The Study Of The Correlation Between CAPS Gene Mutation And Recurrent Miscarriage

Part 1 Identification of the pathogenic gene and mutation in a consanguineous family with RPLObjective:This study aimed to identify the pathogenic gene and mutation of a consanguineous family with recurrent pregnancy loss(RPL)through whole exome sequencing(WES)technology,and provide new clues and evidence for understanding the cause of RPL.Materials and methods:A consanguineous family,including the parents who were cousins with no history of spontaneous abortion and their three daughters who had been diagnosed as non-syndromic RPL,was recruited in this study.The three patients were chosen for WES on illumina hiseq2500 platform.All WES data were filtered and the rare nonsynonymous homozygous variants shared by all three patients were preserved.Then,through further comprehensive screening and analysis,the optimal candidate pathogenic gene and mutation of this RPL family was identified and the genotype-disease phenotype co-segregation analysis was performed among the family members.Sanger sequencing for coding regions and splicing sites of CAPS was performed in another 264 unrelated RPL patients and rare variants were identified.Results:Pedigree analysis suggested the presence of a recessive inheritance model.After filtering all WES data,a total of six rare nonsynonymous homozygous variants shared by 3 patients were preserved.Further analysis indicated that a rare homozygous nonsense mutation,CAPS(NM 004058.5:c.377delC,p.Leu127Trpfs),might be the potential pathogenic gene and mutation for this RPL family.Sanger sequencing confirmed that the three patients carried the homozygous p.Leu127Trpfs while their parents carried the heterozygous p.Leu127Trpfs.We identified another three additional rare heterozygous variants in three unrelated RPL cases,while no rare homozygous or compound heterozygous variants were found.Conclusions:Our results indicated that the autosomal homozygous nonsense mutation,p.Leu127Trpfs,in CAPS might be a maternal effect causative mutation of RPL pathogenesis.Part 2 Biological effects of CAPS in RPLObjective:This study aimed to explore the possible biological effects of Calcyphosine(CAPS)in RPL.Materials and methods:The expression and distribution of CAPS in decidual and placental villi tissues from patients who underwent voluntary termination of early pregnancy were measured by immunofluorescence.The expression of CAPS in decidua and placental villi tissues from unrelated RPL patients and controls was measured by qPCR.For human endometrial epithelial RL95-2 cell,the CAPS-specific siRNA was verified by qPCR and Western blot;the expression of Secreted Phosphoprotein 1(SPP1)and Matrix Metallopeptidase 9(MMP9)were also measured by qPCR;the concentration of Prostaglandin E2(PGE2)in cell culture supernatant was evaluated by ELIS A;intracellular calcium ion(Ca2+)concentration was determined by Fluo-3,AM combined with flow cytometry.For human placenta choriocarcinoma JAR cell:cell migration and invasion were detected by transwell;cell apoptosis was measured by flow cytometry.Human decidual gland epithelial cells in early pregnancy were separated and cultured,then confirmed by immunofluorescence.Double-label immunofluorescence with laser confocal microscopy were used to detect the subcellular localization of CAPS and investigate if it can be co-localized with endoplasmic reticulum marker PDIA3.Results:In human decidual tissue during early pregnancy,CAPS was only expressed in decidual gland epithelial cells.In human placental villus tissue,CAPS was only expressed in syncytiotrophoblasts.Compared to the controls,the expression of CAPS was down-regulated in the decidua and placental villi tissues from unrelated RPL patients.Knockdown of CAPS expression in RL95-2 cell might promote the expression of SPP1 and MMP9 which are endometrial receptivity related markers,and the release of PGE2.Meantime,it might also decrease the intracellular Ca2+concentration.Besides,knockdown of CAPS might lead to a slight decrease in JAR cell migration and invasion,but might not affect JAR cell apoptosis.CAPS could colocalize with PDIA3 in the primary decidual glandular epithelial cells,suggesting that CAPS distributed in the endoplasmic reticulum.Conclusions:CAPS was expressed in human decidual gland epithelial cells from early pregnancy and distributed in the endoplasmic reticulum.Knockdown CAPS expression might promote endometrial receptivity-related factors by reducing Ca2+ concentration in decidual glandular epithelial cells,leading to the occurrence of RPL.