Objective:To determine the carrier frequency and hot-spot variants of a custom-designed expanded carrier screening (ECS) panel with 216 diseases (216-ECS panel) within a Chinese population of childbearing age.Methods:Whole-exome sequencing data from a cohort of 3 097 unrelated healthy individuals (including 1 424 couples) from Peking Union Medical College Hospital between January 2013 and December 2023 were analyzed. Totally 220 genes which inherited in a recessive manner of 216-ECS panel were included in the analysis. The analysis included variant carrier rate, gene carrier rate, cumulative carrier rate, at-risk couple rates, and variant spectrum.Results:(1) Pathogenic variants were identified in 1 472 (47.53%, 1 472/3 097) individuals, with an average of 0.65 pathogenic variants per individual. The rate of at-risk couples was 3.93% (56/1 424). (2) A total of 180 genes were identified, with 16 genes exhibiting a gene carrier rate of ≥1% and 33 genes having a rate of ≥0.5%, most of which were associated with inherited metabolic diseases. Noteworthy genes with higher gene carrier rates and high-frequency variants included GJB2: c.235del, PAH: c.728G>A, ATP7B: c.2333G>T, SLC26A4: c.919-2A>G, GALC: c.1901T>C, POLG: c.2890C>T, SLC22A5: c.1472C>G, USH2A: c.2802T>G, SLC25A13: c.852_855del, GAA: c.761C>T and c.752C>T. Conclusion:This study offers a focused analysis of carrier frequencies and hot-spot variants of 216 diseases of the ECS panel constructed by our laboratory among the Chinese population, laying a foundation for the development of ECS programs tailored to the Chinese population.

OBJECTIVE: To carry out genetic analysis for a fetus with confined placental mosaicism (CPM) for trisomy 2 (T2) in conjunct with fetal uniparental disomy (UPD). METHODS: Amniocentesis and chromosomal karyotyping was carried out for a pregnant woman with a high risk for chromosome 2 anomalies indicated by non-invasive prenatal testing (NIPT). Single nucleotide polymorphism array (SNP-array) and trio-whole exome sequencing (Trio-WES) were carried out. Ultrasonography was used to closely monitor the fetal growth. Multifocal sampling of the placenta was performed after delivery for copy number variation sequencing (CNV-seq). RESULTS: The fetus was found to have a normal chromosomal karyotype. SNP-array has revealed multiple regions with loss of heterozygosity (LOH) on chromosome 2. Trio-WES confirmed the presence of maternal UPD for chromosome 2. Ultrasonography has revealed intrauterine growth restriction and oligohydramnios. Intrauterine fetal demise had occurred at 23⁺⁴ weeks of gestation. Pathological examination had failed to find salient visceral abnormality. The placenta was proved to contain complete T2 by CNV-seq. CONCLUSION T2 CPM can cause false positive result for NIPT and may be complicated with fetal UPD, leading to adverse obstetric outcomes such as intrauterine growth restriction, oligohydramnios and intrauterine fetal demise.
Female ; Humans ; Pregnancy ; Amniocentesis ; Chromosomes, Human, Pair 2/genetics* ; DNA Copy Number Variations ; Fetal Death ; Fetal Growth Retardation/genetics* ; Fetus ; Mosaicism ; Oligohydramnios ; Placenta ; Trisomy/genetics* ; Uniparental Disomy/genetics*

Understanding the regulatory networks for germ cell fate specification is necessary to developing strategies for improving the efficiency of germ cell production in vitro. In this study, we developed a coupled screening strategy that took advantage of an arrayed bi-molecular fluorescence complementation (BiFC) platform for protein-protein interaction screens and epiblast-like cell (EpiLC)-induction assays using reporter mouse embryonic stem cells (mESCs). Investigation of candidate interaction partners of core human pluripotent factors OCT4, NANOG, KLF4 and SOX2 in EpiLC differentiation assays identified novel primordial germ cell (PGC)-inducing factors including BEN-domain (BEND/Bend) family members. Through RNA-seq, ChIP-seq, and ATAC-seq analyses, we showed that Bend5 worked together with Bend4 and helped mark chromatin boundaries to promote EpiLC induction in vitro. Our findings suggest that BEND/Bend proteins represent a new family of transcriptional modulators and chromatin boundary factors that participate in gene expression regulation during early germline development.
Animals ; Cell Differentiation/genetics* ; Chromatin/metabolism* ; Embryonic Stem Cells ; Germ Cells/metabolism* ; Germ Layers/metabolism* ; Mice

Objective: To investigate the impact of maternal X chromosome aneuploidies on cell free DNA (cf-DNA) prenatal screening. Methods: After genetic counseling, invasive prenatal diagnosis was provided for the 124 cases with high risk of sex chromosome aneuploidie (SCA) indicated by cf-DNA prenatal screening. For cases with discordant results of fetal prenatal diagnosis and cf-DNA prenatal screening, maternal leukocyte was collected for copy number variation sequencing (CNV-seq) to detect whether the maternal X chromosome was carrying variations. Results: Totally, 124 cases with high risks of SCA indicated by cf-DNA prenatal screening, 9 cases refused to take invasive prenatal diagnosis, while the remaining 115 cases received. Among the 115 cases, 41 cases received accordant results with cf-DNA prenatal screening while 74 cases discordant. Among the 74 cases with discordant results, 19 cases were indicated with maternal X chromosome variations by maternal leukocyte CNV-seq, which accounting for 25.7% (19/74) of the SCA false positive cases, and 15.3% (19/124) of all SCA cases. Conclusions Pregnant women with X chromosome variations may affect the results of cf-DNA prenatal screening, resulting in false positive or false negative outcomes, it should be emphasized that the cf-DNA results may be affected by maternal X chromosome variations. In cases with discordant results of prenatal diagnosis and cf-DNA prenatal screening, maternal leukocyte CNV-seq is recommended to find the reasons of false positive or negative results. And cf-DNA prenatal screening is not recommended for pregnant women who are already known with X chromosome variations.

Objective To investigate the prenatal diagnosis and genetic counseling of fetal nuchal fold (NF) thickening.Methods This study retrospectively analyzed 17 fetuses with increased NF detected by prenatal ultrasound examination in Peking Union Medical College Hospital,Peking Union Medical College & Chinese Academy of Medical Sciences from December 1,2016 to December 1,2017.All cases were divided into isolated (isolated group) or non-isolated increased NF group (non-isolated group) according to whether the fetus had concomitant ultrasonographic abnormalities or not.Karyotype and chromosomal microarray analysis (CMA) were performed on all cases.Clinical data,prenatal genetic testing results and pregnancy outcomes were analyzed.Results Of those twelve cases in the isolated group,two were terminated due to the identification of chromosomal abnormalities and pathogenic copy number variations (CNVs) and the fetal autopsy results were consistent with the prenatal diagnosis.The rest 10 pregnancies were all continued including one fetus carrying a variant of unknown significance,which was proved to be a paternal heredity by CMA,and nine without genetic abnormalities and all-these infants were healthy during follow-up.Among the five non-isolated cases,one was diagnosed as trisomy 21 by karyotyping and CMA,and the other four were found to have structural abnormalities under ultrasound scan,but without genetic abnormalities in karyotyping and CMA.And all the five pregnancies were terminated after genetic counseling and three of them chose whole exome sequencing (WES) for further test.One homozygous mutation in CHRNA 1 gene and one de novo mutation in SETD2 gene were found in two cases,respectively,while no abnormality was identified in the other one case.Conclusions Once increased NF were indicated by ultrasound examination,prenatal genetic testing should be offered to the patients,including CMA,regardless of other ultrasonographic abnormalities,and WES should also be offered when necessary.Considering a thickened NF is associated with increased risks of structural defects,a close follow-up with fetal echocardiography and ultrasound is required even the prenatal tests are normal.

Adenine ; Animals ; Gene Editing ; Mice ; Zygote ; metabolism

[Abstrcat] Objectives To analyze 3 cases of 17q12 microdeletion syndrome diagnosed prenatally, and to demonstrate clinical phenotype of the syndrome in prenatal setting.Methods From January 2013 to July 2017,1 370 women received invasive prenatal diagnosis and chromosome microarray analysis(CMA)in Peking Union Medical College Hospital. Among them, 3 fetuses were diagnosed as 17q12 microdeletion syndrome.All 3 cases were low-risk pregnancies.Abnormal structures in fetal kidney were found in all 3 cases, including 1 case of multiple renal cysts,2 cases of bilateral hyperechogenic kidneys.These women accepted invasive prenatal diagnosis followed by karyotyping, parental fluorescence in situ hybridization or CMA validation.Results The second and third trimester ultrasound showed that all 3 fetuses had bilateral renal structural abnormalities, including hyperechogenic kidney, multiple cysts and renal pelvis dilatation. The karyotyping of the 3 fetuses were normal.CMA examination showed that each case had 1.4-1.6 Mb deletion in 17q12 region.Two cases were de novo deletion and 1 case was inherited from the mother who had mild symptoms. The 3 women decided to terminate pregnancies after genetic counseling. Conclusion 17q12 microdeletion syndrome is a recurrent chromosome microdeletion syndrome, and the unique phenotype in prenatal setting is the abnormal structure of bilateral kidneys.A few cases of 17q12 microdeletion syndrome even inherited normally phenotypical parents, and prenatal genetic counseling of 17q12 microdeletion syndrome is relatively difficult.

β-Thalassemia is a global health issue, caused by mutations in the HBB gene. Among these mutations, HBB -28 (A>G) mutations is one of the three most common mutations in China and Southeast Asia patients with β-thalassemia. Correcting this mutation in human embryos may prevent the disease being passed onto future generations and cure anemia. Here we report the first study using base editor (BE) system to correct disease mutant in human embryos. Firstly, we produced a 293T cell line with an exogenous HBB -28 (A>G) mutant fragment for gRNAs and targeting efficiency evaluation. Then we collected primary skin fibroblast cells from a β-thalassemia patient with HBB -28 (A>G) homozygous mutation. Data showed that base editor could precisely correct HBB -28 (A>G) mutation in the patient's primary cells. To model homozygous mutation disease embryos, we constructed nuclear transfer embryos by fusing the lymphocyte or skin fibroblast cells with enucleated in vitro matured (IVM) oocytes. Notably, the gene correction efficiency was over 23.0% in these embryos by base editor. Although these embryos were still mosaic, the percentage of repaired blastomeres was over 20.0%. In addition, we found that base editor variants, with narrowed deamination window, could promote G-to-A conversion at HBB -28 site precisely in human embryos. Collectively, this study demonstrated the feasibility of curing genetic disease in human somatic cells and embryos by base editor system.
APOBEC-1 Deaminase ; genetics ; metabolism ; Base Sequence ; Blastomeres ; cytology ; metabolism ; CRISPR-Cas Systems ; Embryo, Mammalian ; metabolism ; pathology ; Female ; Fibroblasts ; metabolism ; pathology ; Gene Editing ; methods ; Gene Expression ; HEK293 Cells ; Heterozygote ; Homozygote ; Humans ; Point Mutation ; Primary Cell Culture ; Promoter Regions, Genetic ; Sequence Analysis, DNA ; beta-Globins ; genetics ; metabolism ; beta-Thalassemia ; genetics ; metabolism ; pathology ; therapy

Targeted point mutagenesis through homologous recombination has been widely used in genetic studies and holds considerable promise for repairing disease-causing mutations in patients. However, problems such as mosaicism and low mutagenesis efficiency continue to pose challenges to clinical application of such approaches. Recently, a base editor (BE) system built on cytidine (C) deaminase and CRISPR/Cas9 technology was developed as an alternative method for targeted point mutagenesis in plant, yeast, and human cells. Base editors convert C in the deamination window to thymidine (T) efficiently, however, it remains unclear whether targeted base editing in mouse embryos is feasible. In this report, we generated a modified high-fidelity version of base editor 2 (HF2-BE2), and investigated its base editing efficacy in mouse embryos. We found that HF2-BE2 could convert C to T efficiently, with up to 100% biallelic mutation efficiency in mouse embryos. Unlike BE3, HF2-BE2 could convert C to T on both the target and non-target strand, expanding the editing scope of base editors. Surprisingly, we found HF2-BE2 could also deaminate C that was proximal to the gRNA-binding region. Taken together, our work demonstrates the feasibility of generating point mutations in mouse by base editing, and underscores the need to carefully optimize base editing systems in order to eliminate proximal-site deamination.
APOBEC-1 Deaminase ; genetics ; metabolism ; Animals ; Bacterial Proteins ; genetics ; metabolism ; Base Sequence ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Cytidine ; genetics ; metabolism ; Embryo Transfer ; Embryo, Mammalian ; Endonucleases ; genetics ; metabolism ; Gene Editing ; methods ; HEK293 Cells ; High-Throughput Nucleotide Sequencing ; Humans ; Mice ; Mice, Inbred C57BL ; Microinjections ; Plasmids ; chemistry ; metabolism ; Point Mutation ; RNA, Guide ; genetics ; metabolism ; Thymidine ; genetics ; metabolism ; Zygote ; growth & development ; metabolism ; transplantation

Objective To evaluate the efficacy and safety of pelvic arterial embolization (PAE) in women with intractable primary postpartum hemorrhage (PPH). Methods Clinical data of 36 cases were analyzed retrospectively in which women underwent PAE for intractable primary PPH in Peking Union Medical College Hospital between Jan 2006 and Jan 2015. The success rate of PAE were measured and possible predictive risk factors associated with treatment failure were analyzed. The complications secondary to PAE were also recorded. Results (1)The etiology of PPH. Among the 36 cases, 21 patients delivered viginally (Group VD) and 15 received cesarean section (Group CS). The most frequent cause of PPH was uterine atony (72%, 26/36). The less common causes were placental problems (28%, 10/36), genital tract trauma (6%, 2/36) and coagulation defects (3%, 1/36) in turn. Three patients (8%, 3/36) had combined causes.(2)Interventions before PAE. Uterotonic medications were used in all patients. 31 patients received carboprost methylate suppositorites,27 received carbetocin and 31 received carboprost tromethamine. Besides, 20 patients received one or more surgical interventions before PAE. PAE was performed when these interventions failed. (3) Characteristics of PAE. Altogether 78 arteries were embolized in 36 cases. Embolization of bilateral uterine arteries was performed in 31 cases, right internal iliac artery and bilateral inferior epigastric arteries were embolized in one case. Right internal pudendal artery, bilateral uterine arteries and bilateral internal iliac arteries were embolized in one case. And bilateral uterine arteries, bilateral internal iliac arteries were embolized in one case. In the other 2 cases, bilateral internal iliac arteries were embolized.(4)Efficacy of PAE. The overall technical success rate of PAE was 100%(36/36), while the clinical success rate was 94%(34/36). All patients survived.(5)Complications of PAE. 15 patients were transferred to ICU after PAE for 1 to 7 days. Except self-limited fever, no puncture site hematoma, buttock necrosis or vessel rupture was observed. The effect on menstrual cycle and fertility were followed in 25 patients. 17 (68%, 17/25) reported resumption of normal menses and 8 (32%, 8/25) reported amenorrhea. Three pregnancies after PAE were observed. Conclusion PAE is a safe and effective treatment for intractable primary PPH which can prevent hysterectomy and preserve fertility of patients.