SRSF1 haploinsufficiency is responsible for a syndromic developmental disorder associated with intellectual disability

Abstract

SRSF1 (also known as ASF/SF2) is a non-small nuclear ribonucleoprotein (non-snRNP) that belongs to the arginine/serine (R/S) domain family. It recognizes and binds to mRNA, regulating both constitutive and alternative splicing. The complete loss of this proto-oncogene in mice is embryonically lethal. Through international data sharing, we identified 17 individuals (10 females and 7 males) with a neurodevelopmental disorder (NDD) with heterozygous germline SRSF1 variants, mostly de novo, including three frameshift variants, three nonsense variants, seven missense variants, and two microdeletions within region 17q22 encompassing SRSF1. Only in one family, the de novo origin could not be established. All individuals featured a recurrent phenotype including developmental delay and intellectual disability (DD/ID), hypotonia, neurobehavioral problems, with variable skeletal (66.7%) and cardiac (46%) anomalies. To investigate the functional consequences of SRSF1 variants, we performed in silico structural modeling, developed an in vivo splicing assay in Drosophila, and carried out episignature analysis in blood-derived DNA from affected individuals. We found that all loss-of-function and 5 out of 7 missense variants were pathogenic, leading to a loss of SRSF1 splicing activity in Drosophila, correlating with a detectable and specific DNA methylation episignature. In addition, our orthogonal in silico, in vivo, and epigenetics analyses enabled the separation of clearly pathogenic missense variants from those with uncertain significance. Overall, these results indicated that haploinsufficiency of SRSF1 is responsible for a syndromic NDD with ID due to a partial loss of SRSF1-mediated splicing activity.

Keywords: Drosophila; SRSF1; epigenetic signature; haploinsufficiency; neurodevelopmental disorder; splicing.

Figure 1

Clinical variants in SRSF1 cause syndromic developmental disorder associated with intellectual disability (A) Gene, transcript, and protein structure of SRSF1. Clinical variants were shown on the protein level. Evolutionary conservation of the RRM domains is shown with bold amino acids showing evolutionarily conserved residues. Missense variants are indicated by green arrows, nonsense variants are indicated in red, and frameshift variants in orange. (B) Pedigrees of the 16 families reported in this cohort. (C) Photographs of individuals with SRSF1 variants. Nonspecific facial features were observed in the individuals. Individuals 4 and 15 were referred for marfanoid features: they presented dolichostenomelia, arachnodactyly, and pectus deformity.

Figure 2

Bio-informatic pathogenicity predictions and structural modeling of missense variants (A) Pathogenicity prediction scores for the seven SRSF1 missense variants generated by eight meta-prediction tools (BayesDel with AF, BayesDel without AF, MetaLR, MetaRNN, MetaSVM, REVEL, CADD, Eigen) as implemented in the VarSome human genomic search engine. Colored bars on the right represent the number of meta-tools supporting pathogenic (red), uncertain (brown), and benign (green) predictions. (B) Structural prediction of SRSF1 using AlphaFold and PyMol. (C) Left: missense variants superimposed on the structural prediction. Right: Surface rendering of the SRSF1 protein structure. Arrows indicate p.Asp44Asn and p.His183Arg, two residues closer to the protein surface. (D) Structural prediction of the seven missense variants. The underlined variants, p.Asp44Asn and p.His183Arg, are located at the protein surface. The other variants are more oriented toward the internal structure of the protein. Carbons are represented in yellow, nitrogens in blue, and oxygens in red for the wild-type amino acids; carbons are represented in purple in the modelized alterations. Asterisks indicate potential steric clashes in the mutated structures.

Figure 3

Eye and neuronal splicing read-outs of clinical variants (A) Representative eye picture of flies expressing luciferase, SF2, SRSF1, and SRSF1 clinical variants in the fly eye under the control of the GMR-GAL4 enhancer. (B) The irregularity score or regularity index of flies expressing luciferase (negative control), SRSF1 (positive control), a SRSF1 splicing-deficient protein (F56D/F58D/K138A) (negative control), and SRSF1 clinical variants. n > 10, ^∗p < 0.01, data are represented as mean ± SEM. p.Asp44Asn and p.His183Arg display a lower IREG score similar to the SRSF1-overexpressing flies. (C) Pigmentation score measuring the depigmentation in the different controls and the clinical variants. n > 10, ^∗p < 0.01, data are represented as mean ± SEM. (D) Offspring frequencies were measured in flies expressing luciferase, SRSF1, a splicing-deficient SRSF1 protein, and the clinical variants pan-neuronally. n > 10, ^∗p < 0.01.

Figure 4

Episignature assessment of SRSF1 VUSs p.Asp44Asn and p.His183Arg (A) Heatmap indicates that the two VUS samples (orange) are clustering with controls (blue) and away from the SRSF1 samples with confirmed pathogenic variants (individuals I1–I6, I11, I14, and I15 used for episignature discovery) (red). Each row represents one of the 107 probes selected as the episignature, and each column represents an individual with either an SRSF1 variant (red or orange) or a control (blue). (B) Multidimensional scaling plot (MDS) also shows clustering of the SRSF1 VUS samples with controls. (C) Support vector machine classifier model (SVM) shows that the VUSs have a probability score (methylation variant pathogenicity score, MVP) of close to 0 compared with the SRSF1 samples carrying confirmed pathogenic variants with MVP scores of close to 1. The model is trained using the 107 selected SRSF1 episignature probes and 75% of controls and other neurodevelopmental disorder samples on EpiSign (blue circles). The 25% remaining are used as testing samples (gray circles). (D) Circos plot representing the differentially methylated probes (DMPs) shared between each pair of cohorts. The thickness of the connecting lines indicates the number of probes shared between the paired cohorts. SRSF1 cohort is indicated by the green arrow. (E) Tree-and-leaf visualization of Euclidean clustering of the SRSF1 cohort alongside the 56 other EpiSign disorders using the top n DMPs for each cohort, where n = 500 or the max number of DMPs available if <500. Cohort samples are aggregated using the median value of each probe within a group. Each leaf (node) represents a cohort, with node sizes illustrating relative scales of the number of selected DMPs for the corresponding cohort, and node colors indicative of the global mean methylation difference where blue is more hypomethylated and red hypermethylated. The SRSF1 cohort with confirmed pathogenic variants is highlighted in green. ADCADN, cerebellar ataxia deafness and narcolepsy syndrome; AUTS18, susceptibility to autism 18; BEFAHRS, Beck-Fahrner syndrome; BFLS, Borjeson-Forssman-Lehmann syndrome; BISS, blepharophimosis intellectual disability SMARCA2 syndrome; CdLS, Cornelia de Lange syndrome; CHARGE, CHARGE syndrome; Chr16p11.2del, chromosome 16p11.2 deletion syndrome; CSS, Coffin-Siris syndrome; CSS4, Coffin-Siris syndrome 4; CSS9, Coffin-Siris syndrome 9; Down, Down syndrome; Dup7, 7q11.23 duplication syndrome; DYT28, dystonia 28; EEOC, epileptic encephalopathy-childhood onset; FLHS, Floating-Harbor syndrome; GTPTS, genitopatellar syndrome; HMA, Hunter McAlpine craniosynostosis syndrome; HVDAS, Helsmoortel-van der Aa syndrome; ICF, immunodeficiency-centromeric instability-facial anomalies syndrome; IDDSELD, intellectual developmental disorder with seizures and language delay; Kabuki, Kabuki syndrome; KDVS, Koolen-De Vries syndrome; Kleefstra, Kleefstra syndrome; LLS, Luscan-Lumish syndrome; MKHK, Menke-Hennekam syndrome; MLASA2, myopathy lactic acidosis and sideroblastic anemia 2; MRD23, intellectual developmental disorder 23; MRD51, intellectual developmental disorder 51; MRX93, intellectual developmental disorder X-linked 93; MRX97, intellectual developmental disorder X-linked 97; MRXSA, intellectual developmental disorder X-linked syndromic Armfield type; MRXSCH, intellectual developmental disorder X-linked syndromic Christianson type; MRXSCJ, intellectual developmental disorder X-linked syndromic Claes-Jensen type; MRXSN, intellectual developmental disorder X-linked syndromic Nascimento type; MRXSSR, intellectual developmental disorder X-linked syndromic Snyder-Robinson type; PHMDS, Phelan-McDermid syndrome; PRC2, PRC2 complex (Weaver and Cohen-Gibson) syndrome; RENS1, Renpenning syndrome; RMNS, Rahman syndrome; RSTS, Rubinstein-Taybi syndrome; SBBYSS, Ohdo syndrome; Sotos, Sotos syndrome; TBRS, Tatton-Brown-Rahman syndrome; WDSTS, Wiedemann-Steiner syndrome; WHS, Wolf-Hirschhorn syndrome; Williams, Williams syndrome.