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• W3045621188 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. 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. The regulation of protein-coding gene transcription is a highly complex process that is crucial for proper gene expression. RNA polymerase II (RNAPII) interacts with many other complexes and factors to ensure accurate pre-initiation, initiation, elongation, and termination of mRNA transcription (reviewed in Cramer,1Cramer P. RNA polymerase II structure: from core to functional complexes.Curr. Opin. Genet. Dev. 2004; 14: 218-226Crossref PubMed Scopus (68) Google Scholar Orphanides and Reinberg,2Orphanides G. Reinberg D. A unified theory of gene expression.Cell. 2002; 108: 439-451Abstract Full Text Full Text PDF PubMed Scopus (704) Google Scholar Kornberg,3Kornberg R.D. Eukaryotic transcriptional control.Trends Cell Biol. 1999; 9: M46-M49Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar and Lee and Young4Lee T.I. Young R.A. Transcription of eukaryotic protein-coding genes.Annu. Rev. Genet. 2000; 34: 77-137Crossref PubMed Scopus (628) Google Scholar). Mutations in several genes and proteins involved in mRNA processing have been implicated in human diseases. For example, heterozygous pathogenic variants in POLR2A (MIM: 180660), which encodes the largest subunit of RNAPII, have recently been identified as the cause of a neurodevelopmental disorder (NDD) with hypotonia and variable intellectual and behavioral anomalies (NEDHIB [MIM: 618603]).5Haijes H.A. Koster M.J.E. Rehmann H. Li D. Hakonarson H. Cappuccio G. Hancarova M. Lehalle D. Reardon W. Schaefer G.B. et al.De Novo Heterozygous POLR2A Variants Cause a Neurodevelopmental Syndrome with Profound Infantile-Onset Hypotonia.Am. J. Hum. Genet. 2019; 105: 283-301Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar Similarly, pathogenic variants in several subunits of the transcription factor IID complex, which plays a key role in transcriptional initiation,6Cormack B.P. Struhl K. The TATA-binding protein is required for transcription by all three nuclear RNA polymerases in yeast cells.Cell. 1992; 69: 685-696Abstract Full Text PDF PubMed Scopus (273) Google Scholar,7Mizzen C.A. Yang X.-J. Kokubo T. Brownell J.E. Bannister A.J. Owen-Hughes T. Workman J. Wang L. Berger S.L. Kouzarides T. et al.The TAF(II)250 subunit of TFIID has histone acetyltransferase activity.Cell. 1996; 87: 1261-1270Abstract Full Text Full Text PDF PubMed Scopus (619) Google Scholar have been implicated in NDDs. These include pathogenic variants in TATA binding protein associated factors, e.g., an X-linked NDD (MRXS33 [MIM: 300966]) is caused by variants in TAF1 (MIM: 313650) and an autosomal-recessive NDD (MRT60 [MIM: 617432]) by variants in TAF13 (MIM: 600774).8O’Rawe J.A. Wu Y. Dörfel M.J. Rope A.F. Au P.Y. Parboosingh J.S. Moon S. Kousi M. Kosma K. Smith C.S. et al.TAF1 Variants Are Associated with Dysmorphic Features, Intellectual Disability, and Neurological Manifestations.Am. J. Hum. Genet. 2015; 97: 922-932Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar,9Tawamie H. Martianov I. Wohlfahrt N. Buchert R. Mengus G. Uebe S. Janiri L. Hirsch F.W. Schumacher J. Ferrazzi F. et al.Hypomorphic Pathogenic Variants in TAF13 Are Associated with Autosomal-Recessive Intellectual Disability and Microcephaly.Am. J. Hum. Genet. 2017; 100: 555-561Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar Furthermore, bi-allelic variants in two subunits of the RNAPII interacting Integrator complex, INTS1 (MIM: 611345) and INTS8 (MIM: 611351), have been shown to be associated with NDDs (NDCAGF [MIM: 618571] and NEDCHS [MIM: 618572]) and lead to both altered splicing patterns and differential gene expression in cells derived from affected individuals.10Oegema R. Baillat D. Schot R. van Unen L.M. Brooks A. Kia S.K. Hoogeboom A.J.M. Xia Z. Li W. Cesaroni M. et al.Human mutations in integrator complex subunits link transcriptome integrity to brain development.PLoS Genet. 2017; 13: e1006809Crossref PubMed Scopus (51) Google Scholar Using trio exome sequencing on an Illumina HiSeq 2500 platform and data analysis with an in-house pipeline as described previously,11Hauer N.N. Popp B. Schoeller E. Schuhmann S. Heath K.E. Hisado-Oliva A. Klinger P. Kraus C. Trautmann U. Zenker M. et al.Clinical relevance of systematic phenotyping and exome sequencing in patients with short stature.Genet. Med. 2018; 20: 630-638Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar we identified the de novo splice-site variant c.321+1G>T in SCAF4 (MIM: 616023) (GenBank: NM_020706.2) in an individual with mild intellectual disability (ID) and seizures. SCAF4 consists of 20 exons and encodes SR (serine and arginine)-related CTD (C-terminal domain)-associated factor 4, consisting of 1,147 amino acids and containing an N-terminal conserved CTD-interacting domain and an RNA recognition motif (Figures 1A and 1B ).13Yuryev A. Patturajan M. Litingtung Y. Joshi R.V. Gentile C. Gebara M. Corden J.L. The C-terminal domain of the largest subunit of RNA polymerase II interacts with a novel set of serine/arginine-rich proteins.Proc. Natl. Acad. Sci. USA. 1996; 93: 6975-6980Crossref PubMed Scopus (289) Google Scholar SCAF4 interacts with the C-terminal domain of the largest subunit of RNAPII, and together with SCAF8, is required for correct polyA site selection and mRNA termination.13Yuryev A. Patturajan M. Litingtung Y. Joshi R.V. Gentile C. Gebara M. Corden J.L. The C-terminal domain of the largest subunit of RNA polymerase II interacts with a novel set of serine/arginine-rich proteins.Proc. Natl. Acad. Sci. USA. 1996; 93: 6975-6980Crossref PubMed Scopus (289) Google Scholar,14Gregersen L.H. Mitter R. Ugalde A.P. Nojima T. Proudfoot N.J. Agami R. Stewart A. Svejstrup J.Q. SCAF4 and SCAF8, mRNA Anti-Terminator Proteins.Cell. 2019; 177: 1797-1813.e18Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar So far, SCAF4 has not been implicated in NDDs or other diseases. According to gnomAD15Karczewski K.J. Francioli L.C. Tiao G. Cummings B.B. Alföldi J. Wang Q. Collins R.L. Laricchia K.M. Ganna A. Birnbaum D.P. et al.Variation across 141,456 human exomes and genomes reveals the spectrum of loss-of-function intolerance across human protein-coding genes.bioRxiv. 2019; https://doi.org/10.1101/531210Crossref Scopus (0) Google Scholar constraint scores, SCAF4 is intolerant toward loss-of-function variants (pLI = 1, o/e = 0.03 (0.01–0.11)), thus supporting a pathogenic relevance of the identified splice-site variant. We used GeneMatcher16Sobreira N. Schiettecatte F. Valle D. Hamosh A. GeneMatcher: a matching tool for connecting investigators with an interest in the same gene.Hum. Mutat. 2015; 36: 928-930Crossref PubMed Scopus (821) Google Scholar to identify eight other unrelated individuals with NDDs and likely pathogenic variants in SCAF4 and two individuals with variants of unknown significance (Table 1, Table S1). Variants were identified through routine diagnostic testing or in a research setting approved by the ethical review boards of the respective institutions (Table S1). Informed consent was obtained from the parents or legal guardians of the affected individuals.Table 1Clinical DetailsIndividual1234567891011GenderfmmmfmmmfmmAge3y 3mo20mo16y18y13y6y 6mo6y 5mo11y4.5y4y10yVariant cDNAc.321+1G>Tc.453_456delTGAAc.1028delCc.1301C>Ac.1423C>Tc.1614+1G>Cc.1649dupTc.1812G>Ac. 1889G>Ac.3200_3201delAGc.783G>TVariant RNA/proteinr.spl?p.Asn151Lysfs∗8p.Pro343Hisfs∗3p.Ser434∗p.Arg475∗r.spl?p.Met550Ilefs∗4p.Trp604∗p.Trp630∗p.Glu1067Valfs∗3p.Leu261PheDe novoyesyesyesyesyesyesyesmaternalyesunknown (also in affected sister)yesHeight cm/SD101 / 0.6684.5 / −0.28162.6 / −1.6165.5 / −1.51157.5 / −0.41126.5 / 1114 / −0.22 (5y)136 / −1.69125 / −1.24 (8.5y)N/A142 / −0.25Weight kg/SD17.2 / 1.0412.5 / +0.1067.5 / 0.4056.9 / −1.2544.4 / −0.6124.9 / 026 / 1.56 (5y)38.2 / −0.1421 / 1.19N/A62.7 / 2.39OFC cm/SD49.2 / −0.3847.5 / −0.8N/A59 / 1.4251.8 / −1.952.8 / 0N/A55 / 0.5449.2 / −0.92N/A57.6 / 2.77DD/IDmild-moderate (SON-IQ 1x67, 1x50)mildmildseveremildmild (tIQ 91 WISC-V)language delaylearning diff.mild-moderate (DQ 56)moderateyesWalking14mo17mo14mo4–5y14mo12mo18mo14–15mo29moN/A15moFirst words12mo18mo2.5y5y14mo16mo3y2–3y3.7ynot yet3–4ySpeech3y 3mo: 30 wordsN/A3y: speech therapy10 words>2y: 2-word comb.delayN/A11y: 20 wordssimple sentences4y: no speechN/ARegressionstagnation with seiz.nonoN/Apossibly in speechnononoyes (10–12mo)N/ApossiblySeizures, onset age4x, 27–30mononointractable (myoclonic/tonic), 10momyoclonus, 12ynomyoclonic astatic epilepsy, 4yintractable, 18monoN/Ayes, 2yMRI anom.subcort., periventr. hypomyelinationN/DN/D (normal US)possible focal cortical dysplasia, volume lossnormalN/Dnonspecific FLAIR hyperintensities frontal subcortical area left > rightwhite matter changes, cerebellar atrophyPCH, HCCN/AHCCMuscular hypotoniayesyesnoyesnoN/RN/RhypertoniayesN/AyesBehavioral anom.autistic featuresN/Aautistic features in infancyautism, aggression, hyperactivityautismautism, hyperactivity, aggressionnoself-injurous, aggressionaggressionautism, hyperactivitydisruptive behavior, autismCardiac defectN/Dmurmur, US normalVSD, bicuspid aortic valve, hypoplastic aortic arch, dilated cardiomyopathyN/AVSD (self-resolved)NDN/RN/RVSD (self-resolved), PFON/AN/RRenal ano.N/DN/Dunilateral agenesismulticystic kidneysunilateral hydronephrosisnoN/RN/Rmulticystic kidneysN/AN/RUrogenital anom.nonocryptorchidismcryptorchidismnonoN/RN/Ringuinal herniaN/AN/RGI anomaliesnonoTE fistula, imperforate anuss/p pyloroplasty, nissen fundoplication, G-tubenonenoN/RN/RnoN/AN/RSkeletal anom.nonosacrum segmentation anom., brachydactylykyphosis, scoliosislordosis, hallux valgus, toe syndactyly II/IIIkyphosisN/Rbilateral ankle rotation, pectus excavatumantevertion of femurN/Ascoliosis, pronation of feetOthernonenonetethered cord, hypothyroidismsleep apnea, chronic lung disease, bronchopulmonary dysplasia, Sotos syndromepremature adrenarchenonenonedelayed teeth eruptionnonenonenonef, female; m, male; y, years; mo, months; SD, standard deviation; OFC, occipito-frontal head circumference; DD, developmental delay; ID, intellectual disability; N/A, not available or not applicable; N/D, not done; N/R, not reported; anom., anomaly; HCC, hypoplasia of corpus callosum; VSD, ventricle septum defect; PFO, persistent foramen ovale; PCH, pontocerebellar hypoplasia; TE, tracheesophageal; US, ultrasound. Open table in a new tab f, female; m, male; y, years; mo, months; SD, standard deviation; OFC, occipito-frontal head circumference; DD, developmental delay; ID, intellectual disability; N/A, not available or not applicable; N/D, not done; N/R, not reported; anom., anomaly; HCC, hypoplasia of corpus callosum; VSD, ventricle septum defect; PFO, persistent foramen ovale; PCH, pontocerebellar hypoplasia; TE, tracheesophageal; US, ultrasound. The nine variants that were considered likely pathogenic included two splice-site and seven truncating variants (Figures 1A and 1B). None of them were present in gnomAD.15Karczewski K.J. Francioli L.C. Tiao G. Cummings B.B. Alföldi J. Wang Q. Collins R.L. Laricchia K.M. Ganna A. Birnbaum D.P. et al.Variation across 141,456 human exomes and genomes reveals the spectrum of loss-of-function intolerance across human protein-coding genes.bioRxiv. 2019; https://doi.org/10.1101/531210Crossref Scopus (0) Google Scholar Eight occurred de novo, and one nonsense variant c.1812G>A (p.Trp604∗) was inherited from a healthy mother. gnomAD15Karczewski K.J. Francioli L.C. Tiao G. Cummings B.B. Alföldi J. Wang Q. Collins R.L. Laricchia K.M. Ganna A. Birnbaum D.P. et al.Variation across 141,456 human exomes and genomes reveals the spectrum of loss-of-function intolerance across human protein-coding genes.bioRxiv. 2019; https://doi.org/10.1101/531210Crossref Scopus (0) Google Scholar contains 14 presumably truncating variants in SCAF4, of which 10 are located in the last exon and probably are benign due to escaping nonsense-mediated mRNA decay. In contrast, the two splice-site and seven truncating variants in our cohort are predicted to affect the N-terminal two thirds of the protein, and therefore likely trigger nonsense-mediated mRNA-decay or produce a severely truncated protein. Nonsense-mediated mRNA decay was indicated by reverse transcription PCR (RT-PCR) on cDNA/RNA from a PaxGene (PreAnalytiX, BD and QIAGEN) blood sample of I4 with the c.1301C>A (p.Ser434∗) variant (Figure S1A). In contrast, the mutant c.1889G>A (p.Trp630∗) allele in I9 was still equally visible. However, as RNA sequencing (see below) indicated reduced SCAF4 expression also in this individual (Figure S1B, Table S2), a later decay process might occur. Furthermore, we confirmed aberrant splicing for the splice-site variant in I1 by showing several aberrant transcripts with complete or partial loss of exons 3, 4, or 5 (Figures S2A and S2B). RNA sequencing in I1 additionally revealed another isoform with intron 4 retained (Figures S2C and S2D). For all truncating variants in the N-terminal part of SCAF4, a general loss-of-function mechanism or haploinsufficiency is therefore likely. The Decipher database17Firth H.V. Richards S.M. Bevan A.P. Clayton S. Corpas M. Rajan D. Van Vooren S. Moreau Y. Pettett R.M. Carter N.P. DECIPHER: Database of Chromosomal Imbalance and Phenotype in Humans Using Ensembl Resources.Am. J. Hum. Genet. 2009; 84: 524-533Abstract Full Text Full Text PDF PubMed Scopus (1167) Google Scholar,18Deciphering Developmental Disorders StudyPrevalence and architecture of de novo mutations in developmental disorders.Nature. 2017; 542: 433-438Crossref PubMed Scopus (765) Google Scholar contains several deletions including SCAF4, but all encompass a large number of additional genes, thereby preventing specific deductions regarding SCAF4 haploinsufficiency in these individuals. Additionally, we identified two variants of unknown significance. The frameshifting variant c.3200_3201del (p.Glu1067Valfs∗3) segregated in two siblings with neurodevelopmental phenotypes. Parents were not available for testing. However, it is located in the C-terminal part of the gene/protein, where most of the truncating variants in gnomAD reside. The de novo missense variant c.783G>T (p.Leu261Phe) was identified in an individual with a consistent NDD phenotype. Though SCAF4 is not particularly intolerant toward missense variants (gnomAD constraint scores: z = 1.94, o/e = 0.79 (0.73–0.85)) and though this variant is not located in any of the known functional domains, there is some evidence pointing to a possible pathogenic relevance. This variant is not present in gnomAD, affects a highly conserved amino acid (Figure S3A), is predicted to be deleterious by Mutation Taster,19Schwarz J.M. Rödelsperger C. Schuelke M. Seelow D. MutationTaster evaluates disease-causing potential of sequence alterations.Nat. Methods. 2010; 7: 575-576Crossref PubMed Scopus (2123) Google Scholar PP2,20Adzhubei I. Jordan D.M. Sunyaev S.R. Predicting functional effect of human missense mutations using PolyPhen-2.Curr. Protocols Human Genet. 2013; Chapter 7 (Unit7.20-Unit27.20)PubMed Google Scholar SIFT,21Sim N.-L. Kumar P. Hu J. Henikoff S. Schneider G. Ng P.C. SIFT web server: predicting effects of amino acid substitutions on proteins.Nucleic Acids Res. 2012; 40: W452-7Crossref PubMed Scopus (1345) Google Scholar and M-CAP22Jagadeesh K.A. Wenger A.M. Berger M.J. Guturu H. Stenson P.D. Cooper D.N. Bernstein J.A. Bejerano G. M-CAP eliminates a majority of variants of uncertain significance in clinical exomes at high sensitivity.Nat. Genet. 2016; 48: 1581-1586Crossref PubMed Scopus (446) Google Scholar (Figure S3B), and resulted in reduced SCAF4 expression in RNA sequencing in I11, while mutant and wild-type alleles were equally visible in RT-PCR (Figure S1C). The clinical features of the affected individuals are summarized in Table 1. For phenotypic delineation, we excluded I10 and I11 due to their unclear variant status and I4 who additionally has Sotos syndrome (SOTOS1 [MIM: 117550]) due to a pathogenic variant in NSD1 (MIM: 606681), confounding the clinical picture. The remaining eight individuals all had developmental delay and intellectual disability, mostly in the mild range. Speech was more severely affected than motor development. While age of walking was within a normal range of 12–18 months in seven individuals, only two individuals had normal speech development (first words before 15 months). Speech was severely delayed (first words after 2 years) in four individuals. Developmental stagnation co-occurring with seizures was reported in one and possible developmental regression in two individuals. Behavioral anomalies were reported in five individuals (63%) and included autistic features, hyperactivity, and aggressive behavior. Seizures occurred in four individuals (50%) and included myoclonic seizures in two and intractable seizures in one. Brain MRIs were performed in five individuals showing nonspecific white matter anomalies in three of them. I9 was diagnosed with pontocerebellar hypoplasia and a thin corpus callosum. Variable other features included renal (38%), cardiac (38%), or skeletal (63%) anomalies. I3 presented with multiple malformations and anomalies. Minor but rather unspecific facial dysmorphism were noted in most of the individuals. Shared facial features in two individuals (I1, I2) were epicanthus, a flat nasal bridge, a bulbous nasal tip, and a deep philtrum (Figure 1C). Taken together, likely gene-disruptive variants in SCAF4 are associated with a variable neurodevelopmental phenotype with predominantly mild developmental delay and intellectual disability and frequently with seizures and behavioral anomalies. As increasingly observed for other genes associated with mild NDDs, putatively pathogenic, autosomal-dominant variants in SCAF4 may be inherited from a mildly affected or presumably healthy parent, as observed for I8. SCAF4 interacts with RNAPII. Interestingly, also variants in POLR2A, encoding the largest subunit of RNAPII, have been identified to cause a neurodevelopmental disorder, possibly due to a dominant-negative effect on RNA transcription.5Haijes H.A. Koster M.J.E. Rehmann H. Li D. Hakonarson H. Cappuccio G. Hancarova M. Lehalle D. Reardon W. Schaefer G.B. et al.De Novo Heterozygous POLR2A Variants Cause a Neurodevelopmental Syndrome with Profound Infantile-Onset Hypotonia.Am. J. Hum. Genet. 2019; 105: 283-301Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar While variable developmental delay, behavioral abnormalities, and white matter anomalies in brain imaging overlap between individuals with either SCAF4 or POLR2A variants, the POLR2A-associated phenotype appears to be more severe with profound muscular hypotonia as a prominent feature.5Haijes H.A. Koster M.J.E. Rehmann H. Li D. Hakonarson H. Cappuccio G. Hancarova M. Lehalle D. Reardon W. Schaefer G.B. et al.De Novo Heterozygous POLR2A Variants Cause a Neurodevelopmental Syndrome with Profound Infantile-Onset Hypotonia.Am. J. Hum. Genet. 2019; 105: 283-301Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar In the context of its interaction with RNAPII, an important role for SCAF4 in mRNA polyA recognition and mRNA termination was characterized recently.14Gregersen L.H. Mitter R. Ugalde A.P. Nojima T. Proudfoot N.J. Agami R. Stewart A. Svejstrup J.Q. SCAF4 and SCAF8, mRNA Anti-Terminator Proteins.Cell. 2019; 177: 1797-1813.e18Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar CRISPR-Cas9-mediated knockout of SCAF4 in HEK293 cells resulted in increased transcriptional read-through or in the usage of alternative last exons. This, in turn, was visible as an increase in the number of genes with an elongated 3′ sequence over genes with a truncated one.14Gregersen L.H. Mitter R. Ugalde A.P. Nojima T. Proudfoot N.J. Agami R. Stewart A. Svejstrup J.Q. SCAF4 and SCAF8, mRNA Anti-Terminator Proteins.Cell. 2019; 177: 1797-1813.e18Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar To investigate whether SCAF4 defects also result in incorrect mRNA transcription termination and mRNA processing in cells from affected individuals, we performed paired-end RNA sequencing on RNA from peripheral blood, collected and extracted with the PaxGene system (PreAnalytiX, BD and QIAGEN), of four individuals with SCAF4 variants (I1, I4, I9, I11) and eight healthy control subjects. In the affected individuals, we then determined deregulated genes, performed principal component analysis (PCA) using DESeq2 package v.1.24.0,23Love M.I. Huber W. Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2.Genome Biol. 2014; 15: 550Crossref PubMed Scopus (32370) Google Scholar and determined differentially used isoforms using salmon v.1.0.24Martin M. Cutadapt removes adapter sequences from high-throughput sequencing reads.EMBnetjournal. 2011; 17: 10-12Crossref Google Scholar,25Patro R. Duggal G. Love M.I. Irizarry R.A. Kingsford C. Salmon provides fast and bias-aware quantification of transcript expression.Nat. Methods. 2017; 14: 417-419Crossref PubMed Scopus (3607) Google Scholar More details are available in Supplemental Material and Methods. Of note, this approach detects only known and annotated isoforms and indicates shifts in their respective usage. We found that a very large number of genes (n = 9,038) were differentially expressed between the groups of four affected individuals and the eight control subjects, with an adjusted p value < 0.01 (FDR corrected). Of those, 4,328 were upregulated and 4,710 were downregulated (Table S2). Affected individuals and control subjects readily clustered within their group (Figures 2A and 2B ), including I11 with the missense variant, thus pointing to a similar loss-of-function mechanism as for the truncating variants and supporting its pathogenic relevance. However, additional data as well as direct comparison to expression profiles of individuals with other NDDs would be required to draw any further conclusions on the possible pathogenicity of this or other missense variants in SCAF4. Enrichment of Gene Ontology (GO) terms as well as Reactome Pathways26Fabregat A. Sidiropoulos K. Viteri G. Forner O. Marin-Garcia P. Arnau V. D’Eustachio P. Stein L. Hermjakob H. Reactome pathway analysis: a high-performance in-memory approach.BMC Bioinformatics. 2017; 18 (142–142)Crossref PubMed Scopus (322) Google Scholar among down- and upregulated genes were analyzed using the PANTHER v.14.027Mi H. Muruganujan A. Ebert D. Huang X. Thomas P.D. PANTHER version 14: more genomes, a new PANTHER GO-slim and improvements in enrichment analysis tools.Nucleic Acids Res. 2019; 47: D419-D426Crossref PubMed Scopus (1427) Google Scholar enrichment tool from Gene Ontology28Ashburner M. Ball C.A. Blake J.A. Botstein D. Butler H. Cherry J.M. Davis A.P. Dolinski K. Dwight S.S. Eppig J.T. et al.The Gene Ontology ConsortiumGene ontology: tool for the unification of biology.Nat. Genet. 2000; 25: 25-29Crossref PubMed Scopus (27012) Google Scholar,29The Gene Ontology ConsortiumExpansion of the Gene Ontology knowledgebase and resources.Nucleic Acids Res. 2017; 45: D331-D338Crossref PubMed Scopus (1229) Google Scholar with the following settings: test type: “Fisher’s Exact;” correction: “Bonferroni correction for multiple testing.” A list of all expressed genes within control subjects and the affected individuals with a base mean > 2 was used as background. In this analysis, we found that downregulated genes were enriched for GO terms that included regulation of gene expression and regulation of nucleobase-containing compound metabolic processes (Figure 2C). Furthermore, they were enriched for Reactome pathways that included translation, gene expression, and RNA Polymerase II Transcription (Table S2). Upregulated genes were enriched for GO terms like ribosome biogenesis and organonitrogen compound biosynthetic processes (Figure 2D), as well as for Reactome pathways like translation, metabolism of RNA, and rRNA processing (Table S2). In addition to broad transcriptional deregulation, we also detected differential splicing of 2,942 genes (adjusted p value < 0.05) between affected individuals and control subjects, supporting the role of SCAF4 in mRNA processing. We selected two alternatively spliced genes and confirmed a shift in the usage of alternative transcripts using RT-PCR with primers flanking differentially spliced regions (Figure S4). The most prominent splicing effect observed was the increased fraction of short transcripts in more than 70% of all events (2,095 out of 2,942 total) (Figure 3A). This was mainly due to usage of alternative down-stream transcription start sites (TSSs) (70%) and/or alternative up-stream transcription termination sites (TTSs) (37%) and/or exon loss (75%) (Figure 3B). Interestingly, for most of the transcripts, truncation was caused by a combination of two or more of these alterations with all three modifications occurring simultaneously in 17.8% of all altered and in 25.1% of shortened transcripts. As an example, the isoform switch of BTLA (MIM: 607925) is shown. Here, two shorter isoforms due to usage of downstream TSS, upstream TTS, and exon loss have an increased expression level (Figure 3C). Similarly to the observations from differentially expressed genes, differentially spliced genes were enriched for GO terms like nucleic acid metabolic processes and gene expression as well as for Reactome pathways like chromatin modifying enzymes, chromatin expression, and gene expression (transcription) (Figure 3D, Table S2). We compared the differentially spliced genes in individuals with variants in SCAF4 with the 565 differentially spliced genes after SCAF4 knockout in HEK293 cells14Gregersen L.H. Mitter R. Ugalde A.P. Nojima T. Proudfoot N.J. Agami R. Stewart A. Svejstrup J.Q. SCAF4 and SCAF8, mRNA Anti-Terminator Proteins.Cell. 2019; 177: 1797-1813.e18Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar and found an overlap of 25.7% (145 out of 565). A total of 66 (45.5%) of these genes used an alternative last exon (ALE). As various cell types usually have unique transcriptome profiles, usage of HEK293 versus human blood cells might contribute to discrepancies between differentially spliced genes. Also, complete SCAF4 knockout in HEK293 cells versus a heterozygous variant in affected individuals could play a role, since the remaining allele might still cover some of the SCAF4 function. Nevertheless, the overlap between differe" @default.
• W3045621188 created "2020-08-03" @default.
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• W3045621188 date "2020-09-01" @default.
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• W3045621188 title "Variants in SCAF4 Cause a Neurodevelopmental Disorder and Are Associated with Impaired mRNA Processing" @default.
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• W3045621188 doi "https://doi.org/10.1016/j.ajhg.2020.06.019" @default.
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