Evolution of human-specific neural SRGAP2 genes by incomplete segmental duplication.

Gene duplication is an important source of phenotypic change and adaptive evolution. We leverage a haploid hydatidiform mole to identify highly identical sequences missing from the reference genome, confirming that the cortical development gene Slit-Robo Rho GTPase-activating protein 2 (SRGAP2) duplicated three times exclusively in humans. We show that the promoter and first nine exons of SRGAP2 duplicated from 1q32.1 (SRGAP2A) to 1q21.1 (SRGAP2B) ∼3.4 million years ago (mya). Two larger duplications later copied SRGAP2B to chromosome 1p12 (SRGAP2C) and to proximal 1q21.1 (SRGAP2D) ∼2.4 and ∼1 mya, respectively. Sequence and expression analyses show that SRGAP2C is the most likely duplicate to encode a functional protein and is among the most fixed human-specific duplicate genes. Our data suggest a mechanism where incomplete duplication created a novel gene function-antagonizing parental SRGAP2 function-immediately "at birth" 2-3 mya, which is a time corresponding to the transition from Australopithecus to Homo and the beginning of neocortex expansion.

Telomere-to-telomere assembly of a complete human X chromosome.

After two decades of improvements, the current human reference genome (GRCh38) is the most accurate and complete vertebrate genome ever produced. However, no single chromosome has been finished end to end, and hundreds of unresolved gaps persist1,2. Here we present a human genome assembly that surpasses the continuity of GRCh382, along with a gapless, telomere-to-telomere assembly of a human chromosome. This was enabled by high-coverage, ultra-long-read nanopore sequencing of the complete hydatidiform mole CHM13 genome, combined with complementary technologies for quality improvement and validation. Focusing our efforts on the human X chromosome3, we reconstructed the centromeric satellite DNA array (approximately 3.1 Mb) and closed the 29 remaining gaps in the current reference, including new sequences from the human pseudoautosomal regions and from cancer-testis ampliconic gene families (CT-X and GAGE). These sequences will be integrated into future human reference genome releases. In addition, the complete chromosome X, combined with the ultra-long nanopore data, allowed us to map methylation patterns across complex tandem repeats and satellite arrays. Our results demonstrate that finishing the entire human genome is now within reach, and the data presented here will facilitate ongoing efforts to complete the other human chromosomes.

Single-nucleotide polymorphism array and fluorescence in situ hybridization analysis to decode the cytogenetic profile of atypical partial hydatidiform moles diagnosed by short tandem repeat polymorphism analysis.

Accurate diagnosis of partial hydatidiform moles (PHMs) is crucial for improving outcomes of gestational trophoblastic neoplasia. The use of short tandem repeat (STR) polymorphism analysis to distinguish between PHM and hydropic abortuses is instrumental; however, its diagnostic power has not been comprehensively assessed. Herein, we evaluated the diagnostic efficacy of STR in differentiating between PHM and hydropic abortus, thus providing an opportunity for early measurement of human chorionic gonadotropin for PHMs. We reviewed charts of STR polymorphism analysis performed on fresh villous specimens and patient blood samples using a commercial kit for 16 loci. The genetic classification of 79 PHMs was confirmed. STR was reliable in differentiating PHMs when at least 15 loci were available. Typically, PHMs are characterized by their triploidy, including two paternal and one maternal haploid contribution. In our sample, seven PHMs lacked the three-allelic loci, requiring fluorescence in situ hybridization (FISH) analysis to investigate imbalanced biparental conceptus and single-nucleotide polymorphism array analysis to reveal cytogenetic details. Of these PHMs, two, three, and one were identified as androgenetic/biparental mosaics (diploids), monospermic diandric monogynic triploids, and a typical dispermic diandric monogynic triploid, respectively. The remaining case was monospermic origin, but its ploidy details could not be available. Therefore, STR differentiated PHM from a biparental diploid abortus in most cases. However, PHM diagnosis may be compromised when STR is used as the sole method for cases displaying distinct cytogenetic patterns lacking the three-allelic loci, including androgenetic/biparental mosaicism. Therefore, FISH should be considered to confirm the diagnosis.

Spermatogonium-Derived Complete Hydatidiform Mole.

A complete hydatidiform mole (CHM) is a conceptus with only sperm-derived chromosomes. Here, we report on a CHM with genomic DNA identical to that of the paternal somatic cells. The CHM developed in a woman who had undergone intrauterine implantation of a blastocyst obtained through in vitro injection of a presumed round spermatid into one of her oocytes. The CHM was genetically identical to peripheral white cells of her husband and contained no maternally derived nuclear DNA. We hypothesize that a spermatogonium, rather than a round spermatid, was inadvertently selected for the procedure. The CHM developed into a gestational trophoblastic neoplasia, which resolved after chemotherapy. (Funded by the Japan Society for the Promotion of Science.).

Novo pathogenic variations of NLRP7 increasing the risk of gestational trophoblastic neoplasia.

Identification of novo mutations of NLRP7 in HM patients. NLRP7 mutations increasing the risk of HM progression.

Diagnostic Utility of TSSC3 and RB1 Immunohistochemistry in Hydatidiform Mole.

The general notion of complete hydatidiform moles is that most of them consist entirely of paternal DNA; hence, they do not express p57, a paternally imprinted gene. This forms the basis for the diagnosis of hydatidiform moles. There are about 38 paternally imprinted genes. The aim of this study is to determine whether other paternally imprinted genes could also assist in the diagnostic approach of hydatidiform moles. This study comprised of 29 complete moles, 15 partial moles and 17 non-molar abortuses. Immunohistochemical study using the antibodies of paternal-imprinted (RB1, TSSC3 and DOG1) and maternal-imprinted (DNMT1 and GATA3) genes were performed. The antibodies' immunoreactivity was evaluated on various placental cell types, namely cytotrophoblasts, syncytiotrophoblasts, villous stromal cells, extravillous intermediate trophoblasts and decidual cells. TSSC3 and RB1 expression were observed in all cases of partial moles and non-molar abortuses. In contrast, their expression in complete moles was identified in 31% (TSSC3) and 10.3% (RB1), respectively (p < 0.0001). DOG1 was consistently negative in all cell types in all cases. The expressions of maternally imprinted genes were seen in all cases, except for one case of complete mole where GATA3 was negative. Both TSSC3 and RB1 could serve as a useful adjunct to p57 for the discrimination of complete moles from partial moles and non-molar abortuses, especially in laboratories that lack comprehensive molecular service and in cases where p57 staining is equivocal.

DNA analysis of partial hydatidiform mole revealing triandric monogynic tetraploidy.

The authors present a case of a partial hydatidiform mole where DNA analysis (STR - short tandem repeat genotyping) showed a triandric monogynic tetraploid genome composition with a XXXY gonosomal complement. This genetic finding clinicopathologically correlates with a partial hydatidiform mole, although it is rare in comparison with the typical, diandric monogynic triploid partial moles. The genetic analysis definitively confirmed the suspected diagnosis of a partial mole. To exclude the possibility that molar pregnancy represented retained products of conception after elective pregnancy termination, STR profiles from molar pregnancy and previous products of conception were compared. Short tandem repeats genotyping is a useful molecular genetic method in the differential diagnosis of partial hydatidiform moles, where clinical-pathological findings are frequently ambiguous.

MicroRNA profiling in complete and partial hydatidiform moles.

INTRODUCTION: Hydatidiform mole is one of the gestational trophoblastic disease and comprises complete (CM) and partial moles (PM), which carries a risk of developing persistence disease, invasive mole or choriocarcinoma. MicroRNAs (miRNAs) have been discovered in various tissues, including neoplastic tissues. Its role in the pathogenesis of molar pregnancy or as biomarkers are still largely uncertain. The aim of this study is to identify the differentially expressed miRNAs in CM and PM. MATERIALS AND METHODS: Using next-generation sequencing, the miRNAs profiles of CM (n=3) and PM (n=3) moles, including placenta of non-molar abortus (n=3) as control were determined. The differentially expressed miRNAs between each group were analysed. Subsequently, bioinformatics analysis using miRDB and Targetscan was utilised to predict target genes. RESULTS: We found 10 differentially expressed miRNAs in CMs and PMs, compared to NMAs, namely miR- 518a-5p, miR-423-3p, miR-503-5p, miR-302a-3p, and miR-1323. The other 5 miRNAs were novel, not listed in the known database. The 3 differentially expressed miRNAs in CMs were predicted to commonly target ZTBT46 and FAM73B mRNAs. DISCUSSION: miR-518 was consistently observed to be downregulated in CM versus PM, and CM versus NMA. Further bioinformatic analysis to provide insight into the possible role of these miRNAs in the pathogenesis of HMs, progression of disease and as potential diagnostic biomarkers as well as therapeutic targets for HMs is needed.

Immunohistochemical expression of BCL-2 in hydatidiform moles: a tissue microarray study.

Background: Hydatidiform moles (HM) are members of gestational trophoblastic diseases (GTD) and, in some cases, might progress to gestational trophoblastic neoplasia (GTN). HMs are either partial (PHM) or complete (CHM). Some HMs are challenging in arriving at a precise histopathological diagnosis. This study aims to investigate the expression of BCL-2 by immunohistochemistry (IHC) in HMs as well as in normal trophoblastic tissues "products of conception (POC) and placentas" using Tissue MicroArray (TMA) technique. Methods: TMAs were constructed using the archival material of 237 HMs (95 PHM and 142 CHM) and 202 control normal trophoblastic tissues; POC and unremarkable placentas. Sections were immunohistochemically stained using antibodies against BCL-2. The staining was assessed semi-quantatively (intensity and percentage of the positive cells) in different cellular components (trophoblasts and stromal cells). Results: BCL-2 showed cytoplasmic expression in more than 95% of trophoblasts of PHM, CHM and controls. The staining showed a significant reduction of the intensity from controls (73.7%), PHMs (76.3%) to CHM (26.9%). There was a statistically significant difference between PHM and CHM in the intensity (p-value 0.0005) and the overall scores (p-value 0.0005), but not the percentage score (p-value > 0.05). No significant difference was observed in the positivity of the villous stromal cells between the different groups. All cellular components were visible using the TMA model of two spots/case (3 mm diameter, each) in more than 90% of cases. Conclusions: Decreased BCL-2 expression in CHM compared to PHM and normal trophoblasts indicates increased apoptosis and uncontrolled trophoblastic proliferation. Construction of TMA in duplicates using cores of 3 mm diameter can overcome tissue heterogeneity of complex lesions.

Hydatidiform mole.

Hydatidiform mole is the most common form of gestational trophoblastic disease. It is an abnormally formed placental tissue with characteristic changes in karyotype, arising in fertilization disorders. The presence of abundant paternal genetic information plays a key role in the pathogenesis of complete and partial hydatidiform moles. These lesions are characterized by a relatively wide spectrum of morphological changes that may not be fully expressed, especially in the early stages of pregnancy. In addition, some changes can be observed in non-molar gravidities, which, unlike hydatidiform moles, lack any risk of malignant transformation. Although conventional histological examination still plays a key role in the diagnosis, it should be supplemented by other methods that reliably differentiate individual lesions. Accurate diagnosis of molar gravidities is important not only for determining the correct therapeutic approach, but the obtained data may also contribute to further research of these pathological entities.

Molecular diagnosis of complete and partial hydatidiform moles.

Complete and partial hydatidiform moles are abnormal products of conception that can be identified by clinical, ultrasonographic, morphologic, histologic, and genetic methods. The diagnosis is usually confirmed only by histological examination. However, accurate diagnosis based on morphological criteria is difficult and some studies have shown that misclassifications are common, even when analysed by highly experienced pathologists. Misdiagnosis may mean that women are either not included in adequate ß-hCG follow-up with the risk that the hydatidiform mole progresses to choriocarcinoma or, conversely, are included in follow-up unnecessarily. A reliable complementary method to pathological interpretation may be genetic analysis of the conceptus to eliminate the diagnostic dilemma by distinguishing non-molar spontaneous abortions from hydatidiform moles and defining the type of hydatidiform mole. The aim of our short paper is to introduce the routine molecular analysis used in our laboratory to a wider range of clinical pathologists.

Biallelic NLRP7 variants in patients with recurrent hydatidiform mole: A review and expert consensus.

Hydatidiform mole (HM) is an abnormal human pregnancy characterized by excessive growth of placental trophoblasts and abnormal early embryonic development. Following a first such abnormal pregnancy, the risk for women of successive molar pregnancies significantly increases. To date variants in seven maternal-effect genes have been shown to cause recurrent HMs (RHM). NLRP7 is the major causative gene for RHM and codes for NOD-like receptor (NLR) family pyrin domain containing 7, which belongs to a family of proteins involved in inflammatory disorders. Since its identification, all NLRP7 variants have been recorded in Infevers, an online registry dedicated to autoinflammatory diseases (https://infevers.umai-montpellier.fr/web/). Here, we reviewed published and unpublished recessive NLRP7 variants associated with RHM, scored their pathogenicity according to the American College of Medical Genetics classification, and recapitulated all functional studies at the level of both the patients and the conceptions. We also provided data on further variant analyses of 32 patients and genotypes of 36 additional molar pregnancies. This comprehensive review integrates published and unpublished data on NLRP7 and aims at guiding geneticists and clinicians in variant interpretation, genetic counseling, and management of patients with this rare condition.

Auxiliary and experimental diagnostic techniques for hydatidiform moles.

Hydatidiform moles are classified into complete hydatidiform moles (CHMs), which are androgenetic and diploid, and partial hydatidiform moles (PHM), which are triploid with two paternal chromosomes and one maternal chromosome. The incidence of gestational trophoblastic neoplasia differs substantially between CHM and PHM. However, they are occasionally difficult to diagnose. In this review, auxiliary and experimental methods based on cytogenetic features and advanced molecular detection techniques applied to the diagnosis and analysis of hydatidiform moles are summarized, including basic principles, characteristics, and clinical implications. Short tandem repeat polymorphism analysis is considered the gold standard for the genetic diagnosis of hydatidiform moles. In clinical settings, immunohistochemical analyses of p57KIP2 , an imprinted gene product, are widely used to differentiate CHMs from other conceptuses, including PHMs. Recently, new molecular genetic techniques, such as single nucleotide polymorphism arrays, have been applied to research on hydatidiform moles. In addition to insights from classical methods, such as chromosome analysis, recently developed approaches have yielded novel findings related to the mechanism underlying the development of androgenetic CHMs.

Genetic screening of Chinese patients with hydatidiform mole by whole-exome sequencing and comprehensive analysis.

PURPOSE: We aim to explore if there are any other candidate genetic variants in patients with a history of at least one hydatidiform mole (HM) besides the well-known variants in NLRP7 and KHDC3L. METHODS: The diagnosis of HM type was based on histopathology, and available HM tissues were collected for short tandem repeat (STR) genotyping to verify the diagnosis. DNA extracted from blood samples or decidual tissues of the 78 patients was subjected to whole-exome sequencing (WES). RESULTS: We identified five novel variants in NLRP7, two novel variants in KHDC3L, and a chromosome abnormality covering the KHDC3L locus among patients with HM. We found that patients with HM who carried heterozygous variants in KHDC3L had a chance of normal pregnancy. We also detected four novel genetic variants in candidate genes that may be associated with HM. CONCLUSION: Our study enriched the spectrum of variants in NLRP7 and KHDC3L in Chinese HM patients and provided a new outlook on the effects of heterozygous variants in KHDC3L. The novel candidate genetic variants associated with HMs reported in this study will also contribute to further research on HMs.

[Analysis of three Chinese pedigrees affected with recurrent hydatidiform mole due to variants of NLRP7 gene].

OBJECTIVE: To explore the genetic etiology of recurrent hydatidiform mole (RHM) and provide accurate guidance for reproduction. METHODS: Peripheral venous blood samples of the probands with RHM and members from 5 unrelated pedigrees were collected. Genomic DNA was extracted by using routine method, and whole exome sequencing was carried out to detect variants of RHM-associated genes including NLRP7 and KHDC3L. Sanger sequencing and real-time quantitative PCR (RT-qPCR) were used to validate the candidate variants and delineate their parental origin. RESULTS: Homozygous or compound heterozygous variants of the NLRP7 gene were identified in four patients from three pedigrees, which included a homozygous deletion of exon 1 to 4 of NLRP7 in patient P1 and her elder sister, compound heterozygous variants of NLRP7 c.939delG (p.Q314Sfs*6) pat and c.1533delG (p.N512Tfs*4) mat in patient P2, and compound heterozygous variants of NLRP7 c.2389_2390delTC (p.A798Qfs*6) pat and c.2165A>G (p.D722G) mat in patient P4. All variants were interpreted as pathogenic or likely pathogenic according to the American College of Medical and Genomics (ACMG) guidelines. Among these, NLRP7 exons 1 to 4 deletion, c.939delG (p.Q314Sfs*6), c.1533delG (p.N512Tfs*4) and c.2389_2390delTC (p.A798Qfs*6) were unreported previously. CONCLUSION: Variants of the NLRP7 gene probably underlay autosomal recessive RHM in the three pedigrees, and definitive molecular diagnosis is beneficial for accurate genetic counseling. Above finding has also enriched the spectrum of the NLRP7 variants underlying RHM.

Detection of Parental Contribution to Molar Genome Leads to Diagnosis of Recurrent Hydatidiform Mole in a Family with NLRP7 Variants.

Introduction: Genetically, complete hydatidiform mole (CHM) is androgenetic diploid, containing two sets of paternal chromosomes. In most cases, recurrent HM (RHM) is CHM but has diploid biparental chromosome constitution. Case report: We report a mother with RHM, both with biparental diploidy. The mother was compound heterozygous for two variants, c.1720dup, p.(C574Lfs*4) and c.2165A > G, p.(D722G) of the NLRP7 gene, as was a brother who fathered 2 normal pregnancies. Conclusion: The genotype study should be obtained for patients of CHM, even in their first pregnancy, followed by genetic screening for maternal-effect variants in those with biparental moles. This strategy will identify patients in their first pregnancy with HM that have a decreased chance for a normal pregnancy, to allow genetic counseling, perhaps utilizing a donor egg.

[Twin gestation with regressive partial hydatidiform mole and coexisting live fetus]. / Nerazvivayushchiisya chastichnyi puzyrnyi zanos v slitnoi dikhorial'noi platsente s normal'nym razvitiem vtorogo bliznetsa.

The case of dichorionic twin pregnancy is described, with a fused placenta, one part of which is represented by a tissue of partial hydatidiform mole (PHM) with signs of regression, the second part is a placenta of a common structure with a normal development of the second twin. The delivery took place at the term of 38 weeks with a live healthy girl weighing 3250 g. A single placental disc consisted of two fused placentas with a clear boundary between them. The placenta of a live-born girl was mature, with focal chorangiosis, the second part of the disc was represented by the PHM tissue with avascular giant bizarre villi, some of them with central cisterns, with stromal fibrosis, low proliferative activity of the villous trophoblast and a significant narrowing of the intervillous space. A genetic study was carried out on the material of paraffin blocks from two parts of the placental disc containing the tissue of the villous chorion, and the blood of the parents. Comparative analysis of DNA isolated from the paraffin block of PHM with the DNA of the parents revealed the presence of diandric dispermic triploidy. No chromosomal pathology was found in the placenta of a living girl. For hydatidiform mole in the case of multiple pregnancy, an increase in the volume of the affected placenta is characteristic compared to the normal placenta of the twin. In our observation, the presence in the placenta with PHM signs characteristic of placentas with antenatal fetal death, stromal fibrosis of the villi and low proliferative activity of the trophoblast suggests a regression of PHM.

NLRP7: From inflammasome regulation to human disease.

Nucleotide-binding oligomerization domain (NOD) and leucine-rich repeat (LRR)-containing receptors or NOD-like receptors (NLRs) are cytosolic pattern recognition receptors, which sense conserved microbial patterns and host-derived danger signals to elicit innate immune responses. The activation of several prototypic NLRs, including NLR and pyrin domain (PYD) containing (NLRP) 1, NLRP3 and NLR and caspase recruitment domain (CARD) containing (NLRC) 4, results in the assembly of inflammasomes, which are large, cytoplasmic multiprotein signalling platforms responsible for the maturation and release of the pro-inflammatory cytokines IL-1ß and IL-18, and for the induction of a specialized form of inflammatory cell death called pyroptosis. However, the function of other members of the NLR family, including NLRP7, are less well understood. NLRP7 has been linked to innate immune signalling, but its precise role is still controversial as it has been shown to positively and negatively affect inflammasome responses. Inflammasomes are essential for homeostasis and host defence, but inappropriate inflammasome responses due to hereditary mutations and somatic mosaicism in inflammasome components and defective regulation have been linked to a broad spectrum of human diseases. A compelling connection between NLRP7 mutations and reproductive diseases, and in particular molar pregnancy, has been established. However, the molecular mechanisms by which NLRP7 mutations contribute to reproductive diseases are largely unknown. In this review, we focus on NLRP7 and discuss the current evidence of its role in inflammasome regulation and its implication in human reproductive diseases.

Causative Mutations and Mechanism of Androgenetic Hydatidiform Moles.

Androgenetic complete hydatidiform moles are human pregnancies with no embryos and affect 1 in every 1,400 pregnancies. They have mostly androgenetic monospermic genomes with all the chromosomes originating from a haploid sperm and no maternal chromosomes. Androgenetic complete hydatidiform moles were described in 1977, but how they occur has remained an open question. We identified bi-allelic deleterious mutations in MEI1, TOP6BL/C11orf80, and REC114, with roles in meiotic double-strand breaks formation in women with recurrent androgenetic complete hydatidiform moles. We investigated the occurrence of androgenesis in Mei1-deficient female mice and discovered that 8% of their oocytes lose all their chromosomes by extruding them with the spindles into the first polar body. We demonstrate that Mei1-/- oocytes are capable of fertilization and 5% produce androgenetic zygotes. Thus, we uncover a meiotic abnormality in mammals and a mechanism for the genesis of androgenetic zygotes that is the extrusion of all maternal chromosomes and their spindles into the first polar body.