TBX1

IntroductionIntroduction

Insights into TBX1Insights into TBX1

The gene locus for TBX1 in humans is on 22q11.2 (chromosome 22). This is the region of a common mutation (named 22q11.2 deletion syndrome) characterised by a deletion of 3Mb of DNA (30 genes) leading to the phenotype of the DiGeorge syndrome. By examining patients with DiGeorge syndrome but without the 3Mb deletion syndrome, tbx1 has been flagged as one which has a high mutation rate, and is therefore implicated in DiGeorge syndrome. However, mutations in the protein TBX1 alone do not implicate mental retardation. Mutants of TBX1 are responsible for at least five major phenotypes:

• Conotruncal anomaly face
• Cardiac defects
• Thymic hypoplasia
• Velopharyngeal insufficiency with cleft palate
• Parathyroid disfunction with hypocalcaemia

TBX1 has been found to be haploinsufficient, i.e. dosage-specific: mice which carry too few or too many copies of TBX1 typically show most of the signs of DiGeorge syndrome. However, TBX1 may not be the only implicated gene: Crkl mutations causes some of the DiGeorge phenotype abnormalities.

The TBX1 gene consists of twelve exons: 1-8, 9A, 9B, 10 and 9C, and has three alternative spliced forms of the gene. Thus, when screening for mutants the protein must be screened in all three alternative splice forms. One example of such a screening is for the -39 C→T mutation through a technique known as ARMS (amplification refractory mutation system). A screening of 38 patients with DiGeorge syndrome without the deletion uncovered this mutant. A functional analysis of this mutation is discussed below.

Functions and pathways of TBX1Functions and pathways of TBX1

TBX1 and CRKL genes interact in a dose dependent manner during development. In mice, Tbx1 activates the transcription of an Fgf10 promoter dependent on a T-box binding element (TBE). Fgf8 has also been postulated as a possible taret of Tbx1. Transcription of TBX1 in humans depends on an enhancer with a forkhead binding site responsive to Foxc1, Foxc2 and Foxa2, but links in these pathways are uncertain.

The molecular basis of dosage sensitivity is unknown, however it is thought that activation requires heterodimerisation but overexpression leads to homodimerisation.

Known malfunctional mutationsKnown malfunctional mutations

• Lysine 411 → Proline in exon 9C
• tbx1 1223delC (eliminates nuclear localisation signal and produces frameshift for 51 codons)
• -39C → T (exon 2) - lies within untranslated region and affects secondary structure of mRNA
• Phenylalanine 148 → Tyrosine - conserved residue in the T-box domain
• Glycine 310 → Serine (conserved residue)
• 1274-1281 delACTATCTC → translational frameshift mutation

-39C → T-39C → T

The -39C → T mutant change affects the secondary structure of the mRNA. It lies outside of the 129 bp 5'-UTR, which ordinarily contains 76% GC content. Molecular modelling predicts a stable secondary structure for the wild-type TBX1, but the presence of T instead of C at -39 reduces the stability of this fold, reuslting in a different optimal folding from that of the wild type. Using luciferase as a marker, experiments showed that this mutation greatly increases the amount of TBX1 present in the cell by about 2-fold. This results in the phenotype caused by overexpression of TBX1.