Variants in SCAF4 Cause a Neurodevelopmental Disorder and Are Associated with Impaired mRNA Processing

Abstract

RNA polymerase II interacts with various other complexes and factors to ensure correct initiation, elongation, and termination of mRNA transcription. One of these proteins is SR-related CTD-associated factor 4 (SCAF4), which is important for correct usage of polyA sites for mRNA termination. Using exome sequencing and international matchmaking, we identified nine likely pathogenic germline variants in SCAF4 including two splice-site and seven truncating variants, all residing in the N-terminal two thirds of the protein. Eight of these variants occurred de novo, and one was inherited. Affected individuals demonstrated a variable neurodevelopmental disorder characterized by mild intellectual disability, seizures, behavioral abnormalities, and various skeletal and structural anomalies. Paired-end RNA sequencing on blood lymphocytes of SCAF4-deficient individuals revealed a broad deregulation of more than 9,000 genes and significant differential splicing of more than 2,900 genes, indicating an important role of SCAF4 in mRNA processing. Knockdown of the SCAF4 ortholog CG4266 in the model organism Drosophila melanogaster resulted in impaired locomotor function, learning, and short-term memory. Furthermore, we observed an increased number of active zones in larval neuromuscular junctions, representing large glutamatergic synapses. These observations indicate a role of CG4266 in nervous system development and function and support the implication of SCAF4 in neurodevelopmental phenotypes. In summary, our data show that heterozygous, likely gene-disrupting variants in SCAF4 are causative for a variable neurodevelopmental disorder associated with impaired mRNA processing.

Keywords: SCAF4; epilepsy; intellectual disability; mRNA processing; neurodevelopmental disorder; seizures.

Figure 1

SCAF4 Variants Observed in Individuals with NDDs (A) Schematic drawing of SCAF4 (longest isoform GenBank: NM_020706.2) with identified variants. Non-coding exonic regions are displayed in white, coding exons in gray, and encoded domains (according to Ensembl¹²) in color. (B) Schematic drawing of the SCAF4 protein. One missense and eight likely gene-disrupting variants (excluding two splice-site variants) are displayed below the scheme. Variants of unknown significance are shaded in gray. (C) Clinical pictures of individuals with pathogenic variants in SCAF4 (I1 at age 3 and 5 years; I2 at age 20 months; I5 at age 13 years; I6 at age 6.5 years; I9 at age 9 years). Note common facial features such as epicanthus, a deep nasal bridge, bulbous nasal tip, and deep philtrum, particularly in I1 and I2. Abbreviations: CID, conserved CTD-interacting domain; SR, Ser/Arg-rich domain; RRM, RNA recognition motif; PQ, Pro/Gln-rich domain.

Figure 2

Transcriptome Analysis in Four Individuals with Variants in SCAF4 (A) Heatmap displaying 9,038 differentially expressed genes between four affected individuals and eight healthy control subjects and depicting the expression similarity between individuals with adjusted p value < 0.01. A total of 4,710 genes were downregulated and 4,328 genes were upregulated in the affected individuals. Heatmap was created using the pheatmap package v.1.0.12 (see Web Resources) applying the standard settings (complete linkage method for hierarchical clustering for both columns and rows). Expression values were normalized, and genes were sorted by adjusted p value. Blue, downregulated; red, upregulated. (B) Principal component analysis (PCA) of affected individuals and control subjects separated by genotype and sex. Affected individuals and control subjects readily clustered within their group. PCA plot was created using the DESeq2 package version 1.24.0. (C and D) Gene Ontology (GO) term analysis of (C) downregulated and (D) upregulated genes depicting the top five GO terms for biological processes.

Figure 3

Impact of SCAF4 Variants on mRNA Processing and Splicing (A) mRNA isoform expression changes of four individuals with variants in SCAF4 detected by IsoformSwitchAnalyzeR, , analysis of RNA-sequencing data. Most frequent changes include length loss, exon loss, and usage of an alternative transcription start site (TSS) more downstream. (B) Venn diagram depicting the most prominent causes for a length loss of isoforms. Truncation results from a combination of exon loss, alternative down-stream transcription start sites (TSS downstream), and alternative up-stream transcription termination sites (TTS upstream). Venn diagram was created using the eulerr package version 6.0.0 (see Web Resources). (C) Example of an alternative spliced gene (BTLA). All expressed transcripts are shown in the upper panel. The isoform usage is shown in the lower panel. In individuals with variants in SCAF4, the two shorter isoforms with downstream TSSs, upstream TTSs, and exon loss showed increased expression while the longest isoform 1 is used less. (D) Gene Ontology (GO) term analysis of differentially spliced genes depicting the top five GO terms for biological processes. Abbreviations: TSS, transcription start site; TTS, transcription termination site. Asterisks indicate statistical significance (^∗p < 0.05, ^∗∗∗p < 0.001).

Figure 4

Impact of CG4266 Knockdown on Drosophila Nervous System (A) Representative pictures of neuromuscular junctions (NMJs) from L3 control (upper panel) and pan-neuronal CG4266 knockdown (lower panel) larvae. The white-framed box indicates the cutout on the right side. NMJ area and length as well as the number of synaptic boutons, islands, and branches were determined. In the RNAi_1 larva, an increased number of AZs can be observed in comparison to control larva. Scale bars represent 10 μm. (B) Quantification revealed a significant increase in the number of AZs upon CG4266 knockdown with two RNAi fly lines using the elav-x;dicer II driver (BL#25750). (C) Flies with knockdown of CG4266 in all neurons (elav-Gal4/Cyo, BL#8765) showed significant locomotor impairment in the climbing assay, as measured by the amount of time that 70% of flies in a vial needed to crawl up 8.8 cm after being tapped down. Data represent the mean from a minimum of 170 flies tested per genotype. (D) In the courtship conditioning paradigm, both CG4266 RNAi lines showed significant impairment of learning upon knockdown with a mushroom-body-specific driver line (UAS-Dcr-2;247-Gal4). The short-term memory was reduced significantly in RNAi line 1 with RNAi line 2 showing the same tendency. Graphs display number of animals below the columns per genotype. Differences between learning indices of control and mutant flies were statistically compared by a randomization test with 10,000 bootstrap replicates with a custom R script. Error bars represent the SEM. Asterisks indicate statistical significance (^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001).