Extended Data Fig. 6: Evaluation of potential splice effects by RT-PCR and Sanger or Nanopore sequencing of PED002, PED013, PED017 and PED104.

RNA analysis for interpretation of variant effect in PED002, PED013, PED017 and PED104. (a) RT-PCR and nanopore sequencing results for PED002A (mother; blue) and PED002B (father; green) versus a control blood sample (red). The Sashimi plot shows retention of 8 intronic bases (black arrow) of the 8th intron of DNAJB11 as a result from the intronic c.853-10 G > A variant in both parents. (b) RT-PCR and Sanger sequencing of paternal cDNA shows the synonymous PIBF1 c.954 G > A p.(Lys318 = ) variant identified in PED013 causes skipping of exon 8, predicted to result in a downstream frameshift and premature termination. The right (cDNA) figure shows the initiation of a heteroduplex after exon 7, with the mutant allele continuing to exon 9. (c) RT-PCR and Sanger sequencing of fetal cDNA shows the TPI1 c.544-1 G > C variant identified in PED017 alters the canonical splice acceptor site of exon 6, resulting in an in-frame deletion of 2 amino acids. The right (cDNA) figure shows the start of a heteroduplex after exon 5, with the mutant sequence displaying a 6 bp deletion from the start of exon 6. (d) RT-PCR and nanopore sequencing on a maternal blood sample of PED104 A shows skipping of the (out-of-frame) exon 9 in MECOM as a result from the intronic c.2208 + 4A > T variant (red). The long reads included a synonymous SNP (c.2667 G > A) in exon 14, which allowed separation and comparison of the mutant (red) versus wildtype (blue) alleles.