Report of four new patients with protein-truncating mutations in C6orf221/KHDC3L and colocalization with NLRP7.
ABSTRACT: To date, two maternal-effect genes have been shown to have causative roles in recurrent hydatidiform moles (RHMs); NLRP7 that is mutated in 48-60% of patients with RHMs and C6orf221 (HUGO-approved nomenclature is now KHDC3L), a recently identified gene, that is mutated in 14% of patients with RHMs who are negative for NLRP7 mutations. We sequenced KHDC3L in 97 patients with RHMs and reproductive loss who are mostly negative for NLRP7 mutations. We identified three unrelated patients, each homozygous for one of the two protein-truncating mutations, a novel 4-bp deletion resulting in a frameshift, c.299_302delTCAA, p.Ile100Argfs*2, and a previously described 4-bp deletion, c.322_325delGACT, p.Asp108Ilefs*30, transmitted on a shared haplotype to three patients from different populations. We show that five HM tissues from one of these patients are diploid and biparental similar to HMs from patients with two defective NLRP7 mutations. Using immunofluorescence, we show that KHDC3L protein displays a juxta perinuclear signal and colocalizes with NLRP7 in lymphoblastoid cell lines from normal subjects. Using cell lines from patients, we demonstrate that the KHDC3L mutations do not change the subcellular localization of the protein in hematopoietic cells. Our data highlight the similarities between the two causative genes for RHMs, KHDC3L and NLRP7, in their subcellular localization, the parental contribution to the HM tissues caused by them, and the presence of several founder mutations and variants in both of them indicating positive selection and adaptation.
Project description:Background:Hydatidiform mole (HM) is an abnormal human pregnancy with excessive trophoblastic proliferation and abnormal embryonic development, dividing into two complete HM (CHM) and partial HM (PHM) groups. One subcategory of the CHMs is recurrent and familial, which is known as biparental HM (BiHMs) or recurrent HM (RHM). NLRP7, KHDC3L and PADI6 are maternal-effect genes involved in RHMs. NLRP7 is a major gene responsible for RHMs. This study was performed on patients with molar pregnancies and miscarriage. The aim of this study was to genetic screen for mutations in NLRP7 and KHDC3L genes in an affected woman with previous history of 5RHM and the sibling with history of miscarriage. Materials and Methods:In this experimental study, DNA was extracted from blood samples. KHDC3L and NLRP7 were polymerase chain reaction (PCR) amplified. The PCR products were purified and Sanger sequenced. Results:In this study, there is no mutation in KHDC3L gene but a novel mutation was identified in the NACHT domain of NLRP7 gene. Patient with five recurrent moles had this mutation in the homozygous state while her sister with one miscarriage and one normal child showed this mutation in the heterozygous state. Conclusion:In this study, we identified a new mutation in NLRP7 gene of a patient with recurrent HM. Following egg donation, this patient has a normal boy. The sister of this patient with heterozygous mutation has a spontaneous abortion and one normal child that confirm the impact of a defective allele of NLRP7 on reproductive wastage in a recent finding.
Project description:The objective of this study was to evaluate the mutational spectrum of NLRP7 and KHDC3L (C6orf221) in women with sporadic and recurrent androgenetic complete hydatidiform moles (AnCHM) and biparental hydatidiform moles (BiHM) to address the hypothesis that autosomal recessive mutations in these genes are only or primarily associated with BiHM.We recruited 16 women with suspected recurrent and sporadic AnCHM and five women with suspected BiHM in addition to their reproductive partners into our study. We then sequenced the coding exons of NLRP7 and KHDC3L from DNA isolated from either blood or saliva from the study subjects.Sequence analysis of NLRP7 and KHDC3L revealed previously described single nucleotide polymorphisms in patients with AnCHM. However, in patients with BiHM, we identified a novel homozygous mutation and a previously described intragenic duplication of exons 2 to 5 in NLRP7, both of which are likely to be disease causing. We did not identify mutations in KHDC3L in patients with either form of hydatidiform moles.The absence of mutations in women with AnCHM supports a role for NLRP7 or KHDC3L in BiHM only. The absence of mutations in KHDC3L in women with BiHM is consistent with its minor role in this disease compared with NLRP7, the major BiHM gene.
Project description:Are mutations in NLRP2/7 (NACHT, LRR and PYD domains-containing protein 2/7) or KHDC3L (KH Domain Containing 3 Like) associated with recurrent pregnancy loss (RPL) or infertility?We found no evidence for mutations in NLRP2/7 or KHDC3L in unexplained RPL or infertility.Mutations in NLRP7 and KHDC3L are known to cause biparental hydatidiform moles (BiHMs), a rare form of pregnancy loss. NLRP2, while not associated with the BiHM pathology, is known to cause recurrent Beckwith Weidemann Syndrome (BWS).Ninety-four patients with well characterized, unexplained infertility were recruited over a 9-year period from three IVF clinics in Sweden. Blood samples from 24 patients with 3 or more consecutive miscarriages of unknown etiology were provided by the Recurrent Miscarriage Clinic at St Mary's Hospital, London, UK.Patients were recruited into both cohorts following extensive clinical studies. Genomic DNA was isolated from peripheral blood and subject to Sanger sequencing of NLRP2, NLRP7 and KHDC3L. Sequence electropherograms were analyzed by Sequencher v5.0 software and variants compared with those observed in the 1000 Genomes, single nucleotide polymorphism database (dbSNP) and HapMap databases. Functional effects of non-synonymous variants were predicted using Polyphen-2 and sorting intolerant from tolerant (SIFT).No disease-causing mutations were identified in NLRP2, NLRP7 and KHDC3L in our cohorts of unexplained infertility and RPL.Due to the limited patient size, it is difficult to conclude if the low frequency single nucleotide polymorphisms observed in the present study are causative of the phenotype. The design of the present study therefore is only capable of detecting highly penetrant mutations.The present study supports the hypothesis that mutations in NLRP7 and KHDC3L are specific for the BiHM phenotype and do not play a role in other adverse reproductive outcomes. Furthermore, to date, mutations in NLRP2 have only been associated with the imprinting disorder BWS in offspring and there is no evidence for a role in molar pregnancies, RPL or unexplained infertility.This study was funded by the following sources: Estonian Ministry of Education and Research (Grant SF0180044s09), Enterprise Estonia (Grant EU30020); Mentored Resident research project (Department of Obstetrics and Gynecology, Baylor College of Medicine); Imperial NIHR Biomedical Research Centre; Grant Number C06RR029965 from the National Center for Research Resources (NCCR; NIH). No competing interests declared.
Project description:Background:Recurrent hydatidiform moles (RHMs) are an unusual pregnancy with at least two molar gestations that are associated with abnormal proliferation of trophoblastic tissue and a failure in the embryonic tissues development. Three maternal-effect genes, including NLRP7, KHDC3L, and PADI6 have been identified as the cause of RHMs. The present study aimed to understand the association of a founder mutation with the incidence and prevalence of a disease in different individuals of a population. Methods:14 unrelated Iranian patients with recurrent reproductive wastage, including at least two HMs, entered this study. In order to find a possible mutation in KHDC3L, all the 14 samples were Sanger sequenced. For haplotype analysis, three single nucleotide polymorphisms (SNPs) were selected with highest Minor Allele Frequency along KHDC3L. Results:A common KHDC3L mutation with the same haplotype was identified in four out of 14 patients with RHM. Regarding the present study, c.1A>G is the highest reported mutation in KHDC3L so far and is also the first report of the homozygous state that has led to RHM. Conclusion:c.1A>G mutation in KHDC3L is the highest reported mutation around the world. Our data also demonstrated the presence of founder effects for this particular mutation in Iranian populations. These data suggest that the high frequency of this mutation is potentially responsible for a higher rate of RHM in Iran.
Project description:Gestational trophoblastic disease (GTD) is a group of conditions that originate from the abnormal hyperproliferation of trophoblastic cells, which derive from the trophectoderm, the outer layer of the blastocyst that would normally develop into the placenta during pregnancy. GTDs encompass hydatidiform mole (HM) (complete and partial), invasive mole, gestational choriocarcinoma, placental-site trophoblastic tumor, and epithelioid trophoblastic tumor. Of these, the most common is HM, and it is the only one that has been reported to recur in the same patients from independent pregnancies, which indicates the patients' genetic predisposition. In addition, HM is the only GTD that segregates in families according to Mendel's laws of heredity, which made it possible to use rare familial cases of recurrent HMs (RHMs) to identify two maternal-effect genes, NLRP7 and KHDC3L, responsible for this condition. Here, we recapitulate current knowledge about RHMs and conclude with the role and benefits of testing patients for mutations in the known genes.
Project description:Recurrent hydatidiform mole (RHM) is defined by the occurrence of repeated molar pregnancies in affected women. Two genes, NLRP7 and KHDC3L, play a causal role in RHM and are responsible for 48-80% and 5% of cases, respectively. Here, we report the results of screening these two genes for mutations in one Iranian and one Indian patient with RHM. No mutations in NLRP7 were identified in the two patients. KHDC3L sequencing identified two novel protein-truncating mutations in a homozygous state, a 4-bp deletion, c.17_20delGGTT (p.Arg6Leufs*7), in the Iranian patient and a splice mutation, c.349+1G>A, that affects the invariant donor site at the junction of exon 2 and intron 2 in the Indian patient. To date, only four mutations in KHDC3L have been reported. The identification of two additional mutations provides further evidence for the important role of KHDC3L in the pathophysiology of RHM and increases the diversity of mutations described in Asian populations.
Project description:Familial recurrent hydatidiform mole (RHM) is a maternal-effect autosomal recessive disorder usually associated with mutations of the NLRP7 gene. It is characterized by HM with excessive trophoblastic proliferation, which mimics the appearance of androgenetic molar conceptuses despite their diploid biparental constitution. It has been proposed that the phenotypes of both types of mole are associated with aberrant genomic imprinting. However no systematic analyses for imprinting defects have been reported. Here, we present the genome-wide methylation profiles of both spontaneous androgenetic and biparental NLRP7 defective molar tissues. We observe total paternalization of all ubiquitous and placenta-specific differentially methylated regions (DMRs) in four androgenetic moles; namely gain of methylation at paternally methylated loci and absence of methylation at maternally methylated regions. The methylation defects observed in five RHM biopsies from NLRP7 defective patients are restricted to lack-of-methylation at maternal DMRs. Surprisingly RHMs from two sisters with the same missense mutations, as well as consecutive RHMs from one affected female show subtle allelic methylation differences, suggesting inter-RHM variation. These epigenotypes are consistent with NLRP7 being a maternal-effect gene and involved in imprint acquisition in the oocyte. In addition, bioinformatic screening of the resulting methylation datasets identified over sixty loci with methylation profiles consistent with imprinting in the placenta, of which we confirm 22 as novel maternally methylated loci. These observations strongly suggest that the molar phenotypes are due to defective placenta-specific imprinting and over-expression of paternally expressed transcripts, highlighting that maternal-effect mutations of NLRP7 are associated with the most severe form of multi-locus imprinting defects in humans.
Project description:Complete hydatidiform mole (HM) is a gestational trophoblastic disease resulting in hyperproliferation of trophoblast cells and absence of embryo development. Mutations in the maternal-effect gene NLRP7 are the major cause of familial recurrent complete HM. Here, we established an in vitro model of HM using patient-specific induced pluripotent stem cells (iPSCs) derived trophoblasts harboring NLRP7 mutations. Using whole transcriptome profiling during trophoblast differentiation, we showed that impaired NLRP7 expression results in precocious downregulation of pluripotency factors, activation of trophoblast lineage markers, and promotes maturation of differentiated extraembryonic cell types such as syncytiotrophoblasts. Interestingly, we found that these phenotypes are dependent on BMP4 signaling and BMP pathway inhibition corrected the excessive trophoblast differentiation of patient-derived iPSCs. Our human iPSC model of a genetic placental disease recapitulates aspects of trophoblast biology, highlights the broad utility of iPSC-derived trophoblasts for modeling human placental diseases and identifies NLRP7 as an essential modulator of key developmental cell fate regulators.
Project description:A hydatidiform mole (HM) is a human pregnancy with hyperproliferative placenta and abnormal embryonic development. Mutations in NLRP7, a member of the nucleotide oligomerization domain-like receptor family of proteins with roles in inflammation and apoptosis, are responsible for recurrent HMs. However, little is known about the functional role of NLRP7. Here, we demonstrate that peripheral blood mononuclear cells from patients with NLRP7 mutations and rare variants secrete low levels of IL-1? and TNF in response to LPS. We show that the cells from patients, carrying mutations or rare variants, have variable levels of increased intracellular pro-IL-1? indicating that normal NLRP7 down-regulates pro-IL-1? synthesis in response to LPS. Using transient transfections, we confirm the role of normal NLRP7 in inhibiting pro-IL-1? and demonstrate that this inhibitory function is abolished by protein-truncating mutations after the Pyrin domain. Within peripheral blood mononuclear cells, NLRP7 co-localizes with the Golgi and the microtubule-organizing center and is associated with microtubules. This suggests that NLRP7 mutations may affect cytokine secretion by interfering, directly or indirectly, with their trafficking. We propose that the impaired cytokine trafficking and secretion caused by NLRP7 defects makes the patients tolerant to the growth of these earlier arrested conceptions with no fetal vessels and that the retention of these conceptions until the end of the first trimester contribute to the molar phenotype. Our data will impact our understanding of postmolar choriocarcinomas, the only allograft non-self tumors that are able to invade maternal tissues.
Project description:Complete Hydatidiform Mole (HM) is a gestational trophoblastic disease resulting in hyper proliferation of trophoblast cells and absence of embryo development. Mutations in NLRP7 gene is a major cause of familial recurrent complete HM, where no embryonic tissue is present during the pregnancy. NLRP7 is a novel protein and its function is poorly understood. Lack of NLRP7 in the mouse genome and challenges in studying human embryogenesis as well as human trophoblast differentiation has prevented elucidation of the pathophysiology of this disease and the function of NLRP7 in HM. Here, we established an in vitro model of HM using NLRP7 deficient patient-derived human induced pluripotent stem cells (iPSCs). Whole transcriptome profiling during trophoblast differentiation revealed that NLRP7 deficiency results in precocious downregulation of pluripotency factors, activation of trophoblast genes and promotes maturation of differentiated extraembryonic cell types such as syncytiotrophoblasts. We further showed that these phenotypes are dependent on BMP4 signaling and BMP pathway inhibition prevented excessive trophoblast differentiation of HM-iPSCs. Taken together, this novel human iPSC model of a genetic placental disease recapitulates aspect of trophoblast biology and sheds light on early human embryogenesis by identifying NLRP7 as an important regulator of this process. Overall design: mRNA profiles were obtained from trophoblast-like cells derived from patient specific iPSCs and unrelated healty control iPSCs upon treatment via FGF and ACTIVIN/NODAL inhibitors together with either BMP4 or BMP inhibitor.