Reduced transcription of TCOF1 in adult cells of Treacher Collins syndrome patients

Abstract

Background: Treacher Collins syndrome (TCS) is an autosomal dominant craniofacial disorder caused by frameshift deletions or duplications in the TCOF1 gene. These mutations cause premature termination codons, which are predicted to lead to mRNA degradation by nonsense mediated mRNA decay (NMD). Haploinsufficiency of the gene product (treacle) during embryonic development is the proposed molecular mechanism underlying TCS. However, it is still unknown if TCOF1 expression levels are decreased in post-embryonic human cells.

Methods: We have estimated TCOF1 transcript levels through real time PCR in mRNA obtained from leucocytes and mesenchymal cells of TCS patients (n = 23) and controls (n = 18). Mutational screening and analysis of NMD were performed by direct sequencing of gDNA and cDNA, respectively.

Results: All the 23 patients had typical clinical features of the syndrome and pathogenic mutations were detected in 19 of them. We demonstrated that the expression level of TCOF1 is 18-31% lower in patients than in controls (p < 0.05), even if we exclude the patients in whom we did not detect the pathogenic mutation. We also observed that the mutant allele is usually less abundant than the wild type one in mesenchymal cells.

Conclusions: This is the first study to report decreased expression levels of TCOF1 in TCS adult human cells, but it is still unknown if this finding is associated to any phenotype in adulthood. In addition, as we demonstrated that alleles harboring the pathogenic mutations have lower expression, we herein corroborate the current hypothesis of NMD of the mutant transcript as the explanation for diminished levels of TCOF1 expression. Further, considering that TCOF1 deficiency in adult cells could be associated to pathologic clinical findings, it will be important to verify if TCS patients have an impairment in adult stem cell properties, as this can reduce the efficiency of plastic surgery results during rehabilitation of these patients.

Figure 1

Normalized TCOF1 expression levels in TCS patients (circles) and controls (squares). A) TCOF1 expression levels in leucocytes samples from patients (mean ± SEM = 6.846 ± 0.3278) and controls (mean ± SEM = 8.093 ± 0.3587). B) TCOF1 expression levels in mesenchymal cell samples from patients (mean ± SEM = 0.9687 ± 0.1122) and controls (mean ± SEM = 1.414 ± 0.09010). Mean expression value is represented by horizontal lines.

Figure 2

Genomic DNA (gDNA) and complementary DNA (cDNA) sequencing of mesenchymal stem cells samples from four TCS patients. The pathogenic mutation of TCS patients 16, 21, 22, and 23 are c.218_222insAACC (exon 3), c.4344dupA (exon 24), c.431delC (exon 5), and c.4218dupG (exon 23), respectively. gDNA was sequenced with intronic primers and cDNA with exonic primers. Observing all gDNA samples, we can assume that all analyzed individuals are heterozygous for the pathogenic mutation. Analyzing cDNA samples, we could detect the mutant allele expression in TCS 16 and 23; TCS 21 and 22 express only the wild-type allele. Note that even when the mutant allele is expressed (patients TCS16 and TCS23), the peak heights are lower.