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. 2025 Aug 6;66(11):14. doi: 10.1167/iovs.66.11.14

Repeat Expansion and Somatic Instability in TCF4 in Patients With Fuchs Endothelial Corneal Dystrophy Identified by Small Pool PCR

Sayo Maeno 1, Arisa Yamashita 2, Yoshinori Oie 1,✉, Ryota Koto 1, Chifune Kai 1, Nozomi Nishida 1, Masayuki Nakamori 3, Motokazu Tsujikawa 1,2,✉, Kohji Nishida 1
PMCID: PMC12347246  PMID: 40767442

Abstract

Purpose

The purpose of this study was to investigate trinucleotide repeat (TNR) expansion and somatic repeat instability in the TCF4 gene using small pool polymerase chain reaction (SP-PCR) in patients with Fuchs’ endothelial corneal dystrophy (FECD).

Methods

We selected 15 patients with FECD who had an abnormal CTG extension allele of 50 or more in the TCF4 gene via a short tandem repeat (STR) assay and triplet repeat primed PCR (TP-PCR). Expanded repeat number and variations in the number of repeat expansion within the leukocyte DNA of these 15 patients were examined via SP-PCR. DNA was collected from the corneal endothelium of a patient who underwent keratoplasty. Subsequently, SP-PCR was performed to compare the expanded alleles in the corneal endothelium and leukocytes.

Results

SP-PCR of the patients showed TNR expansion in TCF4, with an average maximum repeat number of 1602 ± 1258. The average expanded allele variation was 19 ± 15, indicating somatic repeat instability. Additionally, the maximum repeat number and somatic repeat instability varied significantly among patients, with a significant correlation between them (R = 0.84, P < 0.05). The maximum number of CTG repeats in the corneal endothelium was greater than that in leukocytes.

Conclusions

Somatic repeat instability was observed in the leukocytes of the patients, demonstrating a correlation with the maximum number of repeats. Our results also indicated that TNR expansion of genomic DNA in the corneal endothelium was higher than that in the leukocytes of patients with FECD.

Keywords: Fuchs endothelial corneal dystrophy (FECD), TCF4, repeat expansion, somatic instability, small pool PCR

Fuchs endothelial corneal dystrophy (FECD) is a bilateral eye disorder that causes damage to the corneal endothelium. FECD is characterized by abnormal deposition of the extracellular matrix (guttae), thickening of Descemet's membrane, and subsequent corneal endothelial dysfunction.1 Visual function declines as the disease progresses.2–6 Although FECD is the most common corneal endothelial dystrophy, keratoplasty remains the only definitive treatment to restore vision in patients with FECD. Consequently, FECD is the primary indication for keratoplasty globally and holds significant clinical relevance due to its prevalence and progressive nature.7⁻9

FECD is reportedly hereditary, with an autosomal dominant trait, although locus heterogeneity has also been reported. One of the important loci is TCF4. Among the genes implicated in late-onset FECD, TCF4 has emerged as the most consistently associated susceptibility gene. Although variants in AGBL1, LOXHD1, SLC4A11, and ZEB1 have been reported in a limited number of cases,10–13 genome-wide association studies have identified several variants within and around TCF4, including the intronic single-nucleotide polymorphism (SNP; rs613872)14,15 and a CTG trinucleotide repeat expansion (CTG18.1) within intron 3.16,17 TNR expansion has a stronger association with FECD than do other previously reported variants.16 Notably, TNR expansion exceeding 50 repeats was reported in 79% of American, 77% of British, 77% of German, and 21% of Japanese patients with FECD.16,18–21 In contrast, only 3% of healthy controls worldwide show repeat expansion. This strongly suggests that FECD is a triplet repeat disease (TRD).

TRDs are a group of diseases caused by increased numbers of specific TNRs in genes, even when located in a non-coding region such as introns or 3′ untranslated regions.22,23 A key feature of TRDs is the instability of repeat elongation in somatic cells. During fertilization, when an abnormally elongated allele is inherited from one parent, there is naturally only one type of repeat length, referred to as the progenitor allele length (PAL). However, during development and differentiation, each somatic cell undergoes different repeat elongations, resulting in repeat lengths that vary from the PAL in each cell.24,25 This variation also occurs between different tissues and may be one of the reasons why most TRDs primarily affect the nervous system. This phenomenon in which each cell has a different repeat length is called somatic repeat instability. The presence, absence, and degree of somatic repeat instability are critical factors in patients with TRDs.26,27

Testing for somatic repeat instability is generally challenging, particularly during polymerase chain reaction (PCR) amplification and observation. Owing to the preferential amplification of short, normal repeat alleles, overextended repeat alleles are often not identified. Genomic Southern blot is a commonly used method; however, it requires large amounts of DNA and cannot identify the number of elongations in each cell type. One approach to identifying the number of elongations is long-read sequencing.28 Using this method, TNR elongations in the corneal endothelium of a Caucasian patient with FECD (>1000 repeats) were reported to be significantly longer than those characterized in leukocytes from the same individual (<90 repeats).29 Long-read sequencing can provide comprehensive mapping and assembly of the genome with high accuracy. However, comprehensive assessment of somatic repeat instability requires a large number of reads, which might pose challenges regarding cost and DNA quantity.30 Furthermore, this method cannot be used directly in tissues such as corneal endothelial cells owing to the limited number of cells and difficulty obtaining sufficient amounts of DNA for long-read sequencing.

Small-pool PCR (SP-PCR) is used to directly assess somatic repeat instability within small amounts of in vivo DNA. This technique amplifies both short and abnormally expanded alleles by diluting DNA to an optimal concentration. Further, it captures the length and variety of expanded alleles, confirming somatic repeat instability without requiring high DNA input.30 Given the limited DNA yield from corneal endothelium, SP-PCR is particularly suitable for studying FECD. Moreover, compared with long-read sequencing, SP-PCR is more cost-effective and does not require specialized equipment or a large amount of DNA, making it more accessible for conducting studies on small clinical samples. Consequently, SP-PCR has been documented as a valuable technique for investigating somatic repeat instability in many TRDs.24,31–33 However, to our best knowledge, no such reports exist for FECD. Therefore, we aimed to investigate the extensive somatic repeat instability of the TNR expansion in the TCF4 gene in Japanese patients with FECD using SP-PCR.

Methods

This observational, cross-sectional study adhered to the tenets of the Declaration of Helsinki. Approval was obtained from the Institutional Review Board/Ethics Committee of Osaka University Hospital. All patients provided informed consent after receiving an explanation about the nature and probable consequences of the study.

Patients

Among 69 consecutive patients with FECD at the Osaka University Hospital Department of Ophthalmology, 15 exhibited TNR expansion. The presence of TNR expansion in these patients was previously reported using Short Tandem Repeat (STR) analysis and Triplet Repeat PCR (TP-PCR).18 Patients with expansions of 50 repeats or more identified by STR analysis and those positive by TP-PCR were selected for this study. The threshold of 50 repeats is widely used not only for FECD,19,34–37 but also for other trinucleotide repeat disorders, such as myotonic dystrophy,38 dentatorubral-pallidoluysian atrophy,39 and spinocerebellar ataxias.40 Cases of repeat lengths of 50 or more were included in the study, whereas those of fewer than 50 repeats were excluded.

These patients underwent comprehensive ophthalmological examinations, from whom peripheral blood samples were collected. Cornea specialists diagnosed all FECD cases based on the presence of longstanding bilateral corneal guttae or a beaten metal appearance, excluding other causative factors such as surgery or inflammation.41 A patient's corneal endothelium, obtained during endothelial keratoplasty, was subjected to genomic DNA analysis.

Ophthalmic Examinations

Corneal specialists determined the modified Krachmer grade for each patient using slit-lamp microscopy.42 Ocular anterior segment examinations were conducted using swept-source anterior segment optical coherence tomography (AS-OCT; CASIA SS-2000; Tomey Corporation, Nagoya, Japan) and specular microscopy (SP-3000P; TOPCON, Tokyo, Japan). We determined three tomography pachymetry maps and posterior elevation map patterns, loss of regular isopaches, displacement of the thinnest point of the cornea, and presence of posterior surface depression in each case.43

DNA Extraction

Genomic DNA was extracted from peripheral blood-derived leukocytes using a DNeasy Blood and Tissue Kit (QIAGEN, Germany). Genomic DNA was extracted from the corneal endothelium using the ISOGEN (Nippon Gene, Japan), following the manufacturer's modified acid guanidinium thiocyanate–phenol–chloroform method. After phase separation, the aqueous phase was precipitated with ethanol, washed with sodium citrate and ethanol solutions, vacuum-dried, and dissolved in DNase-free water. Extracted DNA was stored at −20°C until further analysis. Regarding blood-derived samples, DNA concentration and purity were assessed by a NanoDrop One spectrophotometer (Thermo Fisher Scientific, USA), with 260/280 absorbance ratios ranging between 1.62 and 1.88. Considering corneal endothelium-derived samples, DNA quantity was too low for spectrophotometric measurement; therefore, DNA presence was confirmed using droplet digital PCR (ddPCR). Standardized quality control measures, such as degradation assessment and purity quantification, could not be performed for the corneal endothelium-derived DNA due to limited yields. However, successful PCR amplification from both blood- and endothelium-derived DNA confirmed their suitability for downstream applications. A summary of DNA concentration and purity measurements of peripheral blood-derived samples is presented in Supplementary Table S1.

Droplet Digital PCR

Because the SP-PCR method we used is highly sensitive to DNA concentration, accurate measurement of DNA concentration was crucial. Given the minimal cell count and limited DNA yield from the corneal endothelium of patients with FECD, we used ddPCR to determine the DNA concentration. In ddPCR, PCRs are performed in a water-in-oil droplet emulsion, fractionating the sample into 20,000 droplets for individual PCR amplification.44 This method, typically used to detect rare DNA targets or copy number variations, was applied to measure corneal endothelium-derived DNA concentration based on the control gene, RPP30. The same method was used for peripheral blood-derived DNA comparison.

The PCR reaction mixture (20 µL/reaction) contained 10 µL of ddPCR Supermix for probes (Bio-Rad, CA), 1 µL each of PrimePCR AP3B1 Primer Probe set × 20 (FAM) chr5 and PrimePCR RPP30 Primer Probe set × 20 (HEX) chr10, 0.5 µL of the DNA sample, and 7.5 µL of dH2O. The QX200 Droplet Generator (Bio-Rad, CA) was used to generate PCR droplets, and thermal cycling was conducted using a PTC Tempo 96 Thermal Cycler (Bio-Rad, CA). The PCR cycle included initial denaturation at 95°C for 10 minutes, 40 cycles of denaturation at 94°C for 30 seconds, annealing-extension at 60°C for 60 seconds, and final heating at 98°C for 10 minutes, followed by a hold at 4°C. Amplification signals were read using the QX200 Droplet Reader (Bio-Rad, CA) and analyzed with the QX Manager (Bio-Rad, CA), recorded as copies/µL.

Small Pool PCR

In regard to the DNA of patients without extremely abnormal TNR expansion (less than 90 CTG repeats), both alleles were amplified for STR analysis. However, when an allele with extremely abnormal TNR expansion (more than 90 repeats) was present, only the shorter allele was amplified, resulting in a single band due to the absence of the amplification of the expanded allele (Fig. 1A). Monckton et al. revealed that the expanded mutant allele can be effectively amplified at a specific concentration through sample dilution.25 Subsequently, a PCR method was proposed to amplify alleles with repeat expansions by diluting the sample to an optimal concentration. Figure 1A demonstrates that amplification of templates with heterogeneous repeat lengths often favors shorter alleles. However, this bias can be mitigated by optimizing DNA dilution, enabling detection of abnormal expanded alleles. Figure 1B illustrates differences in expanded allele detection through sample dilution, enhancing the clarity of expanded allele profile. This method was developed to analyze TRDs where the repeat length is unstable between cells.

Figure 1.

Figure 1.

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Schema of small pool PCR (SP-PCR). (A) Principle of SP-PCR: when the sample DNA is not diluted, and the concentration is high, only the normal short alleles are selectively amplified in all PCR reactions (upper panel). However, when the sample is appropriately diluted, reactions occur, which amplify the abnormally expanded alleles (lower panel). (B) Actual results of SP-PCR: in addition to the normal alleles (indicated by the white arrowhead), numerous abnormally expanded alleles were identified. M indicates size markers. SP-PCR, small pool polymerase chain reaction.

SP-PCR was performed following the method described by Gomes-Pereira et al.45 SP-PCR was conducted using primers specific for TCF4. Amplification was performed using a T100TM Thermal Cycler (Bio-Rad, CA) with an Expand Long Template PCR System (Roche, Germany). In respect of peripheral blood-derived DNA, the reaction volume was 25 µL with 1 ng, 750 pg, or 500 pg of genomic DNA and 10 pM of each primer. The PCR cycle consisted of initial denaturation at 94°C for 2 minutes, 25 cycles of denaturation at 94°C for 20 seconds, annealing at 55°C for 30 seconds, and extension at 68°C for 5 minutes and 30 seconds, with a final extension at 68°C for 7 minutes. Regarding corneal endothelium-derived DNA, the reaction volume was 12.5 µL with 100 pg of genomic DNA. The PCR cycle consisted of initial denaturation at 94°C for 2 minutes, 35 cycles of denaturation at 94°C for 20 seconds, annealing at 55°C for 30 seconds, and extension at 68°C for 8 minutes, with a final extension at 68°C for 7 minutes. When comparing TNR expansion in the corneal endothelium with that in peripheral blood, amplification of peripheral blood-derived DNA was conducted under the same conditions.

Southern Blot Analysis of SP-PCR Products

The PCR products were detected by Southern blot analysis, which had a high sensitivity and specificity for amplified DNA. Further, 5 µL of the SP-PCR products were applied to a 0.8% agarose gel buffered with 40 mM Tris-acetate and 1 mM EDTA. The PCR products were electrophoresed in 1 × TAE buffer for 30 minutes at 80 V, followed by 3 hours at 50 V. Following overnight transfer of standard capillary gel to a nylon membrane (Roche, Germany), the membrane was exposed to 254 nm UV light for 1 minute using a DNA crosslinker (UVP, Germany). The membrane was then placed in DIG Easy Hyb (Roche, Germany) and prehybridized at 70°C for 30 minutes. Following prehybridization, the membrane was placed in a reaction solution containing 15 mL of DIG Easy Hyb and 15 µL of denatured digoxigenin (DIG)-labeled (CAG) 7 probe and hybridized for 3.5 hours at 70°C. After hybridization, the membrane was treated according to the manual procedure using the DIG Wash and Block Buffer set (Roche, Germany), and 40 drops of CDP-Star (Roche, Germany) were added as the final step. Luminescence was detected using ChemiDoc XRS+ (Bio-Rad, CA) and analyzed using Image Lab (Bio-Rad, CA). Data on repeat length and variations in the number of repeat expansion was obtained.

DNA Extraction and SP-PCR for Patient-Derived Corneal Endothelium

Corneal endothelial sample was collected from a patient who underwent keratoplasty. Owing to the limited amount of DNA extracted from the corneal endothelium, we performed SP-PCR using a limited number of lanes. SP-PCR of the corneal endothelium and leukocytes from the same patient was performed simultaneously under the same conditions.

Statistical Analyses

Spearman's rank correlation coefficient (ρ) and corresponding P value were used for analyzing the relationship between repeat lengths and variations from peripheral blood. All statistical analyses were performed using R software (version 4.4.1; R Foundation for Statistical Computing, Vienna, Austria) within the RStudio environment (version 2024.12.1). The P values < 0.05 were considered statistically significant.

Results

Variability of TNR Expansion Length and Somatic Repeat Instability in Leukocytes of Patients With FECD

In our previous study,18 we performed STR and TP-PCR analyses including a cohort of 69 Japanese patients with FECD to detect the presence of TNR expansion. Among these patients, 10 showed only one normal allele by STR analysis, accompanied by additional peaks on TP-PCR. In another 5 patients, STR analysis detected 2 distinct alleles, and in each case, one of the alleles had more than 50 repeats, exceeding the commonly accepted threshold. In total, 15 patients were identified to have repeat lengths exceeding the commonly accepted threshold of 50 repeats or more. Consequently, SP-PCR was performed involving these 15 patients. A flowchart of the study is shown in Figure 2.

Figure 2.

Figure 2.

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The flowchart of determining the repeat length in this study . To analyze leukocyte DNA from 59 consecutive cases, a short tandem repeat assay, and triplet repeat primed PCR were initially performed, as previously described. Using this combined approach, the expanded alleles and their approximate sizes were determined. Samples with repeat expansions exceeding 50 or those not reliably detected by these methods were subjected to further analysis using SP-PCR. SP-PCR, sSmall pool polymerase chain reaction.

SP-PCR analysis clearly demonstrated the repeat length expansion and variations in the number of repeat expansion, indicating somatic repeat instability of CTG18.1. Here, we first present a case of long repeats and high instability (Fig. 3) and another of short repeats and low instability (Fig. 4). Figure 3 shows the case of a patient with long repeats and high somatic repeat instability. The patient was a 60-year-old Japanese female individual (patient no. 3) with moderate FECD. The modified Krachmer grade was 4 in both eyes. The best-corrected distance visual acuity (CDVA) was 24/20 in the right eye and 18/20 in the left eye, accompanied by subjective glare symptoms. Using a specular microscope, extensive and confluent guttae were observed, which made determining the exact endothelial cell density challenging (see Figs. 3D, 3E). All three of the characteristic tomographic pachymetry and posterior elevation map patterns were observed in both eyes: loss of regular isopachs, displacement of the thinnest point, and focal posterior surface depression43 (see Figs. 3F, 3G). Bands of various lengths were observed in the SP-PCR results (see Fig. 3A). At least 30 different length bands were detected, ranging from less than 100 to 2807 repeats, indicating the expansion of CTG repeats and high somatic repeat instability among cells as reported in TRDs.46

Figure 3.

Figure 3.

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A case of long repeat expansion and significant somatic repeat instability . (A) SP-PCR from genomic DNA of leukocytes. The maximum repeat number is 2807 (indicated by the black arrowhead). The normal repeat allele bands are indicated by the white arrowhead. Extremely expanded repeat alleles (over 1000 repeats) were frequently observed, and high somatic repeat instability was evident (at least 30 different bands). M indicates marker lanes. (B, C) Slit-lamp examination revealed a central zone of confluent corneal guttae, determined as Krachmer grade 4 in both eyes (B: right eye, C: left eye). (D, E) Specular microscopy images obtained from the central cornea displayed dark areas with confluent central corneal guttae in both eyes (D: right eye, E: left eye). (F, G) Anterior segment optical coherence tomographic findings confirmed the loss of regular isopaches, predominant flattening of the posterior corneal surface compared with the anterior surface, and displacement of the thinnest point away from the central cornea (F: right eye, G: left eye). SP-PCR, small pool polymerase chain reaction.

Figure 4.

Figure 4.

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A case of short repeat expansion without somatic repeat instability . (A) SP-PCR from genomic DNA of leukocytes. The banding pattern is completely different from that of the patient illustrated in Figure 3. Only one pattern of expanded allele band (58 repeats), which was also detected in a short tandem repeat assay (black arrowhead), is present. Somatic repeat expansion was not observed. The normal repeat allele band (11 repeats) appears faintly below the expanded band (white arrowhead). (B, C) Slit-lamp examination revealed central corneal guttae, determined as Krachmer grade 1 and 2 in the right (C) and left eyes (D), respectively. (D, E) Specular microscopic images obtained from the central cornea displayed some dark areas with central corneal guttae in both eyes (D: right eye, E: left eye). (F, G) Anterior segment optical coherence tomographic findings confirmed the loss of regular isopaches and slight displacement of the thinnest point from the central cornea (F: right eye, G: left eye). SP-PCR, small pool polymerase chain reaction.

Figure 4 shows a patient with short repeats and poor somatic repeat instability. She was a 73-year-old Japanese female individual (patient no. 13) with mild FECD. Her modified Krachmer grading was grade 1 for the right eye and grade 2 for the left eye. The best-CDVA was 20/20 in the right eye and 16/20 in the left eye. Mild guttae were observed under a specular microscope (see Figs. 4D, 4E). We confirmed two of the tomography pachymetry maps and posterior elevation map patterns (see Figs. 4F, 4G); however, as shown in the presented clinical data, this patient’s FECD was slightly milder than that of patient no. 3. Furthermore, the maximum number of repeat alleles and somatic repeat instability observed using SP-PCR were considerably different (see Fig. 4A). The abnormal repeat expansion in this patient was short enough to be detected using STR analysis, resulting in 58 repeats. SP-PCR detected only this repeat allele and showed no evidence of somatic repeat instability.

The results of these 15 FECD cases are summarized in the Table. The highest repeat number observed was in patient no. 1, with 3652 repeats. This patient also exhibited marked somatic repeat instability, with the least number of repeats being <100. In zygotes, the abnormal allele length is reportedly close to this minimum; however, through different elongation processes in each cell, SP-PCR detected 20 different repeat expansions. Conversely, patients with abnormal repeat lengths short enough to be detected by STR analysis (patient nos. 12–15) showed minimal somatic repeat instability. However, one patient (patient no. 3), with a repeat length of 93, near the detection limit of the STR analysis, exhibited significant somatic repeat instability (see Fig. 3). This suggests that somatic repeat instability cannot be confirmed solely by STR analysis of leukocyte DNA. Notably, patients with abnormal repeat numbers below 60, detectable by STR analysis, showed less somatic repeat instability, whereas longer repeat numbers appeared to increase instability. To investigate this, we examined the relationship between the longest repeat number identified via SP-PCR and variations in the number of repeat expansion (somatic repeat instability) in 15 patients, observing a significant correlation among them (R = 0.839, 95% confidence interval [CI] = 0.452–0.989, P < 0.001; Fig. 5).

Table.

Demographic Information, CTG Trinucleotide Repeat Length, and Somatic Repeat Instability of Leukocytes

Modified Krachmer Grade TNR Expansion in DNA From Peripheral Blood
Patient No. Age Sex Right Left Repeat Length by STR and TP-PCR Longest Repeat Length by Small Pool PCR Repeat Variation by Small Pool PCR
1 92 F 6 6 11, >50 3652 22
2 73 F 2 6 14, >50 3155 46
3 62 F 4 4 34, 93* 2807 30
4 73 F 2 2 11, >50 2731 36
5 79 M 3 4 11, >50 2724 24
6 70 M 5 6 17, >50 2374 23
7 50 F 4 4 11, >50 2281 40
8 74 M 4 6 11, >50 1831 27
9 65 F 6 6 17, >50 1211 19
10 44 M 4 4 26, >50 593 9
11 56 F 4 4 24, >50 366 6
12 79 F 2 2 24, 56* 127 3
13 73 F 1 2 11, 58* – 2
14 55 F 4 4 24, 57* – 2
15 28 F 2 2 12, 56* – 2
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STR, short tandem repeat assay; TP-PCR, triplet repeat primed polymerase chain reaction; TNR, trinucleotide repeat.

*

Repeat numbers were determined by STR analysis.

Figure 5.

Figure 5.

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Correlations between the repeat lengths and variations from peripheral blood . A significant correlation was observed between repeat length and variation, with the number of repeat variation represented on the x-axis and longest repeat length on the y-axis (R = 0.839, 95% CI = 0.452–0.989, P < 0.001).

Enhanced TNR Expansion in Corneal Endothelium Compared to Leukocyte

Previous somatic repeat instability results were based on the analysis of leukocyte DNA, not the corneal endothelium, which is the primary disease site. In this study, corneal endothelial and leukocyte DNAs were obtained from a 70-year-old Japanese male individual (patient no. 6) with advanced FECD at the time of endothelial keratoplasty (Fig. 6). The best-CDVA was 6/20 in the right eye and 2/20 in the left eye, accompanied by subjective symptoms of glare and blurred vision in the morning than at night. Using ddPCR, the obtained copy numbers of RPP30 from the patient's corneal endothelium and leukocytes were 66.8 and 1560 copies/µL, respectively. The calculated DNA concentrations were 0.2 and 4.73 ng/µL, respectively. The amount of DNA recovered from the corneal endothelium was approximately the minimum amount required for SP-PCR analysis.

Figure 6.

Figure 6.

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Comparison of small pool (SP-)PCR for genomic DNA from leukocytes and corneal endothelium . (A, B) SP-PCR results obtained from the corneal endothelium (A) and peripheral blood (B). The most prominent band in the corneal endothelium (1335 repeats) exceeds the size of the most expanded allele observed in leukocytes (1027 repeats), as indicated by white arrowheads. Notably, the most expanded allele detected in the corneal endothelium (3199 repeats, marked by the black arrowhead) was significantly larger than that in leukocytes. (C, D) Slit-lamp examination revealed central confluent corneal guttae, with corneal edema, determined as Krachmer grade 5 and 6 in the right (C) and left eyes (D). (E, F) Specular microscopic images obtained from the central cornea displayed dark areas with confluent central corneal guttae in both eyes (E: right eye, F: left eye). (G, H) Anterior segment optical coherence tomographic findings confirmed the loss of regular isopaches, predominant flattening of the posterior corneal surface compared with the anterior surface, and displacement of the thinnest point away from the central cornea (G: right eye, H: left eye). SP-PCR, small pool polymerase chain reaction.

SP-PCR analysis revealed somatic repeat instability in both the patient's corneal endothelium and leukocytes (see Fig. 6A). The most prominent band in the corneal endothelium was 1335 repeats, which was larger than the highest number of repeats in leukocytes (1027 repeats). The longest repeat length in the corneal endothelium was 3199 repeats. These results indicate a more pronounced TNR expansion in the corneal endothelium than in the peripheral blood.

Discussion

This study is the first to demonstrate somatic repeat instability and abnormal TNR expansions in the TCF4 gene in patients with FECD using SP-PCR, revealing significant variability in repeat length and instability among patients. We detected TNR expansions of up to 3000 repeats in both the peripheral blood and corneal endothelium in patients with FECD. Although other studies have explored somatic repeat instability through long-read sequencing approaches, our study underscores the practicality of SP-PCR, offering a direct assessment even in tissues such as the corneal endothelium, where only a small amount of DNA can be obtained. Thus, SP-PCR is cost-effective and does not require specialized equipment or a large amount of DNA, making it a practical and accessible method for investigating somatic repeat instability. Although the previous study evaluated the repeat length of the corneal endothelium using primary culture cells,29 it is possible that we have confirmed direct repeat lengths without any alterations due to the culture process.

In our study, both the number of CTG18.1 repeat expansions and somatic repeat instability varied significantly among the patients. The reasons for these variations remain unclear; however, repeat expansions reportedly occur during DNA replication and RNA transcription and are related to DNA repair mechanisms.24,27,47,48 The number of DNA replications from the zygote and TCF4 gene transcription is expected to differ among individuals, tissues, and cells, which could explain the variations in repeat expansions. Our SP-PCR analysis may detect these differences and the possibility of repeat expansion. In addition, the number of replications and transcriptions increases with age, which might explain why many TRDs, including FECD, have a late onset.

In many TRDs, including myotonic dystrophy type 1,49,50 repeat expansions and instability in the affected tissues are greater than those in leukocytes.51 In our study, repeat expansions in the corneal endothelium were observed to be significantly longer than those in leukocytes in a single case. This observation is consistent with findings in other TRDs and suggests that CTG18.1 repeat expansion might exert pathogenic effects specifically in the corneal endothelium in FECD. However, we acknowledge that Figure 6 shows data of only one patient, which might limit the generalizability of the findings. Furthermore, repeat expansion and instability vary among individuals with TRDs. Our SP-PCR analysis showed similar interindividual variability in patients with FECD, supporting the relevance of somatic instability in this disorder and, despite the limited sample size, providing a foundation for future studies on CTG18.1 repeat dynamics.

As in other repeat expansion disorders, such as myotonic dystrophy type 1, several mechanisms have been proposed to explain how CTG18.1 expansion in TCF4 leads to cellular dysfunction in patients with FECD.30 These include dysregulation of TCF4 isoform expression,21 RNA-mediated toxicity via sequestration of MBNL proteins,52 repeat-associated non-AUG (RAN) translation of toxic peptides,53 and age- and tissue-specific somatic instability.28 The overlap with DM1 pathogenesis suggests that similar molecular processes might underlie the degeneration observed in corneal endothelial cells.

The relationship between individual differences in CTG18.1 repeat expansions and the severity or prognosis of FECD remains debatable. Saade et al. demonstrated that phenotypic anticipation, a characteristic of TRDs, is observed in patients with FECD across generations, indicating that CTG18.1 repeats are longer in children than in their parents.54 However, they stated that “the relationship between repeat expansion over generations and phenotypic severity is unclear.” In our study, although based on comparison of the two cases, patients with long repeats and high somatic repeat instability appeared to have more severe symptoms.

The small sample size is a limitation of this study. The limited sample size restricted the statistical power and might have introduced potential sampling bias. The number of patients with TNR expansion in Japan is relatively small; therefore, including a large number of patients with TNR expansion was challenging. In the future, we aim to conduct further studies with larger cohorts to evaluate the relationship between TNR expansion and disease severity, as well as the correlation between repeat expansion in the corneal endothelium and peripheral blood. Given the limited DNA yield from corneal endothelium samples, we used a high-yield ISOGEN-based extraction method and ddPCR to enable precise quantification of DNA input for SP-PCR. In contrast, peripheral blood-derived DNA was extracted using a conventional column-based method, and its concentration was determined using spectrophotometry. We empirically optimized the conditions for each sample type based on their specific characteristics and constraints. These methodological differences were essential to ensure reliable amplification and detection of repeat expansions. Moreover, as shown in Figure 3, two-fold variation in DNA concentration did not appear to significantly affect the maximum repeat length, suggesting that minor variations in input levels are unlikely to compromise the validity of the results. Additionally, the use of different DNA extraction methods for leukocyte and corneal endothelial samples might have influenced the observed repeat length distributions. This methodological difference remains a potential confounder. Future studies should aim to standardize DNA extraction methods to improve the somatic repeat instability measurements.

A further limitation of this study, is the possibility that DNA fragmentation, which might have differed between sample types due to the use of different extraction methods, might have affected amplification efficiency. Although careful handling and the successful detection of expanded alleles suggest that sufficient intact DNA was present, the inability to perform standard quality control measures for DNA from corneal endothelial cells (e.g. gel electrophoresis) remains a constraint.

The findings in this study might have important implications for future research and clinical applications regarding gene editing. Previous studies have shown that abnormally expanded TNRs can form stable secondary structures that interfere with editing efficiency and impair homology-directed repair, potentially leading to increased somatic instability after gene editing.55⁻57 These findings underscore the importance for assessing repeat length prior to therapeutic intervention. In this context, SP-PCR might be valuable for presurgical screening for gene editing. Moreover, if future research elucidates that specific single-nucleotide variants or other genetic markers are found to be reliably associated with extremely expanded alleles, they could serve as surrogate indicators of repeat expansion. This would enable a simpler and more scalable screening approach, helping identify appropriate candidates for genome editing without requiring SP-PCR in every case.

In conclusion, we successfully investigated TNR repeat expansion in the TCF4 gene of patients with FECD using SP-PCR, confirming the exact repeat lengths in the corneal endothelium and leukocytes. Somatic repeat instability was observed even in the peripheral blood of patients with FECD and correlated with the maximum number of repeats. Our findings suggest that TNR expansion in the corneal endothelium is greater than that in leukocytes, which might result in cornea-specific abnormalities.

Supplementary Material

Supplement 1
iovs-66-11-14_s001.pdf (129.6KB, pdf)

Acknowledgments

Supported by AMED (grant number: JP22ek0109590), MHLW Research on rare and intractable diseases program (grant number: 23FC1044), and JSPS KAKENHI (grant number: JP21K09718).

Disclosure: S. Maeno, None; A. Yamashita, None; Y. Oie, None; R. Koto, None; C. Kai, None; N. Nishida, None; M. Nakamori, None; M. Tsujikawa, None; K. Nishida, None

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