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Hypertrophic chondrocytes and their specific marker, the type X collagen gene (Col10a1), are critical components of endochondral bone formation during skeletal development. We previously found that Runx2 is an indispensable mouse Col10a1 gene regulator and identified many other transcription factors (TFs) that potentially interact with the 150-bp Col10a1 cis-enhancer. However, the roles of these candidate TFs in Col10a1 expression and chondrocyte hypertrophy have not been elucidated. Here, we focus on 32 candidate TFs recently identified by analyzing the 150-bp Col10a1 enhancer using the transcription factor affinity prediction (TRAP) program. We found that 12 TFs (Hoxa3, Lsx, Evx2, Dlx5, S8, Pax2, Egr2, Mef2a, Barhl2, GKlf, Sox17, and Crx) were significantly upregulated and four TFs (Lhx4, Tbx5, Mef2c, and Hb9) were significantly downregulated in hypertrophic MCT cells, which show upregulation of Col10a1 expression. Most of the differential expression pattern of these TFs conformed with the results obtained from ATDC5 cell model and primary mouse chondrocytes. Notably, Tbx5 was downregulated upon Col10a1 upregulation, overexpression of Tbx5 decreased Col10a1 expression, and knock-down of Tbx5 increased Col10a1 expression in hypertrophic chondrocytes, suggesting that Tbx5 is a negative regulator of Col10a1. We further generated a stable Tbx5-overexpressing ATDC5 cell line and ColX-Tbx5 transgenic mice driven by Col10a1-specific enhancers and promoters. Tbx5 overexpression decreased Col10a1 expression in ATDC5 cells cultured as early as day 7 and in limb tissue on post-natal day 1. Slightly weaker alkaline phosphatase staining was also observed in cell culture on day 7 and in limb digits on embryonic day 17.5, indicating mildly delayed ossification. Further characterization of these candidate Col10a1 transcriptional regulators could help identify novel therapeutic targets for skeletal diseases associated with abnormal chondrocyte hypertrophy.

Circulating fetal cell-free DNA (cfDNA) is generally shorter than maternal cfDNA. Size selection of shorter cfDNA in total cfDNA could significantly increase the fetal fraction, but there are few reports of using this method to decrease the false negative rate for NIPT. In this study, nine false negative cases were retrospectively analyzed by NIPT retesting and E-gel based size-selection NIPT and the fetal cfDNA fraction in maternal total cfDNA was evaluated by calculating the proportion of reads from chromosome Y. Fetal placenta karyotypes were confirmed by CNVplex assays to analysis the reasons for false negative cases. Of the 81,601 pregnancies who underwent NIPT, nine false negative cases (0.01%) were found. Of eight retested cases, two (25%) had positive NIPT retest results, and five (62.5%) had positive size-selection NIPT results. For fetal cfDNA fraction, 100% cases had improvement after size-selection NIPT compared with the initial NIPT and retest results, and the fetal cfDNA fraction growth ratio ranged from 99 to 359%. For one twin pregnancy with one T18 fetus, size selection improved the fetal cfDNA fraction to 23.10%, and successfully detected the T18 fetus in NIPT. Placental tissue analysis results for two cases indicated both had confined placental mosaicism (CPM), which was confirmed with size-selection NIPT. In conclusion, size selection can significantly enrich the fetal cfDNA fraction and decrease the false negative rate of NIPT, especially for CPM and twin pregnancies.