Familial STAG2 germline mutation defines a new human cohesinopathy

Abstract

We characterize a novel human cohesinopathy originated from a familial germline mutation of the gene encoding the cohesin subunit STAG2, which we propose to call STAG2-related X-linked Intellectual Deficiency. Five individuals carry a STAG2 p.Ser327Asn (c.980 G > A) variant that perfectly cosegregates with a phenotype of syndromic mental retardation in a characteristic X-linked recessive pattern. Although patient-derived cells did not show overt sister-chromatid cohesion defects, they exhibited altered cell cycle profiles and gene expression patterns that were consistent with cohesin deficiency. The protein level of STAG2 in patient cells was normal. Interestingly, STAG2 S327 is located at a conserved site crucial for binding to SCC1 and cohesin regulators. When expressed in human cells, the STAG2 p.Ser327Asn mutant is defective in binding to SCC1 and other cohesin subunits and regulators. Thus, decreased amount of intact cohesin likely underlies the phenotypes of STAG2-SXLID. Intriguingly, recombinant STAG2 p.Ser327Asn binds normally to SCC1, WAPL, and SGO1 in vitro, suggesting the existence of unknown in vivo mechanisms that regulate the interaction between STAG2 and SCC1.

Fig. 1

Pedigree. a Pedigree shows typical X-linked recessive inheritance of the c.980 G > A (p.Ser327Asn) in the STAG2 gene (NM_001042749.1) on the X chromosome. The variant was studied in the proband (arrow) and in male and female relatives by allele-specific PCR and confirmed in the proband and all affected male relatives by Sanger sequencing. b For Sanger sequencing we used the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems^®) and the Applied Biosystems (ABI) 3130 Genetic Analyzer. Sequencing data were analyzed using the software Sequencher version 4.1.4 (Gene Code Corporation)

Fig. 2

Sister chromatid cohesion assay and immunoreaction experiments. a p.Ser327Asn STAG2 does not show effect on the sister chromatid phenotype. Open (gray) and closed (black) sister chromatids are shown for the patient and four controls with WT STAG2 (C1-4). A total of 100 metaphases were analyzed for each individual. The amount of closed sister chromatids are indicated at the bottom of each individual column. Open and closed sister chromatid phenotypes in metaphase cells from the patient are demonstrated. b Immunofluorescence of patient fibroblasts. Cultured fibroblasts from the proband were incubated overnight with anti-STAG2 (J-12) at 4 °C followed by Alexa 488-conjugated second antibody (green) and nuclear counterstain with DAPI (blue). The nuclear localization of STAG2 was confirmed in the double label immunofluorescence merged picture. Similar results were observed in three independent experiments. c Western blot of STAG2 from patient fibroblasts. Protein extracts from patient and control individuals were subjected to immunoblotting using the anti-STAG2 (J-12). After visualization of STAG2, the blot membrane was stripped and re-probed with alpha-tubulin antibody, which was used as a quantitative control. Similar results were observed between the patient and the two controls in three independent experiments

Fig. 3

Cell cycle profiles of STAG2 WT and Ser327Asn fibroblasts. a Two independent lines of WT fibroblasts (WT-1 and WT-2) and STAG2 Ser327Asn fibroblasts were cultured and stained with propidium iodide and anti-MPM2 antibody. Cells were harvested and analyzed by flow cytometry. Representative graphs are shown, with the cell populations with 2N and 4N DNA contents indicated. b Quantification of the fibroblasts in G1, S, and G2/M in a (n = 2 independent experiments)

Fig. 4

Enrichment analyses of Gene Ontology terms in cultured STAG2 Ser327Asn fibroblast. Bar plots represent the statistical significance of the enrichment (−log₁₀(p-value)) in STAG2 Ser327Asn fibroblasts. Expression of genes in cell division, mitosis, DNA replication, and DNA damage repair pathways, shown in blue bars, was upregulated in the STAG2 Ser327Asn fibroblasts

Fig. 5

STAG2 residues predicted to be putative interacting sites for protein–protein interfaces based on consensus classifier. a Residue colors based on Spider score from white to blue, with residues closer to blue with the greater the likelihood to be involved in mediating a protein–protein interaction. SCC1 is shown as cartoon in red. b Non-covalent interactions made by WT residue. Ser327 forms a hydrogen bond with Asn325 that is substituted to a donor-pi interaction with Tyr328 in the mutant model. SCC1 is shown as cartoon in blue. The mutant residue is shown with dark gray carbons

Fig. 6

STAG2 p.Ser327Asn retains binding to WAPL and SGO1 in vitro. a Cartoon diagram of the crystal structure of human STAG2–SCC1, with Ser327 and neighboring residues shown in sticks. b In vitro binding of STAG2–SCC1 to GST-WAPL or GST-Sgo1. GST was used as the negative control. Top and bottom panels show the autoradiograph and Coomassie staining of the binding reactions, respectively. c Quantification of the relative WAPL-binding and SGO1-binding activities of STAG2–SCC1 WT, p.Ser327Asn (Ser327Asn), and Lys330Glu (K330E) (normalized to WT) in b. Mean ± SD, n = 3 independent experiments. For WAPL binding, WT vs. S327N, p = 0.6271, not significant; WT vs. K330E, p < 0.0001. For SGO1 binding, WT vs. S327N, p = 0.0002, p < 0.05; WT vs. K330E, p = 0.0007, p < 0.05. All p-values were based on t-test. d ITC graphs of the binding between pSGO1 and SA2–SCC1 complexes (WT and p.Ser327Asn), with the dissociation constants (K _d) indicated

Fig. 7

STAG2 p.Ser327Asn (S327N) is defective in binding to cohesin and regulators in human cells. a Anti-Myc immunoprecipitates from HeLa cells transfected with the indicated plasmids were blotted with the indicated antibodies. b Quantification of the relative SCC1 intensities in blots in a. Mean ± SD, n = 3 independent experiments. WT vs. S327N, p = 0.0004, p < 0.05; WT vs. D793K, p < 0.0001 (t-test). c In vitro binding between ³⁵S-SCC1 and GST-STAG2 proteins. GST was used as the negative control. Top and bottom panels show the autoradiograph and Coomassie staining of the binding reaction, respectively