TCOF1 is the primary pathogenic gene for TCS in approximately 78–93% of cases10. More than 250 mutations have been identified in TCOF1, while the causative pathogenic variants in 11% of TCS cases are still unknown11–13 (Fig. 3). We studied a Chinese family with TCS and identified a potential pathogenic mutation (TCOF1 c.1562_1574del), which can provide a reference for future research on TCS.
TCOF1 encodes a nucleolar protein (Treacle) that contains three domains: unique amino Lis1 homology domain, C-terminus, and a characteristic CRD2 (Fig. 4A). Treacle participates in the transcription of ribosomal DNA (rDNA) genes, which are essential for the formation and proliferation of NCCs and the development of craniofacial tissues14. During transcription, the CRD and C-terminus of TCOF1 interact with RNA polymerase I (Pol I) and upstream binding factor (UBF), respectively15,16 (Fig. 4B). Two pathogenic mechanisms for TCOF1 leading to TCS have been proposed: decreasing ribosomal RNA (rRNA) or disrupting the stability of telomeres, both of which could alter the cell fate of NCCs and result in the malformation of craniofacial tissues17,18. Valdez et al.16 found that downregulated TCOF1 expression would cause insufficient production of rRNA and abnormal formation of ribosomes during NCC development, ultimately contributing to abnormal NCC formation and proliferation. Xin et al.19 found that the lack of TCOF1 results in telomere replication defects, causing fragile telomeres and genome instability, which may damage the survival and proliferation of NCCs and give rise to TCS. In this study, TCOF1 c.1562_1574del was a heterozygous mutation. Haploinsufficiency may also be a possible pathogenic mechanism because Dixon et al.20 found that Tcof1 haploinsufficient embryos represented with severe cranio-skeletal defects. Thus, Tcof1 affects NCC formation and proliferation, causing a deficiency in the number of cranial NCCs.
The TOCF1 protein has ~ 73% intrinsically disordered regions that are able to extensively interact with other proteins through phase separation, to perform certain or even complex physiological and pathological processes in cells21–25. In ribosome synthesis, TCOF1 binding with Pol I and chromatin-bound UBF is implicated in the process of transcription through phase separation23,26. The TCOF1 c.1562_1574del (p.A521fs) mutation damaged the CRD, causing the failure of Pol I binding, which may result in abnormal phase separation (Fig. 4A, B). Additionally, the absence of a C-terminus eliminates recognition of the rRNA promoter and UBF recruitment, which may disrupt the formation of CUL3KBTBD8-NOLC1-TCOF1 platform and lead to deficient ribosome biogenesis in NCCs27 (Fig. 4B).
Antenatal examination of TCS is still limited by the broad spectrum of identified pathogenic variants of TCS and the mismatch between genotypes and phenotypes. Therefore, it is urgent to continue to uncover pathogenic mutations of TCS, which will improve the accuracy of antenatal examination and provide better guidance for genetic counseling. Meanwhile, a clear understanding of the sequence-structure-function relationship of TCOF1 and the mechanism of protein truncation leading to abnormal phase separation may reveal the etiology of TCS more accurately.
A de novo frameshift mutation in TCOF1 (c.1562-1574del, p.A521fs) was identified in a Chinese TCS family. The deletion in TCOF1 may cause the failure in interaction of TCOF1 with other transcriptional factors and may inhibit the development of NCCs. The discovery of the novel mutation expands the mutation spectrum of TCOF1 in TCS and provides a reference for antenatal examination and future gene therapy.