Figure 4: Detection and validation of MAE of rare variants.

(a) Distribution of heterozygous single nucleotide variants (SNVs) across samples for different consecutive filtering steps. Heterozygous SNVs detected by exome sequencing (black), SNVs with RNA-seq coverage of at least 10 reads (grey), SNVs where the alternative allele is mono-allelically expressed (alternative allele frequency >0.8 and Benjamini-Hochberg corrected P value <0.05, blue), and the rare subset of those (ExAC minor allele frequency <0.001, red). (b) Fold change between alternative (ALT+1) and reference (REF+1) allele read counts for the patient #80256 compared to total read counts per SNV within the sample. Points are coloured according to the groups defined in a. (c) Gene-wise comparison of RNA and protein fold changes of the patient #80256 compared to the average across the fibroblast cell lines of all other patients. The position of the gene ALDH18A1 is highlighted. Reliably detected proteins that were not detected in this sample are shown separately with their corresponding RNA fold changes (points below solid horizontal line). (d) Relative intensity for metabolites of the proline biosynthesis pathway (inlet) for the patient #80256 and 16 healthy controls of matching age. Equi-tailed 95% interval (whiskers), 25th, 75th percentile (boxes) and median (bold horizontal line) are indicated. Data points belonging to the patient are highlighted (red circles, P values were computed using the Student’s t-test). (e) Cell counts under different growth conditions for the NHDF and patient #80256. Both fibroblasts were grown in fetal bovine serum (FBS), dialysed FBS (without proline) and dialysed FBS with proline added. Boxplot as in d. P values are based on a two-sided Wilcoxon test. (f) Intron retention for MCOLN1 in patient #62346. Tracks from top to bottom: genomic position on chromosome 19, amino acid translation (red for stop codons), RefSeq gene model, coverage of WES of patient #62346, RNA-seq based coverage for patients #62346 and #85153 (red and orange shading, respectively). SNVs are indicated by non-reference coloured bars with respect to the corresponding reference and alternative nucleotide.