0106(1.0052–1.0161) 0.0001 Female 73(60.33%) 37(34.58%) History Family history of gallbladder stone, n (%) 35(28.93%) 10(9.35%) 1.0138(1.0061–1.0215) 0.0004 Smoking, n (%) a 17(14.05%) 21(19.63%) 0.996(0.9891–1.003) 0.2614 Alcohol drinking, n (%) b 6(4.96%) 9(8.41%) 0.9944(0.9838–1.005) 0.2992 Regular sports, n (%) c 24(19.84%) 40(37.38%) 0.9912(0.9854–0.9971) 0.0037 Regular breakfast, n (%) d 88(72.73%) 90(84.11%) 0.9932(0.9866–0.9996) 0.0401 Diabetes, n (%) 12(9.92%) 4(3.74%) 1.0105(0.9988–1.0223) 0.0791 Hypertension, n (%) 26(21.49%) 21(19.63%) 1.0011(0.9947–1.0076) 0.7289 Antilipemic agents, n (%) 9(7.44%) 1(0.93%) 1.0217(1.0006–1.0431) 0.0438 LDL (mmol/L ± SD) 2.62 ± 0.72 2.62 ± 0.72 0.9999(0.9958–1.004) 0.9741 HDL (mmol/L ± SD) 1.19 ± 0.34 1.25 ± 0.31 0.9949(0.9858–1.0041) 0.2725 TC (mmol/L ± SD) 4.31 ± 0.82 4.35 ± 0.85 0.9994(0.9958–1.003) 0.7555 TG (mmol/L ± SD) 1.39 ± 0.67 1.4 ± 0.85 0.9998(0.9958–1.0038) 0.934 DBIL (umol/L ± SD) 5.18 ± 6.42 3.85 ± 1.58 1.0008(0.9999–1.0017) 0.0733 IBIL (umol/L ± SD) 8.49 ± 11.17 7.86 ± 3.77 1.0001(0.9997–1.0005) 0.5944 TBIL (umol/L ± SD) 13.72 ± 13.92 11.7 ± 5.19 1.0002(0.9999–1.0005) 0.1866 Number of stones, n (%) Single-shot 34(28.1%) Frequent 87(71.9%) Stone diameter (mm ± SD) 10.51 ± 7.80 BMI  Body Mass Index = weight (kg)/height (m) 2 , LDL  Low Density Lipoprotein, HDL  High Density Lipoprotein, TC  Total Cholesterol, TG  Triglyceride, DBIL  Direct Bilirubin, IBIL  Indirect Bilirubin,  TBIL  Total Bilirubin, OR  Odds ratio, CI  Confidence interval a Smoke at least 1 cigarette per day for 1 year, defined as non-smoker if you have quit for more than 6 months b Alcoholic products such as liquor, beer or wine at least once a week for 1 year, if abstained from alcohol for more than 6 months is defined as non-drinker c Eat breakfast no less than 5 times a week d More than 150 min per week of physical activity of moderate intensity or higher * p  < .05 indicates statistical significance Clinical characteristics of the study subjects Stone diameter (mm ± SD) BMI  Body Mass Index = weight (kg)/height (m) 2 , LDL  Low Density Lipoprotein, HDL  High Density Lipoprotein, TC  Total Cholesterol, TG  Triglyceride, DBIL  Direct Bilirubin, IBIL  Indirect Bilirubin,  TBIL  Total Bilirubin, OR  Odds ratio, CI  Confidence interval a Smoke at least 1 cigarette per day for 1 year, defined as non-smoker if you have quit for more than 6 months b Alcoholic products such as liquor, beer or wine at least once a week for 1 year, if abstained from alcohol for more than 6 months is defined as non-drinker c Eat breakfast no less than 5 times a week d More than 150 min per week of physical activity of moderate intensity or higher * p  < .05 indicates statistical significance Inclusion criteria for the stone group: patients with gallbladder stones confirmed by imaging, with surgical indications for concomitant transperitoneal laparoscopic cholecystectomy. Exclusion criteria: patients with cystic dilatation of the bile ducts, hepatitis B, malnutrition, severe hepatic and renal insufficiency, chronic obstructive pulmonary disease (COPD), Bronchial Asthma, Bronchiectasis, Ulcerative Colitis (UC), Crohn's Disease (CD), a history of malignant neoplasm, a history of previous biliary tract surgery, or a history of major gastric resection. Inclusion criteria for the control group: healthy medical check-up patients with gallbladder stones ruled out by imaging or patients who underwent surgery for diseases other than gallbladder stones in our hospital, such as cholecystectomy for adenomatous polyps of the gallbladder, hernia repair, appendicectomy, excision of body masses, etc. Exclusion criteria: patients with cystic dilatation of the bile ducts, hepatitis B, malnutrition, severe liver and renal insufficiency, chronic obstructive pulmonary disease (COPD), Bronchial Asthma, Bronchiectasis, Ulcerative Colitis (UC), Crohn's Disease (CD), history of malignant tumours, history of previous biliary surgery or history of gastric resection, or patients with history of biliary surgery. patients with a history of resection. This study was approved by local Institutional Review Boards (approval number: LL-KY-2021912). Patients were consented by an informed consent process that was reviewed by local Institutional Review Boards and certify that the study was performed in accordance with the ethical standards as laid down in the 1964 Declaration of Helsinki. Chemical reagents were purchased from Genesky (China), TransGen Biotech (China), Takara biotech (Japan),Applied Biosystems (USA). Sample processing: 40 μL of proteinase K solution at a concentration of 10 mg/mL was added to a 1.5 mL test tube, then the cryo-frozen blood sample was slowly dissolved and 400 μL of the blood sample was added to the test tube. To extract DNA using the Blood Genomic DNA Extraction Kit(Concert Biotechnology, China): Preheat the Nucleic Acid Purifier for 20 min. After confirming the correct information of the reagent strip code, sample type, sample volume, elution volume, etc., the instrument will automatically extract the DNA, and the liquid in the tube of the instrument will be collected as the extracted DNA. Take 1ul of DNA sample and electrophoresis it in 1% agarose gel condition for quality control and concentration evaluation, dilute the DNA sample to working concentration 10-30ng/μl based on the estimated concentration. Diluted DNA sample PCR amplification using 2720 Thermal Cycler (Applied Biosystems, USA), amplification conditions consisted of 1step: initial denaturation at 95ºC for 2 min; 2step: 35 cycles at 94ºC for 20 s, 60ºC for 40 s(-0.5ºC/cycle), and 72ºC for 1.5 min; 3step: at 94ºC for 20 s, 59ºC for 30 s, and 72ºC for 1.5 min, with one additional cycle at 72ºC for 3 min; 3step: one additional cycle at 72ºC for 2 min. Multiplex PCR reaction: PCR primer preparation. Preparation of PCR conditions: Concentration of each pair of primers (µM) in the multiplex PCR primer (Probe Mix): except for rs59257210 and rs28415193 , the concentration of the primers for the other SNP loci are all 1µM. PCR reaction: Shake the prepared PCR reaction premix(10 × PerfectStart® Taq buffer 2ul, 10 × GC Enhancer6ul, dNTP 2.4ul, MgCl2 0.8ul, Probe Mix 2.0ul, Hot Star Taq 0.4ul, ddH2O 5.4ul) well, then put it into the centrifuge and centrifuge to mix well, each reaction well was divided into 19 ul. 1ul of DNA sample was then added into each reaction well, after mixing well, the centrifuge was centrifuged at 3,000 rpm for 0.5 min, and then put it into the 2720 Thermal Cycler (Applied Biosystems, USA), amplification conditions consisted of 1step: initial denaturation at 95ºC for 2 min; 2step: 35 cycles at 94ºC for 20 s, 60ºC for 40 s(-0.5ºC/cycle), and 72ºC for 1.5 min; 3step: at 94ºC for 20 s, 59ºC for 30 s, and 72ºC for 1.5 min, with one additional cycle at 72ºC for 3 min; 3step: one additional cycle at 72ºC for 2 min. Multiplex PCR product purification: Add 5U of SAP enzyme and 2U of Exonuclease I enzyme to 20μl of PCR product, warm bath at 37℃ for 1 h, and then inactivate at 75℃ for 15min. Ligation reaction: Ligation primer preparation. Reaction conditions: Shake the prepared the pre-mixed solution for ligation reaction (10 × ligase buffer 1.00ul, Labelp Mix 0.25ul, Ligase Primer Mix 0.4ul, DNA ligase 1.0ul, ddH2O 3.35ul) well, load it into 96-well plate, 6ul for each reaction well, add 4ul of multiplexed PCR purification product to each reaction well, mix it well and put it into centrifuge at 3000 rpm for 0.5min, then put the 96-well plate into the 2720 Thermal Cycler immediately and with 38 cycle at 94ºC for 1 min and 56ºC for 4 min. Ligation product on ABI3730XL sequencer(Applied Biosystems, USA): After tenfold dilution of the ligation product, take 1 μl of the ligation product and mix it with 0.5 μl of Liz500 SIZE STANDARD, 8.5 μl of Hi-Di, denature at 95 degrees Celsius for 5 min and then put it on the ABI3730XL sequencer. Take 1ul of DNA sample and electrophoresis it in 1% agarose gel condition for quality control and concentration evaluation, dilute the DNA sample to working concentration 10-30ng/μl based on the estimated concentration. Diluted DNA sample PCR amplification using 2720 Thermal Cycler (Applied Biosystems, USA), amplification conditions consisted of 1step: initial denaturation at 95ºC for 2 min; 2step: 35 cycles at 94ºC for 20 s, 60ºC for 40 s(-0.5ºC/cycle), and 72ºC for 1.5 min; 3step: at 94ºC for 20 s, 59ºC for 30 s, and 72ºC for 1.5 min, with one additional cycle at 72ºC for 3 min; 3step: one additional cycle at 72ºC for 2 min. Multiplex PCR reaction: PCR primer preparation. Preparation of PCR conditions: Concentration of each pair of primers (µM) in the multiplex PCR primer (Probe Mix): except for rs59257210 and rs28415193 , the concentration of the primers for the other SNP loci are all 1µM. PCR reaction: Shake the prepared PCR reaction premix(10 × PerfectStart® Taq buffer 2ul, 10 × GC Enhancer6ul, dNTP 2.4ul, MgCl2 0.8ul, Probe Mix 2.0ul, Hot Star Taq 0.4ul, ddH2O 5.4ul) well, then put it into the centrifuge and centrifuge to mix well, each reaction well was divided into 19 ul. 1ul of DNA sample was then added into each reaction well, after mixing well, the centrifuge was centrifuged at 3,000 rpm for 0.5 min, and then put it into the 2720 Thermal Cycler (Applied Biosystems, USA), amplification conditions consisted of 1step: initial denaturation at 95ºC for 2 min; 2step: 35 cycles at 94ºC for 20 s, 60ºC for 40 s(-0.5ºC/cycle), and 72ºC for 1.5 min; 3step: at 94ºC for 20 s, 59ºC for 30 s, and 72ºC for 1.5 min, with one additional cycle at 72ºC for 3 min; 3step: one additional cycle at 72ºC for 2 min. PCR primer preparation. Preparation of PCR conditions: Concentration of each pair of primers (µM) in the multiplex PCR primer (Probe Mix): except for rs59257210 and rs28415193 , the concentration of the primers for the other SNP loci are all 1µM. PCR reaction: Shake the prepared PCR reaction premix(10 × PerfectStart® Taq buffer 2ul, 10 × GC Enhancer6ul, dNTP 2.4ul, MgCl2 0.8ul, Probe Mix 2.0ul, Hot Star Taq 0.4ul, ddH2O 5.4ul) well, then put it into the centrifuge and centrifuge to mix well, each reaction well was divided into 19 ul. 1ul of DNA sample was then added into each reaction well, after mixing well, the centrifuge was centrifuged at 3,000 rpm for 0.5 min, and then put it into the 2720 Thermal Cycler (Applied Biosystems, USA), amplification conditions consisted of 1step: initial denaturation at 95ºC for 2 min; 2step: 35 cycles at 94ºC for 20 s, 60ºC for 40 s(-0.5ºC/cycle), and 72ºC for 1.5 min; 3step: at 94ºC for 20 s, 59ºC for 30 s, and 72ºC for 1.5 min, with one additional cycle at 72ºC for 3 min; 3step: one additional cycle at 72ºC for 2 min. Multiplex PCR product purification: Add 5U of SAP enzyme and 2U of Exonuclease I enzyme to 20μl of PCR product, warm bath at 37℃ for 1 h, and then inactivate at 75℃ for 15min. Ligation reaction: Ligation primer preparation. Reaction conditions: Shake the prepared the pre-mixed solution for ligation reaction (10 × ligase buffer 1.00ul, Labelp Mix 0.25ul, Ligase Primer Mix 0.4ul, DNA ligase 1.0ul, ddH2O 3.35ul) well, load it into 96-well plate, 6ul for each reaction well, add 4ul of multiplexed PCR purification product to each reaction well, mix it well and put it into centrifuge at 3000 rpm for 0.5min, then put the 96-well plate into the 2720 Thermal Cycler immediately and with 38 cycle at 94ºC for 1 min and 56ºC for 4 min. Ligation primer preparation. Reaction conditions: Shake the prepared the pre-mixed solution for ligation reaction (10 × ligase buffer 1.00ul, Labelp Mix 0.25ul, Ligase Primer Mix 0.4ul, DNA ligase 1.0ul, ddH2O 3.35ul) well, load it into 96-well plate, 6ul for each reaction well, add 4ul of multiplexed PCR purification product to each reaction well, mix it well and put it into centrifuge at 3000 rpm for 0.5min, then put the 96-well plate into the 2720 Thermal Cycler immediately and with 38 cycle at 94ºC for 1 min and 56ºC for 4 min. Ligation product on ABI3730XL sequencer(Applied Biosystems, USA): After tenfold dilution of the ligation product, take 1 μl of the ligation product and mix it with 0.5 μl of Liz500 SIZE STANDARD, 8.5 μl of Hi-Di, denature at 95 degrees Celsius for 5 min and then put it on the ABI3730XL sequencer. SNP data were obtained from the 1000genomes database, and the selection criteria were R2 ≥ 0.8 and the minor allele frequency (MAF) ≥ 10% in the Chinese Han population, with preference given to loci located in exonic regions and functional loci, and combined with relevant literature studies in the field for loci screening. Finally, 28 mucin polymorphism loci ( MUC1 rs4072037, MUC2 rs41349846/ rs41532344/ rs2856111, MUC4 rs712005/ rs2246980/ rs2258447 /rs2259292, MUC5AC rs28415193/ rs56047977, MUC5B rs2037089/ rs2075854/ rs3829224/ rs2672785/ rs2735709, MUC6 rs10902268/ rs573849895/ rs59257210/ rs61869016/ rs7396383/ rs74644072/ rs7481521/ rs9704308,MUC17 rs10229731/ rs4729655/ rs55903219/ rs73168398/ rs74974199 ) were identified, which were derived from 7 mucin genes that were considered to be possibly related to gallbladder stones.

Mucins are expressed and play a critical role in various parts of the body, closely linked to several inflammatory diseases. Human saliva contains a range of mucins, which lubricate and maintain a moist environment while also preventing the invasion of harmful bacteria, thereby contributing significantly to oral health maintenance [ 34 ]. Mucins present on the surface of the respiratory tract act as a natural physical barrier, trapping pathogens and foreign substances in the airway, which can be more easily removed by cilia [ 35 ]. Several respiratory viruses require the secretion of specific proteases to disrupt the mucin-coating and invade the body [ 36 ]. Mucins secreted by the intestinal mucosa are closely associated with the distribution of intestinal microflora, and abnormal mucin expression has been observed in the intestinal mucosal tissues of patients with Crohn's disease and ulcerative colitis [ 35 ]. Mucins are involved in the formation of different types of gallbladder stones; animal experiments have shown that mice with excessive mucin secretion in the gallbladder mucosa had increased cholesterol concentrations in the gallbladder wall tissues, impairing gallbladder function and promoting the development of cholesterol crystals [ 37 ]. Furthermore, mice with defects in the mucin gene showed a significant reduction in the incidence of cholesterol stones, as a result of delayed cholesterol crystallisation in the bile due to reduced mucin secretion caused by gene defects [ 38 ]. The causal relationship between excessive mucin secretion and biliary cholesterol supersaturation remains unclear, although it is hypothesized that elevated biliary cholesterol concentrations may stimulate the gallbladder wall to secrete more mucin. Similar studies on bile pigmented gallbladder stones have confirmed the stimulatory effect of gallbladder inflammation on mucin expression, suggesting a joint involvement of inflammatory factors and mucin in the formation of pigmented stones [ 39 ]. The MUC family in this study includes MUC1, 2, 4, 5AC, 5B, 6, and 17. MUC1 is the first membrane-bound mucin in the mucin family to be detected, and it can be expressed apically in the glandular luminal or proximal tubular luminal surface of epithelial cells in many tissues and organs.) MUC1 is closely related to malignant tumours, such as breast, pancreatic, and ovarian cancers, and its expression is up-regulated in tumour cells and closely associated with several biological characteristics, such as growth of tumour cells, metastasis, and epigenetic regulation. The expression of MUC1 is up-regulated in tumour cells and is closely related to the growth, metastasis, immune escape and epigenetic regulation of tumour cells [ 40 ]. MUC1 also plays a role in inflammatory diseases, its N-terminal glycosylation site can play a role in glycosylation and thus participate in signal transduction and immune regulation and other cellular biological activities, MUC1 can activate the NF-κB inflammatory pathway by directly increasing the proportion of NF-κB in the gene, and inhibition of intestinal mucosal MUC1 expression can not only inhibit the NF-κB pathway and thus alleviate inflammation, but also improve the inflammation in the intestinal mucosa, and can also improve the inflammation in the mucosa [ 41 ]. It also improves the barrier function of the intestinal mucosa. In an animal study, mice carrying the human MUC1 gene showed a significantly higher rate of gallbladder cholesterol absorption and lower gallbladder emptying rate, and developed gallbladder stones earlier than wild-type mice when fed a lithogenic diet, implying that MUC1 in mice promotes the formation of cholesterol crystals in the gallbladder by facilitating cholesterol uptake and impairing gallbladder dynamics [ 37 ]. The sticky mucus covering the mucosal surface of the intestine plays a role in forming a defence barrier against pathogens and blocking the invasion of external stimuli in the intestine. Among them, MUC2 protein secreted by cup cells is the main macromolecular component of intestinal mucus. MUC2 covers the intestinal surface to form a barrier mucus layer, which acts as the first line of defence to protect the intestinal mucosa from damage [ 42 ]. When inflammatory progressive diseases, such as ulcerative colitis, occur in the human gut, the expression of MUC2 in the gut is significantly reduced, which leads to a weakening of the protective capacity of the intestinal mucosa, which in turn increases the susceptibility of the gut to inflammation [ 43 ]. In addition MUC2 deficiency may also lead to an imbalance in the intestinal flora, spontaneous enteritis, and intestinal tumours, which may be related to the pro-inflammatory factor TNF-α. Abnormalities in TNF-α activate the JNK pathway, which is associated with cellular stress and affects the secretion of mucins by the cuprocytes, as well as inducing a breach in the intestinal defences by decreasing the expression of tight junction proteins [ 44 ]. Although the mechanism of action of MUC2 in the gallbladder is unknown, it can be speculated that MUC2 may play an equally important protective function in the gallbladder epithelium. Previously, MUC5 was thought to have three isoforms, namely MUC5A, MUC5C and MUC5B, but through gene sequencing, it has been learnt that MUC5A and MUC5C are the same protein, and therefore the two proteins are jointly named MUC5AC [ 45 ]. MUC5AC has been detected to be up-regulated in the serum of patients with gallbladder cancer, and is also highly expressed in intrahepatic bile duct stone disease and benign gallbladder diseases, including chronic cholecystitis [ 46 , 47 ]. In gallbladder stone disease, MUC5AC aggregates to form a mucus-like gel in the gallbladder. To further investigate the formation mechanism of MUC5AC in gallbladder stone disease, it was found that the expression level of MUC5AC increased with the elevation of cholesterol concentration gradient, and cholesterol crystals might be a key factor leading to the overexpression of MUC5AC, and that cholesterol crystals might be a key factor leading to the overexpression of MUC5AC. In addition, the extent of MUC5AC overexpression induced by cholesterol crystals was also attenuated when the downstream MyD88/NF-κB pathway was inhibited using an IL-1 antagonist, implying that it is the cholesterol crystals-induced IL-1β/IL-1R/MyD88 inflammatory pathway that is one of the mechanisms leading to the overexpression of MUC5AC in the gallbladder [ 48 ]. MUC6, as a secreted mucin, is expressed to varying degrees in a number of organs in the human body, including the fundus of the stomach, duodenum, gallbladder, and cervical epithelium, and in the gastric mucosa, MUC6 can inhibit the growth of Helicobacter pylori (HP) in the stomach through the expression of acetylglucosaminide residues, in addition to the secretion of mucus for the protection of the gastric mucosa, which can be seen to play a protective role in the mucosa [ 49 , 50 ]. MUC6 also plays an important role in malignant tumours. In a study on gastric cancer, the expression of MUC6 in gastric cancer was lower than that in pre-cancerous tissues, and considering the negative correlation between its expression level and the size of tumour, depth of infiltration, and chorioallantoic metastasis, the down-regulation of the expression of MUC6 may be related to the malignant transformation of gastric epithelial cells as a result of the diminished protection of gastric epithelium [ 50 ]. MUC17 as a novel membrane-tethered mucin, has been studied in gastrointestinal inflammation and intestinal cancer because it is widely present in intestinal epithelial cells [ 51 , 52 ]. Expressed as a transmembrane mucin in Normal human conjunctival epithelium [ 53 ]. Therefore, we hypothesise that it is also present in the epithelial cells of the gallbladder mucosa and plays a role in gallbladder stone formation. In the current research, the distribution of gene frequencies and genotype frequencies of all mucin polymorphic loci in the control group adhered to the Hardy–Weinberg equilibrium, suggesting that the samples in this study were a true representation. Following adjustment for gender and age, polymorphisms were separately analyzed for different types of gallbladder stones. The risk of cholesterol stone prevalence was found to be significantly higher in carriers of MUC4 rs712005 G and MUC5B rs2037089 C alleles; the MUC6 rs10902268 A and rs61869016 C alleles reduced the risk of pigment gallstone; MUC5AC rs28415193 G and MUC2 rs41532344 T alleles increased the risk of mixed stones. The risk of cholesterol stone prevalence was found to be significantly higher in carriers of MUC2 rs2856111 C/T genotype, and the risk of cholesterol stone prevalence could be reduced by the MUC17 rs10229731 A/C and rs73168398 G/A genotypes; MUC6 rs10902268 G/A genotype carriers reduced the risk of mixed stones. The various types of gallbladder stones are composed of different stone components, indicating that the factors and mechanisms involved in their formation may differ, or the extent of their involvement may vary. The mucin gene polymorphisms associated with different types of gallbladder stones indicate that the stone-forming mechanisms may vary. For mixed gallbladder stones, not all loci associated with other types of stones were found, suggesting the complex formation process of mixed gallbladder stones and the potential interaction and influence of nucleating factors. Further studies can be conducted to analyze the role of mucin in mixed gallbladder stones with different combinations of components after elucidating the stone-forming mechanisms of the various components. Many diseases are now believed to result from a combination of genetic and environmental factors, and gallbladder stone disease is no different. In addition to the impact of dietary habits, lifestyle, and other external factors, the influence of heredity and genetic mutations in the development of gallbladder stones is increasingly being emphasized. A study conducted by Taiwanese researchers indicated that various mucin gene variations have different effects on male and female patients with gallbladder stones, and that the MUC1 rs4072037 and MUC2 rs7396030 loci are risk factors for gallbladder stones in males but do not significantly increase the risk of gallbladder stones in females [ 29 ]. After categorizing the stones based on their composition, this study revealed that the proportion of mixed stones was higher in male patients compared to females, while the proportion of cholesterol stones was higher in female patients compared to males. Furthermore, no significant difference was observed at the MUC1 rs4072037 locus when comparing all types of gallbladder stones with the control group. If we aim to compare gender differences in mucin gene polymorphisms, the comparison should be based on the same type of gallbladder stones to ensure more precise results. Due to the small sample size in each gender group after further categorization, gender differences could not be further analyzed in this study. Mutations in mucin gene polymorphic sites may impact the transcription and translation process of mucin genes, alter the spatial structure or characteristics of various mucins, or even enhance or reduce mucin expression levels, thereby affecting the formation of gallbladder stones. This requires further confirmation through additional research. The next step is to investigate whether different prevention and treatment methods are needed for various types of stones, and to identify susceptibility genes in high-risk individuals to determine the types of stones prone to gallbladder stone formation, in order to develop personalized prevention and treatment plans. In this study, we conducted an analysis of mucin gene polymorphisms in different types of gallbladder stones from a genetics perspective. We confirmed the association between various categories of gallbladder stones and sex polymorphisms in mucin genes. This serves as the basis for identifying susceptibility genes for different types of gallbladder stones and for further studying the underlying mechanisms. However, there are some limitations. The sample size for each stone type was relatively small. In the future, it will be necessary to expand the sample size of the gallbladder stone group and even conduct a multi-racial, multi-regional, and multi-center study to further validate the conclusions. This study only explored the genetic association of mucin with gallbladder stones and did not deeply investigate the specific pathways and mechanisms through which genetic polymorphisms influence the development of gallbladder stones. These limitations need to be addressed, and more comprehensive and in-depth studies are needed to further explore the specific mechanisms by which mucin contributes to the development of different types of gallbladder stones.

The statistical analysis of all data and information was operated by SPSS 26.0 statistical software. Measurement information conforming to normal distribution was expressed as mean ± standard deviation, information not conforming to normal distribution was expressed as median (interquartile spacing), and counting information was expressed as number and percentage; t test was used to test the continuous variables, chi-square test was used to test the categorical variables, and the differences in genotype frequency and allele frequency between two groups and the Hardy–Weinberg equilibrium test was used as the chi-square test. The associations of mucin single nucleotide polymorphisms, clinical data and laboratory indicators with different types of gallbladder stones were evaluated by logistic regression analysis, and the corresponding odds ratios (OR) and 95% confidence intervals (95% CI) were calculated. P -values less than 0.05 was considered statistically significant.

Gallstone disease is a common surgical condition worldwide. In recent years, with changes in people's lifestyle and diet, the incidence of gallstones has gradually increased [ 1 , 2 ]. According to statistics, the incidence rate of gallstones in the European population has reached 20% [ 2 , 3 ], and China's incidence of gallstones is also showing an upward trend year by year. However, there has been little progress in the study of the mechanism of gallstone formation, as well as in the methods of prevention and treatment. Contemporary consensus attributes the genesis of gallbladder stones to various factors including aberrant cholesterol and bilirubin metabolism [ 4 – 8 ], diminished gallbladder motility [ 9 ], insulin resistance [ 10 ], biliary tract infections [ 11 – 13 ], and genetic predispositions [ 14 ]. Notably, emerging research has implicated mucins, high molecular weight glycoproteins synthesized and secreted by gallbladder epithelial cells, in the pathogenesis of gallbladder stones [ 2 , 15 , 16 ]. These proteins, characterized by oligosaccharide side chains and gel-like properties [ 17 ], are integral to bile composition and are pivotal in stone nucleation. Mucins found within gallbladder tissue can be categorized into membrane-bound and secretory types. While membrane-bound mucins adhere to the cell membrane, secretory mucins are released into bile, where they form a protective mucus-like layer on the gallbladder mucosa. This layer not only lubricates the mucosa but also shields it from microbial invasion and toxins, thereby conferring a degree of protection [ 17 ]. During gallbladder stone formation, the overproduction of mucins acts as a nucleation facilitator, accelerating the nucleation process of cholesterol crystals [ 18 , 19 ]. Studies have identified over 20 mucin genes, some of which exhibit close associations with gallbladder stone formation [ 17 ]. Specifically, the expression of MUC1, MUC3, MUC5B, and MUC6 is augmented in gallbladder tissues with stones, with MUC2 and MUC4 also detected, the latter potentially linked to cholesterol stone calcification [ 20 – 22 ]. There are different views on the expression of mucins in different types of gallbladder stones. Korean scholars analysed the stone composition and showed that there was no significant difference in the expression of MUC3, MUC5AC and MUC6 in the gallbladders of patients with cholesterol and bile pigment stones groups [ 23 ]. Another study noted significant differences in the expression of MUC5AC and MUC5B in gallbladders with different types of stones, with higher expression especially in patients with bile pigment stones [ 10 ]. In a study on mucin expression in cholesterol stones, researchers found a significant increase in MUC3 and MUC5B in the gallbladder epithelium of patients with cholesterol stones, gallbladder cholesterol deposits, or cholecystitis when performing experiments on gallbladder tissues from patients with cholesterol stones, as well as when performing experiments on LPS-treated canine gallbladder cells, it was found that on LPS-treated canine gallbladder epithelial cells MUC3 and MUC5B also exhibited upregulated expression, all of which suggest that inflammatory stimuli are associated with increased mucin secretion [ 20 ]. In a study of gallbladder mucin in bile pigment stones, MUC5AC expression was found to be more pronounced in bile pigment stones compared to cholesterol stones, which also positively correlated with inflammatory scores in gallbladder pathology [ 24 ]. It has been demonstrated that genetic factors play a significant role in the development of various types of gallbladder stones. Nevertheless, there is a paucity of research investigating the genetics of mucin in gallbladder stones, and the role of this in different types of gallbladder stones remains a valuable area for further investigation. This study aims to probe the role of MUC gene mutations in gallbladder stone formation and explore the correlation between mucin gene polymorphisms and different stone types. Clinical samples, encompassing venous blood specimens and pertinent demographic and laboratory data, were scrutinized for single nucleotide polymorphisms (SNPs) in MUC genes via polymerase chain reaction (PCR) and DNA sequencing. Mutations in SNP loci in the genes ABCG5, ABCG8, UGT1A1, ABCB4, ABCB11, CFTR, CYP7A1, FGFR4, TEAD2, PKD1L3, PNPLA3 have been shown to be associated with gallbladder stone formation [ 25 – 28 ]. Some scholars have explored MUC1 (rs4072037), 2 (rs2856111) are associated with gallstone disease in Chinese men [ 29 ]. Previous studies was focused on the impact of MUC2 (rs2856111), 4 (rs2258447), 17 (rs4729655, rs74974199) on endometriosis development and associated clinical features [ 30 – 32 ]. Hepatocellular carcinoma-related study suggests MUC6 (rs61869016, rs7481521) reduces risk of HCC [ 33 ]. However, less research has been done on Mucin gene polymorphisms in the field of gallbladder stones, and ultimately the 28 relevant SNPs ( MUC1 rs4072037, MUC2 rs41349846/ rs41532344/ rs2856111, MUC4 rs712005/ rs2246980/ rs2258447 /rs2259292, MUC5AC rs28415193/ rs56047977, MUC5B rs2037089/ rs2075854/ rs3829224/ rs2672785/ rs2735709, MUC6 rs10902268/ rs573849895/ rs59257210/ rs61869016/ rs7396383/ rs74644072/ rs7481521/ rs9704308,MUC17 rs10229731/ rs4729655/ rs55903219/ rs73168398/ rs74974199) in gallbladder stone-associated mucin were additionally selected in this study in the hope that they can provide a new reference for the screening of different gallbladder stone susceptibility genes.

Supplementary Material 1: Table S1. PCR primer. Supplementary Material 2: Table S2. Ligation primer. Supplementary Material 3: Table S2. Ligation primer. Supplementary Material 1: Table S1. PCR primer. Supplementary Material 2: Table S2. Ligation primer. Supplementary Material 3: Table S2. Ligation primer.