Mutations in TCF8 cause posterior polymorphous corneal dystrophy and ectopic expression of COL4A3 by corneal endothelial cells

Abstract

Posterior polymorphous corneal dystrophy (PPCD, also known as PPMD) is a rare disease involving metaplasia and overgrowth of corneal endothelial cells. In patients with PPCD, these cells manifest in an epithelial morphology and gene expression pattern, produce an aberrant basement membrane, and, sometimes, spread over the iris and nearby structures in a way that increases the risk for glaucoma. We previously mapped PPCD to a region (PPCD3) on chromosome 10 containing the gene that encodes the two-handed zinc-finger homeodomain transcription factor TCF8. Here, we report a heterozygous frameshift mutation in TCF8 that segregates with PPCD in the family used to map PPCD3 and four different heterozygous nonsense and frameshift mutations in TCF8 in four other PPCD probands. Family reports of inguinal hernia, hydrocele, and possible bone anomalies in affected individuals suggest that individuals with TCF8 mutations should be examined for nonocular anomalies. We detect transcripts of all three identified PPCD genes (VSX1, COL8A2, and TCF8) in the cornea. We show presence of a complex (core plus secondary) binding site for TCF8 in the promoter of Alport syndrome gene COL4A3, which encodes collagen type IV alpha 3, and we present immunohistochemical evidence of ectopic expression of COL4A3 in corneal endothelium of the proband of the original PPCD3 family. Identification of TCF8 as the PPCD3 gene provides a valuable tool for the study of critical gene regulation events in PPCD pathology and suggests a possible role for TCF8 mutations in altered structure and function of cells lining body cavities other than the anterior chamber of the eye. Thus, this study has identified TCF8 as the gene responsible for approximately half of the cases of PPCD, has implicated TCF8 mutations in developmental abnormalities outside the eye, and has presented the TCF8 regulatory target, COL4A3, as a key, shared molecular component of two different diseases, PPCD and Alport syndrome.

Figure  1

Haplotype and mutational analysis of the TCF8 2916_2917delTG mutation in family UM:139. a, Based on sequencing of cloned versions of the two alleles present in UM:139, the frameshift mutation (M) sequence trace is shown on the left, and the corresponding reference (R) sequence, NM_030751, is shown on the right. b, Haplotyping over the 8.55-cM PPCD3 region, with the 2916_2917delTG mutation designated M and the reference allele designated R. All relationships of genotyped individuals are correct as depicted, as determined from the previously reported UM:139 genome scans (Moroi et al. ; Shimizu et al. 2004). A large number of nonparticipating descendants of members of generation II do not appear in this figure. Open symbols indicate absence of PPCD, filled symbols indicate affected individuals, flags indicate individuals with hernia, and crossed symbols indicate individuals considered to have indeterminate phenotypes, since they do not have PPCD but do have other corneal findings, such as guttae or FECD. c, Traits that did not cosegregate with the mutation include late-onset hearing loss, migraine headaches, lupus, and arthritis. The locus map shows the order of markers and intermarker distances. The germline of II-8 contains three distinct versions of chromosome 10 shown among his progeny: unaffected haplotype without deletion (III-3 and III-17), affected haplotype plus deletion (III-1, III-5, III-7, III-10, III-11, and III-12), and affected haplotype without deletion (III-15 and IV-28). The three possible explanations include back mutation, a double recombination event, and germline mosaicism in individual II-9.

Figure  2

Structure of the TCF8 transcript. Locations of the mutations and the sequence variant relative to exons encoding functional domains are shown. TCF8 encodes a homeodomain (HD) flanked by two zinc-finger clusters (Z1–Z4 and Z5–Z7). An acidic activation domain (AAD) is encoded after the last zinc-finger domain.

Figure  3

Additional PPCD families with TCF8 mutations. The genotype of each individual at the relevant locus is displayed. M indicates the mutant allele and R indicates the reference allele. In each case, the mutant sequence trace is on the left and the sequence trace corresponding to the reference allele is on the right. a and b, Families with nonsense mutations. c and d, Families with frameshift mutations. Symbols are as described in figure 1. Relatives in UM:A01 are shown as having indeterminate ocular phenotype because we have not examined them and have no records on them, but since the proband’s father has been confirmed and has neither PPCD nor the TCF8 mutation, we do not expect to find PPCD or the TCF8 mutation in that branch of the family. Relationships are confirmed as depicted for all genotyped individuals.

Figure  4

Quantitation of transcript levels. Real-time sqPCR on cDNA from various mouse tissues used primers for mouse genes Tcf8 (NM_011546), Vsx1 (NM_054068), and Col8a2 (NM_199473). Eye posterior includes retinal pigment epithelium, choroid, and sclera.

Figure  5

Immunohistochemical detection of type IV collagens in corneal endothelium. The corneal endothelium, as marked by vertical arrows, is on the right in each panel. Positive intercytoplasmic staining for COL4A3 in the UM:139 proband’s corneal endothelial cells is indicated by horizontal arrows in the sample from the proband COL4A3 panel. In the proband panels, there is a retrocorneal fibrous membrane interposed between the endothelium on the right and the well-defined Descemet membrane on the left. This retrocorneal fibrous membrane is not present in the control panels. COL4A1 and COL4A5 signals are not seen in samples from either the proband or the control.