ONLINE MUTATION REPORT GATA4 zinc finger mutations as a molecular rationale for septation defects of the human heart

G ATA4 is localised on human chromosome 8p23.1-p22 and codes for a zinc finger transcription factor. Mice lacking Gata4 suffer from defective ventral morphogenesis and heart tube formation. 2 This factor therefore targets various genes important for heart development and basic cardiac function. Recently, three GATA4 mutations have been detected in families with congenital heart disease. 4 A mutation affecting a residue next to the C-terminal zinc finger has been identified in family members with atrial septal defects, and resulted in the disruption of a physical interaction between Gata4 and TBX5. There is growing evidence that the two zinc fingers of Gata4 display distinct functions so as to facilitate protein–DNA and protein–protein interactions. To further our understanding of the specific role of GATA4 in human congenital heart disease, we searched for mutations in the sequences coding for the zinc fingers in diseased heart tissues of 68 patients with complex heart malformations, encompassing atrial (ASD), ventricular (VSD), and atrioventricular septal defects (AVSD). These patients mostly died in early infancy as a result of the severity of the cardiac abnormalities. We isolated genomic DNA from the formalin fixed malformed hearts, amplified exons 3 and 4 from DNA fragments of about 2 kb, and carried out double strand direct sequencing. Mutations were confirmed by polymerase chain reaction restriction fragment length polymorphism (PCRRFLP) or cloning and subsequent sequencing to separate alleles (fig 1A–F). We identified 23 mutations, of which several are located in the zinc fingers and basic regions of GATA4 (table 1). Except for three, the genotypes observed in our collection of 68 hearts were mostly heterozygous, as seen on sequence electropherograms. Some patients had two to four mutations, including four with mutations in both zinc fingers of GATA4. Cloning of a fragment from a patient heterozygous for two zinc N-finger mutations showed location on different alleles. Additional cloning of a fragment heterozygous for three nonsynonymous mutations (c.631TRC, c.687GRT, c.731ARG) gave three haplotypes after sequencing of four clones—that is, one clone with all three reference alleles (c.631T, c.687G, c.731A), two clones with one mutant allele (c.631T, c.687T, c.731A), and one clone with two mutant alleles (c.631C, c.687G, c.731G). These mutations were absent in DNA isolated from 10 normal formalin fixed hearts, five frozen normal hearts, and 50 blood samples from unrelated healthy individuals used as controls.