Novel Synonymous and Deep Intronic Variants Causing Primary and Secondary Pyruvate Dehydrogenase Complex Deficiency

Figure 2

RNA analyses of PDHA1 in RNA from blood and cultured fibroblasts. (a) PCR products of PDHA1 cDNA generated from RNA extracted from patient 1 (P1) and control (C) fibroblasts, treated with (+) and without (-) emetine 7 hours before harvesting, and RNA extracted from blood from P1 and C. Extra bands in P1 correspond to mRNA lacking exon 5 and exon 5+6. (b) PCR products of PDHA1 cDNA generated from RNA extracted from blood from patient 2 (P2), his mother, father, and control (C) and RNA extracted from fibroblasts from P2 and control, treated with (+) and without (-) emetine 7 hours before harvesting. Extra band in P2 corresponds to mRNA lacking exon 6. (c) Schematic picture of variants identified in P1 and P2 PDHA1 on DNA level and the resulting aberrant spliced mRNAs. (d) Gene expression of PDHA1 in blood and total expression/all different known transcripts separately of 135 healthy controls, P1, and P2; framed box plot indicates MANE Select transcript (NM_000284.4, ENST00000422285.7); transcripts with high expression in P2 are naturally occurring transcripts without exon 6: ENST00000379804.1 (exons 8-9-10-11), ENST00000379805.3 (exons 1-2-3-4-5), ENST00000481733.1 (exons 7-8-9-10), ENST00000492364.1 (exons 1-2-3-4), and ENST00000540249.5 (exons 1-2-3-4-5-7-8-9-10-11). (e) Gene expression of PDHA1 in fibroblasts and total expression/all different known transcripts separately of 21 healthy controls, P1, and P2; framed box plot indicates MANE Select transcript (NM_000284.4, ENST00000422285.7); transcript with high expression in P2 is naturally occurring transcript without exon 6: ENST00000540249.5 (exons 1-2-3-4-5-7-8-9-10-11). (f) Sashimi plot of RNAseq data from fibroblast RNA from P1, P2, and the control group consisting of the median number of reads in 3 healthy controls. RNA coverage is given on the axis, and a number of split reads spanning introns are indicated on the exon junction lines. Bottom graph depicts MANE Select transcript (NM_000284.4, ENST00000422285.7) and known transcript (NM_001173456.2, ENST00000540249.5) lacking exon 6 with reference exons as black boxes. Plot shows a low proportion of aberrantly spliced mRNA lacking exon 5 in P1 and high proportion of aberrantly spliced mRNA lacking exon 6 in P2. The control group has a small number of splice junctions directly from exons 5 to 7, corresponding to the known transcript lacking exon 6.

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