[Prenatal genetic diagnosis of the fetuses with isolated corpus callosum abnormality].

Objective: To explore the application value of chromosome karyotype analysis, chromosomal microarray analysis (CMA) and whole exome sequencing (WES) in prenatal diagnosis of isolated corpus callosum abnormality (CCA) fetus. Methods: Fetuses diagnosed with isolated CCA by ultrasound and MRI and receiving invasive prenatal diagnosis in Guangzhou Women and Children's Medical Center and Qingyuan People's Hospital from January 2010 to April 2021 were selected. Karyotype analysis and/or CMA [or copy number variation sequencing (CNV-seq)] were performed on all fetal samples, and WES was performed on fetal samples and their parents whose karyotype analysis and/or CMA (or CNV-seq) results were not abnormal. Results: Among 65 fetuses with isolated CCA, 38 cases underwent karyotype analysis, and 3 cases were detected with abnormal karyotypes, with a detection rate of 8% (3/38). A total of 49 fetuses with isolated CCA underwent CMA (or CNV-seq) detection, and 6 cases of pathogenic CNV were detected, the detection rate was 12% (6/49). Among them, the karyotype analysis results were abnormal, and the detection rate of further CMA detection was 1/1. The karyotype results were normal, and the detection rate of further CMA (or CNV-seq) detection was 14% (3/21). The detection rate of CMA as the first-line detection technique was 7% (2/27). A total of 25 fetuses with isolated CCA with negative results of karyotyping and/or CMA were tested by WES, and 9 cases (36%, 9/25) were detected with pathogenic genes. The gradient genetic diagnosis of chromosomal karyotyping, CMA and WES resulted in a definite genetic diagnosis of 26% (17/65) of isolated CCA fetuses. Conclusions: Prenatal genetic diagnosis of isolated CCA fetuses is of great clinical significance. The detection rate of CMA is higher than that of traditional karyotyping. CMA detection could be used as a first-line detection technique for fetuses with isolated CCA. WES could increase the pathogenicity detection rate of fetuses with isolated CCA when karyotype analysis and/or CMA test results are negative.

[Analysis of families with fetal congenital abnormalities but negative prenatal diagnosis by whole exome sequencing].

Objective: To evaluate the value of whole exome sequencing (WES) in prenatal clinical application. Methods: A total of 1 152 cases of congenital abnormal [including structural malformation, nuchal translucency (NT) thickening and intrauterine growth restriction] with traditional prenatal diagnosis [including G-band karyotype analysis and chromosome microarray analysis (CMA)] negative were analyzed. The congenital abnormal fetuses were divided into retrospective group and prospective group according to the time of WES detection, that is whether the pregnancy termination or not. According to the specific location of fetal malformation and their family history, the cohort was divided into subgroups. The clinical prognosis of all fetuses were followed up, and the effect of WES test results on pregnancy decision-making and clinical intervention were analyzed. According to the follow-up results, the data of fetuses with new phenotypes in the third trimester or after birth were re-analyzed. Results: Among 1 152 families who received WES, 5 families were excluded because of nonbiological parents. Among the remaining 1 147 families, 152 fetuses obtained positive diagnosis (13.3%,152/1 147), including 74 fetuses in the retrospective group (16.1%,74/460) and 78 fetuses in the prospective group (11.4%,78/687). In fetuses with negative CMA and G-band karyotype analysis results but new phenotypes in the third trimester or after birth, the positive rate by WES data re-analysis was 4.9% (8/163). A total of 34 (21.3%, 34/160) fetuses were directly affected by the corresponding positive molecular diagnosis. Among 68 cases of live births with diagnostic variation grade 4, 29 cases (42.7%, 29/68) received appropriate medical intervention through rapid review of WES results. Conclusions: WES could increase the detection rate of abnormal fetuses with negative G-banding karyotype analysis and CMA by 13.3%. Prenatal WES could guide pregnancy decision-making and early clinical intervention. It might be an effective strategy to pay attention to the special follow-up of the third trimester and postnatal fetus and to re-analyze the WES data.

AXIN2 gene silencing reduces apoptosis through regulating mitochondria-associated apoptosis signaling pathway and enhances proliferation of ESCs by modulating Wnt/ß-catenin signaling pathway.

OBJECTIVE: Embryonic stem cells (ESCs) mainly originate from totipotent cells in early-stage of mammalian embryo and could proliferate in a manner of un-limitation. This study aimed to investigate roles of Axin2 in proliferation of ESCs and explore the associated mechanisms. MATERIALS AND METHODS: Axis inhibition protein 2 (AXIN2) over-expression (LV5-AXIN2) and AXIN2 RNA interfere (LV3-AXIN2-RNAi) vectors were structured and transfected into H9 cells. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) was used to evaluate cell proliferative activity. Flow cytometry analysis was employed to measure apoptosis of H9 cells. AXIN2, ß-catenin, transcription factor 4 (TCF4), c-myc, c-jun and Cyclin D mRNA levels and protein expressions were determined using quantitative real-time PCR (qRT-PCR) and Western blotting assay. RESULTS: LV5-AXIN2 and LV3-AXIN2-RNAi were successfully structured with higher transfecting efficacy. AXIN2 gene silencing remarkably increased proliferative activity and AXIN2 treatment significantly induced apoptosis of H9 cells, comparing with blank vector group (p<0.05). AXIN2 gene silencing significantly enhanced B-cell lymphoma-2 (Bcl-2) expression and remarkably inhibited cleaved caspase-3 expression comparing to that in blank vector group (p<0.05). AXIN2-RNAi treatment significantly enhanced and AXIN2 over-expression significantly reduced ß-catenin and TCF4 expression, comparing to that in blank vector group (p<0.05). AXIN2 gene silence activated down-stream molecules of Wnt/ß-catenin signaling pathway, including c-jun, c-myc, and Cyclin D1 (p<0.05). CONCLUSIONS: AXIN2 gene silencing reduced apoptosis by regulating mitochondria-associated apoptosis signaling pathway and enhanced proliferation by modulating molecules in Wnt/ß-catenin signaling pathway. Therefore, targeting of aberrant apoptosis and AXIN2 might be a novel clinical strategy to inhibit aging and enhance self-renewal of ESCs.

[Application of chromosomal microarray analysis for fetuses with talipes equinovarus].

OBJECTIVE: To investigate the application of fetuses with talipes equinovarus(TE)using chromosomal microarray analysis(CMA)technology. METHODS: From May 2012 to June 2015, 54 fetuses were found with TE and with or without other structural anomalies by prenatal ultrasound. Karyotyping was taking for them all, and the fetuses with normal karyotypes took another CMA test. The data were analyzed with CHAS software. Finally all the cases were followed up to know about their pregnancy outcomes. RESULTS: One of the 54 cases was detected with abnormal karyotype which was trisomy 18(2%, 1/54). CMA was undertaken to the remaining fetuses, they were divided into 2 groups, including isolated TE group(n= 38)and complex TE group(n=15). The detection rate of clinical significant copy number variations(CNV)by CMA was 11%(6/53), while isolated and complex TE group were 5%(2/38)and 4/15, respectively(P= 0.047). Of the 53 cases, 51 cases were successfully followed up. Eleven cases were found without TE after birth, and the false positive rate(FPR)of TE was 22%(11/51). CONCLUSIONS: Whole-genome high-resolution CMA increased the detection rate by 11% in fetuses with TE. With the FPR and the detection rate of the clinical significant CNV of 2 groups, whole-genome CMA could be recommended to the fetuses with complex TE group but normal karyotypes. A series of ultrasonic tests should be suggested to the isolate TE group, while with the abnormal ultrasound, fetuses would be suggested to have CMA test for decreasing the rates of invasive prenatal diagnosis and FPR.