Fig 1.
Location of FOXC2 and PITX2 variants identified in congenital glaucoma patients in this study.
(A, B) Cartoons correspond to FOXC2 and PITX2C mRNAs indicating the localization of different encoded protein domains. Rectangles the solid lines represent coding and 3’UTR sequences, respectively. FHD, forkhead domain; HD: homeodomain; ID, inhibitory domain; TA, transactivation domain. The green box in PITX2C mRNA indicates the 14-aminoacid OAR (Otp and ARistaless) domain [39]. (C) FOXC2 and FOXC2-AS1 gene structure as a notated in Ensembl. Note the presence of a promoter (regulatory region, red rectangle) overlapping the two genes.
Fig 2.
Prediction of conformational changes induced by the identified coding FOXC2 and PITX2 variants.
Predicted model structures of wild type (left) (A, C, E and G) and mutant (right) proteins (B, D, F and H) were obtained with QUARK as described in Materials and Methods.
Table 1.
FOXC2, PITX2 and CYP1B1 variants identified in congenital glaucoma patients.
Table 2.
Rare FOXC2 and PITX2 gene variations identified in PCG and associated clinical features.
Fig 3.
Evidence for modifier effect of rare PITX2 variants on age at onset and severity in CYP1B1 congenital glaucoma patients.
All mutations were detected in the heterozygous state. Note that subject II:1 in family PCG-133 was diagnosed at the age of 3 months and carried the de novo p.(P179T) PITX2 variant, whereas his brother, who did not carry this variant, was diagnosed at the age of 10 years. The proband in family PCG-139 also carried a rare PITX2 variant (p.(A188T)) and presented glaucoma diagnosed at the age of seven days. Both probands required more surgical operations to control IOP than the rest of patients. Below symbols are indicated genotypes for CYP1B1 and PITX2, age at diagnosis and number or surgical operations per eye, respectively. M1, CYP1B1: p.(A179fs*18). M2, CYP1B1: p.(E387K). M3, CYP1B1: p.(E173*). M4, PITX2: p.(P179T). M5, PITX2: p.(A188T). Arrows show the index cases. +: wild-type allele. The asterisk indicates a de novo PITX2 variant.
Fig 4.
Multiple amino acid and nucleotide sequence alignment of FOXC2 and PITX2 from different vertebrate species.
Sequence alignment was generated by ClustalW (http://www.ebi.ac.uk/clustalw/). Residues affected by mutations are indicated by arrows.
Fig 5.
Altered transcriptional activity of FOXC2 and PITX2 variants identified in this study.
cDNA constructs encoding the different variants were transiently co-expressed in HEK-293T cells along with cDNAs constructs encoding luciferase coupled to the CXCR4 promoter (transactivation reporter) and the red fluorescent protein (RFP) as a transfection control. The transcriptional activity was normalized to the amount of the corresponding wild type protein and represented as percentage of the luciferase activity of the wild-type protein. Each lane contained 15 μg of total protein obtained from the cell lysates. The protein levels of the different FOXC2 and PITX2 versions present in HEK-293T cells 24 h after transfection were determined by western immunoblot using a monoclonal anti-myc antibody (1:300) (Santa Cruz). RFP was analyzed via western immunoblot using an anti-RFP antibody (1:500) (Evrogen). The sample loading control, endogenous LDH, was also detected via immunoblot using an anti-LDH antibody (1:1000) (Chemicon). Error bars correspond to the SD of two independent experiments carried out in triplicate. Asterisks indicate statistical significance as compared to the control: P<0,01 (**); P<0,001 (***). Significance was calculated by one-way ANOVA followed by Tukey multiple-comparison test and t-student test.
Fig 6.
FOXC2 p.H395N variant decreases protein stability.
Time course stability analysis of FOXC2 (A) and PITX2 (C) coding variants found in PCG patients was carried out by transient expression in HEK-293T cells. Transfected cells were treated with the protein synthesis inhibitor cycloheximide and the different recombinant proteins were detected by Western immunoblot using an anti-myc monoclonal antibody (Santa Cruz) at the indicated time points, as explained in Materials and Methods. Relative amounts of FOXC2 are expressed as a percentage of levels at time 0 h. The rate of decay and half-lives of the recombinant FOXC2 (B) and PITX2 (D) coding variants at the indicated time-points were determined from linear regression analysis as described in the Materials and Methods. Error bars correspond to the SD of three independent experiments carried out in triplicate. Asterisks indicate statistical significance compared to the control: p<0.01 (**). Two-way ANOVA followed by Tukey multiple-comparison test.
Fig 7.
Coding FOXC2 and PITX2C variants identified in this study do not alter subcellular localization in cells in culture.
cDNA constructs encoding the indicated recombinant versions of FOXC2 and PITX2C were transiently expressed in HEK-293T cells. The recombinant proteins were detected by fluorescent immunocytochemistry with an anti-myc antibody (Santa Cruz). Nuclei were visualized by fluorescent DAPI staining. Original magnification: X600.