First Report of a Single Exon Deletion in TCOF1 Causing Treacher Collins Syndrome

Abstract

Treacher Collins syndrome (TCS) is a rare craniofacial disorder characterized by facial anomalies and ear defects. TCS is caused by mutations in the TCOF1 gene and follows autosomal dominant inheritance. Recently, mutations in the POLR1D and POLR1C genes have also been identified to cause TCS. However, in a subset of patients no causative mutation could be found yet. Inter- and intrafamilial phenotypic variability is high as is the variety of mainly family-specific mutations identified throughout TCOF1. No obvious correlation between pheno- and genotype could be observed. The majority of described point mutations, small insertions and deletions comprising only a few nucleotides within TCOF1 lead to a premature termination codon. We investigated a cohort of 112 patients with a tentative clinical diagnosis of TCS by multiplex ligation-dependent probe amplification (MLPA) to search for larger deletions not detectable with other methods used. All patients were selected after negative screening for mutations in TCOF1, POLR1D and POLR1C. In 1 patient with an unequivocal clinical diagnosis of TCS, we identified a 3.367 kb deletion. This deletion abolishes exon 3 and is the first described single exon deletion within TCOF1. On RNA level we observed loss of this exon which supposedly leads to haploinsufficiency of TREACLE, the nucleolar phosphoprotein encoded by TCOF1.

Fig. 1

The patient at the age of 26 years, showing the characteristic facial anomalies of TCS: down-slanting palpebral fissures, a lower eyelid coloboma, hypoplasia of the zygomatic complex and low-set, small ears. Earlier photographs were published previously [Teber et al., 2004].

Fig. 2

MLPA analysis. a Result for the patient. b Result for the father. Each square represents 1 MLPA probe. The bars enclose the range for a normal dosage. The probe for exon 3 shows a reduced dosage in the patient.

Fig. 3

a Result of the long-range PCR. PCR from the patient (p), a normal person (co) as control, the healthy father (f) of the patient and the negative control (H₂O). Product length of the wild-type allele was 6,840 bp. For the patient only the preferentially amplified product for the deleted allele of 3.367 kb was visible, confirming the deletion detected by MLPA. b Sequencing of the breakpoint and scheme of TCOF1. The figure shows the result of the sequence analysis of the patient's long-range PCR product under a scheme of TCOF1 (upper part: wild type with exon 3, lower part: with deletion of exon 3). The dashed lines indicate the position of the deletion. The 3 underlined nucleotides in the sequence can be aligned to both sides of the breakpoint (chr5: 149,741,531–149,741,534: 149,744,897–149,744,900, GRCh37/hg19). For comparison, part of the wild-type sequence is shown above.

Fig. 4

Results on RNA level. a Expression analysis of TCOF1 in peripheral blood RNA from the patient (M18662). The integrity of the RNA samples was demonstrated by amplification of an RNA-specific fragment of the ACTB locus. The upper TCOF1 product was derived from the wild-type allele (WT), while the lower product was derived from the allele harboring the deletion of exon 3 (del). +RT = RT-PCR with reverse transcriptase, –RT = RT-PCR without reverse transcriptase, H₂O = RT-PCR without RNA. b Sequencing of the breakpoint on RNA level and scheme of TCOF1. The result of the sequencing of the patient's transcripts with the forward (F_Ex1) and reverse (R_Ex4) primer is shown. The schemes above depict the WT mRNA of TCOF1 and the transcript lacking exon 3. The breakpoint is indicated by a dashed line. The thus introduced premature stop codon is indicated by an asterisk in exon 5 together with the truncation. The results confirm that exon 2 is spliced directly onto exon 4. Black and grey squares = exons, small grey squares = UTR, horizontal dashed line indicates that not all exons are shown.