Destabilized SMC5/6 complex leads to chromosome breakage syndrome with severe lung disease
Saskia N. van der Crabben, … , Johanne M. Murray, Gijs van Haaften
Saskia N. van der Crabben, … , Johanne M. Murray, Gijs van Haaften
Published July 18, 2016
Citation Information: J Clin Invest. 2016;126(8):2881-2892. https://doi.org/10.1172/JCI82890.
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Concise Communication Genetics

Destabilized SMC5/6 complex leads to chromosome breakage syndrome with severe lung disease

Abstract

The structural maintenance of chromosomes (SMC) family of proteins supports mitotic proliferation, meiosis, and DNA repair to control genomic stability. Impairments in chromosome maintenance are linked to rare chromosome breakage disorders. Here, we have identified a chromosome breakage syndrome associated with severe lung disease in early childhood. Four children from two unrelated kindreds died of severe pulmonary disease during infancy following viral pneumonia with evidence of combined T and B cell immunodeficiency. Whole exome sequencing revealed biallelic missense mutations in the NSMCE3 (also known as NDNL2) gene, which encodes a subunit of the SMC5/6 complex that is essential for DNA damage response and chromosome segregation. The NSMCE3 mutations disrupted interactions within the SMC5/6 complex, leading to destabilization of the complex. Patient cells showed chromosome rearrangements, micronuclei, sensitivity to replication stress and DNA damage, and defective homologous recombination. This work associates missense mutations in NSMCE3 with an autosomal recessive chromosome breakage syndrome that leads to defective T and B cell function and acute respiratory distress syndrome in early childhood.

Authors

Saskia N. van der Crabben, Marije P. Hennus, Grant A. McGregor, Deborah I. Ritter, Sandesh C.S. Nagamani, Owen S. Wells, Magdalena Harakalova, Ivan K. Chinn, Aaron Alt, Lucie Vondrova, Ron Hochstenbach, Joris M. van Montfrans, Suzanne W. Terheggen-Lagro, Stef van Lieshout, Markus J. van Roosmalen, Ivo Renkens, Karen Duran, Isaac J. Nijman, Wigard P. Kloosterman, Eric Hennekam, Jordan S. Orange, Peter M. van Hasselt, David A. Wheeler, Jan J. Palecek, Alan R. Lehmann, Antony W. Oliver, Laurence H. Pearl, Sharon E. Plon, Johanne M. Murray, Gijs van Haaften

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Figure 2

NSMCE3 mutations destabilize the SMC5/6 complex.

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NSMCE3 mutations destabilize the SMC5/6 complex. (A) Schematic of the S...
(A) Schematic of the SMC5/6 complex. (B) Structure of NSMCE1/NSMCE3, highlighting the positions of the Leu264Phe (L264F) and Pro209Leu (P209L) mutants that destabilize the WH/B subdomain of NSMCE3. Loops missing from the deposited structure were modelled and are shown in gray (24). (C) Yeast 2-hybrid analysis showing interactions between NSMCE3 and NSMCE1 or NSMCE4, scored on His– plates containing 5 mM 3-amino-1,2,4-triazol (-LTH). WT denotes WT NSMCE3; L97A: NSMCE3 Leu97Ala, which abrogates NSMCE1 binding (25); F266A: NSMCE3 Phe266Ala, which reduces binding to NSMCE4 (21). L264F only interacts with NSMCE1; P209L has reduced interaction with NSMCE1 and no interaction with NSMCE4. Controls: c1, BD-NSMCE3 with empty AD vector; c2, empty BD vector with AD-NSMCE4 (top) or NSMCE1 (bottom). (D) NSMCE1 and His-tagged NSMCE3 (WT, P209L, and L264F were coexpressed in, and purified from, E. coli. Left: Coomassie-stained SDS-PAGE shows copurification of NSMCE1 and NSMCE3. Right: Immunodetection of His-tagged NSMCE3. P209L is unstable; L264F is more stable. (E) Size exclusion chromatography column. NSMCE1 and NSMCE3 co-elute, forming a stable complex. NSMCE1 and L264F co-elute, over a broader range of volume and non-uniform distribution, indicating either unfolding of L264F or interaction with the chromatography resin. (F) GFP-tagged NSMCE3 (WT or L264F) and Myc-tagged NSMCE1 were ectopically expressed in human U20S osteosarcoma cells; proteins were stable and full length in input samples. Proteins were immunopurified using anti-GFP beads. Purification of NSMCE1 was reduced when copurified with L264F, which indicated reduced affinity. (G) GFP-tagged NSMCE3 (WT or L264F) was ectopically expressed in U20S cells. Proteins were immunopurified using anti-GFP beads. Reduced copurification indicates a compromised integration of the mutated NSMCE3 into the SMC5/6 complex. (H) Western blot analysis of SMC5/6. NSMCE3 is undetectable in fibroblasts from individual B (GVH02) and individual D (FCP488) compared with primary fibroblast cell lines (48BR and GM05757). High exposure of anti-SMC5 blot shows reduction of SMC5. Levels of both SMC5 and SM6 were reduced in patient fibroblasts, indicating destabilization of the complex. Loading control: tubulin.