Voltar
Artigo Acesso aberto Revisado por pares
BIBTEX RIS

CAPS Mutations Are Potentially Associated with Unexplained Recurrent Pregnancy Loss

2018; Elsevier BV; Volume: 189; Issue: 1 Linguagem: Inglês

10.1016/j.ajpath.2018.09.010

ISSN

1525-2191

Autores

Hong Pan, Huifen Xiang, Jing Wang, Zhaolian Wei, Yiran Zhou, Beihong Liu, Tengyan Li, Xu Ma, Yunxia Cao, Binbin Wang,

Tópico(s)

Pregnancy and preeclampsia studies

Resumo

Recurrent pregnancy loss (RPL) is a major concern for women's reproductive health. Several studies have proved that genetics is a major factor leading to unexplained RPL, but the maternal pathogenic genes involved in RPL remain largely unknown. A consanguineous family, including the parents who were cousins and their three daughters who had been diagnosed as having nonsyndromic unexplained RPL, was recruited in this study. A rare homozygous variant in calcyphosine (CAPS; ENST00000588776: c.377delC, p.Leu127Trpfs) might be the potential candidate variant for this RPL family through whole-exome sequencing. Sanger sequencing confirmed that the three affected sisters carried the homozygous p.Leu127Trpfs, whereas their parents carried the heterozygous p.Leu127Trpfs. CAPS encodes a Ca2+-binding protein and may play a role in the regulation of Ca2+ transport. Although the precise underlying mechanisms remain unclear, the previous study suggested that they may be involved in cross talk between Ca2+ signaling and cAMP–protein kinase A pathways, which are crucial to embryo implantation and pregnancy maintenance. Knockdown of CAPS expression might promote the expression of secreted phosphoprotein 1 and matrix metalloproteinase 9, and the release of prostaglandin E2, which all played important roles in embryo implantation and early pregnancy maintenance. These results indicated that the autosomal recessive homozygous mutation, p.Leu127Trpfs, in CAPS might be a maternal effect causative mutation of RPL pathogenesis. Recurrent pregnancy loss (RPL) is a major concern for women's reproductive health. Several studies have proved that genetics is a major factor leading to unexplained RPL, but the maternal pathogenic genes involved in RPL remain largely unknown. A consanguineous family, including the parents who were cousins and their three daughters who had been diagnosed as having nonsyndromic unexplained RPL, was recruited in this study. A rare homozygous variant in calcyphosine (CAPS; ENST00000588776: c.377delC, p.Leu127Trpfs) might be the potential candidate variant for this RPL family through whole-exome sequencing. Sanger sequencing confirmed that the three affected sisters carried the homozygous p.Leu127Trpfs, whereas their parents carried the heterozygous p.Leu127Trpfs. CAPS encodes a Ca2+-binding protein and may play a role in the regulation of Ca2+ transport. Although the precise underlying mechanisms remain unclear, the previous study suggested that they may be involved in cross talk between Ca2+ signaling and cAMP–protein kinase A pathways, which are crucial to embryo implantation and pregnancy maintenance. Knockdown of CAPS expression might promote the expression of secreted phosphoprotein 1 and matrix metalloproteinase 9, and the release of prostaglandin E2, which all played important roles in embryo implantation and early pregnancy maintenance. These results indicated that the autosomal recessive homozygous mutation, p.Leu127Trpfs, in CAPS might be a maternal effect causative mutation of RPL pathogenesis. Recurrent pregnancy loss (RPL) is a serious reproductive health problem; according to the American Society for Reproductive Medicine, RPL is defined as two or more clinical pregnancy losses that affect 2% to 5% of all couples.1Practice Committee of American Society for Reproductive MedicineEvaluation and treatment of recurrent pregnancy loss: a committee opinion.Fertil Steril. 2012; 98: 1103-1111Abstract Full Text Full Text PDF PubMed Scopus (668) Google Scholar, 2El Hachem H. Crepaux V. May-Panloup P. Descamps P. Legendre G. Bouet P.E. Recurrent pregnancy loss: current perspectives.Int J Womens Health. 2017; 9: 331-345Crossref PubMed Scopus (182) Google Scholar The causes of RPL are various, including uterine factors, antiphospholipid syndrome, inherited thrombophilias, and endocrine, cytogenetic, environmental, and psychological factors.2El Hachem H. Crepaux V. May-Panloup P. Descamps P. Legendre G. Bouet P.E. Recurrent pregnancy loss: current perspectives.Int J Womens Health. 2017; 9: 331-345Crossref PubMed Scopus (182) Google Scholar However, after testing for all these causes, some women have unidentified causes and are considered as having unexplained RPL (URPL).3Kutteh W.H. Novel strategies for the management of recurrent pregnancy loss.Semin Reprod Med. 2015; 33: 161-168Crossref PubMed Scopus (38) Google Scholar Many studies have proved that genetics is a major factor leading to URPL, and many candidate genes have been identified. A recent meta-analysis, including 428 case-control studies, identified 21 variants of 13 genes that might contribute to URPL.4Pereza N. Ostojic S. Kapovic M. Peterlin B. Systematic review and meta-analysis of genetic association studies in idiopathic recurrent spontaneous abortion.Fertil Steril. 2017; 107: 150-159.e2Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar However, owing to the large differences in sample collection between the studies, the associations were all moderate with RPL. Recently, whole-exome sequencing (WES) was used to identify novel causative genes in RPL patients. A study using WES identified compound heterozygous mutations in dynein cytoplasmic 2 heavy chain 1 (DYNC2H1; NM_001080463) and arachidonate 15-lipoxygenase (ALOX15; NM_001140) in miscarriages from two RPL families.5Qiao Y. Wen J. Tang F. Martell S. Shomer N. Leung P.C. Stephenson M.D. Rajcan-Separovic E. Whole exome sequencing in recurrent early pregnancy loss.Mol Hum Reprod. 2016; 22: 364-372Crossref PubMed Scopus (54) Google Scholar Another recent study using WES in 49 independent URPL patients identified 27 coding heterozygous variants in 22 genes that belonged to distinct pathways related to implantation and pregnancy outcomes.6Quintero-Ronderos P. Mercier E. Fukuda M. Gonzalez R. Suarez C.F. Patarroyo M.A. Vaiman D. Gris J.C. Laissue P. Novel genes and mutations in patients affected by recurrent pregnancy loss.PLoS One. 2017; 12: e0186149Crossref PubMed Scopus (42) Google Scholar However, owing to miscarriages or the lack of segregation analysis, limited progress has been made in identifying the maternal pathogenic genes for RPL. Calcyphosine (CAPS; NM_004058) encodes a Ca2+-binding protein, belonging to the EF hand motif family, that may play a role in the regulation of Ca2+ transport.7Dong H. Li X. Lou Z. Xu X. Su D. Zhou X. Zhou W. Bartlam M. Rao Z. Crystal-structure and biochemical characterization of recombinant human calcyphosine delineates a novel EF-hand-containing protein family.J Mol Biol. 2008; 383: 455-464Crossref PubMed Scopus (16) Google Scholar Although the precise mechanism of CAPS remains unclear, the previous study suggested that it may be involved in the cross talk between Ca2+ signaling and cAMP–protein kinase A (PKA) pathways.8Lefort A. Lecocq R. Libert F. Lamy F. Swillens S. Vassart G. Dumont J.E. Cloning and sequencing of a calcium-binding protein regulated by cyclic AMP in the thyroid.EMBO J. 1989; 8: 111-116Crossref PubMed Scopus (40) Google Scholar Therefore, CAPS might be related to RPL because of the regulation of Ca2+ signaling and cAMP-PKA pathways, which are crucial to embryo implantation and pregnancy maintenance.9Kusama K. Yoshie M. Tamura K. Imakawa K. Isaka K. Tachikawa E. Regulatory action of calcium ion on cyclic AMP-enhanced expression of implantation-related factors in human endometrial cells.PLoS One. 2015; 10: e0132017Crossref PubMed Scopus (19) Google Scholar, 10Salker M.S. Singh Y. Durairaj R. Yan J. Alauddin M. Zeng N. Steel J.H. Zhang S. Nautiyal J. Webster Z. Brucker S.Y. Wallwiener D. Anne C.B. Brosens J.J. Lang F. LEFTY2 inhibits endometrial receptivity by downregulating Orai1 expression and store-operated Ca(2+) entry.J Mol Med. 2018; 96: 173-182Crossref PubMed Scopus (11) Google Scholar Herein, a consanguineous family, including the parents who were cousins and all three daughters who had been diagnosed as having nonsyndromic URPL, was recruited. The phenotype of this family suggested a maternal homozygous mutation shared by the three sisters. To identify the likely causal mutation, WES was performed in the three affected sisters for shared rare homozygous variants, and an autosomal recessive frameshift mutation in CAPS was identified. The aim of this study is to identify a potential maternal effect causative mutation of RPL pathogenesis using WES technology in an RPL family. All subjects were recruited from the Center for Reproductive Medicine at The First Affiliated Hospital of Anhui Medical University between 2015 and 2018. According to the American Society for Reproductive Medicine's criteria, the peripheral blood of the three sisters diagnosed as having nonsyndromic URPL and their parents, who were cousins, was collected. In addition, 264 independent RPL patients were included in the study. All clinical information available is shown in Table 1. The decidual and placental villi tissues from five independent URPL patients (cases) and five deliberate pregnancy termination cases with no history of spontaneous abortions (controls) were also collected. The study was approved by the Institute Ethics Committee, National Research Institute for Family Planning, and written informed consent was obtained from all subjects.Table 1Clinical Features of Familial and Independent RPL Patients with Mutations in CAPSPatient numberCAPS mutation (ENST00000588776)GenotypeSpontaneous abortion, nAge at diagnosis, yearsMenstrual historyUterusHormonalKaryotypeTORCHAnti-β2GP1ACA, ANA, and anti-dsDNAHepatitisInfectionsChildbearingMale factorNucleotideAmino acidFamily members II-1c.377delCp.Leu127TrpfsHOM530NormalNormalNormal46, XX−−−−No1None II-2c.377delCp.Leu127TrpfsHOM226NormalNormalNormal46, XX−−−−No1None II-3c.377delCp.Leu127TrpfsHOM223NormalNormalNormal46, XX−−−−No0NoneIndependent RPL cases 1c.274G>Ap.A92THET226NormalNormalNormal46, XX−−−−No1None 2c.685C>Tp.R229CHET229NormalNormalNormal46, XX−−−−No0None 3c.764_765insTp.S256Efs*8HET231NormalNormalNormal46, XX−−−−No0None−, negative; ACA, anticardiolipin antibody; ANA, antinuclear antibody; dsDNA, double-stranded DNA; β2GP1, β2 glycoprotein 1; HET, heterozygous; HOM, homozygous; RPL, recurrent pregnancy loss; TORCH, Toxoplasma gondii others rubella virus cytomegalovirus herpes simplex viruses 1 and 2. Open table in a new tab −, negative; ACA, anticardiolipin antibody; ANA, antinuclear antibody; dsDNA, double-stranded DNA; β2GP1, β2 glycoprotein 1; HET, heterozygous; HOM, homozygous; RPL, recurrent pregnancy loss; TORCH, Toxoplasma gondii others rubella virus cytomegalovirus herpes simplex viruses 1 and 2. Genomic DNA samples from the three sisters were used to perform WES. Exomes were captured with SureSelect Human All Exon V5 Kit (Agilent Technologies, Santa Clara, CA), and DNA sequencing was performed on the HiSeq 2500 platform (Illumina, San Diego, CA). BWA version 0.7.12 (http://bio-bwa.sourceforge.net; OMICS_00654)11Li H. Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform.Bioinformatics. 2009; 25: 1754-1760Crossref PubMed Scopus (26628) Google Scholar was used for mapping. The variants were referenced to hg19 genome (http://genome.ucsc.edu, last accessed May 2, 2018) and annotated using SAMtools version 0.1.19 (http://samtools.sourceforge.net).12Li H. Handsaker B. Wysoker A. Fennell T. Ruan J. Homer N. Marth G. Abecasis G. Durbin R. 1000 Genome project data processing subgroup. The sequence alignment/map (SAM) format and SAMtools.Bioinformatics. 2009; 25: 2078-2079Crossref PubMed Scopus (31532) Google Scholar Rare homozygous mutations [missense, nonsense, frameshift, or splice site variants; frequency, <1% in 1000 Genomes Project database, Single Nucleotide Polymorphism Database, National Heart, Lung, and Blood Institute Exome Sequencing Project database, Exome Variant Server (ExAC) database, and Genome Aggregation Database r2.0.2 (http://gnomad.broadinstitute.org)] were retained. Then, the genotypes of the candidate variants were confirmed in the family members by Sanger sequencing. All the coding regions of candidate CAPS gene were sequenced in another 264 independent RPL patients. The primer sequences are shown in Table 2. Mutationtaster (http://www.mutationtaster.org, last accessed April 14, 2018),13Schwarz J.M. Cooper D.N. Schuelke M. Seelow D. MutationTaster2: mutation prediction for the deep-sequencing age.Nat Methods. 2014; 11: 361-362Crossref PubMed Scopus (2419) Google Scholar PolyPhen-2 version 2.2.2 (http://genetics.bwh.harvard.edu/pph2, last accessed April 14, 2018),14Adzhubei I.A. Schmidt S. Peshkin L. Ramensky V.E. Gerasimova A. Bork P. Kondrashov A.S. Sunyaev S.R. A method and server for predicting damaging missense mutations.Nat Methods. 2010; 7: 248-249Crossref PubMed Scopus (9284) Google Scholar and PROVEAN version 1.1.3 (http://provean.jcvi.org/index.php, last accessed August 17, 2018)15Choi Y. Sims G.E. Murphy S. Miller J.R. Chan A.P. Predicting the functional effect of amino acid substitutions and indels.PLoS ONE. 2012; 7 (e46688)Crossref Scopus (1937) Google Scholar were used to predict the effect of the mutations on protein structure and function. Conservation analysis was performed using Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo, last accessed August 14, 2018).16Sievers F. Wilm A. Dineen D.G. Gibson T.J. Karplus K. Li W. Lopez R. McWilliam H. Remmert M. Söding J. Thompson J.D. Higgins D.G. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega.Mol Syst Biol. 2011; 7: 539Crossref PubMed Scopus (9092) Google ScholarTable 2Primers for Sanger Sequencing and Real-Time RT-PCRVariableIdentificationSequencesLength, bpSanger sequencingCAPS-1F5′-GAGCCAGCCCACTAAGTCACGT-3′903CAPS-1R5′-CTGTGGCACTGCATCCCTTCCT-3′CAPS-2F5′-CTGTCCAGGGTCACACAGGAATCAT-3′858CAPS-2R5′-CTCCGCATCTCAGCATCCTCTGT-3′CAPS-3F5′-GGACTCCTCCCTAAGAGCTGCTGT-3′765CAPS-3R5′-CTGGTCAGCATGGCGTGGGT-3′CAPS-4F5′-GCAGGACTGGCTAGACCAGGT-3′790CAPS-4R5′-CCAGCTAGTGACGAGTACAACGAGAT-3′Real-time RT-PCRβ-Actin-F5′-CTCCATCCTGGCCTCGCTGT-3′1175′-ACTAAGTCATAGTCCGCCTAGA-3′CAPS-qPCR-F5′-GTCCAGGGTCACACAGGAATC-3′1845′-GACTGCTTACTCAACTGCCAG-3′SPP1-qPCR-F5′-CTCCATTGACTCGAACGACTC-3′2305′-CAGGTCTGCGAAACTTCTTAGAT-3′MMP9-qPCR-F5′-TGTACCGCTATGGTTACACTCG-3′975′-GGCAGGGACAGTTGCTTCT-3′ Open table in a new tab The mRNA levels of CAPS in decidual and placental villi tissues from the five URPL patients and the five controls were analyzed by real-time RT-PCR (RT-qPCR). Each sample was analyzed in triplicate, and data were analyzed using the 2−△△Ct method. Melting curve analysis and agarose gel electrophoresis confirmed the amplification specificity. Primers were picked from PrimerBank (https://pga.mgh.harvard.edu/primerbank, last accessed April 5, 2018), and the sequences are shown in Table 2. The human endometrial epithelial RL95-2 (CRL 1671; ATCC, Manassas, VA) and human placenta choriocarcinoma JAR (HTB 144; ATCC) cell lines were maintained in medium supplemented with 10% fetal bovine serum, 100 mg/mL penicillin, and 100 mg/mL streptomycin in 5% CO2 at 37°C. For transient transfection, the cells were seeded in 6-well culture dishes and transfected with siRNA-CAPS using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA), according to the manufacturer's protocol. The siRNA set for CAPS, including three pairs of siRNA-CAPS, positive control, negative control (NC), and FAM-labeled NC, was purchased from Genepharma (Shanghai, China). Real-time RT-PCR and Western blot analysis were used to detect the expression of CAPS. Each assay was run in triplicate with each set of primers, and data were analyzed using the 2−△△Ct method. Melting curve analysis and Sanger sequencing confirmed the amplification specificity. The mRNA level of each sample was normalized to β-actin mRNA. Densitometry of bands was analyzed using the ImageJ software version k 1.45 (NIH, Bethesda, MD; https://imagej.nih.gov/ij). The CAPS–homo-919 duplexes were as follows: 5′-GGAUCUGGAGGAGUUCCUUTT-3′ (forward) and 5′-AAGGAACUCCUCCAGAUCCTT-3′ (reverse). Cells were seeded in 6-well plates and transfected with CAPS–homo-919 and NC in the following assays. The human endometrial epithelial RL95-2 cell line was used to detect the biomarkers related to endometrial receptivity and implantation using real-time RT-PCR. Each assay was run in duplicate with each set of primers. The mRNA level of each sample was normalized to β-actin mRNA. The sequences of the primers for secreted phosphoprotein 1 (SPP1; NM_001251830) and matrix metalloproteinase-9 (MMP9; NM_004994) are listed in Table 2. The human endometrial epithelial RL95-2 cell line was used to detect the content of prostaglandin E2 (PGE2) release. After treatment, cell-free supernatant was collected and PGE2 content was measured using a PGE2 enzyme-linked immunosorbent assay kit, monoclonal (Cayman Chemical, Ann Arbor, MI). Fetal bovine serum–free JAR cells were added to the upper chamber, and 500 μL medium with 25% fetal bovine serum was placed in the lower compartment. After incubation, the nonmigrated cells in the upper chamber were removed with a cotton swab. The migrated cells were fixed with 100% methanol, stained with 0.5% crystal violet, and washed with phosphate-buffered saline. The membranes were observed under a light microscope, and three fields were randomly selected to represent each membrane. As for the invasion assay, 70 μL Matrigel diluted with Dulbecco's modified Eagle's medium was used to plate each transwell chamber and was incubated for 4 hours at 37°C, and the same procedure for migration assay was conducted. Statistical analysis was performed using GraphPad Prism 5 (GraphPad Inc., San Diego, CA). The independent-sample t-test was applied to compare the data, and P < 0.05 was considered statistically significant. Three sisters (II-1, II-2, and II-3), from a consanguineous Chinese family (Figure 1A), experienced unexplained RPL. Their parents, who were cousins, had given birth to three daughters and one son, without a history of spontaneous miscarriages. The peripheral blood and clinical information from all of them were collected in 2015 (Table 1). The proband (II-1) with the age at diagnosis of 30 years, who had been married for 8 years and had a healthy girl during her second pregnancy, miscarried her first and third to sixth pregnancies for unknown reasons. Her sister (II-2) with the age at diagnosis of 26 years, who had been married for 6 years and had a healthy girl during her second pregnancy, miscarried her first and third pregnancies for unknown reasons. Another sister (II-3) with the age at diagnosis of 23 years, who had been married for 2 years, miscarried twice for unknown reasons. They all had normal karyotype, hormone levels, menstrual history, and uterus. They were negative for TORCH (Toxoplasma gondii others rubella virus cytomegalovirus herpes simplex viruses 1 and 2), anti-β2 glycoprotein 1, anticardiolipin antibody, antinuclear antibody, anti–double-stranded DNA, hepatitis, and infections during screening. Male factor was excluded as well. In addition, the mean age of the 264 independent RPL patients was 29.26 years, and they all experienced at least two spontaneous miscarriages with unknown reasons (Table 1). In the consanguineous RPL family, three affected sisters (II-1, II-2, and II-3) were chosen for WES. Pedigree analysis suggested the presence of a recessive inheritance model. Therefore, rare homozygous mutations (missense, nonsense, frameshift, or splice site variants; frequency, <1% in 1000 Genomes Project database, Single Nucleotide Polymorphism Database, Exome Sequencing Project database, ExAC database, and Genome Aggregation Database) that were shared by all three sisters were retained (Supplemental Table S1). Six nonsynonymous homozygous variants in CAPS (NM_004058), patatin-like phospholipase domain containing 6 (PNPLA6; NM_001166114), semaphorin 6B (SEMA6B; NM_032108), TBC1 domain family member 25 (TBC1D25; NM_002536), WWC family member 3 (WWC3; NM_015691), and zinc finger protein 441 (ZNF441, NM_152355) were identified (Supplemental Table S28Lefort A. Lecocq R. Libert F. Lamy F. Swillens S. Vassart G. Dumont J.E. Cloning and sequencing of a calcium-binding protein regulated by cyclic AMP in the thyroid.EMBO J. 1989; 8: 111-116Crossref PubMed Scopus (40) Google Scholar, 17Synofzik M. Gonzalez M.A. Lourenco C.M. Coutelier M. Haack T.B. Rebelo A. Hannequin D. Strom T.M. Prokisch H. Kernstock C. Durr A. Schols L. Lima-Martinez M.M. Farooq A. Schule R. Stevanin G. Marques W.J. Züchner S. PNPLA6 mutations cause Boucher-Neuhauser and Gordon Holmes syndromes as part of a broad neurodegenerative spectrum.Brain. 2014; 137: 69-77Crossref PubMed Scopus (151) Google Scholar, 18Collet P. Domenjoud L. Devignes M.D. Murad H. Schohn H. Dauca M. The human semaphorin 6B gene is down regulated by PPARs.Genomics. 2004; 83: 1141-1150Crossref PubMed Scopus (21) Google Scholar, 19Itoh T. Kanno E. Uemura T. Waguri S. Fukuda M. OATL1, a novel autophagosome-resident Rab33B-GAP, regulates autophagosomal maturation.J Cell Biol. 2011; 192: 839-853Crossref PubMed Scopus (126) Google Scholar, 20Han Q. Lin X. Zhang X. Jiang G. Zhang Y. Miao Y. Rong X. Zheng X. Han Y. Han X. Wu J. Kremerskothen J. Wang E. WWC3 regulates the Wnt and Hippo pathways via Dishevelled proteins and large tumour suppressor 1, to suppress lung cancer invasion and metastasis.J Pathol. 2017; 242: 435-447Crossref PubMed Scopus (50) Google Scholar, 21Wang Y. Jiang M. Yao Y. Cai Z. WWC3 inhibits glioma cell proliferation through suppressing the Wnt/beta-catenin signaling pathway.DNA Cell Biol. 2018; 37: 31-37Crossref PubMed Scopus (16) Google Scholar). Given the frequency and location of the mutations, the effect on protein structure and function predicted by Mutationtaster, PolyPhen-2, PROVEAN, and SIFT, the gene expression profile, the conservation among several species, the published phenotype of knockout mice from Mouse Genome Informatics, the variants reported in the ClinVar database (https://www.ncbi.nlm.nih.gov/clinvar), the function of the genes, and the three sisters' phenotypes, all six rare nonsynonymous homozygous variants were thoroughly screened. The SEMA6B and WWC3 variants were predicted to not alter the protein structure and function and were not conserved among species. The ZNF441 variant was not conserved and had not been previously reported. The TBC1D25 variant was located near the C-terminal end of the TBC1D25 protein, which may not affect the protein features. Furthermore, all these variants have not been reported to relate to human reproductive diseases so far. PNPLA6 was thought to function in the nervous system, and PNPLA6 variants were always related to severe syndromes, such as spastic paraplegia 39 [Online Mendelian Inheritance in Man (OMIM) number 612020],22Rainier S. Bui M. Mark E. Thomas D. Tokarz D. Ming L. Delaney C. Richardson R.J. Albers J.W. Matsunami N. Stevens J. Coon H. Leppert M. Fink J.K. Neuropathy target esterase gene mutations cause motor neuron disease.Am J Hum Genet. 2008; 82: 780-785Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar Boucher-Neuhauser syndrome (OMIM number 215470),17Synofzik M. Gonzalez M.A. Lourenco C.M. Coutelier M. Haack T.B. Rebelo A. Hannequin D. Strom T.M. Prokisch H. Kernstock C. Durr A. Schols L. Lima-Martinez M.M. Farooq A. Schule R. Stevanin G. Marques W.J. Züchner S. PNPLA6 mutations cause Boucher-Neuhauser and Gordon Holmes syndromes as part of a broad neurodegenerative spectrum.Brain. 2014; 137: 69-77Crossref PubMed Scopus (151) Google Scholar Laurence-Moon syndrome (OMIM number 245800),23Hufnagel R.B. Arno G. Hein N.D. Hersheson J. Prasad M. Anderson Y. Krueger L.A. Gregory L.C. Stoetzel C. Jaworek T.J. Hull S. Li A. Plagnol V. Willen C.M. Morgan T.M. Prows C.A. Hegde R.S. Riazuddin S. Grabowski G.A. Richardson R.J. Dieterich K. Huang T. Revesz T. Martinez-Barbera J.P. Sisk R.A. Jefferies C. Houlden H. Dattani M.T. Fink J.K. Dollfus H. Moore A.T. Ahmed Z.M. Neuropathy target esterase impairments cause Oliver-McFarlane and Laurence-Moon syndromes.J Med Genet. 2015; 52: 85-94Crossref PubMed Scopus (74) Google Scholar Oliver-McFarlane syndrome (OMIM number 275400),23Hufnagel R.B. Arno G. Hein N.D. Hersheson J. Prasad M. Anderson Y. Krueger L.A. Gregory L.C. Stoetzel C. Jaworek T.J. Hull S. Li A. Plagnol V. Willen C.M. Morgan T.M. Prows C.A. Hegde R.S. Riazuddin S. Grabowski G.A. Richardson R.J. Dieterich K. Huang T. Revesz T. Martinez-Barbera J.P. Sisk R.A. Jefferies C. Houlden H. Dattani M.T. Fink J.K. Dollfus H. Moore A.T. Ahmed Z.M. Neuropathy target esterase impairments cause Oliver-McFarlane and Laurence-Moon syndromes.J Med Genet. 2015; 52: 85-94Crossref PubMed Scopus (74) Google Scholar and Gordon Holmes syndrome (OMIM number 212840).24Topaloglu A.K. Lomniczi A. Kretzschmar D. Dissen G.A. Kotan L.D. McArdle C.A. Koc A.F. Hamel B.C. Guclu M. Papatya E.D. Eren E. Mengen E. Gurbuz F. Cook M. Castellano J.M. Kekil M.B. Mungan N.O. Yuksel B. Ojeda S.R. Loss-of-function mutations in PNPLA6 encoding neuropathy target esterase underlie pubertal failure and neurological deficits in Gordon Holmes syndrome.J Clin Endocrinol Metab. 2014; 99: E2067-E2075Crossref PubMed Scopus (78) Google Scholar However, the three sisters were healthy and had no sign of any identified disease, except URPL. Furthermore, hypogonadotropic hypogonadism is one of the classic characteristics of Boucher-Neuhauser syndrome and Gordon Holmes syndrome, which is associated with underdevelopment of the gonads and infertility.17Synofzik M. Gonzalez M.A. Lourenco C.M. Coutelier M. Haack T.B. Rebelo A. Hannequin D. Strom T.M. Prokisch H. Kernstock C. Durr A. Schols L. Lima-Martinez M.M. Farooq A. Schule R. Stevanin G. Marques W.J. Züchner S. PNPLA6 mutations cause Boucher-Neuhauser and Gordon Holmes syndromes as part of a broad neurodegenerative spectrum.Brain. 2014; 137: 69-77Crossref PubMed Scopus (151) Google Scholar, 24Topaloglu A.K. Lomniczi A. Kretzschmar D. Dissen G.A. Kotan L.D. McArdle C.A. Koc A.F. Hamel B.C. Guclu M. Papatya E.D. Eren E. Mengen E. Gurbuz F. Cook M. Castellano J.M. Kekil M.B. Mungan N.O. Yuksel B. Ojeda S.R. Loss-of-function mutations in PNPLA6 encoding neuropathy target esterase underlie pubertal failure and neurological deficits in Gordon Holmes syndrome.J Clin Endocrinol Metab. 2014; 99: E2067-E2075Crossref PubMed Scopus (78) Google Scholar Therefore, the homozygous variant in CAPS (ENST00000588776: c.377delC, p.Leu127Trpfs), a frameshift mutation caused by a single-base deletion, was the potential candidate for this RPL family (Figure 1B). The CAPS variant was not previously reported in 1000 Genomes Project, Exome Sequencing Project, and ExAC databases, and was likely to cause a loss-of-function mutation. This amino acid residue is highly conserved across human, chimpanzee, cattle, rabbit, and dog (Figure 1C). Furthermore, no homozygous loss-of-function mutation has been registered in the ExAC database, and the minor allele frequency of any loss-of-function mutation in CAPS is A, p.A92T; c.685C>T, p.R229C; c.764_765insT, p.S256Efs*8) in three patients; however, no homozygous or compound heterozygous variants were found (Table 1). The p.A92T and p.S256Efs*8 variants were not previously reported. The p.R229C variant was reported once in the ExAC database. Among them, a truncated protein would result from the p.S256Efs*8 variant. The decidual and placental villi tissues were collected from five independent URPL patients who had experienced two consecutive spontaneous abortions because of unknown reasons. The couples' karyotypes were all normal. Tissues were also collected from five deliberate pregnancy termination cases that had no history of spontaneous abortions as controls. Compared with controls, the expression of CAPS was down-regulated in decidual and placental villous tissues of RPL patients (P < 0.05) (Figure 2). Real-time RT-PCR and Western blot analysis were used to detect CAPS expression and select the most effective CAPS siRNA. According to the data, CAPS expression in CAPS–homo-919–transfected cells showed a significant decrease compared with the other groups, and the effectiveness of CAPS–homo-919 was best among the three CAPS siRNAs (data not shown). Therefore, CAPS–homo-919 was used for CAPS knockdown in the following experiments. To explore the possible role of CAPS in embryo implantation, the expression of several endometrial receptivity markers was detected after CAPS–homo-919 transfection. Compared with controls, Real-time RT-PCR showed that the expression of SPP1 and MMP9 was up-regulated in RL95-2 cells after down-regulation of CAPS expression (Figure 3A). SPP1 and MMP9 play important roles in implantation and early pregnancy establishment.25Johnson G.A. Burghardt R.C. Bazer F.W. Spencer T.E. Osteopontin: roles in implantation and placentation.Biol Reprod. 2003; 69: 1458-1471Crossref PubMed Scopus (264) Google Scholar, 26Vu T.H. Werb Z. Matrix metalloproteinases: effectors of development and normal physiology.Genes Dev. 2000; 14: 2123-2133Crossref PubMed Scopus (1045) Google Scholar The expression levels of SPP1 and MMP9 were up-regulated by 2.04- and 2.82-fold, respectively. At the epithelial-stromal interface, PGE2, released from endometrial epithelial cells driven by Ca2+ influx, was shown to be essential for stromal cell decidualization and embryo implantation.27Ruan Y.C. Guo J.H. Liu X. Zhang R. Tsang L.L. Dong J.D. Chen H. Yu M.K. Jiang X. Zhang X.H. Fok K.L. Chung Y.W. Huang H. Zhou W.L. Chan H.C. Activation of the epithelial Na+ channel triggers prostaglandin E(2) release and production required for embryo implantation.Nat Med. 2012; 18: 1112-1117Crossref PubMed Scopus (121) Google Scholar The human endometrial epithelial RL95-2 cells are generally used as a model for providing secretory products and receptive epithelial cells.28Hannan N.J. Paiva P. Dimitriadis E. Salamonsen L.A. Models for study of human embryo implantation: choice of cell lines?.Biol Reprod. 2010; 82: 235-245Crossref PubMed Scopus (227) Google Scholar An enzyme-linked immunosorbent assay showed that CAPS–homo-919 could induce a dramatic increase in PGE2 release from the RL95-2 cells (P < 0.05), compared with NC (Figure 3B). Trophoblast migration and invasion are critical features of implantation and placental development. To examine the possible effect of CAPS knockdown on cell migration and invasion, the transwell assays were performed using JAR cells transfected with CAPS–homo-919 or NC. The transwell assay showed that CAPS–homo-919 could inhibit JAR cell migration compared with NC (P < 0.01) (Supplemental Figure S1A). In addition, the transwell assay with Matrigel revealed that CAPS–homo-919 could affect JAR cell invasion compared with NC (P < 0.01) (Supplemental Figure S1B). These results indicated that knockdown of CAPS might lead to a slight decrease in JAR cell migration and invasion. In a consanguineous Chinese family of three sisters with nonsyndromic URPL, an autosomal recessive frameshift mutation was identified in CAPS (ENST00000588776: c.377delC, p.Leu127Trpfs) by WES, which is a possible link to URPL. The results indicated that the homozygous mutation, p.Leu127Trpfs, in CAPS might be the maternal effect causative mutation of RPL pathogenesis. These data demonstrated that CAPS might be the maternal recessive causative mutation in this nonsyndromic URPL family. Three sisters from a consanguineous Chinese family experienced at least two spontaneous miscarriages for unknown reasons. Pedigree analysis suggested there might be a maternal recessive homozygous mutation shared by the three sisters. Through WES and subsequent bioinformatic analysis, it was found that the homozygous variant in CAPS, a frameshift mutation caused by a single-base deletion, was the candidate causative mutation in this RPL family. The CAPS variant (ENST00000588776: c.377delC, p.Leu127Trpfs) had never been reported in 1000 Genomes Project, Exome Sequencing Project, and ExAC databases, and was likely to cause a loss-of-function mutation. Besides, no homozygous loss-of-function mutation has been registered in the ExAC database, and the minor allele frequency of any loss-of-function mutation in CAPS is low enough to explain the autosomal recessive model of this rare condition. To determine the contribution of the CAPS gene to RPL, all the exons of CAPS were screened by Sanger sequencing in 264 independent cases with RPL, and no homozygous or compound heterozygous variants were identified. Three heterozygous mutations in three independent cases with RPL were identified, which might not be responsible for RPL pathogenesis because of heterozygosity because the mother of the three sisters also carried the heterozygous p.Leu127Trpfs and gave birth to four children without a history of RPL. Thus, these data suggested that a biallelic CAPS mutation is a possible link to URPL and that further screening the biallelic CAPS mutation from unrelated families would strengthen the genetic evidence. The Ca2+ signaling pathway is known to participate in the regulation of several implantation-related events, such as adhesion of the blastocyst to the endometrium,29Thie M. Denker H.W. In vitro studies on endometrial adhesiveness for trophoblast: cellular dynamics in uterine epithelial cells.Cells Tissues Organs. 2002; 172: 237-252Crossref PubMed Scopus (60) Google Scholar prostaglandin release from endometrial epithelial cells and the process of stromal cell decidualization,27Ruan Y.C. Guo J.H. Liu X. Zhang R. Tsang L.L. Dong J.D. Chen H. Yu M.K. Jiang X. Zhang X.H. Fok K.L. Chung Y.W. Huang H. Zhou W.L. Chan H.C. Activation of the epithelial Na+ channel triggers prostaglandin E(2) release and production required for embryo implantation.Nat Med. 2012; 18: 1112-1117Crossref PubMed Scopus (121) Google Scholar and the expression of implantation factors.30Banerjee P. Ghosh S. Dutta M. Subramani E. Khalpada J. Roychoudhury S. Chakravarty B. Chaudhury K. Identification of key contributory factors responsible for vascular dysfunction in idiopathic recurrent spontaneous miscarriage.PLoS One. 2013; 8: e80940Crossref PubMed Scopus (59) Google Scholar Similar to the Ca2+ signaling pathway, the cAMP-PKA pathway is also essential for embryo implantation progression.31Kusama K. Yoshie M. Tamura K. Daikoku T. Takarada T. Tachikawa E. Possible roles of the cAMP-mediators EPAC and RAP1 in decidualization of rat uterus.Reproduction. 2014; 147: 897-906Crossref PubMed Scopus (19) Google Scholar CAPS encodes a Ca2+-binding protein, which might be involved in cross-signaling between Ca2+ signaling and the cAMP-PKA pathway.8Lefort A. Lecocq R. Libert F. Lamy F. Swillens S. Vassart G. Dumont J.E. Cloning and sequencing of a calcium-binding protein regulated by cyclic AMP in the thyroid.EMBO J. 1989; 8: 111-116Crossref PubMed Scopus (40) Google Scholar Moreover, real-time RT-PCR results revealed that the expression of CAPS might be down-regulated in decidual and placental villous tissues of RPL patients. These data suggested that CAPS might be involved in embryo implantation and early pregnancy maintenance. The prolonged receptivity state will cause poor embryo implantation and miscarriage. Thus, a proper transient window of endometrial receptivity is essential for embryo implantation.32Salker M.S. Nautiyal J. Steel J.H. Webster Z. Sucurovic S. Nicou M. Singh Y. Lucas E.S. Murakami K. Chan Y.W. James S. Abdallah Y. Christian M. Croy B.A. Mulac-Jericevic B. Quenby S. Brosens J.J. Disordered IL-33/ST2 activation in decidualizing stromal cells prolongs uterine receptivity in women with recurrent pregnancy loss.PLoS One. 2012; 7: e52252Crossref PubMed Scopus (148) Google Scholar, 33Achache H. Revel A. Endometrial receptivity markers, the journey to successful embryo implantation.Hum Reprod Update. 2006; 12: 731-746Crossref PubMed Scopus (631) Google Scholar SPP1, expressed by endometrial epithelial cells, was reported to act as an important component of the uteroplacental microenvironment and play an essential role in implantation.25Johnson G.A. Burghardt R.C. Bazer F.W. Spencer T.E. Osteopontin: roles in implantation and placentation.Biol Reprod. 2003; 69: 1458-1471Crossref PubMed Scopus (264) Google Scholar Highly expressed MMP9 can promote the degradation of endometrial extracellular matrix during embryo implantation and may play an important role in the occurrence of RPL.34Bischof P. Endocrine, paracrine and autocrine regulation of trophoblastic metalloproteinases.Early Pregnancy. 2001; 5: 30-31PubMed Google Scholar, 35Pereza N. Ostojic S. Volk M. Kapovic M. Peterlin B. Matrix metalloproteinases 1, 2, 3 and 9 functional single-nucleotide polymorphisms in idiopathic recurrent spontaneous abortion.Reprod Biomed Online. 2012; 24: 567-575Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar In addition, increased expression of SPP1 may increase the expression of MMP9.36Desai B. Rogers M.J. Chellaiah M.A. Mechanisms of osteopontin and CD44 as metastatic principles in prostate cancer cells.Mol Cancer. 2007; 6: 18Crossref PubMed Scopus (118) Google Scholar Using the human endometrial epithelial RL95-2 cell line, which is generally used as a model for providing secretory products and receptive epithelial cells, knockdown of CAPS expression was found to increase the expression of SPP1 and MMP9. Besides, the enzyme-linked immunosorbent assay also showed that knockdown of CAPS expression could induce a significant increase in PGE2, an important factor for embryo implantation27Ruan Y.C. Guo J.H. Liu X. Zhang R. Tsang L.L. Dong J.D. Chen H. Yu M.K. Jiang X. Zhang X.H. Fok K.L. Chung Y.W. Huang H. Zhou W.L. Chan H.C. Activation of the epithelial Na+ channel triggers prostaglandin E(2) release and production required for embryo implantation.Nat Med. 2012; 18: 1112-1117Crossref PubMed Scopus (121) Google Scholar and endometrial decidualization, release from the epithelial RL95-2 cells. Therefore, these results indicated that CAPS might be a link to URPL, by regulating the expression of SPP1 and MMP9 and the release of PGE2. However, the pathways and mechanisms linking CAPS and RPL require further elucidation. Besides, to determine the effect of CAPS knockdown on trophoblast cells, a first-trimester trophoblast cell model of JAR choriocarcinoma cells was used. The results confirmed that knockdown of CAPS expression in JAR cells could lead to a slight decrease in cell migration and invasion, which might impair trophoblast infiltration ability. Because the mother of the three sisters gave birth to three daughters carrying homozygous p.L127Wfs*73, the effect of this mutation from the placental side might be limited. In sum, the results indicated that the autosomal recessive homozygous mutation, p.Leu127Trpfs, in CAPS might be a maternal effect causative mutation of RPL pathogenesis. We thank the patients and their family members for participating in this study. Download .doc (.03 MB) Help with doc files Supplemental Table S1 Download .doc (.05 MB) Help with doc files Supplemental Table S2

Referência(s)