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. 2014 Sep;124(9):4028-38.
doi: 10.1172/JCI73264. Epub 2014 Aug 8.

Hypomorphism in human NSMCE2 linked to primordial dwarfism and insulin resistance

Hypomorphism in human NSMCE2 linked to primordial dwarfism and insulin resistance

Felicity Payne et al. J Clin Invest. 2014 Sep.

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.

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Figures

Figure 5
Figure 5. Modeling of NSMCE2/MMS21 deficiency in zebrafish.
Injection of splice-site MOs targeted to Nsmce2 markedly reduce body size compared with that of control-injected or uninjected WT embryos. (A) Representative images of some arbitrarily defined length categories: normal, –1 to 1 SD; short, –1 to –2 SD; dwarf, –2 to –3 SD; severe, < –3 SD. (B) Quantification of length of MO-injected zebrafish (uninjected, n = 18; control, n = 28; MO, n = 37). (CE) Coinjection of WT human NSMCE2/MMS21 mRNA (WT rescue) with nsmce2-targeting MOs attenuated the dwarf phenotype observed in the nsmce2 morphant alone (morph). In contrast, coinjection of either (C) human NSMCE2/MMS21 S116Lfs*18, (D) human NSMCE2/MMS21 A234Efs*4, or (E) SUMO LD human NSMCE2/MMS21 mRNAs together with nsmce2-targeting MOs did not affect the severity of the dwarf phenotype. Uninj, uninjected; con, control. Graphs show SD scores relative to those of uninjected WT zebrafish embryos from the same mating. n = 12–15 for each condition. P values were calculated by ANOVA with post-hoc Tukey test; **P < 0.01, ***P < 0.001.
Figure 4
Figure 4. Increased micronucleus formation, impaired S phase progression, and increased SCE in LCLs with NSMCE2/MMS21 mutations.
(A) Binucleates in LCLs from WT, P1, and from a Bloom syndrome patient, either untreated (Unt) or following repeated exposure to HU (50 μM/d for 4 days). (B) HU-induced MN formation in Cyt-B–induced binucleate LCLs. (C) BrdU flow cytometry profiles from WT and P1 LCLs either untreated or following treatment with low-dose HU (250 μM). S phase content (boxed area) following a BrdU pulse 30 minutes prior to each time point is shown. (D) Normalized total S phase content compared with that of untreated cells at each time point. Means ± SD are shown (n = 3). (E) BLM foci formation in P1 LCLs relative to WT following treatment with HU (1 mM for 16 hours) or IR (10 Gy for 16 hours). (F) BLM foci formation in primary dermal fibroblasts from P1 compared with WT following HU exposure (1 mM for 24 hours). (G) UV radiation–induced SCEs in WT and P1 LCLs compared with untreated cells. These effects, although significant, were not as profound as those observed in BLM patient LCLs, which exhibited 23 ± 6 SCEs/metaphase in untreated cells and 38 ± 4 SCEs/metaphase following UV irradiation. *P < 0.05 (unpaired t test).
Figure 3
Figure 3. HU-induced nuclear abnormalities in primary fibroblast cells are rescued by expression of WT but not SUMO LD NSMCE2/MMS21.
Typical micrographs of binucleated dermal fibroblasts showing (A and B) MN or (C) NPB. Scale bars: 5 μm. (D) Increased frequency of MN or (E) NPB in Cyt-B–treated dermal fibroblasts from P1 relative to age-matched healthy controls (WT) following a 4-hour block in 1 mM HU. (F) DOX-induced expression of WT or SUMO LD NSMCE2/MMS21 in P1 or control dermal fibroblasts (WT fibroblasts, 2 samples shown) analyzed by immunoblotting with antibodies specific to human NSMCE2/MMS21 (WB: α-NSMCE2). Asterisk indicates a crossreactive band that serves as an internal loading control. (G and H) DOX-induced ectopic expression of WT NSMCE2/MMS21 reduces frequencies of MN (G) and NPB (H) to levels seen in control cells. In contrast, DOX-induced ectopic expression of NSMCE2/MMS21 (LD) significantly increases levels of both MN (G) and NPB (H). Frequencies of MN per 100 or NPB per 1,000 binucleated cells are shown for each experimental condition, as indicated. All bar graphs represent mean ± SEM (n = 4). *P < 0.05; **P < 0.01; ***P < 0.001 (unpaired 1-tailed t test).
Figure 2
Figure 2. Expression and auto-SUMOylation of NSMCE2/MMS21 frameshift mutations.
(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).
Figure 1
Figure 1. A syndrome of primordial dwarfism and extreme insulin resistance associated with compound heterozygous NSMCE2/MMS21 frameshift mutations.
(AG) P1 at (A) 4 months, (B) 2 years, (C) 12 years, and (D) 24 years of age. (E) Profile at 12 years showing prominent midface and small lower jaw. (F) Severe axillary acanthosis nigricans with skin tags. (G) Severe acanthosis nigricans on the lateral neck. (H) Pedigree diagram for P1 with adult heights, where available, shown in meters. NSMCE2 genotypes are shown as WT, 116fs (p.Ser116Leufs*18), and 234fs (p.Ala234Glufs*4). (IK) P2 shown at 27 years old. (I) Whole-body appearance illustrating dwarfism and paucity of adipose tissue and muscle. (J) Profile showing prominent midface and small lower jaw. (K) Detail of arm at antecubital fossa showing severe acanthosis nigricans. (L) Pedigree diagram for P2. Written, informed consent was obtained from patients or their families for publication of these images.

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