Interpretation
Previous studies have detected genetic mutations to ICP primarily by Sanger sequencing or offspring studies based on a limited number of individuals. The rare (MAF < 0.01) variations that affect ICP have been more challenging to access. Fortunately, WES emerged as an efficient approach to dig for all the mutations of targeted genes. Exonic variants, particularly missense, nonsense, loss-of-function variants, incline to show the most dramatic effect sizes, possessing the greater power for detection. In recent years, in obstetrics and gynecology, there are a number of examples of using WES method to search for the key candidate genes and causative variants. For instance, Huusko et al employed WES revealing HSPA1L as a genetic risk factor for spontaneous preterm birth.22 To our knowledge, this study is the largest analysis to date of the role of mutations of ABC series genes in 151 ICP susceptibility.
Intriguingly, seven novel pathogenic loci in ABCB4 , ABCB11and ABCC2 gene was simultaneously assigned to damaging group, suggesting that the accuracy of our results. Several studies have shown that the heterozygous missense mutations in ABCB4 , especially low-frequency and rare variations, is common responsible for the occurrence of ICP disease, which is consistent with our result.24-26 After filtering the frequency of the data, we identified a total of eight mutations having a MAF < 0.01, seven of which were heterozygous missense and one was nonsense in ABCB4 . The role ofABCB11 in ICP has also been clearly identified, although its contribution seems less than ABCB4 . A comprehensive analysis of multiple previous studies have concluded that up to 5% of ICP cases harbor a monoallelic mutations in ABCB11 .27 Our study confirmed the role of ABCB11 and further expanded the role of ABCB11 gene. Previous studies have shown that the presence of Arg696Trp mutation in ICP samples, which was confirmed in our samples.
The attention and genetic contribution of ABCC2 to ICP was less than that of ABCB4 and ABCB11 . We didn’t detected any mutations which were previously reported in ABCC2 gene associated ICP in this cohort. The reasonable explanation of this discrepancy may be due to distinct genetic background and genetic heterogeneity of populations. Our results demonstrated a small number of variations, especially that one novel pathogenic heterozygous (ABCC2Ser1342Tyr) variant was detected. The function of ABCC2 involved in bile formation. In placental tissue, the expression of ABCC2upregulated, indicating that ABCC2 is also likely overexpressed in the liver tissue of ICP patients, resulting in increased bile formation, thus, implying that Ser1342Tyr most likely belonged to function-gained mutation. More et al also found that the expression ofABCC2 was more closely related in the livers from alcohol cirrhosis cohort, indicating that ABCC2 expression changed in liver related disease.28, which is consistent with the fact that ICP is a liver disease. As for other three genes which expressed differently, we found ABCC6 having a novel mutation His1043Gln which was located in probable damaging group. Therefore, the gene ABCC6 and the loci His1043Gln was of in interest in the development of ICP disease. In our study, we failed to detect the novel mutations ABCE1 and ABCG5 genes, therefore, we conjectured it is probably due to the fact that the known mutations in these two genes associated with ICP.
Because there have been relatively few studies on the genetic basis of ICP diseases, which leaded to many functional genes have not been mined. However, based on previous studies, several researchers found some evidence that the genes may predispose to liver or ICP disease, such asABCB9 associated with Hepatocellular Carcinoma andABCG2associated with ICP.29, 30 Therefore, we hypothesized that the genetic mutations in these unknown function genes excludingABCB4 , ABCB11 and ABCC2 confers susceptibility to ICP, especially damaging group. These findings extended the role of mutations and strengthens the understanding of genetic basis of ICP disease, along with many other ABC family genes.
Bacq et al suggested that the elevations in serum TBA, ALT and AST activity were identified among ICP patients who have mutations in ABC transporter genes.31 In agreement with that, our results confirmed that the mutation group containing 42 novel variants was associated with higher TBA, AST, DBIL, CHOL, TG and HDL (Table S2; and Figure 3). Furthermore, Piatek K et al suggested that the analysis of genotypes’ co-existence pointed to the possibility of the mutated variants of polymorphism of genes having a summation effect on the development of ICP disease.32 Consistent with this result, in our study, we have six ICP patients contain two mutations at once. Notably, ICP harbored both two mutations increased the average TBA level by 17.33, 20.22 and 50.32 μmol/L, respectively, based on the difference in average TBA levels in ICP with two mutations, one mutation, no mutation in ABC transporter genes and no mutation in healthy controls. As expected, so did the AST, DBIL, CHOL, TG and HDL. The reasonable explanation of this situation is that double mutation has additive effect on TBA level, and this accumulation of TBA level results in ICP exacerbation.
Moreover, the significant difference between wild type and mutant type was biochemical index, which suggested that the accumulation of TBA could lead to lipid abnormality. Consistent with this result, researchers found that, bile acids (BA), which well known for its amphipathic nature, is essential to lipid absorption and energy balance in human.33-35 Thus, taken together, our study provides additional support for effect of mutations in the ABC family genes on ICP disease and lipid metabolism.
Not surprisingly, to date, there are now only a handful of reports genetic analysis studies for ICP disease. The present findings not only enrich the molecular basis of the known functional genes (such asABCB4 , ABCB11 and ABCC2 ) but also expand the new candidate genes (ABCA, ABCB, ABCC, ABCD-ABCG) associated with ICP. Our results support the facts the mutations in ABC transporters’ genes lead to ICP disease. Therefore, further work on the genetic mutations involved in ICP pathogenesis has potential to inspire novel therapies for ICP patients.
Recently, there is currently much interest in whether variation in bile salt concentrations and mutations statue of ABCB4 /ABCB11 /ABCC2 /other new candidate genes could be a biomarker for various forms of drug-induced liver injury.36 Furthermore, considering the negative effect of the ABC mutations on mother and fetal outcomes, it is important to genotyped these mutations to timely genetic diagnosed, which, in order to get immediate attention and treatment for ICP susceptible person. Our study provides the evidence that these mutations have potential contribution to the ICP disease.