case_images: PMC4338276_01
case_images: GATA6 variant proband 1.. A) DNA sequence of proband and parents showing a single base pair change from G in the parents to a T in the proband at nucleotide position c.1428+1 G>T. B) Diagram of the GATA6 protein containing a GATA family homeodomain and two DNA binding zinc fingers. Arrows indicate locations of GATA6 variants discovered in proband 1 and 2.
case_images: pone.0118449.g001.jpg
case_images: PMC4338276_01_pone.0118449.g001.jpg
case_images: pone.0118449.g001
case_images: T transition in GATA6 at position c.1428+1, which was confirmed by Sanger sequencing (Fig. 1A). This nucleotide change in proband 1 is located in the exon 4 splice donor site on GATA6 c.1428+1 G>T. The location of this variant likely abrogates proper splicing, resulting in a run-on transcript with downstream exons out of frame or could possibly result in skipping exon 4, which would truncate part of a zinc-finger DNA binding domain (Fig. 1B). Translation of the c.964_970delACGTACC GATA6 sequence we identified in proband 2 is predicted to produce a frame shift mutation in exon 2 at tyrosine 323 changing it to a threonine (Y323T) and to introduce a premature stop codon 21 amino acids downstream of the frame shift (Y323fsX21) (Fig. 1B). The deleted C-terminal region of GATA6 contains both zinc finger DNA binding domains and the nuclear localization sequence (Fig. 1B).
case_images: GATA6 variant proband 2.. DNA Sanger sequencing of proband 2 demonstrates he has a normal allele (upper) and a mutant allele (lower). The wild-type GATA6 sequence c964_970 (underlined region) is deleted in the mutant allele (arrow).
case_images: pone.0118449.g002.jpg
case_images: PMC4338276_01_pone.0118449.g002.jpg
case_images: pone.0118449.g002
case_images: Our sequencing confirmed that proband 2 has a deletion of seven nucleotides (c.964_970delACGTACC) in the GATA6 gene (Fig. 2).
case_images: Loss of Hnf4alpha promoter activation by GATA6 with GATA6
  Y323fsX21.. A) Human wild-type GATA6 and/or GATA6
  Y323fsX21 labeled 'GATA6 variant' were co-expressed with Hnf4alpha promoter in INS-1 cells. Hnf4alpha promoter luciferase activity was measured and levels normalized to vector alone. *P = 0.00001. B) Western blot analysis of human GATA6 protein (W lane) and GATA6
  Y323fsX21 (M lane) expressed in HEK 293T cells. Vector (V lane) only was a negative control and to approximate the size a protein ladder (L lane) was utilized. GATA6 was detected with a GATA6 antibody against the N-terminal of the protein and beta actin was measured as a loading control.
case_images: pone.0118449.g003.jpg
case_images: PMC4338276_01_pone.0118449.g003.jpg
case_images: pone.0118449.g003
case_images: Western blotting revealed that cells transfected with mutant GATA6 generate a truncated protein (Fig. 3B). We found that wild-type GATA6 transactivates the HNF4alpha promoter as expected (Fig. 3A). Conversely, GATA6 Y323fsX21 failed to transactivate HNF4alpha promoter (Fig. 3A). In addition, co-transfection of GATA6 Y323fsX21 with wild-type GATA6 did not inhibit HNF4alpha-Luc activation (Fig. 3A).