Co-occurrence of frameshift mutations in smad6 and tcf12 in a child with complex craniosynostosis

ABSTRACT Non-syndromic craniosynostosis (CS) affects 1 in 2350 live births. Recent studies have shown that a significant fraction of cases are caused by de novo or rare transmitted mutations

that promote premature osteoblast differentiation in cranial sutures. Rare heterozygous loss-of-function (LOF) mutations in _SMAD6_ and _TCF12_ are highly enriched in patients with

non-syndromic sagittal and coronal CS, respectively. Interestingly, both mutations show striking incomplete penetrance, suggesting a role for modifying alleles; in the case of _SMAD6_, a

common variant near _BMP2_ drastically increases penetrance of sagittal CS. Here, we report a proband presenting with both sagittal and coronal craniosynostosis with the highly unusual

recurrence of CS within two months of initial surgery, requiring a second operation to re-establish suture patency at six months of age. Exome sequencing revealed a rare transmitted

frameshift mutation in _SMAD6_ (p. 152 fs*27) inherited from an unaffected parent, absence of the common _BMP2_ risk variant, and a de novo frameshift mutation in _TCF12_ (p.E548fs*14).

_SMAD6_ and _TCF12_ independently inhibit transcriptional targets of BMP signaling. The findings are consistent with epistasis of these mutations, increasing penetrance and severity of CS in

this proband. They also add to the list of composite phenotypes resulting from two Mendelian mutations, and support the utility of exome sequencing in atypical CS cases. The female proband

was delivered at term after an uncomplicated pregnancy, and was referred to the pediatric neurosurgical service at birth due to her abnormal head shape. On physical exam, the patient had

flattening of the left frontal bone with contralateral frontal bossing and associated harlequin deformity of the left orbit, consistent with left coronal synostosis. In addition, the

calvarium posterior to the coronal sutures was elongated and narrow (scaphocephalic), consistent with sagittal synostosis (Fig. 1a, b). A CT scan confirmed sagittal and left coronal

synostosis, and the child underwent endoscopic strip craniectomy at nine weeks of age. The child was subsequently followed biweekly in neurosurgery clinic and by an orthotist for helmet

adjustments to shape skull growth. Two months after surgery, the orthotist noted that the child’s left forehead remained flattened and was not rounding as expected. At the child’s subsequent

neurosurgical clinic visit, the parietal and occipital regions had rounded and expanded as expected; however, the left frontal region had stopped rounding. A repeat head CT was performed,

which demonstrated rapid healing and patency of the sagittal suture and re-fusion of the left coronal suture along with complete fusion of the right coronal suture (Fig. 1c–e). Such rapid

recurrence of craniosynostosis is extremely unusual. To correct the anterior deformity, the child underwent a cranial vault reconstruction with fronto-orbital advancement at six months of

age. The child is developing normally to date, and it is unknown at present if she will need further surgical cranial reconstruction. To explore potential genetic contributions to her

condition, we performed whole exome sequencing of the case-parent trio using DNA prepared from buccal swab samples according to standard protocols. Exome capture was performed using the IDT

xGen capture reagent, which was followed by 99 base paired-end sequencing on the Illumina HiSeq 2000 instrument. Sequence reads were aligned to the GRCh37/hg19 human reference genome using

BWA-Mem. Local realignment and quality score recalibration were performed using the GATK pipeline, after which variants were called using the GATK Haplotype Caller. A Bayesian algorithm,

TrioDeNovo, was used to call de novo mutations1. VQSR ‘PASS’ variants with an ExAC allele frequency ≤10−3 sequenced to a depth of eight or greater in the proband and 10 or greater in each

parent with Phred-scaled genotype likelihood scores >30 and de novo quality scores (log10(Bayes factor)) >6 were considered. Independent aligned reads at variant positions were

visualized in silico to remove false calls. All retained calls had de novo genotype quality scores of 100. Transmitted variants were called as per above, and all variants were annotated

using ANNOVAR2 with allele frequencies assigned to each variant from the ExAC database3. Analysis showed that the proband had rare heterozygous LOF mutations in both of the two predominant

non-syndromic CS genes, _SMAD6_ and _TCF12_4,5. The mutation in _SMAD6_ was an early frameshift mutation (p. 152 fs*27), which was transmitted from an unaffected parent (Fig. 2, Table 1).

The mutation in _TCF12_ was also a frameshift mutation (p.K548fs*14), which was de novo. Both mutations were absent from the ExAC and GnomAD databases, which contain >240,000 alleles3,

and both mutations were confirmed by Sanger sequencing (Fig. 2). No other compelling heterozygous rare LOF or damaging missense variants were identified, and no rare recessive genotypes were

identified (Table 1, Supplementary Table 1). Heterozygous _TCF12_ mutations have been previously shown to cause coronal CS with considerable phenotypic overlap with Saethre–Chotzen

syndrome4, which is caused by LOF mutation in _TWIST1_, which heterodimerizes with _TCF12_ to inhibit transcription downstream of BMP signaling. Similar LOF mutations in _TCF12_ were

subsequently identified in patients with non-syndromic coronal craniosynostosis6. De novo or transmitted LOF mutations in _SMAD6_ are found in ~6% of non-syndromic midline craniosynostosis

cases5. LOF mutations in both _TCF12_ and _SMAD6_ both show striking incomplete penetrance (~40 and 20% penetrance, respectively)4,7. In the case of _SMAD6_, epistatic interaction with a

common risk variant near BMP2, which by itself has modest effect on risk, increases penetrance to >90%5,7. The _BMP2_ rs1884302 locus was genotyped in the proband and both parents, and no

family members harbored the CS risk allele ‘C’, consistent with the parent harboring the _SMAD6_ mutation being free of CS (Fig. 2). The combination of rare LOF mutations at established

Mendelian loci (_SMAD6_ and _TCF12_) in the proband was particularly interesting8. While _SMAD6_ has long been known as an inhibitory-SMAD that negatively regulates BMP signaling, _TCF12_

silencing in mesenchymal stem cells was only recently shown to result in increased phosphorylation of receptor-SMADs, implying that loss of TCF12 function also augments BMP signaling via the

BMP/SMAD axis9. This finding suggests that the combination of _SMAD6_ and _TCF12_ haploinsufficiency increases BMP signaling to levels substantially greater than those seen with either

mutation alone, sufficient to ensure penetrance at both the sagittal and coronal sutures (Fig. 3). Moreover, while neither _SMAD6_ nor _TCF12_ haploinsufficiency in isolation has been

associated with increased rates of reoperation4,5, we propose that these mutations together promote sufficiently high osteogenic drive to promote the very unusual rapidity of recurrent

synostosis after surgery (Figs. 1, 3). The combination of a common _BMP2_ variant with LOF variants in SMAD6 is sufficient to push BMP/SMAD signaling to levels sufficient to cause suture

fusion5. The present results suggest that alleles other than the common _BMP2_ risk variant can have epistasis with rare _SMAD6_ alleles. It seems compelling that the combination of a

_SMAD6_ LOF mutation with loss of an independent inhibitor of BMP signaling via _TCF12_ mutation produces particularly high BMP/SMAD signaling and a strikingly more severe phenotype than

_SMAD6_ LOF mutation alone (Figs. 1, 3). It will be interesting to see whether other patients with non-syndromic complex CS also have mutations in these two genes. While several factors,

such as patient age at presentation, suture fusion pattern, and patient co-morbidities, play a role in which type of surgery (endoscopic versus open) is offered to craniosynostosis patients,

knowing the genetic results for specific patients could prove useful in guiding operative planning for this complex patient population. The goals of cranial vault reconstruction are to

obtain an aesthetically pleasing shape of the skull that will allow adequate growth of the brain in ideally one operation. Identification of high risk genotypes prior to surgery—particularly

in cases with unusual clinical features—may prove useful in guiding surgical management in the future and may enable more informed discussions with patients’ families. HGV DATABASE The

relevant data from this Data Report are hosted at the Human Genome Variation Database at https://doi.org/10.6084/m9.figshare.hgv.2330 https://doi.org/10.6084/m9.figshare.hgv.2333. REFERENCES

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(2017). Article  CAS  Google Scholar  Download references ACKNOWLEDGEMENTS The study protocol was approved by the Yale Human Investigation Committee Institutional Review Board, and consent

for use of patient photographs was obtained by the treating physicians. This project was supported by the Yale Center for Mendelian Genomics (NIH Grant M#UM1HG006504-05), the NIH Medical

Scientist Training Program (NIH/National Institute of General Medical Sciences Grant T32GM007205), and the Howard Hughes Medical Institute. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS *

Department of Genetics, Yale University School of Medicine, New Haven, CT, USA Andrew T. Timberlake, Carol Nelson-Williams, Charuta G. Furey & Richard P. Lifton * Section of Plastic and

Reconstructive Surgery, Yale University School of Medicine, New Haven, CT, USA Andrew T. Timberlake, Robin Wu & John A. Persing * Division of Pediatric Neurosurgery, University of North

Carolina School of Medicine, Chapel Hill, NC, USA Kristi I. Hildebrand & Scott W. Elton * Division of Plastic and Reconstructive Surgery, University of North Carolina School of Medicine,

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Nelson-Williams, C. _et al._ Co-occurrence of frameshift mutations in _SMAD6_ and _TCF12_ in a child with complex craniosynostosis. _Hum Genome Var_ 5, 14 (2018).

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