title: Sequence level genotyping at TCF4 CTG repeats associated with late onset Fuchs endothelial corneal dystrophy
Creators: name: Alkhateeb, Mariam Abdulaziz
Publisher: name: University of Glasgow
Year: 2019

description: Fuchs endothelial corneal dystrophy is an eye disease that affects the endothelium, the innermost layer of the cornea. Fuchs endothelial corneal dystrophy affects ~ 5% of the general population over the age of 40, and Fuchs is the leading indication for corneal transplantation. The hallmark of the disease is the presence of guttae which can be observed using slit lamp biomicroscopy. The severity of the disease can be graded using the Krachmer scale from 0 to 6 (0 means no guttae; and 6 severe confluent guttae). Late onset FECD is associated with the expansion of a CTG repeat in the 3rd intron of the transcription factor 4 gene (TCF4) on chromosome 18, termed CTG18.1 locus. The unexpanded length of the repeats is &lt; 40 CTG repeats, whereas the expanded alleles are ≥ 40 CTG repeats. The Scottish population was screened for the presence of expanded alleles. Southern blot hybridisation was used to detect the expansions with a frequency of 4.72%. Genotyping CTG18.1 alleles using Southern blot is of limited utility due to the assumption that the length of the fragment is equal to the repeat number. This might not always be correct due to the presence of another repeat (CTC1)n(CTT)n(CTC2)n downstream of the CTG repeat. High-throughput next generation sequencing was therefore used to determine the structure of the CTG18.1 locus, to genotype germline alleles, and to find sequence variants. Individuals with larger alleles underwent further investigation. Small pool PCR was used to investigate somatic instability. Allele lengths between 40 to 79 CTG repeats were relatively stable. However, large alleles ≥ 80 CTG repeats were highly unstable. Corneal and saliva DNA samples were obtained from severely affected patients with late onset Fuchs endothelial corneal dystrophy at the time of the corneal transplant. The progenitor allele length for each patient was estimated. Somatic instability and factors modifying disease severity were investigated using small pool PCR. All alleles were also sequenced using MiSeq next generation sequencing to genotype CTG, CTC1 and CTC2 repeats, and to quantify somatic instability. Sequencing revealed CTG and CTC1 were both polymorphic. GTG and CTC variants were detected in the CTG region of the normal alleles. Using MiSeq data, there was allele length effect on somatic instability for alleles between 40 to 70 CTG repeats (r2 = 0.51039 p-value = 0.002). The correlation between estimated progenitor allele length and somatic instability using PacBio RS II data was also significant (r2 =0.40416, p-value &lt;0.001). Testing the CTG18.1 locus is relatively laborious and time consuming. Finding haplotypes that are associated with either unexpanded or expanded alleles by using SNPs around the repeat region is an alternative way to detect individuals carrying a repeat expansion. The data from this project highlighted the importance of utilising next generation sequencing to determine the structure of the CTG18.1 locus, genotyping both repeats which is CTG and CTC repeats, and finding variants. Somatic instability for the larger alleles was investigated using SP-PCR, MiSeq, and PacBio sequencing. The presence of variant repeats in the larger alleles, and differences in somatic instability might mediate age-dependent reduced penetrance in late onset Fuchs endothelial corneal dystrophy caused by repeat expansion. In conclusion, mechanisms mediate age-dependent reduced penetrance in late onset Fuchs endothelial corneal dystrophy caused by repeat expansion was not described by the presence of variant repeats or somatic instability.