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

ABSTRACT: 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.

SUBMITTER: Liu X

PROVIDER: S-EPMC7499841 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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

Liu Xianxian X Lai Hua H Zeng Xiaoming X Xin Siming S Nie Liju L Liang Zhenyi Z Wu Meiling M Chen Yu Y Zheng Jiusheng J Zou Yang Y

BMC pregnancy and childbirth 20200917 1

<h4>Background</h4>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.<h4>Methods</h4>Twenty-six clinical data points were recorded for 151 Chinese ICP patients. The data generated from whole-exome sequencing (W ...[more]

PMID: 32942997

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Project description:Preterm birth is a leading cause of morbidity and mortality in infants. Genetic and environmental factors play a role in the susceptibility to preterm birth, but despite many investigations, the genetic basis for preterm birth remain largely unknown. Our objective was to identify rare, possibly damaging, nucleotide variants in mothers from families with recurrent spontaneous preterm births (SPTB). DNA samples from 17 Finnish mothers who delivered at least one infant preterm were subjected to whole exome sequencing. All mothers were of northern Finnish origin and were from seven multiplex families. Additional replication samples of European origin consisted of 93 Danish sister pairs (and two sister triads), all with a history of a preterm delivery. Rare exonic variants (frequency <1%) were analyzed to identify genes and pathways likely to affect SPTB susceptibility. We identified rare, possibly damaging, variants in genes that were common to multiple affected individuals. The glucocorticoid receptor signaling pathway was the most significant (p<1.7e-8) with genes containing these variants in a subgroup of ten Finnish mothers, each having had 2-4 SPTBs. This pathway was replicated among the Danish sister pairs. A gene in this pathway, heat shock protein family A (Hsp70) member 1 like (HSPA1L), contains two likely damaging missense alleles that were found in four different Finnish families. One of the variants (rs34620296) had a higher frequency in cases compared to controls (0.0025 vs. 0.0010, p = 0.002) in a large preterm birth genome-wide association study (GWAS) consisting of mothers of general European ancestry. Sister pairs in replication samples also shared rare, likely damaging HSPA1L variants. Furthermore, in silico analysis predicted an additional phosphorylation site generated by rs34620296 that could potentially affect chaperone activity or HSPA1L protein stability. Finally, in vitro functional experiment showed a link between HSPA1L activity and decidualization. In conclusion, rare, likely damaging, variants in HSPA1L were observed in multiple families with recurrent SPTB.

Project description:BackgroundIntrahepatic cholestasis of pregnancy (ICP) can cause premature delivery and stillbirth. Previous studies have reported that mutations in ABC transporter genes strongly influence the transport of bile salts. However, to date, their effects are still largely elusive.MethodsA whole-exome sequencing (WES) approach was used to detect novel variants. Rare novel exonic variants (minor allele frequencies: MAF < 1%) were analyzed. Three web-available tools, namely, SIFT, Mutation Taster and FATHMM, were used to predict protein damage. Protein structure modeling and comparisons between reference and modified protein structures were performed by SWISS-MODEL and Chimera 1.14rc, respectively.ResultsWe detected a total of 2953 mutations in 44 ABC family transporter genes. When the MAF of loci was controlled in all databases at less than 0.01, 320 mutations were reserved for further analysis. Among these mutations, 42 were novel. We classified these loci into four groups (the damaging, probably damaging, possibly damaging, and neutral groups) according to the prediction results, of which 7 novel possible pathogenic mutations were identified that were located in known functional genes, including ABCB4 (Trp708Ter, Gly527Glu and Lys386Glu), ABCB11 (Gln1194Ter, Gln605Pro and Leu589Met) and ABCC2 (Ser1342Tyr), in the damaging group. New mutations in the first two genes were reported in our recent article. In addition, compared to the wild-type protein structure, the ABCC2 Ser1342Tyr-modified protein structure showed a slight change in the chemical bond lengths of ATP ligand-binding amino acid side chains. In placental tissue, the expression level of the ABCC2 gene in patients with ICP was significantly higher (P < 0.05) than that in healthy pregnant women. In particular, the patients with two mutations in ABC family genes had higher average values of total bile acids (TBA), aspartate transaminase (AST), direct bilirubin (DBIL), total cholesterol (CHOL), triglycerides (TG) and high-density lipoprotein (HDL) than the patients who had one mutation, no mutation in ABC genes and local controls.ConclusionsOur present study provide new insight into the genetic architecture of ICP and will benefit the final identification of the underlying mutations.

Project description:Background: Intrahepatic cholestasis of pregnancy (ICP) is associated with a high incidence of fetal morbidity and mortality. Therefore, revealing the mechanisms involved in ICP and its association with fetal complications is very important. Methods: Here, we used a whole-exome sequencing (WES) approach to detect novel mutations of organic anion transporting polypeptide (OTAP) genes, ATP-binding cassette transporter (ABC) genes, and receptor genes associated with ICP in 249 individuals and 1,029 local control individuals. Two available tools, SIFT and PolyPhen-2, were used to predict protein damage. Protein structuremodeling and comparison between the reference and modified protein structures were conducted by SWISS-MODEL and Chimera 1.14rc software, respectively. Results: A total of 5,583 mutations were identified in 82 genes related to bile acid transporters and receptors, of which 62 were novel mutations. These novel mutations were absent in the 1,029 control individuals and three databases, including the 1,000 Genome Project (1000G_ALL), Exome Aggregation Consortium (ExAC), and Single-Nucleotide Polymorphism Database (dbSNP). We classified the 62 novel loci into two groups (damaging and probably damaging) according to the results of SIFT and PolyPhen-2. Out of the 62 novel mutations, 24 were detected in the damaging group. Of these, five novel possibly pathogenic variants were identified that were located in known functional genes, including ABCB4 (Ile377Asn), ABCB11 (Ala588Pro), ABCC2 (Ile681Lys and Met688Thr), and NR1H4 (Tyr149Ter). Moreover, compared to the wild-type protein structure, ABCC2 Ile681Lys and Met688Thr protein structures showed a slight change in the chemical bond lengths of ATP-ligand binding amino acid side chains. The combined 32 clinical data points indicate that the mutation group had a significantly (p = 0.04) lower level of Cl ions than the wild-type group. Particularly, patients with the 24 novel mutations had higher average values of alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), total bile acids (TBA), high-density lipoprotein (HDL), and low-density lipoprotein (LDL) than patients with the 38 novel mutations in the probably damaging group and the local control individuals. Conclusion: The present study provides new insights into the genetic architecture of ICP involving these novel mutations.

Project description:Study hypothesisExome sequencing can identify genetic causes of idiopathic recurrent pregnancy loss (RPL).Study findingWe identified compound heterozygous deleterious mutations affecting DYNC2H1 and ALOX15 in two out of four families with RPL. Both genes have a role in early development. Bioinformatics analysis of all genes with rare and putatively pathogenic mutations in miscarriages and couples showed enrichment in pathways relevant to pregnancy loss, including the complement and coagulation cascades pathways.What is known alreadyNext generation sequencing (NGS) is increasingly being used to identify known and novel gene mutations in children with developmental delay and in fetuses with ultrasound-detected anomalies. In contrast, NGS is rarely used to study pregnancy loss. Chromosome microarray analysis detects putatively causative DNA copy number variants (CNVs) in ∼2% of miscarriages and CNVs of unknown significance (predominantly parental in origin) in up to 40% of miscarriages. Therefore, a large number of miscarriages still have an unknown cause.Study design, samples/materials, methodsWhole exome sequencing (WES) was performed using Illumina HiSeq 2000 platform on seven euploid miscarriages from four families with RPL. Golden Helix SVS v8.1.5 was used for data assessment and inheritance analysis for deleterious DNA variants predicted to severely disrupt protein-coding genes by introducing a frameshift, loss of the stop codon, gain of the stop codon, changes in splicing or the initial codon. Webgestalt (http://bioinfo.vanderbilt.edu/webgestalt/) was used for pathway and disease association enrichment analysis of a gene pool containing putatively pathogenic variants in miscarriages and couples in comparison to control gene pools.Main results and the role of chanceCompound heterozygous mutations in DYNC2H1 and ALOX15 were identified in miscarriages from two families with RPL. DYNC2H1 is involved in cilia biogenesis and has been associated with fetal lethality in humans. ALOX15 is expressed in placenta and its dysregulation has been associated with inflammation, placental, dysfunction, abnormal oxidative stress response and angiogenesis. The pool of putatively pathogenic single nucleotide variants (SNVs) and small insertions and deletions (indels) detected in the miscarriages showed enrichment in 'complement and coagulation cascades pathway', and 'ciliary motility disorders'. We conclude that CNVs, individual SNVs and pool of deleterious gene mutations identified by exome sequencing could contribute to RPL.Limitations, reasons for cautionThe size of our sample cohort is small. The functional effect of candidate mutations should be evaluated to determine whether the mutations are causative.Wider implications of the findingsThis is the first study to assess whether SNVs may contribute to the pathogenesis of miscarriage. Furthermore, our findings suggest that collective effect of mutations in relevant biological pathways could be implicated in RPL.Study funding and competing interestsThe study was funded by Canadian Institutes of Health Research (grant MOP 106467) and Michael Smith Foundation of Health Research Career Scholar salary award to ERS.

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

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

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