The pedigree came from Wuhan, Hubei Province, China. The female proband (II-1), the elder monochorionic diamniotic (MCDA) twin (Fig. 2a-c and Addition file: Fig. S1-3), was diagnosed with sagittal craniosynostosis at the age of 9 months in the Department of Neurosurgery, Children’s Hospital of Nanjing Medical University. She was born to non-consanguineous parents without family history of craniosynostosis. Her mother, a 30-year-old Chinese female, conceived MCDA twins by frozen embryo transfer (FET) (Addition file: Fig. S1-3). Her father was 36-year-old at that time. The 26 days after FET, four-dimensional ultrasound scan confirmed two embryos were inside a gestational sac (approximately 22 mm × 13 mm); one embryo was 2.8 mm in length and the fetal heart rate was 107 per minute, the other embryo was 3.3 mm in length and the fetal heart rate was 118 per minute (Addition file: Fig. S1).
At 13 weeks of gestation, IgG of cytomegalovirus and herpes simplex virus were detected in the mother’s serum by the toxoplasmosis, rubella, cytomegalovirus and herpes simplex (TORCH) test (Addition file: Table S1). At 14 weeks of gestations, maternal dietary assessment indicated that the mother had inadequate intakes of energy, protein, fat, vitamins and several other minerals (Addition file: Table S2); and non-invasive prenatal testing demonstrated low risks for Trisomy 13, 18 and 21 (Addition file: Table S3). At 17 weeks of gestations, elevated levels of thyroid-stimulating hormone (TSH) and urinary iodine were detected (Addition file: Table S4). At 27 weeks of gestations, the mother was diagnosed with gestational diabetes mellitus (fasting blood glucose level, 7.38 mmol/L; postprandial one-hour blood glucose level, 15.30 mmol/L; postprandial two-hour blood glucose level, 11.03 mmol/L).
At 28 weeks of gestations, the proband (II-1) and her twin younger sister (II-2) were born via spontaneous vaginal delivery (Fig. 2a, b). The proband (II-1), who had been in persistent breech position on the left side of mother’s uterus (Fig. 2b), was first recorded by ultrasound scan at 23.5 weeks of gestation; her birth weight was 880 g (50-90 centile). The younger sister (II-2), who had been in cephalic position on the right side of mother’s uterus (Fig. 2b), weighted 990 g (90-97 centile) at birth. Only the proband presented the typical appearance of sagittal craniosynostosis (Fig. 2c). While no signs of craniofacial deformity were found in her parents (I-1, I-2) and sister (II-2) (Fig. 2c). Cranial index, which represents the ratio of maximum cranial width to maximum cranial length, is decreased in the head shape of sagittal craniosynostosis. In our case, the cranial index of the proband and her sister are 70.9%, 83.2%, respectively. Taken together, the clinical findings indicate that the proband, who developed sagittal craniosynostosis, had additional persistent breech presentation and intrauterine growth restriction, except for the perinatal risk factors shared by the twins.
The whole-exome sequencing (WES) was applied to uncover the potential genetic etiology leading to sagittal craniosynostosis in our case. Due to the low incidence rate of sagittal craniosynostosis, we focused on private and/or rare (minor allele frequency, MAF < 0.01) variants on exons or splicing sites (Fig. 1). However, none of candidate germline or somatic mutations were proband-specific. As gene-environment interactions have been revealed in the pathogenesis of craniosynostosis [5, 15], we wonder whether the adverse intrauterine exposures (environment) triggered the susceptible individual (II-1) to develop sagittal craniosynostosis. Based on this hypothesis, we re-analyzed our sequencing data and identified a heterozygous missense mutation of AXIN2 (c.1181G>A, p.R394H) in the leukocytes of subjects I-1, II-1 and II-2, and skull periosteum tissue of subject II-1. The findings were further validated by Sanger sequencing (Fig. 3a-b). The nucleotide sequence showed a heterozygous G to A transition at nucleotide 1181 (c.1181 G>A) of the coding sequence, which resulted in the replacement of Arg at residue 394 by His of AXIN2 protein.
Arg394, located at the GSK3β binding domain (amino acid 327 to 413 according to the UniProt Consortium) of the AXIN2 protein (Fig. 3c), is conserved across species (Fig. 3d, e) and was predicted to be potentially deleterious by in silico analysis (Table 1).
I-TASSER structural analysis predicted that the p. R394H substitution may affect the spatial structure of the GSK3β binding domain in AXIN2 protein (Fig. 4 a, b). Taken together, our results indicate that AXIN2 (c.1181G>A, p.R394H) mutation may confer deleterious of sagittal craniosynostosis.