Cracking rare disorders: a new minimally invasive RNA-seq protocol

Abstract

RNA sequencing (RNA-seq) has become key to complementing exome and genome sequencing for variant interpretation. We present a minimally invasive RNA-seq protocol using short-term cultured peripheral blood mononuclear cells (PBMCs) with and without cycloheximide treatment, enabling detection of transcripts subject to nonsense-mediated decay. While broadly applicable, this protocol is particularly suited for neurodevelopmental disorders, as up to 80% of the genes in our intellectual disability and epilepsy gene panel are expressed in PBMCs. Applied to 46 affected individuals and 15 parents, RNA-seq revealed splicing defects in six of nine individuals with splice variants, allowing reclassification of seven variants. Targeted cDNA analysis confirmed aberrant splicing in four individuals but missed intron retention in two. Global analyses (FRASER, OUTRIDER, and monoallelic expression) supported findings but did not yield new diagnoses. We propose a flowchart integrating RNA-seq into diagnostic workflows. Overall, our protocol is easily implementable, captures complex splicing events, and enhances variant classification.

Fig. 1. Gene expression in four different CATs and upon PUR or CHX treatment in LCLs.

A, B Upset plot of the genes within A the Mendeliome gene panel (n = 4732) and B the ID and epilepsy gene panel (n = 1689) and their expression (>1 TPM) in four different CATs. C, D Relative expression levels of CXPC (NM_004628.5) and D the NMD sensitive SRSF2 transcript (ENST00000452355.7) in LCL samples with, respectively, PPT1 (darkblue bars), TPP1 (brown bars), and XPC (heterozygous variant: gray bars; homozygous variant: blue bars) protein truncating variants. For each sample, the relative expression is given in the untreated situation, after 6 h treatment with puromycin, and after 6 h treatment with cycloheximide. The theoretical effect of the heterozygous (left) and homozygous (right) PTV in XPC (XPC (NM_004628.5): c.1290_1295del & p.(Tyr430_Glu432delinsTer) is given in (C) based on the position of the qPCR primers (as indicated by the blue convergent arrows). UNT untreated, CHX treated with cycloheximide, PUR treated with puromycin, C control (i.e., samples without XPC PTV).

Fig. 2. Effect on splicing of intronic WDR26 variant: WDR26 (NM_025160.6): c.1644 + 5G > A.

A Zoomed in IGV plot of exome data showing the heterozygous +5 intronic variant downstream of exon 11. B Sanger profile showing the heterozygous exon 11 skip. C Sashimi plot of exons 10–12 of WDR26 in untreated and treated PBMCs of the affected individual (red = untreated (UNT) and blue = CHX treated (CHX)) and a control PBMC sample, both untreated (green) and CHX treated (brown). The skipping of exon 11 can clearly be observed in both the untreated and CHX-treated PBMCs. D Schematic representation of the splicing effect(s) caused by the variant. While in the untreated PBMCs, the exon skip was observed in 27% of the splicing events from the exon 10 donor site, the aberrant splicing was observed in approximately 50% of the splicing events at that site in the CHX-treated PBMCs.

Fig. 3. Effect on splicing of intronic NIPBL variant: NIPBL (NM_133433.3): c.64 + 6T > C.

A Zoomed in IGV plot of exome data showing the heterozygous +6 intronic variant downstream of exon 2. B Sanger profile with background noise, skip of exon 2 is suggestive but difficult to interpret due to the background noise. C Sashimi plot of exons 2–3 of NIPBL in untreated and treated PBMCs of the affected individual (red = untreated (UNT) and blue = CHX treated (CHX)) and a control PBMC sample, both untreated (green) and CHX treated (brown). The skipping of exon 2 can be observed in both the untreated and CHX-treated PBMCs. D Schematic representation of the splicing effect(s) caused by the variant. The exon skip was observed in 14% and 15% of the splicing events starting from the exon 1 donor site in the untreated and CHX-treated PBMCs.

Fig. 4. Effect on splicing of intronic STAG1 variant: STAG1 (NM_005862.2): c.395-2A > T.

A Zoomed in IGV plot of exome data showing the heterozygous-2 intronic variant upstream of exon 6. B Sanger profile showing the heterozygous exon 6 skip. C Sashimi plot of exons 5–7 of STAG1 in untreated and treated PBMCs of the affected individual (red = untreated (UNT) and blue = CHX treated (CHX)) and a control PBMC sample, both untreated (green) and CHX treated (brown). The skipping of exon 6 can clearly be observed in both the untreated and CHX-treated PBMCs. D Schematic representation of the splicing effect caused by the variant. While the effect was observed in 31% of the splicing events from the exon 5 donor site in the untreated PBMCs, the aberrant splicing was observed in approximately 44% of the splicing events at that site, in the CHX-treated PBMCs.

Fig. 5. Effect on splicing of intronic SMARCC2 variant: SMARCC2 (NM_001330288.1): c.2186-3C > G.

A Zoomed in IGV plot of exome data showing the heterozygous-3 intronic variant upstream of exon 22. B The Sanger profile is difficult to interpret due to the background noise, exon 22 skip is suggestive. C Sashimi plot of exons 20–23 of SMARCC2 in untreated and treated PBMCs of the affected individual (red = untreated (UNT) and blue = CHX treated (CHX)) and a control PBMC sample, both untreated (green) and CHX treated (brown). Different split reads are observed, for the canonical transcript (NM_001330288.1): skip of exon 22, as well as intron retention in intron 21 in untreated PBMCs. Intron retention is not present in CHX-treated cells. D Schematic representation of the splicing effects caused by the variant. Transcript NM_001330288.1: from the exon 21 splice donor, an exon 22 skip was observed in 45% of the reads of CHX-treated PBMCs vs 38% for the untreated cells. Intron retention was only observed in the untreated cells in 4%. Transcript XM_017019886.1: from the exon 20 donor site, 6% of splice events appeared to skip exon 21 in untreated PBMCs, and 17% in cells treated with CHX.

Fig. 6. Effect on splicing of intronic DDX3X variant: DDX3X (NM_001356.5): c.1770-2 A > G.

A Zoomed in IGV plot of exome data of the proband showing the hemizygous-2 intronic variant downstream of exon 16. B Sanger sequencing on cDNA from the mother, a heterozygous carrier of the DDX3X variant: Normal sequence until the end of exon 15, from then on, multiple peaks per nucleotide, the exact effect is difficult to decipher. C Sashimi plot of exons 14–17 of DDX3X in untreated and treated PBMCs of the mother (red = untreated (UNT) and blue = CHX treated (CHX)) and a control PBMC sample, both untreated (green) and CHX treated (brown). From the exon 15 donor, three arches can be distinguished. A cryptic splice acceptor site is recognized in both untreated and treated PBMCs from the patient’s mother (junctions indicated in bold). In the controls, two arches starting from the exon 15 donor are present. D Schematic representation of the splicing effects caused by the variant. A cryptic acceptor was recognized in both untreated and CHX-treated PMBCs, in 37% and 40% of the reads, respectively. In an alternative transcript (NM_001193416.3), exon 16 is shorter and starts 3 bp later than the canonical transcript.

Fig. 7. Effect on splicing of exonic SETD1A variant: SETD1A (NM_014712.2): c.639G > A, p.(Thr213 = ).

A Zoomed in IGV plot of exome data showing the heterozygous c.639G > A in exon 5. B Sanger's profile is indicative of exon 5 skip. C Sashimi plot of exons 4–6 of SETD1A in untreated and treated PBMCs of the affected individual (red = untreated (UNT) and blue = CHX treated (CHX)) and a control PBMC sample, both untreated (green) and CHX treated (brown). The skipping of exon 5 can clearly be observed in both the untreated and CHX-treated PBMCs. In addition, intron 5 retention can be observed. D Schematic representation of the splicing effects caused by the variant. The exon 5 skip effect was observed in 27% and 33% of the splicing events starting from the exon 4 donor site in the untreated and CHX-treated PBMCs, respectively. A cryptic splice donor was also observed, resulting in intron retention in 8% and 20% of the splicing events starting from exon 6 in untreated cells and cells treated with CHX.

Fig. 8. Overview of our proposed diagnostic workup for RNA-sequencing.

A Wet lab workflow. B–D Decision trees for variant classification/evaluation for respectively FRASER (B 1/ pre-set parameters and 2/gene-specific evaluation), OUTRIDER (C), and mono allelic expression (D).