Mutation in NSUN2, which encodes an RNA methyltransferase, causes autosomal-recessive intellectual disability

Abstract

Causes of autosomal-recessive intellectual disability (ID) have, until very recently, been under researched because of the high degree of genetic heterogeneity. However, now that genome-wide approaches can be applied to single multiplex consanguineous families, the identification of genes harboring disease-causing mutations by autozygosity mapping is expanding rapidly. Here, we have mapped a disease locus in a consanguineous Pakistani family affected by ID and distal myopathy. We genotyped family members on genome-wide SNP microarrays and used the data to determine a single 2.5 Mb homozygosity-by-descent (HBD) locus in region 5p15.32-p15.31; we identified the missense change c.2035G>A (p.Gly679Arg) at a conserved residue within NSUN2. This gene encodes a methyltransferase that catalyzes formation of 5-methylcytosine at C34 of tRNA-leu(CAA) and plays a role in spindle assembly during mitosis as well as chromosome segregation. In mouse brains, we show that NSUN2 localizes to the nucleolus of Purkinje cells in the cerebellum. The effects of the mutation were confirmed by the transfection of wild-type and mutant constructs into cells and subsequent immunohistochemistry. We show that mutation to arginine at this residue causes NSUN2 to fail to localize within the nucleolus. The ID combined with a unique profile of comorbid features presented here makes this an important genetic discovery, and the involvement of NSUN2 highlights the role of RNA methyltransferase in human neurocognitive development.

Figure 1

Pedigree, Mapping, and Mutation Screening for Family MR14 (A) Pedigree of family MR14 from the Khairpur district. Filled circles indicate affected girls. (B) HomozygosityMapper analysis of microarray SNP data: Genome-wide significant regions of homozygosity-by-descent (HBD) are seen only on 5p and 14q. The 14q locus was excluded because one of the unaffected siblings was also homozygous at this locus, whereas the unaffected sibling was genotyped as heterozygous at the 5p locus. (C) Ideogrammatic representation of the critical autozygous or HBD locus in region 5p15.31, as determined from this study and in relation to the MRT5 locus identified by Najmabadi et al.⁵ (D) The c.2035G>A substitution encoding the p.Gly679Arg change in a heterozygous carrier and an affected homozygote. (E) ClustalW alignment of NSUN2 across multiple species showing conservation of the p.Gly679 residue in vertebrates and also in nonvertebrate animal species.

Figure 2

Cellular Localization of WT and Mutant NSUN2 in Human Breast Cancer Cell Line HCC1954 WT (A) and mutant (B) NSUN2 constructs in the vector pcDNA-Myc transfected into the human breast cancer cell line HCC1954. A. WT NSUN2 (A) but not mutant NSUN2 (B) protein colocalizes with nucleophosmin, a nucleolar marker. The scale bar represents 20 μm. (C) Costaining for endogenous NSUN2 confirms exclusion of mutant NSUN2 (Myc-labeled) from the nucleoli. Arrows indicate nucleoli (A–C). Costaining with DAPI shows nuclear localization. The scale bar represents 20 μm.

Figure 3

Cytoplasmic, Nuclear, and Nucleolar Localization of WT and Mutant NSUN2 in HCC1954 and HeLa Cells (A) Immunostaining shows nucleolar localization of WT NSUN2 in HCC1954 cells. Costaining with DAPI shows nuclear localization. (B and C) Cellular localization of NSUN2 carrying the 679^Arg variant (p.Gly679Arg) in the nucleoplasm (B) and cytoplasm (C). Costaining with DAPI shows nuclear localization. (D) Quantification of cellular localizations shown in (A–C). Error bars represent the standard deviation. (E) Nucleolar localization of GFP-tagged WT NSUN2 in HeLa cells. White arrow heads indicate nucleoli. (F) Nuclear and cytoplasmic localization in 679^Arg mutant NSUN2 (p.Gly679Arg). White arrow heads indicate nucleoli.

Figure 4

Localization of Endogenous WT Nsun2 in Mouse Cerebellum (A) Sections of mouse cerebellum labeled with an antibody against NSUN2. The following abbreviations are used: NF, nerve fibers; GCL, granule cell layer; and ML, molecular layer. (B) A higher magnification of an insert in (A) shows NSUN2 in Purkinje cells. (C) Sections of the cerebellum labeled with L7/Pcp-2, a marker for Purkinje cells. (D) Colocalization of NSUN2 with nucleophosmin (Npm1) in the nucleoli of Purkinje cells. Nuclei are counter stained with DAPI.