Table 1.
Patient demographics.
Table 2.
Summary of repeat length in TCF4 in leukocytes and corneal endothelium.
Fig 1.
Iso-Seq reads from the corneal endothelium include repeat expansions greater than 6 kb in length.
Iso-Seq reads spanning 11 kb from the IGV of FECD(1) (top two rows) and Cont(1) (bottom two rows). Locations of the CAG repeats (CAGn) and exons in this region as defined previously [12] are labelled. Each sample is shown twice to show the structure and diversity of different read types. The blue-green rich regions in the second FECD row are CAG expansions.
Fig 2.
Representative assessment of CAG expanded repeats.
Magnified view of the same samples as shown in Fig 1, showing the reference sequence (Ref) and the actual CAG sequence of the expanded repeats in FECD(1). The single antisense read in this view is shown with the pink background.
Fig 3.
Repeat length size distribution of TCF4 transcripts originating from FECD expanded alleles.
Various repeat lengths were identified in transcripts originating from each FECD patients expanded allele. Note that the shortest expanded repeat reads from Iso-Seq were 435 [FECD(1)], 210 [FECD(2)], and 320 [FECD(3)], which are >2.5-fold larger than TNR length in leukocyte DNA.
Fig 4.
Southern blot of peripheral blood leukocyte and primary HCEC lines established from control and FECD patients.
(A) TCF4 repeats in DNA isolated from peripheral blood leukocytes (Lanes 1–3) and primary HCECs (Lanes 4–6). Lanes 1 [Cont(4)] and 4 [Cont(5)] represent samples obtained from different control patients. Lanes 2, 5 [FECD(2)] and 3, 6 [FECD(1)] are samples from two FECD patients with expanded repeats. Arrows indicate large molecular weight bands in FECD1 and FECD2 corresponding to approximately 15,000 base pairs and approximately 4500 repeats. For analysis, 5 μg leukocyte DNA and 20 μg primary HCEC DNA was digested with EcoRI and separated on an 0.8% agarose gel. (B) Southern blot of control and FECD primary HCEC cell lines showing large molecular weight bands ranging from approximately 5500—>15,000 base pairs. This corresponds to all FECD samples having at least one allele with a TCF4 expansion of >1100 repeats. Genotype of TCF4 expansion size as determined in leukocytes for each FECD HCEC line is as follows: FECD(4)—16/69; FECD(5)—25/71; FECD(6)—73/82; FECD(7)—18/52; FECD(8)—12/64; FECD(9)—12/66; FECD(10)—100/123; and FECD(11)—17/98. Note that FECD(2) was utilized as a positive control in both (A) and (B). For analysis, 5 μg of primary HCEC DNA was digested with EcoRI and separated on an 0.8% agarose gel.
Table 3.
TCF4 promoter usage.