Two locus inheritance of non-syndromic midline craniosynostosis via rare SMAD6 and common BMP2 alleles
- Yale University School of Medicine, United States
- Howard Hughes Medical Institute, Yale University School of Medicine, United States
- Yale Center for Genome Analysis, United States
- Craniosynostosis and Positional Plagiocephaly Support, United States
- The Rockefeller University, United States
Figures
Figure 1
Phenotypes of midline craniosynostosis.
(a) Normal infant skull with patent sagittal (S) and metopic (M) sutures. (b) Three-dimensional reconstruction of computed tomography (3D CT) demonstrating premature fusion of both the sagittal and …
Figure 2 with 2 supplements
Segregation of SMAD6 mutations and BMP2 SNP genotypes in pedigrees with midline craniosynostosis.
(a) Domain structure of SMAD6 showing location of the MH1 and MH2 domains. The MH1 domain mediates DNA binding and negatively regulates the functions of the MH2 domain, while the MH2 domain is …
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Figure 2—source data 1
Variants identified in SMAD6.
Highlighted variants indicate de novo mutations; 'D' and 'T' respectively denote damaging and tolerated missense variants called by MetaSVM.
- https://doi.org/10.7554/eLife.20125.007
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Figure 2—source data 2
PCR primer sequences for Sanger sequencing of reported variants.
Source Data for Figure 2—figure supplement 2.
- https://doi.org/10.7554/eLife.20125.008
Figure 2—figure supplement 1
Plots of independent Illumina sequencing reads in a parent-offspring trio showing de novo SMAD6 mutation.
The reference sequence of a segment of SMAD6 that includes base 15:67073502 (denoted by arrow) is shown in the top row, with red, blue, green and yellow squares representing A, C, G, T, …
Figure 2—figure supplement 2
Confirmation of SMAD6 mutations by Sanger sequencing of PCR products.
Sanger sequencing traces of PCR amplicons containing SMAD6 mutations identified by exome sequencing are shown. Above each trace or set of traces, the kindred ID, mutation identified in the DNA …
Figure 3 with 3 supplements
Quantile-quantile plots of observed versus expected p-values comparing the burden of rare LOF and damaging (LOF + D-mis) variants in protein-coding genes in craniosynostosis cases.
Rare (allele frequency <2 × 10–5 in the ExAC03 database) loss of function (LOF) and damaging missense (D-mis) variants were identified in 191 probands. The probability of the observed number of …
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Figure 3—source data 1
Source data for Figure 3—figure supplement 3.
- https://doi.org/10.7554/eLife.20125.013
Figure 3—figure supplement 1
Quantile-quantile plots comparing all transmitted, damaging variants in protein-coding genes in 191 probands with midline craniosynostosis to the expected binomial distribution.
De novo variants were excluded from this analysis, leaving 1122 rare (ExAC allele frequency < 2 x10−5), transmitted LOF variants and 3115 transmitted damaging (LOF + D-mis) variants. All genes …
Figure 3—figure supplement 2
Principal-component analysis of 191 probands and 3337 European autism controls.
(a) Principal component analysis of exome sequence genotypes from 191 probands with sagittal, metopic, or combined sagittal and metopic craniosynostosis clustered along with HapMap subjects. Results …
Figure 3—figure supplement 3
Quantile-quantile plot of observed versus expected p-values comparing the burden of damaging (LOF + D-mis) variants in protein-coding genes in craniosynostosis cases and controls.
The frequency of rare (allele frequency < 2 × 10–5 in the ExAC03 database) loss of function and D-mis variants in each gene was compared in 172 European probands with midline craniosynostosis and …
Figure 4
SMAD6 inhibits osteoblast differentiation by inhibiting BMP-mediated SMAD signaling (Salazar et al., 2016).
(a) BMP ligands activate BMP receptors, leading to phosphorylation of receptor-regulated SMADs (R-SMADs), which complex with SMAD4 and enter the nucleus, cooperating with RUNX2 to induce osteoblast …
Figure 5
A de novo variant identified in SMURF1.
(a) Sanger sequence electropherogram of a PCR product amplified from the genomic DNA of a proband with metopic craniosynostosis, confirming a de novo R468W mutation in SMURF1, a SMAD6 binding …
Figure 6
De novo loss-of-function mutations in Sprouty genes.
(a) Pedigree and Sanger sequencing traces for kindred SAG150, demonstrating a de novo nonsense mutation in SPRY4 (p.E160*) in the proband. (b) Pedigree and Sanger sequencing traces in a kindred with …
Tables
Table 1
Enrichment of protein-altering de novo mutations in 132 subjects with sagittal and/or metopic craniosynostosis.
| Observed | Expected | Enrichment | p-value | |||
|---|---|---|---|---|---|---|
| Class | # | #/subject | # | #/subject | ||
| All mutations | 144 | 1.09 | 142.8 | 1.08 | 1.01 | 0.47 |
| Synonymous | 21 | 0.16 | 40.4 | 0.31 | 0.52 | 3.0 × 10−4 |
| Protein altering | 123 | 0.93 | 102.4 | 0.78 | 1.17 | 0.03 |
| Total missense | 110 | 0.83 | 89.7 | 0.68 | 1.23 | 0.02 |
| T-mis | 82 | 0.62 | 75.2 | 0.57 | 1.09 | 0.23 |
| D-mis | 28 | 0.21 | 14.5 | 0.11 | 1.93 | 1.0 × 10−3 |
| Loss of function (LOF) | 13 | 0.10 | 12.7 | 0.10 | 1.03 | 0.50 |
| LOF + D-mis | 41 | 0.31 | 27.1 | 0.21 | 1.51 | 7.8 × 10−3 |
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#, number of de novo mutations in 132 subjects; #/subject, number of de novo mutations per subject; Damaging and tolerated missense called by MetaSVM (D-mis, T-mis respectively); Loss of function denotes premature termination, frameshift, or splice site mutation. For mutation classes with enrichment compared to expectation, p-values represent the upper tail of the Poisson probability density function. For mutation classes in which we observed a paucity of mutations compared to expectation, p-values represent the lower tail.
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Table 1—source data 1
De novo mutations in 132 trios with sagittal and/or metopic craniosynostosis.
Mutations highlighted in orange are likely loss of function mutations, those highlighted in blue are likely damaging missense mutations (D-mis) as called by MetaSVM, and those without highlight are predicted to be tolerated (T-mis) or are synonymous (syn).
- https://doi.org/10.7554/eLife.20125.005
Table 2
Probability of observed de novo mutations in SMAD6 and Sprouty genes occurring by chance in 132 subjects using gene-specific mutation probabilities.
| Gene(s) | Mutations | Number of observed mutations | Number of expected mutations | p value |
|---|---|---|---|---|
| SMAD6 | Loss of function | 2 | 0.00026 | 3.31 × 10−8 |
| SMAD6 | Missense | 1 | 0.0046 | 4.67 × 10−3 |
| SPRY1, SPRY2, SPRY3, SPRY4 | Nonsense, splice site, frameshift | 2 | 0.001193 | 7.11 × 10−7 |
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Probabilities calculated from the Poisson distribution using DenovolyzeR. The probability of observing at least 2 LOF and 1 missense mutation in SMAD6 was 3.6 ×10−9 via Fisher’s method.
Table 3
Enrichment of de novo and transmitted damaging variants in SMAD6 in craniosynostosis.
| Observed | Expected | Enrichment | p-value | |
|---|---|---|---|---|
| De novo LOF and D-mis | 3 | 0.0049 | 612 | 3.6 × 10−9 |
| Transmitted LOF and D-mis | 10 | 0.1404 | 71.2 | 7.0 × 10−16 |
| Total | 13 | 0.1453 | 89.5 | 1.4 × 10−22 |
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LOF, loss of function; D-mis, damaging missense variants per MetaSVM; The total number of SMAD6 variants expected in this cohort was calculated by summing the expected number of de novo and transmitted variants. P-value combining probabilities from de novo and transmitted protein damaging SMAD6 variants was determined by Fisher’s method.
Table 4
Distribution of suture involvement in kindreds with and without rare (allele frequency < 2 × 10−5) de novo and transmitted damaging (LOF + D-mis) variants in SMAD6.
| Total # kindreds | Total # SMAD6 mutations (%) | # LOF (%) | |
|---|---|---|---|
| Sagittal | 113 | 3 (2.7) | 2 (1.8) |
| Metopic | 70 | 7 (10) | 3 (3.9) |
| Sagittal and Metopic | 8 | 3 (37.5) | 3 (37.5) |
| Total | 191 | 13 (6.8) | 8 (4.2) |
Table 5
Risk of craniosynostosis in SMAD6 mutation carriers in the presence or absence of a BMP2 risk allele.
| SMAD6/BMP2 Genotypes | Craniosynostosis (+) | Craniosynostosis (−) |
|---|---|---|
| SMAD6 (+) / BMP2 risk allele (+) | 14 | 0 |
| SMAD6 (+) / BMP2 risk allele (−) | 3 | 13 |
| SMAD6 (−) / BMP2 risk allele (+) | 0 | 18 |
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All members of kindreds found to have a mutation in SMAD6 were included. SMAD6(+) indicates the presence of a heterozygous LOF or D-mis allele. The reported BMP2 risk allele is ‘C’ at risk locus rs1884302, found within a gene desert ~345kb downstream of BMP2. p=1.4 × 10−10 by the Freeman-Halton extension of Fisher’s exact test. Odds ratio in favor of disease was incalculable due to the absence of craniosynostosis in SMAD6 (−) individuals in these kindreds.
Additional files
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Supplementary file 1
Supplementary files for "Two locus inheritance of non-syndromic midline craniosynostosis via rare SMAD6 and common BMP2 alleles".
(A) Exome Sequencing Quality Statistics for all members of craniosynostosis kindreds (n = 455) and autism controls (n = 3337). (B) TDT of an intergenic BMP2 risk allele and intronic BBS9 risk allele in SMAD6 mutation carriers with craniosynostosis. (C) Optimized two locus and single locus parametric models of genotype specific penetrances for SMAD6 and BMP2. (D) Family specific lod scores for each kindred under the two locus and single locus models. (E) Clinical features and BMP2 genotypes in craniosynostosis patients with rare SMAD6, SMURF1, SPRY1, or SPRY4 mutations. (F) De novo mutations identified per trio.
- https://doi.org/10.7554/eLife.20125.023
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Supplementary file 2
Exome sequencing quality statistics.
Exome sequencing quality statistics for all members of craniosynostosis kindreds (n = 455) and autism controls (n = 3337) .
- https://doi.org/10.7554/eLife.20125.024
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Source code 1
R script for two locus and single locus linkage analyses.
- https://doi.org/10.7554/eLife.20125.025
