Fig 2: TIMMDC1 SSO1 and SSO2 antisense oligonucleotides restore normal splicing in the c.597-1340A>G affected fibroblasts.Agarose gels showing semi-quantitative RT-PCR amplicons of normally and alternatively spliced mRNAs in the SSO1, SSO2 or non-specific control NC5 antisense oligonucleotides treated affected (III-6 and III-2; lanes 1–6) and untreated parent (II-1 and II-2) fibroblasts (lanes 7 and 8). Minus RT reactions of the affected fibroblast RNAs (lanes 9 and 10) are also shown. Left panel: Control PCR products from primers (P442/P443) located in Exon 3 and 4 showing amplification from the unaltered mRNA region. The gel showed increased levels of normal TIMMDC1 mRNA in SSO1 and SSO2 (lanes 1, 2 and 4, 5) compared to NC5 (lanes 3, 6) treated affected or untreated (lanes 7, 8) parent fibroblasts. Middle panel: PCR products from primers (P444/448) located in Exon 5 and 6 showing increased levels of normally spliced TIMMDC1 mRNA in SSO1 and SSO2 (lanes 1, 2 and 4, 5) compared to NC5 (lane 3, 6 with predominantly poison exon containing mRNA) treated affected or untreated (lanes 7 and 8 with predominantly normal mRNA) parent fibroblasts. Right panel: PCR products from primers (P446 located within poison exon and P448 within Exon 6) that specifically amplify mRNAs with poison exon sequence. Levels of poison exon containing mRNA is reduced in SSO1 and SSO2 treated (lanes 1, 2 and 4, 5) compared to NC5 (lanes 3 and 6) treated affected fibroblasts but comparable to untreated (lanes 7 and 8) parent fibroblasts.

Fig 3: Aberrant splicing detection and quantification.(a) Aberrant splicing events (Hochberg corrected P value<0.05) for all fibroblasts. (b) Aberrant splicing events (n=175) in undiagnosed patients (n=48) grouped by their splicing category after manual inspection. (c) CLPP Sashimi plot of exon skipping and truncation events in CLPP-affected and CLPP-unaffected fibroblasts (red and orange, respectively). The RNA coverage is given as the log10 RPKM-value and the number of split reads spanning the given intron is indicated on the exon-connecting lines. At the bottom the gene model of the RefSeq annotation is depicted and the aberrantly spliced exon is coloured in red. (d) Same as in c for TIMMDC1. At the bottom the newly created exon is depicted in red within the RefSeq annotation track. (e) Coverage tracks (light red) for patients #35791, #66744, and #91324 based on RNA and WGS. For patient #91324 only WGS is available. The homozygous SNV c.596+2146A>G is present in all coverage tracks (vertical orange bar). The top tracks show the genomic annotation: genomic position on chromosome 3, DNA sequence, amino acid translation (grey, stop codon in red), the RefSeq gene model (blue line), the predominant additional exon of TIMMDC1 (blue rectangle) and the SNV annotation of the 1000 Genomes Project (each black bar represents one variant). (f) Per cent spliced in (Ψ) distribution for different splicing classes and genes. Top: histogram of the genome-wide distribution of the 3′ and 5′ Ψ-values based on all reads over all samples. Middle: The shaded horizontal bars represent the densities (black for high density) of the background, weak and strong splicing class, respectively (Methods section). Bottom: Ψ-values of the predominant donor and acceptor splice sites of genes with private splice sites (that is, found predominant in at most two samples) computed over all other samples.

Fig 4: TIMMDC1 rare intronic variant c.597-1340A>G segregates in the family as a recessive allele.a Family pedigree showing the consanguineous heterozygous (A/G) parents (II-1 and II-2), two affected homozygous (G) individuals (III-2 and III-6) and other unaffected heterozygous (A/G) or homozygous wild type (A) sibs. A slash symbol represents deceased individuals. b Segregation of TIMMDC1 c.597-1340A>G variant. Sanger sequencing chromatograms show the presence or absence of the TIMMDC1 variant in the family members. The TIMMDC1 c.597-1340A>G homozygous variant in affected individuals III-2 and III-6 is marked with a red circle.

Fig 5: TIMMDC1 c.597-1340A>G variant inserts an 80 bp poison exonic sequence between Exon 5 and 6 in TIMMDC1 mRNAs of the affected individuals.a Agarose gel showing semi-quantitative RT-PCR amplicons from normally and alternatively spliced mRNAs from two affected (lanes 1 and 2), carrier parents (lanes 3 and 4) and an unrelated healthy control (lane 5) fibroblast RNAs. Minus RT reactions of the affected fibroblast RNAs (lanes 6 and 7) were also included. Left panel: Control PCR products from primers (P442/P443) located in Exon 3 and 4 showing amplification from the unaltered mRNA region, middle panel: PCR products from primers (P444/448) located in Exon 5 and 6 showing amplification of mRNAs with (lanes 1 and 2) and without (lanes 3–5) poison exon, and right panel: PCR products from primers (P446 located within poison exon and P448 within Exon 6) that specifically amplify mRNAs with poison exon sequence. Note that levels of mRNA with poison exon sequences in the affected individuals and parents (carrying the TIMMDC1 variant; lanes 1–4) are higher than the unrelated control (lane 5) that has low level of aberrant splicing that generates the mRNAs with poison exon sequences. See Fig. 2 for primer location. b Chromatograms showing mRNA sequences with an 80 bp poison exon in fibroblasts of the two affected individuals (see sequence of III-6*, III-2** and III-6•• bands from the gel in a). Parent II-2• band sequence, as expected, showed the absence of poison exon.