Genomic Southern Blot of DNA samples from normal control (lanes 1, 4, 7 and 8) and FECD patients (lanes 2, 3, 5, 6, 9–11).

<p>Lanes 12 and 13 are laboratory control samples that have not been evaluated for FECD. Note that the samples in lanes 2, 3 and 6 are from FECD patients that do not have the repeat expansion. The samples in lanes 5, 9, and 10 are samples from FECD cases with repeat expansion over 1500 repeats. Lane L contains sizing standards.</p

A comparison of TGC repeat length in TCF4 between FECD cases and normal controls.

*<p>p<0.001 for FECD cases vs. controls by Fisher's exact test.</p

STR analysis of PCR amplicons of 3 normal control (A) and 3 FECD patients (B).

<p>The bottom panel is the analysis of a FECD patient with 2 expanded alleles.</p

DNA sequence surrounding the trinucleotide repeat in the intron of the <i>TCF4</i> gene on chromosome 18.

<p>The trinucleotide repeat region is shown in red, and PCR primer sequences used for sizing the repeat region are underlined. This version of the sequence comes from the human reference sequence and contains 25 TGC repeats.</p

Sanger DNA sequencing of DNA samples of a normal control (A) and a FECD patient with two expanded alleles (B).

<p>Sanger DNA sequencing of DNA samples of a normal control (A) and a FECD patient with two expanded alleles (B).</p

Frequency histogram of the TGC repeat length of the longest allele in all 129 samples.

<p>The length of the longest repeat in each sample is shown for both FECD patients (black bars) and normal control subjects (open bars). Note that 3 FECD patients had very long repeat expansions (more than 1500 repeats), as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049083#pone-0049083-g004" target="_blank">Figure 4</a>.</p

Identification of TSPOAP1 variants in a family with RE- FECD.

<p>(A) Pedigree of RE- FECD family. Patient 52 (91 years old) and patient 59 (64 year old) had modified Krachmer scores of 6 in both eyes and TCF4 trinucleotide repeat sizes of 18, 24 and 24, 31 respectively. Patient 53 (66 years old) and patient 62 (52 years old) had modified Krachmer scores of 0 in both eyes and TCF4 trinucleotide repeat sizes of 24, 31 and 18, 32 respectively. Outside of the FECD diagnosis, there were no evident medical conditions or syndromic diseases that were common within the pedigree other than solitary skin cancers in 2 of the 4 family members. (B) Sanger sequencing traces of DNA from the vicinity of the R1058H variant are shown. Both affected family members (I-1 and II-2) are confirmed to be heterozygous for the R1058H variant. (C) Schematic diagram showing the filtering strategy used to identify variants in exome sequencing of 4 family members. The number of variants remaining after each filtering step is shown in the boxes.</p

TSPOAP1 variants in FECD patients.

<p>The locations of two substitution variants in the primary sequence of TSPOAP1 are shown in bold red (positions 1058 and 1738) under a diagram of the structure of the TSPOAP1 gene. The exon that is preferentially excluded from RE+ samples by alternative splicing is shown in red, and the location of this sequence in the TSPOAP1 protein is also shown in red (position 1298–1577). The vertical black arrow designates the start of the region of the TSPOAP1 protein that is thought to interact with TSPO.</p

Differential splicing events.

<p>Differential splicing events.</p

Validation of RNA Sequencing identified mis-splicing events.

<p>(A) RT-PCR amplified products derived from specific primers (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200005#pone.0200005.t002" target="_blank">Table 2</a>) that flanked selected exons in the ADD3, INF2, and CADM1 genes. Amplification of a larger DNA fragment in ADD3 and CADM1 (exon inclusion) and a smaller DNA fragment in INF2 (exon exclusion) are shown in samples obtained from two independent FECD patients that have a TCF4 trinucleotide repeat expansion (denoted with a plus sign). In contrast, these bands are either lacking or in reduced amounts in a sample from a FECD patient that does not contain a TCF4 trinucleotide repeat expansion (denoted with a minus sign) or a non-FECD patient sample (labeled with a C). (B) Numbers in boxes represent percentage of PCR products containing inclusion/exclusion of exons for each sample in (A). Numbers in parentheses are percent spliced in values obtained from RNASeq on the same samples from which PCR was performed. These results confirm exon inclusion and exclusion as identified by RNA sequence and PCR analysis.</p