TBX1 mutation identified by exome sequencing in a Japanese family with 22q11.2 deletion syndrome-like craniofacial features and hypocalcemia

Abstract

Background: Although TBX1 mutations have been identified in patients with 22q11.2 deletion syndrome (22q11.2DS)-like phenotypes including characteristic craniofacial features, cardiovascular anomalies, hypoparathyroidism, and thymic hypoplasia, the frequency of TBX1 mutations remains rare in deletion-negative patients. Thus, it would be reasonable to perform a comprehensive genetic analysis in deletion-negative patients with 22q11.2DS-like phenotypes.

Methodology/principal findings: We studied three subjects with craniofacial features and hypocalcemia (group 1), two subjects with craniofacial features alone (group 2), and three subjects with normal phenotype within a single Japanese family. Fluorescence in situ hybridization analysis excluded chromosome 22q11.2 deletion, and genomewide array comparative genomic hybridization analysis revealed no copy number change specific to group 1 or groups 1+2. However, exome sequencing identified a heterozygous TBX1 frameshift mutation (c.1253delA, p.Y418fsX459) specific to groups 1+2, as well as six missense variants and two in-frame microdeletions specific to groups 1+2 and two missense variants specific to group 1. The TBX1 mutation resided at exon 9C and was predicted to produce a non-functional truncated protein missing the nuclear localization signal and most of the transactivation domain.

Conclusions/significance: Clinical features in groups 1+2 are well explained by the TBX1 mutation, while the clinical effects of the remaining variants are largely unknown. Thus, the results exemplify the usefulness of exome sequencing in the identification of disease-causing mutations in familial disorders. Furthermore, the results, in conjunction with the previous data, imply that TBX1 isoform C is the biologically essential variant and that TBX1 mutations are associated with a wide phenotypic spectrum, including most of 22q11.2DS phenotypes.

Figure 1. The pedigree of this family.

Facial features of subjects III-5 and III-7 are shown.

Figure 2. FISH and array CGH analyses in the proband (III-5).

A. FISH analysis. Two signals are shown for both HIRA at 22q11.2 (red signals indicated by arrows) and ARSA at 22q13 (green signals indicated by arrowheads). B. Array CGH analysis. No copy number change is found for chromosome 10 carrying the second DiGeorge region and chromosome 22 harboring the DGS/VCFS critical region, as well as other chromosomes (not shown). Black, red, and green dots denote signals indicative of the normal, the increased (>+0.5), and the decreased (<−0.8) copy numbers, respectively. Although several red and green signals are seen, there is no portion associated with ≥3 consecutive red or green signals.

Figure 3. TBX1 mutation identified in this family.

A. Genomic structure of TBX1 and the position of the mutation. The color and the white boxes represent the coding regions and the untranslated regions on exons 1–10 (E1–E10), respectively; the red, the purple, and the orange segments indicate the coding regions on the final exons 9C, 9A, and 9B (splice variants), respectively. The T-box is indicated by yellow boxes, the nuclear localization signal (NLA) by a blue segment, and the transactivation domain (TAD) by a green arrow. The c.1253delA (p.Y418fsX459) identified in this family resides on exon 9C. B. Transcripts of TBX1. Three variants are formed by alternative splicing of the final exons 9C, 9A, and 9B. The c.1253delA (p.Y418fsX459) mutation is predicted to yield a truncated TBX1C protein missing the NLS and most of the TAD. The stippled box of p.Y418fsX459 denotes aberrant amino acid sequence produced by the frameshift mutation. C. Electrochromatograms showing the frameshift mutation by Sanger sequencing. The primer sequences used are: 5′-GCGGCCAAGAGCCTTCTCT-3′ and 5′-GGGTGGTAGCCGTGGCCA-3′.