Unexpected role of SIX1 variants in craniosynostosis: expanding the phenotype of SIX1-related disorders

Background Pathogenic heterozygous SIX1 variants (predominantly missense) occur in branchio-otic syndrome (BOS), but an association with craniosynostosis has not been reported. Methods We investigated probands with craniosynostosis of unknown cause using whole exome/genome (n=628) or RNA (n=386) sequencing, and performed targeted resequencing of SIX1 in 615 additional patients. Expression of SIX1 protein in embryonic cranial sutures was examined in the Six1 nLacZ/+ reporter mouse. Results From 1629 unrelated cases with craniosynostosis we identified seven different SIX1 variants (three missense, including two de novo mutations, and four nonsense, one of which was also present in an affected twin). Compared with population data, enrichment of SIX1 loss-of-function variants was highly significant (p=0.00003). All individuals with craniosynostosis had sagittal suture fusion; additionally four had bilambdoid synostosis. Associated BOS features were often attenuated; some carrier relatives appeared non-penetrant. SIX1 is expressed in a layer basal to the calvaria, likely corresponding to the dura mater, and in the mid-sagittal mesenchyme. Conclusion Craniosynostosis is associated with heterozygous SIX1 variants, with possible enrichment of loss-of-function variants compared with classical BOS. We recommend screening of SIX1 in craniosynostosis, particularly when sagittal±lambdoid synostosis and/or any BOS phenotypes are present. These findings highlight the role of SIX1 in cranial suture homeostasis.


Supplementary Information Page
Supplementary case reports 1 Table S1. Primer sequences and methods used for PCR amplification 8 Table S2. Phenotypic composition of cohorts of unsolved CRS probands 9 Table S3. Previously described SIX1 variants in BOS/BOR/HL 10 Box S1. Protein sequence alignments of all human SIX-family paralogues and   Both twins were referred for endocrinological assessment, initially at the age of 3 yr, because of poor weight gain and growth. Aged 4 yr 3 mo their weights were 13.2 kg (-2.24 SD) and 11.5 kg (-3.7 SD), heights 91.5 cm (-2.9 SD) and 88 cm (-3.7 SD), and OFCs 51 cm (+0.3 SD) and 49 cm (-1.1 SD) in twins 1 and 2, respectively. Both boys had a similar triangular-shaped face, with retrognathia, prominent ears and a right-sided scar in the neck and both had bilateral clinodactyly of the 5 th fingers. They had normal developmental milestones and were performing well at a regular school at the age of 7 yr.
Additional investigations included brainstem auditory evoked responses, which were abnormal in the right ear in twin 2, and echocardiogram and abdominal ultrasound, which were normal in both twins. The 2 nd twin, who exhibited more impaired growth, was started on growth hormone treatment at the age of 6 yr.
Genetic investigation using ES did not reveal any rare variant in a known gene associated with craniosynostosis, but identified a heterozygous variant in SIX1, c.31C>T (p.Q11*), in twin 2 (this region was covered by only one normal read in twin 1). The variant was confirmed by dideoxy-sequencing in both twins and in their mother, who presented with a preauricular pit only.
The male proband (ID L112-1) was born full-term to healthy parents after an uncomplicated pregnancy. An abnormal head shape was noted by the child's paediatrician at age 2 yr; a prominent mastoid bulge on the right side prompted a CT scan at this time, demonstrating sagittal and bilateral lambdoid synostosis. The patient underwent calvarial vault remodelling just before age 3 yr. At age 4 yr, the child underwent formal hearing assessment by an audiologist who identified conductive HL in the left ear. Both the family and paediatrician reported normal neurodevelopment at this time, however no formal neurocognitive testing was performed. The proband's only other medical condition is a branchial cyst with fistulization to the medial neck that periodically becomes inflamed and drains purulent material, which has been managed with antibiotic therapy to date. No renal ultrasound has been performed.
At age 4 yr, the parents enrolled their family in a genetic study of lambdoid craniosynostosis through Yale University. The case-parent trio underwent exome sequencing, which did not reveal any exonic de novo variants in the proband, rare recessive genotypes, or pathogenic variants in known craniosynostosis genes. Further evaluation for transmitted damaging variants identified a maternally inherited nonsense mutation in SIX1  This girl (ID 5692) has healthy non-consanguineous Asian parents. There is no family history of renal problems, deafness or congenital abnormality. She presented with an unusual head shape, which was shown by CT scanning to be caused by a combination of sagittal and bilambdoid synostosis and was associated with a type 1 Chiari malformation.
Additional clinical features were long, down-slanting palpebral fissures, prominent eyes and an apparent epibulbar dermoid of the right eye. A diagnosis of Crouzon syndrome was  Although an elongated head shape was noticed at birth, this was not investigated at the time. At the age of 2 yr 2 mo, during a hospital admission for a febrile convulsion, the head shape was again noted and a CT scan demonstrated sagittal synostosis. On craniofacial assessment at the age of 2 yr 6 mo, his OFC was 47 cm (-1.5 SD) and CI 0.72; scaphocephaly with moderate frontal bossing and an occipital bullet were confirmed. The parents mentioned that they had noticed lumps in the neck bilaterally, but were reassured.   Table S1. Primer sequences and methods used for PCR amplification Polymerase (NEB). The indexed PCR products were pooled, and the library was purified and sequenced using MiSeq 500 or 600 cycle reagent kits on the MiSeq platform (Illumina) as previously described. 10 The deleteriousness of identified variants was predicted using CADD scores (CADD GRCh38-v1.6). 11 BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance