Bosma arhinia microphthalmia syndrome (BAMS) is an extremely rare and striking condition characterized by complete absence of the nose with or without ocular defects. We report here that missense mutations in the epigenetic regulator SMCHD1 mapping to the extended ATPase domain of the encoded protein cause BAMS in all 14 cases studied. All mutations were de novo where parental DNA was available. Biochemical tests and in vivo assays in Xenopus laevis embryos suggest that these mutations may behave as gain-of-function alleles. This finding is in contrast to the loss-of-function mutations in SMCHD1 that have been associated with facioscapulohumeral muscular dystrophy (FSHD) type 2. Our results establish SMCHD1 as a key player in nasal development and provide biochemical insight into its enzymatic function that may be exploited for development of therapeutics for FSHD.
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We would like to thank all family members and their relatives for their participation and kind contribution to this study. N. Akarsu was instrumental for recruiting patient 11. Support from the Jean Renny Endowed Chair for Craniofacial Research (M.L.C.) is acknowledged. C.D. is the recipient of a fellowship from the French Ministry of Education and Research. H.F. was supported by a postdoctoral grant from INSERM. B.R. is a fellow of the Branco Weiss Foundation, an A*STAR Investigator, an EMBO Young Investigator and a recipient of the inaugural AAA Fellowship in Amsterdam. This work was supported by funding from the Agence Nationale de la Recherche (ANR-10-IAHU-01, CranioRespiro), the Cancer Council Victoria (fellowship to K.C.), the National Health and Medical Research Council (NHMRC) of Australia to M.E.B. and J.M.M. (1098290 and fellowships 1110206 and 1105754), the Scientific and Technological Research Council of Turkey (TUBITAK) to H.K. (grant 112S398, E-RARE network CRANIRARE-2), the Association Française contre les Myopathies (AFM) to F.M., Victorian State Government Operational Infrastructure Support, an NHMRC IRIISS grant (9000220), the German Federal Ministry of Education and Research (BMBF) to B.W. (grant 01GM1211A, E-RARE network CRANIRARE-2), the German Research Foundation (SFB1002, project D02) to B.W., MACS, VICTA and Baillie Gifford grant support to N. Ragge, Mahidol University and Research Career Development Awards from the Faculty of Medicine Ramathibodi Hospital to D. Wattanasirichaigoon, an A*STAR JCO Career Development grant to A.J., an A*STAR BMRC Young Investigator Grant to S.X. and a Strategic Positioning Fund on Genetic Orphan Diseases from the Biomedical Research Council, A*STAR, Singapore, to B.R.
The authors declare no competing financial interests.
Integrated supplementary information
(a–f) Controls (a–c) and patient 1 (d–f) at 4 years. Patient 1 displays maxillary hypoplasia and absent nasal bones (d,e). Olfactory bulbs and sulci (labeled with red and white arrows, respectively, on the left side in c) are absent in patient 1 (f). (g–i) Patient 5, with right microphthalmia, as shown by MRI (i). (j–l) Skeletal imaging of patient 14 (j,k) and patient 11 (l) indicating midface hyploplasia.
Individuals submitted for exome sequencing are indicated by a red asterisk. Note that Sanger sequencing was unavailable for individual 13.
Residues mutated in BAMS are indicated by pink arrows. Residues mutated in FSHD are indicated by purple arrows. The conserved GHKL-ATPase motifs I–IV are shaded in blue. Hs, Homo sapiens; Mm, Mus musculus; Bt, Bos taurus; Gg, Gallus gallus; Md, Monodelphis domestica; Cm, Chelonia mydas; Xt, Xenopus tropicalis; Dr, Danio rerio; Sc, Saccharomyces cerevisiae. FSHD mutation reference: LOVD SMCHD1 variant database (see URLs).
(a–e and j-q) Embryos. E, embryonic day; gt/+, embryos heterozygous for the Smchd1gt allele expressing LacZ; +/+, wild-type embryos; hf, head folds; npl, nasal placode; ov, optic vesicle; npi, nasal pit; ne, nasal epithelium. (f–i) Coronal sections. (r,s) Transverse sections. An asterisk in p indicates deep nasal staining.
The position of the three different regions analyzed within D4Z4 is indicated above the corresponding column (left, DR1; middle, 5′; right, Mid). For each sample, at least ten cloned DNA molecules were analyzed by Sanger sequencing. Each histogram column corresponds to a single CpG. Black corresponds to the global percentage of methylated CpGs; white corresponds to the global percentage of unmethylated CpGs. The percentage of methylated CpGs out of the total CpGs in each individual analyzed is given in Supplementary Table 3.
See the legend of Supplementary Figure 5 for further information.
Supplementary Figure 7 Comparison of D4Z4 methylation in patients with BAMS or FSHD2, the relatives of patients with BAMS and controls.
Distribution of methylation for the three different regions within the D4Z4 sequence (DR1, 5′ and Mid) in control individuals, patients with FSHD2 carrying an SMCHD1 mutation, and patients with BAMS and their relatives. Means ± s.e.m. are shown. A Kruskal–Wallis multiple-comparisons test was performed, followed by a Dunn’s test and Bonferroni correction, with α = 0.05. ***P < 0.0001, **P < 0.001, *P < 0.05. Blue points represent outliers; red crosses represent medians. The level of methylation is statistically significantly different between controls and patients with FSHD2 for the DR1 (**P < 0.001) and 5′ (***P < 0.0001) regions. The level of methylation is significantly different between controls and patients with BAMS for the 5′ region (*P < 0.05) and between patients with BAMS and their relatives for the DR1 (*P < 0.05) and 5′ (**P < 0.001) regions.
Supplementary Figure 8 Structural model of the N-terminal region of mouse Smchd1, based on the crystal structure of Hsp90.
Residues mutated in BAMS are shown in pink. Residues mutated in FSHD are shown in purple. Motif I is shown in blue. ATP is shown in orange.
Supplementary Figure 9 Fibroblasts derived from patients with BAMS show no defects in NHEJ or in H2AX activation.
(a) A microhomology-mediated end-joining (MMEJ) assay was performed on wild-type (WT), XRCC4-deficient and case 1 and 2 fibroblasts. Whereas XRCC4-deficient fibroblasts show multiple smaller DNA bands after BstXI digestion indicating defects in NHEJ-mediated DNA repair and leading to preferential use of MMEJ-mediated DNA double-strand repair, fibroblasts from patients with BAMS show no defects in NHEJ-mediated DNA repair pathways in comparison to wild-type fibroblasts. (b) Immunoblot analysis of UV- and etoposide-induced phosphorylation of H2AX at Ser139 (γH2AX). Wild-type fibroblasts (WT) and fibroblasts derived from cases 1 and 2 were treated with UV-C (UV) or etoposide (Eto) or left untreated as a control (–). Cells were lysed and subjected to immunoblot analysis with an antibody against γH2AX. Equal protein loading was confirmed by reprobing of the membrane with an antibody against β-actin. Wild-type fibroblasts and those from patients with BAMS did not show significant differences in H2AX activation.
Supplementary Figure 10 ATPase assays performed using wild-type or mutant recombinant mouse Smchd1 protein in the presence of radicicol.
Data are displayed as means ± s.d. from three technical replicates. The data are representative of at least two independent experiments using different batches of protein preparation.
Full-length immunoblot of the cropped blot image in Figure 3g.
Supplementary Figure 12 SMCHD1 overexpression in Xenopus causes dose-dependent craniofacial anomalies.
(a,b) Measurements of eye diameter of Xenopus embryos injected with 240 pg (a) or 500 pg (b) SMCHD1 mRNA. Y353C is an FSHD2 mutation. At least 20 embryos were studied for each condition. (c–f) Representative Xenopus embryos injected with 500 pg of wild-type or FSHD2 mutant SMCHD1 or 120 pg of BAMS mutant mRNA show varying degrees of craniofacial abnormalities as compared to uninjected control tadpoles at 4 days post-fertilization. Data are shown as means ± s.d. P values were calculated by Kruskal–Wallis test followed by Dunn’s post test. n.s., not significant.
Purified recombinant wild-type or mutant proteins were resolved by 4–20% Tris-glycine reducing SDS–PAGE and were stained with SimplyBlue SafeStain. The protein quantities loaded were as follows: left gel, 1.4 μg; middle gel, 1.05 μg; right gel, 0.7 μg. The sizes of molecular weight (MW) markers are as indicated on the left-hand side.
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Gordon, C., Xue, S., Yigit, G. et al. De novo mutations in SMCHD1 cause Bosma arhinia microphthalmia syndrome and abrogate nasal development. Nat Genet 49, 249–255 (2017). https://doi.org/10.1038/ng.3765
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