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. 2012 Jun;49(6):380-5.
doi: 10.1136/jmedgenet-2011-100686. Epub 2012 May 10.

Whole exome sequencing identifies a splicing mutation in NSUN2 as a cause of a Dubowitz-like syndrome

Fernando Jose Martinez  1 , Jeong Ho Lee, Ji Eun Lee, Sandra Blanco, Elizabeth Nickerson, Stacey Gabriel, Michaela Frye, Lihadh Al-Gazali, Joseph G Gleeson
Affiliations

Whole exome sequencing identifies a splicing mutation in NSUN2 as a cause of a Dubowitz-like syndrome

Fernando Jose Martinez et al. J Med Genet. 2012 Jun.

Abstract

Background: Dubowitz syndrome (DS) is an autosomal recessive disorder characterized by the constellation of mild microcephaly, growth and mental retardation, eczema and peculiar facies. Over 140 cases have been reported, but the genetic basis is not understood.

Methods: We enrolled a multiplex consanguineous family from the United Arab Emirates with many of the key clinical features of DS as reported in previous series. The family was analyzed by whole exome sequencing. RNA splicing was evaluated with reverse-transcriptase PCR, immunostaining and western blotting was performed with specific antibodies, and site-specific cytosine-5-methylation was studied with bisulfite sequencing.

Results: We identified a homozygous splice mutation in the NSUN2 gene, encoding a conserved RNA methyltransferase. The mutation abolished the canonical splice acceptor site of exon 6, leading to use of a cryptic splice donor within an AluY and subsequent mRNA instability. Patient cells lacked NSUN2 protein and there was resultant loss of site-specific 5-cytosine methylation of the tRNA(Asp GTC) at C47 and C48, known NSUN2 targets.

Conclusion: Our findings establish NSUN2 as the first causal gene with relationship to the DS spectrum phenotype. NSUN2 has been implicated in Myc-induced cell proliferation and mitotic spindle stability, which might help explain the varied clinical presentation in DS that can include chromosomal instability and immunological defects.

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Figures

Figure 1
Figure 1
Pedigree, phenotype, and predicted effect on splicing of the NSUN2 mutation. (A) Family-1455 presents three affected children from a first-cousin marriage. (B) Photograph of affected individuals, demonstrating blepharophimosis, telecanthus, hypertelorism, high/broad nasal bridge, smooth philtrum, and downturning upper lip in 2 and 3. Eczema on the nape and back are visible in 4 (arrow). (C) Invariant conservation across species of the G residue at the predicted exon 6 splice acceptor site (arrow), with mutant G>C base indicated.
Figure 2
Figure 2
Altered NSUN2 mRNA levels and aberrant splicing in affected individuals from Family-1455. (A) Each experiment compares qRT-PCR levels from healthy unrelated control to mother and two affected members. Severely reduced incorporation of exon 6 was detected from exon 5 to 6 and 6 to 7 reactions, whereas mother displayed about 50% of control. TATA Binding Protein qRT-PCR, used as control. Reactions from exons 5 to 7, 7 to 8 and 8 to 9 showed overall reduction in NSUN2 mRNA levels. (B) Agilent Bioanalyzer visualization of aberrant splicing of NSUN2 mRNA in Family-1455. Amplification from exon 5 to 7 produced a 210 bp product in control and mother, severely reduced in the affected individuals, who instead show a 250 bp product, also evident in mother at reduced level, containing an AluY exon.
Figure 3
Figure 3
Reduced NSUN2 protein levels in affected cell lines from Family-1455. (A) Western blot from whole cell extracts of control, mother and two affected individuals (Aff #1 and Aff #2), loaded in duplicate, show undetectable protein, compared with alpha-tubulin loading control. (B) Immunofluorescence of same cells showing undetectable NSUN2 reactivity (green) but intact Hoechst (blue) staining in affected cells compared with cells from control and mother. Staining is specific to nuclei and is enriched at nucleoli as previously described. Scale bar 10 uM.
Figure 4
Figure 4
Site-specific loss of cytosine-5 methylation (m5C) in two affected members. (A) Secondary structure of tRNAAsp. Prediction is that C47 and C48 require NSUN2 for m5C, whereas C38, methylated by DNMT2, will be unaffected. (B) RNA bisulfite sequencing of tRNAAsp(GTC) in the mother as a control and two affected members. In affected members m5C is specifically lost at NSUN2-dependent sites C47 and C48 (highlighted in bold) whereas m5C of C38 (highlighted with a star) remains unchanged. Blue indicates cytosine-5 not methylated; yellow indicated cytosine-5 methylated.
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Comment in

  • Targeting branch sites of new exons?
    Vorechovsky I. Vorechovsky I. J Med Genet. 2012 Jul;49(7):480. doi: 10.1136/jmedgenet-2012-101055. Epub 2012 Jun 22. J Med Genet. 2012. PMID: 22730556 Free PMC article. No abstract available.

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