Fig. 4.

Analysis of expression of genes at the breakpoints, i.e., PLEKHG3 and TCF4. (a) Graph comparing PLEKHG3 expression between cultured skin fibroblasts of individual II-1 and cultured skin fibroblasts of an unaffected control. Analysis was done by quantitation of transcriptome sequence reads and is shown as fragments per kilobase of exon per million fragments mapped (FPKM). (b) Graph comparing combined levels of all TCF4 transcript variants between cultured skin fibroblasts of individual II-1 and cultured skin fibroblasts of an unaffected control. Analysis was done by quantitation of transcriptome sequence reads. (c) Figure showing the PLEKHG3 : TCF4 fusion transcript generated by the derivative chromosome 14. The first non-coding exon of PLEKHG3 (3’ end chr14:65,171,422) is spliced to a coding exon of TCF4 (5’ end chr18:53,131,349). This coding exon is incorporated into TCF4 transcripts NM_001243227.1, NM_001243226.2, NM_001243228.1, NM_001083962.1, NM_001243230.1, and NM_003199.2. Transcriptome sequencing detected the fusion transcript in cultured skin fibroblasts (data not shown) and RT-PCR and Sanger sequencing detected it in peripheral blood. (d) Diagram showing the 12 TCF4 RefSeq transcripts aligned to chromosome 18 as annotated in GRCh37/hg19. Physical positions and TCF4 exons along chromosome 18 are shown at the top; the exons are labeled as per Sepp et al. [15]. The breakpoint within TCF4 is shown in red. The transcript variant number is shown in parentheses following each RefSeq accession number. nCounter probes detecting each transcript are shown in the right-hand column. (e) Graph showing the composite mRNA level of all 12 TCF4 transcripts in the peripheral blood among individuals II-1, I-2, III-3, two individuals with Pitt-Hopkins Syndrome (PTHS) and pooled unaffected controls. Measurement was done by qRT-PCR. (f) Graph comparing the level of PLEKHG3 : TCF4 fusion mRNA to the composite mRNA level of all 12 TCF4 transcripts in the peripheral blood of individuals II-1, I-2, III-3, and pooled unaffected controls. Measurement was done by qRT-PCR. (g) Graph comparing the mRNA level in peripheral blood for transcripts interrupted by the translocation (NM_001243226.1, NM_001243227.1, NM_001243228.1, NM_001243230.1, NM_003199.2, NM_001083962.1) inclusive of the PLEKHG3-TCF4 fusion transcript. The mRNA levels were measured among individuals II-1, I-2, III-3, two individuals with Pitt-Hopkins Syndrome (PTHS) and pooled unaffected controls by qRT-PCR. (h) Graph comparing mRNA levels in peripheral blood for various TCF4 transcripts as assessed by nCounter Analysis. The mRNA levels were measured for individuals II-1 and III-3, two individuals with Pitt-Hopkins Syndrome (PTHS) and pooled unaffected controls. The transcripts detected by each nCounter probe are defined in panel d