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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.

• ANO3,4,5,7,8,9,10 have been detected in intracellular compartments ANO4,5,9 are also expressed in the plasma membrane. • The so-called intracellular anoctamins are all scramblases and ion channels except of ANO8; they are coexpressed in most cell types. • ANO3,4,8,9,10 shape intracellular Ca2+ signals, some have a role in forming ER/PM membrane contact sides. • ANO5 and ANO10 have specific cellular functions in cell membrane repair and endosomal trafficking, respectively. • A diversity of diseases is caused by genetic variations of these anoctamins, like dystonia (ANO3), obesity (ANO4), limb-girdle muscular dystrophy and gnathodiaphyseal dysplasia (ANO5), chronic kidney disease (ANO9), and ataxia (ANO10). Plasma membrane localized anoctamin 1, 2 and 6 (TMEM16A, B, F) have been examined in great detail with respect to structure and function, but much less is known about the other seven intracellular members of this exciting family of proteins. This is probably due to their limited accessibility in intracellular membranous compartments, such as the endoplasmic reticulum (ER) or endosomes. However, these so-called intracellular anoctamins are also found in the plasma membrane (PM) which adds to the confusion regarding their cellular role. Probably all intracellular anoctamins except of ANO8 operate as intracellular phospholipid (PL) scramblases, allowing for Ca2+-activated, passive transport of phospholipids like phosphatidylserine between both membrane leaflets. Probably all of them also conduct ions, which is probably part of their physiological function. In this brief overview, we summarize key findings on the biological functions of ANO3, 4, 5, 7, 8, 9 and 10 (TMEM16C, D, E, G, H, J, K) that are gradually coming to light. Compartmentalized regulation of intracellular Ca2+ signals, tethering of the ER to specific PM contact sites, and control of intracellular vesicular trafficking appear to be some of the functions of intracellular anoctamins, while loss of function and abnormal expression are the cause for various diseases. [Display omitted] [ABSTRACT FROM AUTHOR]

Communication and material transfer between membranes and organelles take place at membrane contact sites (MCSs). MCSs between the ER and PM, the ER/PM junctions, are the sites where the ER Ca2+ sensor STIM1 and the PM Ca2+ influx channel Orai1 cluster. MCSs are formed by tether proteins that bridge the opposing membranes, but the identity and role of these tethers in receptor‐evoked Ca2+ signaling is not well understood. Here, we identified Anoctamin 8 (ANO8) as a key tether in the formation of the ER/PM junctions that is essential for STIM1‐STIM1 interaction and STIM1‐Orai1 interaction and channel activation at a ER/PM PI(4,5)P2‐rich compartment. Moreover, ANO8 assembles all core Ca2+ signaling proteins: Orai1, PMCA, STIM1, IP3 receptors, and SERCA2 at the ER/PM junctions to mediate a novel form of Orai1 channel inactivation by markedly facilitating SERCA2‐mediated Ca2+ influx into the ER. This controls the efficiency of receptor‐stimulated Ca2+ signaling, Ca2+ oscillations, and duration of Orai1 activity to prevent Ca2+ toxicity. These findings reveal the central role of MCSs in determining efficiency and fidelity of cell signaling. Synopsis: ER and plasma membrane (PM) form tight membrane intersections, where Ca2+ sensor STIM1 and influx channel Orai1 cluster to confer receptor‐stimulated signaling. A new screen identifies Anoctamin 8 (ANO8), a mammalian chloride channel family member, as key tether for ER/PM junctions, regulating STIM1‐Orai1 interactions and determining duration of Orai1‐dependent Ca2+ oscillations. ANO8 associates with STIM1 and Oria1 and controls their interaction.ANO8 increases STIM1‐Orai1 clusters at ER/PM junctions.ANO8 mediates slow, SARAF‐independent Orai1 inactivation at very low cytoplasmic calcium concentrations.ANO8 function depends on interaction with PM lipid PI(4,5)P2.ANO8 assembles all core calcium‐signaling factors at the PI(4,5)P2‐rich compartment. [ABSTRACT FROM AUTHOR]