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Thesis (Ph.D.). / Written for the Division of Experimental Medicine, Dept. of Medicine. Title from title page of PDF (viewed 2008/07/23). Includes bibliographical references.

Background: The 22q11 deletion syndrome (22q11DS), also known as DiGeorge Syndrome, affects ~1/5000 live born children. Congenital heart defects (typically outflow tract and interrupted aortic arch) are present in 75% of individuals with 22q11DS and are the major cause of mortality. Other defects are cleft palate, thymus hypoplasia, inner ear defects and neuropsychiatric abnormalities. Df(16)1 mice carry a ~1 Mb hemizygous deletion on mouse chromosome 16 in a region syntenic with 22q11 and phenocopies 22q11DS. TBX1 is a DNA-binding transcription factor located in this interval and is required for neural crest cell proliferation and migration and for cardiac development. TBX1 point mutations have been identified in patients with DiGeorge syndrome. Thus TBX1 is thought to be a major gene responsible for the cardiac phenotype in 22q11DS. A key unresolved issue is the mechanism of reduced penetrance of cardiac malformations. One possibility is environmental variation during cardiogenesis. A second possibility is that variation in the TBX1 protein interaction network results in variable penetrance of the phenotype. Mutations in TBX1 or interacting partners could affect the structure of this protein interaction network. Aim: The aim of this thesis is to characterize the molecular mechanism of TBX1 function using biochemical and genetic approaches and to define the role of environmental variation on the DiGeorge phenotype. Results First part. Interaction of Df(16)1 with high-fat maternal diet. To determine if a maternal high-fat diet affects the penetrance of cardiac and thymus malformations in the Df1 deletion mouse model, wild-type and Df1 heterozygous embryos from control and high-fat diet groups were analyzed. No significant difference in the penetrance or the severity of cardiac malformations between these groups was found. These results do not support the idea that change in the fat content of maternal diet affects phenotype in this model. Thus, it is possible that high-fat diet interacts specifically with left-right patterning rather than with the genetic control of pharyngeal arch development and neural crest cell migration and survival. Second part. George, a novel ENU induced mutation in Tbx1. The George mutation, identified and mapped to Chr16 between rs4161352 and D16Mit112, results in fully penetrant cleft palate, cardiac malformations (VSD, IAA, CAT), absent cochlea and abnormal semicircular canals, and absent thymus resembling the human DiGeorge phenotype. Tbx1 lies in this interval and sequencing identified a G > A point mutation in exon 3 which is predicted to cause a Arginine to Glutamine change at amino acid position 160. George fails to genetically complement a Tbx1 null allele, confirming that it is causative and that George is functionally a null allele. RT-PCR showed that the George mutation affects splicing, resulting in a transcript lacking exon 3. This causes the loss of 34 amino acids within the TBX1 T-box domain, thus predicting that it affects DNA binding. Transactivation assays show that while the R160Q amino acid substitution significantly reduces the transactivation capacity of TBX1, surprisingly the loss of exon 3 does not affect this function. Analysis of endogenous TBX1 in developing embryos by Western blot showed that the protein expression is absent or significantly reduced. This finding suggests that the observed George phenotype is caused primarily by a loss of TBX1 protein expression. Third part. Investigation of the protein interaction network surrounding TBX1. In order to get a better insight into the protein network surrounding TBX1, a TBX1 split renilla-luciferase protein complementation assay was set up which allowed to test the physical interaction between TBX1 and several putative interactors. It was found that GATA4, SMARCAD1, RBBP5 and PTDSR interact with wild-type TBX1 in HEK293T cells. The R160Q point mutation and the loss of exon 3 affect some of these interactions supporting the idea that variation in the protein interaction network may, at least in part, be responsible for the DGS phenotype.

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