Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2020 Sep 3;107(3):544-554.
doi: 10.1016/j.ajhg.2020.06.019. Epub 2020 Jul 29.

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

Anna Fliedner  1 , Philipp Kirchner  1 , Antje Wiesener  1 , Irma van de Beek  2 , Quinten Waisfisz  2 , Mieke van Haelst  2 , Daryl A Scott  3 , Seema R Lalani  4 , Jill A Rosenfeld  5 , Mahshid S Azamian  4 , Fan Xia  5 , Marina Dutra-Clarke  6 , Julian A Martinez-Agosto  7 , Hane Lee  8 ; UCLA Clinical Genomics Center; Grace J Noh  9 , Natalie Lippa  10 , Anna Alkelai  10 , Vimla Aggarwal  11 , Katherine E Agre  12 , Ralitza Gavrilova  13 , Ghayda M Mirzaa  14 , Rachel Straussberg  15 , Rony Cohen  16 , Brooke Horist  17 , Vidya Krishnamurthy  17 , Kirsty McWalter  18 , Jane Juusola  18 , Laura Davis-Keppen  19 , Lisa Ohden  19 , Marjon van Slegtenhorst  20 , Stella A de Man  21 , Arif B Ekici  1 , Anne Gregor  1 , Ingrid van de Laar  20 , Christiane Zweier  22
Collaborators, Affiliations

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

Anna Fliedner et al. Am J Hum Genet. .

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.

PubMed Disclaimer

Conflict of interest statement

The Department of Molecular and Human Genetics at Baylor College of Medicine receives revenue from clinical genetic testing completed at Baylor Genetics Laboratories. J.J. and K.Mc. are employees of GeneDx, Inc. The remaining authors declare no competing interests.

Figures

Figure 1
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
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
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
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).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Cramer P. RNA polymerase II structure: from core to functional complexes. Curr. Opin. Genet. Dev. 2004;14:218–226. - PubMed
    1. Orphanides G., Reinberg D. A unified theory of gene expression. Cell. 2002;108:439–451. - PubMed
    1. Kornberg R.D. Eukaryotic transcriptional control. Trends Cell Biol. 1999;9:M46–M49. - PubMed
    1. Lee T.I., Young R.A. Transcription of eukaryotic protein-coding genes. Annu. Rev. Genet. 2000;34:77–137. - PubMed
    1. Haijes H.A., Koster M.J.E., Rehmann H., Li D., Hakonarson H., Cappuccio G., Hancarova M., Lehalle D., Reardon W., Schaefer G.B. De Novo Heterozygous POLR2A Variants Cause a Neurodevelopmental Syndrome with Profound Infantile-Onset Hypotonia. Am. J. Hum. Genet. 2019;105:283–301. - PMC - PubMed

Publication types

MeSH terms