<i>TBX1</i> Mutation Identified by Exome Sequencing in a Japanese Family with 22q11.2 Deletion Syndrome-Like Craniofacial Features and Hypocalcemia

<div><p>Background</p><p>Although <i>TBX1</i> 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 <i>TBX1</i> 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.</p><p>Methodology/Principal Findings</p><p>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 <i>in situ</i> 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 <i>TBX1</i> 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 <i>TBX1</i> 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.</p><p>Conclusions/Significance</p><p>Clinical features in groups 1+2 are well explained by the <i>TBX1</i> 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 <i>TBX1</i> isoform C is the biologically essential variant and that <i>TBX1</i> mutations are associated with a wide phenotypic spectrum, including most of 22q11.2DS phenotypes.</p></div

The pedigree of this family.

<p>Facial features of subjects III-5 and III-7 are shown.</p

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

<p><b>A.</b> FISH analysis. Two signals are shown for both <i>HIRA</i> at 22q11.2 (red signals indicated by arrows) and <i>ARSA</i> at 22q13 (green signals indicated by arrowheads). <b>B.</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.</p

Clinical findings of the family members.

<p>Individuals correspond to those shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091598#pone-0091598-g001" target="_blank">Fig. 1</a>.</p><p>i-phosphate: inorganic phosphate; SD: standard deviation; F: female; M: male; and N.E.: not examined.</p>a<p>Reference values: calcium, 9.0–11.0 mg/dL in infants and 8.8–10.2 mg/dL in adults; inorganic phosphate, 4.8–7.5 mg/dL in infants and 2.5–4.5 mg/dL in adults, and intact PTH, 10–65 pg/dL in infants and 14–55 pg/dL in adults.</p><p>Conversion factor to the SI unit: 0.25 for calcium (mmol/L), 0.32 for inorganic phosphate (mmol/L), and 0.106 for intact PTH (pmol/L).</p>b<p>Examined by echocardiography, chest roentgenography, and/or electrocardiography.</p>c<p>Examined by computed tomography.</p>d<p>Received velopharyngeal closure.</p>e<p>On treatment with vitamin D.</p>f<p>Repeated otitis media only.</p>g<p>Received speech therapy.</p>h<p>Required hearing aids.</p>i<p>At the time of diagnosis (11 years of age), serum TSH was <0.01 mIU/L, free T₃ 33.1 pg/mL [51.0 pmol/L], free T₄ 5.11 ng/dL [65.8 nmol/L], and TSH receptor antibody 1284% [normal range <1.9%].</p

<i>TBX1</i> mutation identified in this family.

<p><b>A.</b> Genomic structure of <i>TBX1</i> 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>B.</b> Transcripts of <i>TBX1</i>. 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. <b>C.</b> Electrochromatograms showing the frameshift mutation by Sanger sequencing. The primer sequences used are: 5′-GCGGCCAAGAGCCTTCTCT-3′ and 5′-GGGTGGTAGCCGTGGCCA-3′.</p