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Case Reports
. 2015 Feb 5;96(2):266-74.
doi: 10.1016/j.ajhg.2014.11.019. Epub 2015 Jan 22.

Mutations in DDX58, which encodes RIG-I, cause atypical Singleton-Merten syndrome

Mi-Ae Jang  1 , Eun Kyoung Kim  2 , Hesung Now  3 , Nhung T H Nguyen  3 , Woo-Jong Kim  3 , Joo-Yeon Yoo  3 , Jinhyuk Lee  4 , Yun-Mi Jeong  5 , Cheol-Hee Kim  5 , Ok-Hwa Kim  6 , Seongsoo Sohn  7 , Seong-Hyeuk Nam  8 , Yoojin Hong  8 , Yong Seok Lee  8 , Sung-A Chang  2 , Shin Yi Jang  2 , Jong-Won Kim  1 , Myung-Shik Lee  9 , So Young Lim  10 , Ki-Sun Sung  11 , Ki-Tae Park  12 , Byoung Joon Kim  13 , Joo-Heung Lee  14 , Duk-Kyung Kim  2 , Changwon Kee  15 , Chang-Seok Ki  16
Affiliations
Case Reports

Mutations in DDX58, which encodes RIG-I, cause atypical Singleton-Merten syndrome

Mi-Ae Jang et al. Am J Hum Genet. .

Abstract

Singleton-Merten syndrome (SMS) is an autosomal-dominant multi-system disorder characterized by dental dysplasia, aortic calcification, skeletal abnormalities, glaucoma, psoriasis, and other conditions. Despite an apparent autosomal-dominant pattern of inheritance, the genetic background of SMS and information about its phenotypic heterogeneity remain unknown. Recently, we found a family affected by glaucoma, aortic calcification, and skeletal abnormalities. Unlike subjects with classic SMS, affected individuals showed normal dentition, suggesting atypical SMS. To identify genetic causes of the disease, we performed exome sequencing in this family and identified a variant (c.1118A>C [p.Glu373Ala]) of DDX58, whose protein product is also known as RIG-I. Further analysis of DDX58 in 100 individuals with congenital glaucoma identified another variant (c.803G>T [p.Cys268Phe]) in a family who harbored neither dental anomalies nor aortic calcification but who suffered from glaucoma and skeletal abnormalities. Cys268 and Glu373 residues of DDX58 belong to ATP-binding motifs I and II, respectively, and these residues are predicted to be located closer to the ADP and RNA molecules than other nonpathogenic missense variants by protein structure analysis. Functional assays revealed that DDX58 alterations confer constitutive activation and thus lead to increased interferon (IFN) activity and IFN-stimulated gene expression. In addition, when we transduced primary human trabecular meshwork cells with c.803G>T (p.Cys268Phe) and c.1118A>C (p.Glu373Ala) mutants, cytopathic effects and a significant decrease in cell number were observed. Taken together, our results demonstrate that DDX58 mutations cause atypical SMS manifesting with variable expression of glaucoma, aortic calcification, and skeletal abnormalities without dental anomalies.

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Figures

Figure 1
Figure 1
Families Affected by DDX58 Mutations (A) Pedigree of family A, affected by the c.1118A>C (p.Glu373Ala) DDX58 mutation. An asterisk indicates that exome sequencing was performed. (B) Pedigree of family B, affected by the c.803G>T (p.Cys268Phe) DDX58 mutation. (C) Computed tomographic angiography shows aortic calcification (black arrowheads) and calcification of the aortic valves (white arrowheads) in II:6 (left) and III:7 (right) of family A. (D) Orthopantomograms of II:6 (upper) and III:7 (lower) of family A. These subjects show no dental anomalies except mild dental caries. (E) Photographs of both eyes of family A individual III:7, who was diagnosed with glaucoma at 3 years of age, show severe optic nerve head cupping (upper left and right). Iris defects due to surgical iridectomy, which was performed with filtering surgeries and additional subsequent bilateral glaucoma implant surgeries, are visible (lower left and right). (F) Skeletal findings in the affected families. In family A (FA), foot radiographs of III:2 at age 33 years and his son (IV:3) at age 8 years show acro-osteolysis of the distal phalanges and the resulting triangular shape of the great toes (arrows). Hand radiographs of the son (IV:3) and daughter (IV:4, age 5 years) also reveal erosive changes of the terminal tufts and the resulting hypoplastic appearance of all distal phalanges. Carpal bone ossification is delayed in IV:3, who is approximately 3–4 years of age. Expanded medullary cavities of the metacarpals and metatarsals are not evident in any affected member of family A. In family B (FB), the foot radiograph of II:4 at age 61 years shows marked subluxation of the hallux, erosions, and acro-osteolysis of the distal phalanges and the resulting short toes (upper). Note the similar but lesser degree of erosive changes of the distal phalanges, which show a triangular shape of the great toe, in III:2 of family B (upper, arrow). In the hand radiographs of II:4 and III:2 of family B, note the severe acro-osteolysis of the distal phalanges and flexion contractures of II:4 and mild erosive changes of the distal phalanges in III:2. The medullary cavities of the metatarsals and metacarpals are also not widened in this family.
Figure 2
Figure 2
Ortholog Conservation of DDX58 Mutations and Molecular Helicase Structure of DDX58 (A) Schematic representation of DDX58 mutations relative to the affected protein domains. Also, sequence alignment of DDX58 is shown in vertebrate species. The alignment regions corresponding to the missense mutations are shown. Cys at codon 268 and Glu at codon 373 are highly conserved across all species. The Cys and Glu residues are indicated by the closed white boxes. The Ensembl IDs for DDX58 sequences aligned in this study are as follows: human, ENSP00000369197; chimpanzee, ENSPTRT00000038562; orangutan, ENSPPYT00000022324; mouse, ENSMUSP00000115052; rat, ENSRNOT00000008465; dog, ENSCAFT00000002841; horse, ENSECAT00000023725; cow, ENSBTAP00000053514; and zebrafish, ENSDART00000058276. Abbreviations are as follows: CARD, caspase activation recruitment domain; CTD, C-terminal domain; Hel, helicase domain (Hel-1 and Hel-2 are the two conserved core helicase domains, and Hel-2i is an insertion domain conserved in DDX58-like helicase family); and P, pincer of bridge region connecting Hel-2 to the CTD involved in binding dsRNA. (B) The protein structure is drawn in gray. The RNA and ADP molecules are shown in orange. The locations of the amino acid changes caused by four known SNPs (rs35527044 [p.Thr260Pro], rs951618 [p.Ile406Thr], rs17217280 [p.Asp580Glu], and rs35253851 [p.Phe789Leu]) and two most likely pathogenic variants (p.Cys268Phe and p.Glu373Ala) are shown by blue and red spheres, respectively. The Thr260, Ile406, and Phe789 sites are hidden by the protein. PDB ID 3ZD7 was used for the wild-type (WT) structure of DDX58. This structure contains 73 experimentally missing residues. All protein structural analyses were executed with the program CHARMM. (C and D) Focused views of the WT X-ray structure (C) and 10-ns (ns) molecular dynamics (MD) final structure (D). The 10-ns MD simulation was performed with an implicit solvation model, i.e., the generalized Born model with a simple switch. Two residues (Cys268 and Glu373) affected by mutations are drawn as red wireframe models. Close distances are shown as black dotted lines with distance values. The used atom pairs for the distance measurements are shown in Table S3.
Figure 3
Figure 3
DDX58 Mutations Constitutively Activate RLR-IFN Production Pathways (A) In HEK293FT cells, FLAG-DDX58 (WT), FLAG-DDX58-p.Glu373Ala, or FLAG-DDX58-p.Cys268Phe was transfected along with reporter construct pLuc-PRDIII-I or pLuc-NF-κB for 24 hr. A dual-luciferase assay was performed after 12 hr of polyI:C (1 μg/ml) stimulation. (B and C) HEK293FT cells were transfected with the indicated plasmids for 30 hr and then stimulated with polyI:C (1 μg/ml) for the indicated time. After stimulation, the lysates were separated by SDS-PAGE (B) or on native gels (C). Immunoblot assays were performed with the indicated antibodies. (D) HEK293FT cells were transfected with the indicated plasmids for 24 hr. After stimulation with polyI:C (1 μg/ml) for 12 hr, the expression of IFNB1, TNF, ISG15, and CCL5 was measured by quantitative real-time PCR and normalized to β-actin expression. The primers, which were used for quantitative real-time PCR, were described previously. Statistical analysis using t tests was conducted in GraphPad Prism 4 (∗p < 0.05, ∗∗p < 0.005, ∗∗∗p < 0.001).
Figure 4
Figure 4
Potential Cytotoxicity of Altered DDX58 in HTM cells (A) Phase-contrast photographs of HTM cells transduced with empty vector (null) or expression vectors for the WT, two DDX58 alterations (p.Cys268Phe and p.Glu373Ala), and two other known SNPs (rs11795404 [c.548G>T (p.Ser183Ile)] and rs951618 [c.1217T>C (p.Ile406Thr)]) are shown. The scale bar represents 100 μm. (B) HTM cells were incubated with anti-FLAG antibody diluted 200× or Cy3-conjugated anti-vimentin antibody diluted 500×. The cells were washed and reacted for 2 hr with FITC-conjugated secondary antibody. After the nuclei were counterstained for 20 min with DAPI, the cells were viewed under a fluorescence microscope with the appropriate filter sets and a 200× objective. FITC fluorescence (green) of DDX58 staining merged with DAPI (blue) is shown in the left column, and Cy3 fluorescence (red) of vimentin staining merged with DAPI is shown in the right column. The scale bar represents 100 μm. (C) Cell viability (%) was calculated as the percentage of cells surviving in comparison to the null. The data were statistically analyzed by one-way ANOVA followed by the Newman-Keuls multiple-comparison test, and p < 0.05 was considered significant. The data are presented as the mean percentage ± the SEM (∗p < 0.01 versus WT).
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