Hypomorphism in human NSMCE2 linked to primordial dwarfism and insulin resistance
Felicity Payne, … , Mark O’Driscoll, Robert Semple
Felicity Payne, … , Mark O’Driscoll, Robert Semple
Published August 8, 2014
Citation Information: J Clin Invest. 2014;124(9):4028-4038. https://doi.org/10.1172/JCI73264.
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Research Article Endocrinology

Hypomorphism in human NSMCE2 linked to primordial dwarfism and insulin resistance

Abstract

Structural maintenance of chromosomes (SMC) complexes are essential for maintaining chromatin structure and regulating gene expression. Two the three known SMC complexes, cohesin and condensin, are important for sister chromatid cohesion and condensation, respectively; however, the function of the third complex, SMC5–6, which includes the E3 SUMO-ligase NSMCE2 (also widely known as MMS21) is less clear. Here, we characterized 2 patients with primordial dwarfism, extreme insulin resistance, and gonadal failure and identified compound heterozygous frameshift mutations in NSMCE2. Both mutations reduced NSMCE2 expression in patient cells. Primary cells from one patient showed increased micronucleus and nucleoplasmic bridge formation, delayed recovery of DNA synthesis, and reduced formation of foci containing Bloom syndrome helicase (BLM) after hydroxyurea-induced replication fork stalling. These nuclear abnormalities in patient dermal fibroblast were restored by expression of WT NSMCE2, but not a mutant form lacking SUMO-ligase activity. Furthermore, in zebrafish, knockdown of the NSMCE2 ortholog produced dwarfism, which was ameliorated by reexpression of WT, but not SUMO-ligase–deficient NSMCE. Collectively, these findings support a role for NSMCE2 in recovery from DNA damage and raise the possibility that loss of its function produces dwarfism through reduced tolerance of replicative stress.

Authors

Felicity Payne, Rita Colnaghi, Nuno Rocha, Asha Seth, Julie Harris, Gillian Carpenter, William E. Bottomley, Eleanor Wheeler, Stephen Wong, Vladimir Saudek, David Savage, Stephen O’Rahilly, Jean-Claude Carel, Inês Barroso, Mark O’Driscoll, Robert Semple

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Figure 2

Expression and auto-SUMOylation of NSMCE2/MMS21 frameshift mutations.

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Expression and auto-SUMOylation of NSMCE2/MMS21 frameshift mutations. (A...
(A) Schematic showing positions of patient mutations with respect to the SP-RING SUMO-ligase domain. (B) Schematic of NSMCE2/MMS21 showing disruption of α helix α8 (residues 223–240) by the p.Ala234Glufs*4 mutation. Alignment of human WT (Mms21_human) and p.Ala234Glufs*4 (A234E) MMS21/NSMCE2 based on the crystal structure of S. cerevisiae Mms21 (Mms21_sc; PDB entry: 3HTK; chain C). The 14 amino acids removed by the mutation are marked in boldface. Numbering and secondary structure of S. cerevisiae Mms21 are displayed. The position of the mutation in human MMS21/NSMCE2 is displayed below the sequences. (C) NSMCE2 expression in dermal fibroblasts from P1 assessed using immunoblotting of increasing amounts of whole-cell extract compared with WT fibroblasts. (D) Similar immunoblotting of whole-cell extract from P1 LCLs. (E) Auto-SUMOylation activity of Myc-tagged WT, SUMO LD, and naturally occurring mutant NSMCE2/MMS21 following coexpression with GFP-SUMO1 (upper panels). Auto-SUMOylation was detected following IP using anti-Myc and blotting (WB, Western blot) using anti-GFP antibodies. IgHC, immunoglobulin heavy chain. The lower panel shows relative amounts of immunoprecipitated Myc-NSMCE2. The p.A234Efs*4 mutation appears not to affect protein gel migration under these conditions, unlike p.S116Lfs*18 (Supplemental Figure 3). (F) Quantification of recovered Myc-NSMCE2-GFP-SUMO conjugates by densitometry. Data represent mean ± SD. (n = 3). *P ≤ 0.05 (unpaired, 2-tailed t test).