Identification of Intragenic Exon Deletions and Duplication of TCF12 by Whole Genome or Targeted Sequencing as a Cause of TCF12-Related Craniosynostosis

Abstract

TCF12-related craniosynostosis can be caused by small heterozygous loss-of-function mutations in TCF12. Large intragenic rearrangements, however, have not been described yet. Here, we present the identification of four large rearrangements in TCF12 causing TCF12-related craniosynostosis. Whole-genome sequencing was applied on the DNA of 18 index cases with coronal synostosis and their family members (43 samples in total). The data were analyzed using an autosomal-dominant disease model. Structural variant analysis reported intragenic exon deletions (of sizes 84.9, 8.6, and 5.4 kb) in TCF12 in three different families. The results were confirmed by deletion-specific PCR and dideoxy-sequence analysis. Separately, targeted sequencing of the TCF12 genomic region in a patient with coronal synostosis identified a tandem duplication of 11.3 kb. The pathogenic effect of this duplication was confirmed by cDNA analysis. These findings indicate the importance of screening for larger rearrangements in patients suspected to have TCF12-related craniosynostosis.

Keywords: TCF12-related craniosynostosis; exon duplication; intragenic exon deletion; rearrangements.

Figure 1

Rearrangements identified in TCF12. A–E: Confirmation of deletions. A primer design. F1, forward primer; R1, reverse primer 1; R2, reverse primer 2. F1+R1, wild‐type allele (WT); F1+R2, mutant allele (Δ). B: Results of mutation‐specific deletion PCR analysis. Lane 1, negative control. Lanes 2–4, family 1; wild‐type fragment 1,455 bp, mutant fragment 644 bp; lane 2, II.1; lane 3, II.2; lane 4, III.1. Lanes 5–8, family 2; wild‐type fragment 1,107 bp, mutant fragment 556 bp; lane 5, II.1; lane 6, II.2; lane 7, II.3; lane 8, III.1. Lanes 9–11, family 3; wild‐type fragment 1,303 bp, mutant fragment 646 bp; lane 9, II.1; lane 10, II.2; lane 11, III.2. C–E: Electropherograms of dideoxy sequence analysis. C: Mutant allele in family 1, exons 7–18 deleted. D: Mutant allele in family 2, exon 19 deleted. E: Mutant allele in family 3, exon 20 deleted. Dashed lines indicate where deletions occurred. F–J: Confirmation of duplication. F: PCR from genomic DNA confirming the presence of duplication in index patient of family 4 (III.1), which was inherited from the index patient's clinically unaffected mother (II.2). G: The sequence at the duplication breakpoint is sandwiched between the normal proximal (above) and distal (below) sequences, with the electropherogram underneath (H). I: cDNA amplified from the index patient (III.1) with primers specific to the mutant allele. Two different products were visible on the gel (but absent in two control cDNA samples). J: Sequencing of these products indicated splicing from exon 20 to the duplicated exon 19 in the smaller‐sized product (lower electropherogram). The larger product (upper electropherogram) contains an additional 35 nucleotide neo‐exon between exons 20 and 19. The normal stop codon in exon 20 is highlighted.