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Whole-exome sequencing reveals ANO8 as a genetic risk factor for intrahepatic cholestasis of pregnancy

Background Intrahepatic cholestasis of pregnancy (ICP) is characterized by pruritus and cholestasis in late pregnancy and results in adverse pregnancy outcomes, including preterm delivery and birth weight, which are affected by the genetic and environmental background. However, until now, the genetic architecture of ICP has remained largely unclear. Methods Twenty-six clinical data points were recorded for 151 Chinese ICP patients. The data generated from whole-exome sequencing (WES) using the BGISEQ-500 platform were further analyzed by Burrows-Wheeler Aligner (BWA) software, Genome Analysis Toolkit (GATK), ANNOVAR tool, etc. R packages were used to conduct t-test, Fisher’s test and receiver operating characteristic (ROC) curve analyses. Results We identified eighteen possible pathogenic loci associated with ICP disease in known genes, covering ABCB4, ABCB11, ATP8B1 and TJP2. The loci Lys386Gln, Gly527Gln and Trp708Ter in ABCB4, Leu589Met, Gln605Pro and Gln1194Ter in ABCB11, and Arg189Ser in TJP2 were novel discoveries. In addition, WES analysis indicated that the gene ANO8 involved in the transport of bile salts is newly identified as associated with ICP. The functional network of the ANO8 gene confirmed this finding. ANO8 contained 8 rare missense mutations that were found in eight patients among the 151 cases and were absent from 1029 controls. Out of the eight SNPs, 3 were known, and the remaining five are newly identified. These variants have a low frequency, ranging from 0.000008 to 0.00001 in the ExAC, gnomAD – Genomes and TOPMED databases. Bioinformatics analysis showed that the sites and their corresponding amino acids were both highly conserved among vertebrates. Moreover, the influences of all the mutations on protein function were predicted to be damaging by the SIFT tool. Combining clinical data, it was found that the mutation group (93.36 µmol/L) had significantly (P = 0.038) higher total bile acid (TBA) levels than the wild-type group (40.81 µmol/L). Conclusions To the best of our knowledge, this is the first study to employ WES technology to detect genetic loci for ICP. Our results provide new insights into the genetic basis of ICP and will benefit the final identification of the underlying mutations.


Background
Intrahepatic cholestasis of pregnancy is a pregnancyrelated liver disease that mainly occurs in the second and third trimesters of pregnancy and is characterized by pruritus and abnormal liver functions [1]. The symptoms and biochemical abnormalities usually rapidly disappeared after delivery. The incidence of ICP ranges from below 1% to above 15%, with obvious regional and ethnic differences and familial clustering [2]. In China, it also reaches as high as 5.2% [3]. The recurrence rate of ICP in subsequent pregnancies reaches approximately 40% − 60% [1]. ICP increases the risk for adverse pregnancy and perinatal outcomes, including spontaneous preterm birth, intrauterine distress and amniotic fluid fecal infection [4,5]. The serum bile acid levels in patients increase the risk of adverse perinatal outcomes [6,7]. Therefore, understanding the molecular basis of ICP disease is very important.
Obviously, ICP is a complex disease that depends on multiple interacting factors, including genetics, endocrine hormones, nutrition and the environment [8]. In recent years, whole-genome and whole-exome sequencing have proven to be powerful new approaches to identify disease-associated variants across the full minor allele frequency (MAF) spectrum in animals [9] and humans [10]. Moreover, the 1000 Genomes Project revealed that rare variants constitute the majority of polymorphic sites in human populations [11]. In particular, accumulating evidence has demonstrated that lowfrequency (0.01 ≤ MAF < 0.05) and rare (MAF < 0.01) variations often have a large effect on complex disease etiologies. Increasingly abundant examples of rare variants acting collectively for relevant quantitative traits in medicine have been noted. For example, a previous study revealed that four rare mutations of the IFIH1 gene act independently on type 1 diabetes (TID) risk [12].
Since the first ABCB4 mutation in ICP in Caucasians was reported in 1999, the efforts of many researchers have been dedicated to understanding the mechanism of ICP in many different laboratories across Europe [13]. However, deciphering the genetic basis of ICP disease is still a major challenge. To date, only a handful of causative genes (such as ABCB4 and ABCB11) [14] have been identified via genealogical analysis and Sanger sequencing. In recent years, many studies have addressed the role of the ATP8B1 and TJP2 genes in ICP susceptibility and identified some possible effect loci associated with ICP [14,15]. Identification of the association of these genes with ICP disease is helpful to provide timely diagnosis and appropriate medical intervention for ICP pregnant women to avoid adverse maternal and fetal outcomes. Therefore, it is of great importance to identify a large number of ICP susceptibility genes that remain undiscovered.
The anoctamin family contains 10 members (ANO1-10) with two major functions: Ca 2+ -dependent ion channels (ANO1 and ANO2) and/or Ca 2+ -activated lipid scramblases with nonselective ion channel activity (ANO3-4, ANO6-8) [16][17][18]. The ANO protein family is widely expressed in eukaryotes, exhibits diverse functions in cells throughout the body and is associated with several human diseases [19]. For example, ANO1 plays roles in membrane excitability in olfactory transduction [19] and affects bile secretion and formation [20]. ANO8 encodes the transmembrane protein 16H and plays a role in the transport of glucose and other sugars, bile salts and organic acids, metal ions and amine compounds and ion channel transport, according to the functional annotation of the GeneCards. Moreover, Alaish SM et al. previously reported that ANO8 was differentially expressed in intestinal tissue between AJ (mouse strain) common bile duct ligation (CBDL) and sham-operated mice [21], suggesting that ANO8 plays a role in hepatobiliary disease. Therefore, we extrapolated and hypothesized that mutations in the ANO8 gene might affect the protein expression level and thus the transport function of bile salts.
To the best of our knowledge, only a minority of studies have addressed the genetic loci for ICP disease. However, among them, there have been no papers researching ICP with whole-exome sequencing technology. Thus, the objectives of this work were to analyze genetic mutations and putative pathogenic genes associated with clinical data in a sample of 151 Han Chinese individuals with ICP using WES data. A total of 8 mutations in the ANO8 gene were identified in eight of the 151 individuals.

Samples and clinical features
Peripheral blood samples from 151 Han Chinese ICP patients were collected from the Department of Obstetrics, Jiangxi Provincial Maternal and Child Health Hospital in Nanchang, China. A total of 27 available clinical features, including the age at diagnosis; body mass index (BMI); gestational age; the concentrations of K, Na, Cl, Ca, Mg, and P; white blood cell (WBC), red blood cell (RBC), and platelet (PLT) counts; red blood cell distribution width SD (RDW-SD); alanine transaminase (ALT), aspartate transaminase (AST), total bile acids (TBA), total bilirubin (TBIL), direct bilirubin (DBIL), indirect bilirubin (IDBIL), total cholesterol (CHOL), triglyceride (TG), high-density lipoprotein (HDL), low-density lipoprotein (LDL), and uric acid (UA) levels; newborn birth weight; Apgar score and bleeding amount were recorded. The ion concentration, liver function and lipid index were determined by an AU5800 automatic biochemical analyzer (Beckman Coulter). Routine blood tests were performed using a Sysmex-xn-2000 automatic blood cell analyzer. Summary statistics for all clinical data investigated are shown in Table 1. In addition, 1029 female control individuals without ICP were recruited. Written informed consent was obtained from each participated women in this study.

Whole-exome sequencing
A total of 151 human genomic DNA samples were isolated from peripheral blood using an Axy Prep Blood Genomic DNA Mini Prep Kit (item No. 05119KC3). DNA quality and concentration were determined by a NanoDrop-1000 spectrophotometer (Thermo Fisher, USA) and gel electrophoresis, respectively. Qualified genomic DNA samples were randomly fragmented, and the size of the library fragments was mainly distributed between 150 bp and 250 bp. End repair of DNA fragments was performed, and an "A" base was added at the 3'-end of each strand. Then, adapters were used to ligate to both ends of the end-repaired/dA-tailed DNA fragments for amplification and sequencing. Amplified DNA fragments were then purified and hybridized to a BGI Exon array. The captured products were then further amplified by circularization. Each qualified captured DNA library was then loaded on BGISEQ-500 platforms. Finally, we obtained the raw sequencing data, which were stored in FASTQ format for each individual. The informatics analysis, mainly including quality control, read mapping, variant calling, filtering and annotation, was conducted by using BWA software, GATK and ANNOVAR tool, respectively.

Statistical analysis
The t-test method was performed to analyze the potential significant differences between ANO8 mutations and wild types for the available clinical features. The P value is two sided, and the result was considered significantly different at P < 0.05. Fisher's test was conducted to test the significance of differences in frequencies between different groups. In addition, we performed logistic regression for the IPD (individual patient data) analysis to obtain the area under the ROC curve, e.g., AUC, for the association between premature birth and TBA, ALT and AST. All the above-mentioned analyses were carried out with R software.

Evolutionary conservation analysis
The evolutionary conservation analysis of sites and amino acids was performed in 17 representative vertebrate species, human, chimpanzee, gibbon, macaque, olive baboon, mouse, rat, cow, goat, sheep, pig, dog, dingo, cat, leopard, horse, and elephant, using the genomic alignments of the Ensembl Genome Browser.

The WES data results
We performed whole-exome sequencing of 151 DNA samples with an average of 14003.98 Mb of raw bases. After removing low-quality reads, we obtained an average of 139,940,436 clean reads (13991.34 Mb). The clean reads of each sample had high Q20 and Q30, which showed high sequencing quality. The average GC content was 51.20%. Figure 1 shows the base percentage composition along reads and the distributions of base quality scores on clean reads of one ICP sample (ICP66). The chromosomal positions of SNPs were based on the UCSC GRCh37/hg19. We obtained a total of 72,729 variants, including nonsynonymous, missense, splicing, start lost, stop lost/gained variants. First, we excluded variants with MAF ≥ 0.01 from the 1000 Genomes Project (http://www.internationalgen ome.org/), ExAC (http://exac.broadinstitute.org/) and dbSNP ((https://www.ncbi.nlm.nih.gov/snp) databases, and 22,956 SNPs were included in subsequent analysis. In addition, 3094 variants were preserved using overlapping methods by the 1029 controls. Then, we ranked the genes and their possible damaging loci using the prediction tool SIFT to assess whether a variant affected protein function. The results implied that the gene ANO8 was prominent based on its functional annotation related to bile acid transport and pathogenicity prediction of mutations in genes, in addition to the known functional genes ABCB4, ABCB11, ATP8B1 and TJP2.
The genetic variants of ABCB4, ABCB11, ATP8B1 and TJP2 We identified a total of 61 genetic variants, including 46 intron, 6 synonymous, 8 missense, and 1 nonsense variants, in the ABCB4 gene. Among them, three variants, two missense variants, Lys386Glu and Gly527-Glu, and a nonsense variant, Trp708Ter, were novel and reported for the first time. In addition, another two variants, rs1202754797 and rs201502889, were also identified in the ABCB4 gene (Table 2). For the ABCB11 gene, we observed five variants: Leu589Met, Gln605Pro, Gln1194Ter, Tyr1130Cys and Arg696Trp. The first three were newly identified mutations. After quality control, we also identified 3 and five possible pathogenic loci in ATP8B1 and TJP2, respectively. The 3 loci were Thr9Met, Gly473Arg and Arg628Trp in    Table 2).
The eight variants of the ANO8 gene In addition, interestingly, we found that a total of eight missense mutations in the ANO8 gene in eight out of the 151 samples from patients with ICP disease ( In addition, 122 women of the 151 sampled women delivered their babies. Out of the 122 women, ninety individuals (73.3%, 90/122) gave birth by cesarean section, whereas 32 (26.7%, 32/122) gave birth by vaginal delivery. Thirty-two (26.7%, 32/122) delivered their babies prematurely, and 17 infants' (13.9%, 17/122) birth weights were below 2.5 kg. Three of the 6 babies were born preterm.

Sanger sequencing to validate ANO8 variants and an additional 1029 control individuals
A total of six pairs of primers (Table 4) were designed to amplify PCR products. Then, a comparative analysis of missense mutations of the ANO8 gene was conducted by DNA sequencing from eight ICP patients and an additional 1029 control individuals with WES sequencing. Figure 2 shows the sequencing electropherograms of the known SNP rs1391524054 and the novel mutation rs1.

Assessing the functional impact of rareANO8 variants
These eight ANO8 mutations were absent from the 1000 Genomes Project and 1029 local controls from our hospital. Additionally, the MAFs of these mutations were low, ranging from 8e-6 to 1e-5 in three databases, e.g., ExAC, gnomAD -Genomes and TOPMED. Using Fisher's test method, we found no significant differences in the frequencies of the variants between the 151 cases and 1029 controls (P = 0.13); in contrast, the frequencies in the databases were significantly different. This relatively lower significance between cases and controls (P = 0.13) than between cases and databases might be due to the number of samples involved.
Furthermore, we evaluated the influence of these eight mutations on protein function by using the web-available tool SIFT (http://sift.bii.a-star.edu.sg/) and obtained a score. As a SIFT score less than 0.05 is considered damaging, an amino acid substitution with such a score would be detrimental to the function of ANO8. We found that all these variants were predicted to be damaging (Table 5).

Evolutionary conservation analysis
Evolutionary conservation analysis showed that the rs1 site wild-type nucleotide allele (C) and its corresponding amino acid (proline) were both highly conserved among vertebrates, e.g., pigs, cows, sheep, dogs and cats (Fig. 3).

Tissue expression
We used the human base website (https://hb.flatironin stitute.org) to predict ANO8 gene expression, function, regulation, and interactions in humans. The gene expression results showed that ANO8 was expressed in liver tissue with reasonable confidence (0.71). This result To simultaneously analyze the function of ANO8, we further explored the biological process of ANO8, including the transport of inorganic anions, anions and chloride and the transmembrane transport of the above three ions. In addition, a functional network that captured liver tissuespecific interactions covering 5 data types, namely, coexpression, interaction, TF binding, GSEA microRNA targets and GSEA perturbations, from large data compendia was produced (Fig. 4). The results showed that the genes in the functional network were relevant to transport, such as EPHA1 [22], CELSR3, C10orf71, CDC14B, TM9SF4 [23], and the Wnt signaling pathway, including APC [24], IER5L, OBSL1, and MED12 [25], suggesting that the function of the ANO8 protein was likely to be related to the transport of bile salts.

Correlations between mutations and clinical data
In the 151 ICP samples, regardless of whether the difference was significant, the mutation group tended to be associated with higher Ca 2+ concentrations, platelet counts, TBA levels, TG levels, and bleeding amounts and lower birth weights (Table 6). Notably, the mutation group had significantly (P = 0.038) a higher TBA level than the wild-type group. Moreover, that of the mutation group (93.36 µmol/L) was 2-fold greater than that of the wildtype group (40.81 µmol/L). Moreover, TBA measured by fasting peripheral blood of pregnant women is an important indicator of ICP diagnosis. The IPD analysis (Fig. 5) showed that the TBA level was more highly predictive of premature birth (AUC: 0.670 [95% CI 0548-0.768]) than the ALT and AST levels. The preterm delivery need increased at a TBA cut-off value of 46.05 µmol/L.

Discussion
So far, most reseachers make efforts to dissect the genetic architecture of ICP disease primarily focusing on ABCB4. Previously, three studies simultaneously identified ABCB4 Ile237Ile (rs2109505) as significantly associated with ICP [26][27][28]. These loci were also detected in our population. The MAFs in the 1000 Genomes Project and ExAC databases were 0.26 and 0.27, respectively. We hypothesize that this locus contributes to disease susceptibility by linkage disequilibrium between rs2109505 and the causative variant. Our study confirmed the role of ABCB11 and further expanded the role of ABCB11 gene which encoded the bile salt export pump. Our result confirmed previous studies have shown that the presence of Arg696Trp mutation in ICP population. In addition to the Arg696Trp mutation, other three novel mutations, including one prematurely stop codon Gln1194Ter, and two missense mutations Gln605Pro and Leu589Pro, were predicted  Fig. 2 The sequencing electropherograms of rs1316267732 and rs1 mutations in the ANO8 gene. The mutation location is marked with an arrow   Tables 2 and 3 as pathogenic. Besides, we did not identify any loci corresponding to ATP8B1 and TJP2 in the previous ICP literature [14,29,30], a reasonable explanation for this discrepancy may be the distinct genetic background and genetic heterogeneity of the populations. Combined with clinical data, we found that 80.79% (122/151) delived the baby, in which, 26.7% (32/122) birthed prematurely and 13.9% (17/122) of the newborns weighted less than 2.5 kg. Similarily, the eight patients with ANO8 mutations having 3 newborns delivered prematurely and two spontaneous abortion The above results suggested that women with ICP had increased adverse perinatal outcome incidences, e.g., premature birth, abortion and reduced birth weight, which was consistent with the results of previous studies [2,31]. Besides, the eight patients with ANO8 variants did not carry the possible potential effect loci of the known functional genes, ABCB4, ABCB11, ATP8B1 and TJP2, for ICP disease, implying that these ICP cases with ANO8 mutations are not caused by these mutation of functional known genes.
Bioinformatics analysis suggested that these eight variants in ANO8 gene might play an important role in the etiology of ICP disease. However, ICP disease is regulated by multiple rare variants independently or aggregatively, and further experimental verification is needed. For example, a previous study [32] employed exome array analysis to identify five new loci and lowfrequency variants influencing insulin processing and secretion. Cohen et al. [33] reported that the aggregation of multiple rare variants has been associated with reduced sterol absorption and plasma low-density lipoprotein levels.
Based on the expression and function results of ANO8 combined with literature reports, the function of ANO8 was likely to be related to the transport of bile salts in the liver. Therefore, the mutations in the ANO8 gene identified in ICP women could cause bile acid transport disorder, which leads to bile acid accumulation in liver    tissue. Of course, it is noteworthy that the role of the ANO8 gene and its mutations in cholestasis of pregnancy is based on bioinformatics analysis derived from WES data and network data. It remains to be determined whether ANO8 mutations cause structural and functional defects in ANO8. Therefore, subsequent cell function and in vivo experiments for ANO8 are particularly important. Compared with wild-type group, we found that mutation group of ANO8 gene has higher TBA levels, TG levels and lower birth weights, suggesting that these mutations of the ANO8 gene might be positively involved in the pathogenesis of ICP disease. In addition, recent studies have also reported that TBA levels ≥ 40 µmol/L increased the risk of perinatal complications, such as low Apgar scores, stillbirth and preterm labor [6,34,35], which was consistent with our result, e.g. TBA level of 46.05 µmol/L were a critical value in increasing preterm labor.

Conclusions
In conclusion, by whole-genome sequencing analysis, we identified 18 possible pathogenic loci associated with ICP in the ABCB4, ABCB11, ATP8B1 and TJP2 genes, seven of which were novel loci. Furthermore, 8 missense mutations, including 3 known and five novel mutations, were detected in the ANO8 gene in eight of 151 Han ICP patients. To the best of our knowledge, this study is the first report revealing mutations for ICP disease by WES. By Sanger sequencing, conservation analysis, and protein functional prediction analysis, we confirmed that these variants existed and were associated with ICP. Further research should target the molecular mechanisms of these mutations in ICP disease. Our study provides new insights into the genetic architecture of ICP disease and may contribute to ICP genetic diagnosis.