Association and familial segregation of CTG18.1 trinucleotide repeat expansion of TCF4 gene in Fuchs' endothelial corneal dystrophy

Abstract

Purpose: We tested the association between two intronic polymorphisms (CTG18.1 and rs613872) in TCF4 and Fuchs' endothelial corneal dystrophy (FECD), and analyzed their segregation patterns in families.

Methods: We recruited 120 unrelated Caucasian subjects with FECD and 100 controls. Available family members of probands were recruited. Genotyping of the single nucleotide polymorphism (SNP) rs613872 was performed using Sanger sequencing or real-time allelic discrimination assay. The trinucleotide repeat polymorphism, CTG18.1, was genotyped using a combination of short tandem repeat assay and triplet repeat primed PCR assay. The cytosine-thymine-guanine (CTG) repeat length of ≥40 was classified as an expanded CTG18.1 allele. Association of the two loci with FECD was evaluated. Segregation in 29 families was examined.

Results: The two polymorphisms are in linkage disequilibrium (r(2) = 0.65 in cases and 0.31 in controls). Significant associations were found between FECD and rs613872 (P = 3.1 × 10(-17)), expanded CTG18.1 allele (P = 6.5 × 10(-25)), and their haplotypes (P = 5.9 × 10(-19)). The odds ratio (OR) of each copy of the rs613872 G allele for FECD was estimated to be 9.5 (95% confidence interval [CI], 5.1-17.5). The OR of each copy of the CTG18.1 expanded allele was estimated to be 32.3 (95% CI, 13.4-77.6). The expanded CTG 18.1 allele cosegregated with the trait in 52% (15/29) of families with complete penetrance and 10% (3/29) with incomplete penetrance.

Conclusions: We report, to our knowledge, the first independent replication of the expanded CTG 18.1 allele conferring significant risk for FECD (>30-fold increase). The expanded allele cosegregates with the trait with complete penetrance in a majority of families, but we also document cases of incomplete penetrance.

Keywords: Fuchs' dystrophy; TCF4; genetics.

Figure 1

The intronic TCF4 polymorphisms SNP rs613872 and CTG18.1 trinucleotide repeat alleles are approximately 43 kilobases apart. The TCF4 transcript model is courtesy of Ensembl 2013 (ENST00000565018.2). Reprinted with permission from Flicek P, Ahmed I, Amode MR, et al. Ensembl 2013. Nucleic Acids Res. 2013;41:D48–D55.

Figure 2

Overview of TP-PCR to genotype CTG18.1. Adapted by permission from BMJ Publishing Group Limited. Warner JP, Barron LH, Goudie D, et al. A general method for the detection of large CAG repeat expansions by fluorescent PCR. J Med Genet. 1996,33:1022–1026.²⁶ (A) The STR assay uses primers P1 and P2 that flank the CTG repeat, but the assay can fail to genotype expanded alleles. (B) The trinucleotide repeat specific 3′ end of P4 binds at numerous sites within the CTG repeat in the early rounds of amplification, resulting in a mixture of products. Primer P1 is a 5′-6-FAM locus-specific primer that determines the specificity of the reaction. In the early rounds of amplification, the P4 primer is consumed quickly due to the 33:1 molar ratio of P3 to P4. (C) P3 primer amplifies from end of the mixture of products of the prior cycles. The PCR parameters include a long extension time to ensure complete extension of the longer products within the PCR product mixture and allow for the identification of the expanded CTG18.1 allele(s).

Figure 3

Segregation of SNP rs613872 and CTG18.1 alleles in families with Fuchs' dystrophy. In family F10, all four affected family members are homozygous for rs613872 risk allele G and have the expanded CTG18.1 allele, including two individuals being homozygous for the expanded CTG18.1 allele. Note lack of penetrance of the expanded CTG18.1 allele in individual VVM193 (38-year-old male without any central guttae). Two individuals VVM125 and VVM183 are homozygous and heterozygous for rs613872, respectively. Both lack the expanded CTG18.1 allele and have grade 1 guttae. Family F33 shows cosegregation of the trait with rs613872 and the expanded CTG18.1 allele. Family F14 shows cosegregation of the expanded CTG18.1 allele with the FECD trait with complete penetrance. The SNP rs613872 cosegregates with disease but with incomplete penetrance. Subject VVM209 has grade 1 guttae. Family F32 shows cosegregation of the expanded CTG18.1 allele and rs613872 with the FECD trait, but with incomplete penetrance of both risk alleles. Subject VVM122 is a 95-year-old woman homozygous for the expanded CTG18.1 and rs613872 alleles, but only has grade 1 guttae. Subject VVM175 is heterozygous for both risk alleles and has grade 1 guttae. Subject VVM176 lacks either risk allele and has grade 1 guttae. Family F36 is an example of a family where the trait fails to cosegregate with either rs613872 or expanded CTG18.1 allele. Note VVM208 has one copy of expanded CTG18.1 allele and has only grade 1 guttae. Family F39 is an example of a family where the trait cosegregates with the expanded CTG18.1 and rs613872 alleles, but with incomplete penetrance. Interestingly, VVM149 (52-year-old female) has the expanded allele, but has only grade 1 guttae.

Figure 4

Representative STR analysis and TP-PCR electropherogram tracings. The STR analysis results are shown above the corresponding TP-PCR tracings. The CTG allele sizes are shown with arrows. The TP-PCR tracings have a characteristic ladder with a 3 base pair periodicity. (A) The STR and TP-PCR tracings of unaffected subject heterozygous for two stable CTG18.1 alleles of less than 40 CTG repeats (CTG₁₃, CTG₁₈). On the TP-PCR tracings, the CTG₁₃ and CTG₁₈ alleles give peaks as well as all the intermediate priming sites. Note the slight continuation of the pattern past the CTG₁₈ allele peak due to mispriming at the repeat end. (B) The STR and TP-PCR tracings of unaffected subject homozygous for stable CTG18.1 allele (CTG₁₂, CTG₁₂). (C) The STR and TP-PCR tracings of FECD subject with one stable CTG18.1 allele of 12 CTG repeats and another expanded CTG18.1 allele (CTG₁₂, X). Note STR results revealed only the CTG₁₂ allele and failed to detect the expanded allele. Persistence of the ladder pattern on RP-PCR tracing indicates presence of an expanded allele. (D) The TP-PCR tracing of FECD subject homozygous for expanded CTG18.1 allele (X, X). The STR analysis failed to detect any CTG18.1 allele. This characteristic ladder pattern on the TP-PCR tracing indicates the presence of two expanded CTG18.1 alleles. Note the continuation of the ladder pattern on the TP-PCR tracing.

Figure 5

Linkage analyses on family F10 reveals a signal on chromosome 18q21. A dominant genetic model with penetrance of 0.95, sporadic rate of 0.001, and disease-predisposing allele frequency of 0.001 were assumed.