RNA sequencing (RNA-seq) provides transcript-level evidence for interpreting variants of uncertain significance in monogenic disorders, but its prenatal application is limited by sample availability and processing time. This study evaluated uncultured amniocytes as a practical substrate for prenatal RNA-seq. We performed RNA-seq on 15 matched paired cultured and uncultured amniocyte samples and compared their transcriptomic profiles using principal component analysis, differential expression analysis, and pathway enrichment. We then analyzed 77 uncultured amniocyte samples collected between 16⁺⁶ and 29⁺⁶ weeks of gestation to disease-related gene expression coverage and gestational-age-associated variation. Five selected cases were further analyzed to evaluate transcript-level findings relevant to variant interpretation. Cultured and uncultured amniocytes showed distinct transcriptomic profiles, with 11,234 differentially expressed genes and culture-associated transcriptional and pathway changes. Uncultured amniocytes expressed an average of 63.4% of genes across disease-associated panels, and most disease-related genes were consistently expressed across gestational stages, although stage-associated differences in gene detectability were observed. In selected cases, uncultured amniocyte RNA-seq detected aberrant expression, aberrant splicing, allelic imbalance, and expressed sequence variants. These findings support the feasibility of prenatal RNA-seq using uncultured amniocytes and further evaluation of this approach as a complementary functional assay for prenatal variant interpretation.

Introduction: Congenital duodenal obstruction (CDO) is one of the most common fetal gastrointestinal anomalies, but previous prenatal studies tend to investigate CDO as a portion of gastrointestinal obstruction. Few studies describe the genetic findings of CDO, especially copy number variants, in fetal cohorts with a relatively large sample size. The study aims to investigate the detection rate of genetic causes at different levels and to explore the potential influencing factors of perinatal outcomes of fetuses with CDO. Material and Methods: This retrospective study analyzed karyotype, chromosomal microarray analysis, and trio‐whole exome sequencing (trio‐WES) results of singleton fetuses suspected of CDO in a tertiary center between January 2014 and September 2023. In addition, perinatal outcomes and postnatal medical records of enrolled cases were followed up and analyzed. Results: A total of 98 fetuses were included in the study, of which 69 (70.4%) were classified as isolated CDO and 29 (29.6%) were classified as non‐isolated. The overall rate of genetic anomalies was 20.4% (20/98). Trisomy 21 (9/98 [9.2%]) and microduplication of the 17q12 region (3/98 [3.2%]) were the most common chromosomal numerical abnormalities and pathogenic copy number variants found in this cohort, respectively. Compared to chromosomal microarray analysis, no additional pathogenic or likely pathogenic variants were found in seven cases undergoing trio‐WES. The rate of chromosomal numerical and structural abnormalities was significantly higher in the non‐isolated group (8/29 [27.6%] vs. 6/69 [8.7%], p < 0.05). In terms of perinatal outcomes, the live birth rate was significantly higher in the isolated group (52/69 [75.4%] vs. 15/29 [51.7%], p < 0.05) largely due to fewer terminations of pregnancy. All cases with positive genetic results elected to terminate the pregnancy, and 82% of those with negative results opted to continue the pregnancy. Neonatal mortality was significantly higher in the non‐isolated group (2/15 [13.3%] vs. 0/52 [0.0%], p < 0.05). The overall neonatal survival rate was 97.0% (65/67). Conclusions: The present study highlights the value of prenatal diagnostic testing for fetuses suspected of CDO, both in isolated and non‐isolated cases. Genetic diagnostic outcomes exert substantial influence on pregnancy decision‐making. Perinatal outcome and short‐term prognosis of affected fetuses are reasonably favorable when known genetic causes are excluded.

Background: Dandy–Walker malformation (DWM) is a rare congenital brain defect whose mechanism is still not fully understood. Genetic studies have suggested that NADH Dehydrogenase 1 alpha Subcomplex 4 (NDUFA4) may be associated with DWM; however, the functional consequences of NDUFA4 dysregulation in experimental neural models remain unclear. In this study, we investigated the cellular effects of NDUFA4 deficiency, rather than aiming to establish a causal mechanism for DWM. Methods: NDUFA4‐related differentially expressed proteins were analyzed using iTRAQ tandem mass spectrometry, followed by functional, pathway, and protein interaction network analyses. Then, voltage‐dependent anion Channel 1 (VDAC1), apoptosis‐, endoplasmic reticulum (ER) stress–related proteins, and ETC Complex IV activity were assessed in NDUFA4 knockout mice. After NDUFA4 and VDAC1 knockdown, cell proliferation, apoptosis, mitochondrial status, and ER function were evaluated by CCK‐8, Edu staining, flow cytometry, and Western blot in C8‐D1A cells. In addition, the interaction between NDUFA4 and VDAC1 was evaluated using immunofluorescence and coimmunoprecipitation. Results: NDUFA4 knockout upregulated VDAC1, ER stress‐ and apoptosis‐related proteins, and inhibited ETC complex IV activity in mice. NDUFA4 knockdown inhibited proliferation and promoted apoptosis, ER stress, and mitochondrial damage in C8‐D1A cells, and these changes were partially reversed by VDAC1 knockdown. Moreover, NDUFA4 and VDAC1 colocalized in C8‐D1A cells and mouse cerebellar tissue, and NDUFA4 was found to interact with VDAC1. Conclusions: NDUFA4 deletion was associated with VDAC1 upregulation, mitochondrial damage, ER stress, and apoptosis‐related changes in our experimental models. These findings may provide insight into cellular changes potentially relevant to DWM; however, they do not establish a direct causal relationship.