The overall prevalence of Craniosynotosis was estimated to be 1/2100-2500 births [1-9]. Although most instances of craniosynostosis are nonsyndromic, craniosynostosis often occur together with other anomalies in well-defined patterns as clinically recognized syndromes. The most commonly identified craniosynostosis syndromes include Crouzon, Saethre-Chotzen, Apert, Pfeiffer and Muenke syndromes, which are typically hereditary diseases and most of them, are with autosomal dominant inheritance.

Clinically, these syndromes share some common characteristics in addition to the abnormality of craniofacial features, and the digital anomalies may be the sole differentiating physical finding to allow a clinical diagnosis. High-throughput mutation screening of this group of disorders is now possible by using target enrichment followed by Next-Generation Sequencing (NGS).

In our report, we observed a three-generation family presenting the clinical special features including plagiocephaly, flat face, flat occiput, hypertelorism, ptosis of eyelids, proptosis, deviated nose to left side and especially broad thumbs and toes. Robinow-Sorauf syndrome, an allelic variation of Saethre-Chotzen syndrome was confirmed by typical clinic features and mutation of TWIST1 gene in this Chinese pedigree. This is the first reported cases of RSS in China so far.

A Chinese three generation pedigree with four affected members including two males and two females were recruited in 2017 from the Center of Genetics and Prenatal Diagnosis of the First Affiliated Hospital of Zhengzhou University. Clinical features including skull deformity, facial appearance, and distance between pupils of the 40- years old proband, his mother, sister and his 2-years old nephew were measured [10-16] Radiology of skull, hand and feet of the proband and his nephew were done.

Written consents were taken from the participants or the parents of the patient if the affected individual was under 18 years old. The institutional Ethical Review Board in Zhengzhou University approved the consent forms.

Peripheral blood samples were extracted from the proband, his mother, sister and nephew by Tiangen Extraction kit (Beijing, China). Genomic DNA of 3 μg was used for library preparation according to the manufacturer’s instruction (MyGenostics, Beijing, China), and the final library size ranging from 350 to 450 bp, containing adapter sequences was used in the following experiment.

A panel of genes including FGFR1, FGFR2, FGFR3, POR, MSX2, TWIST1, RECQL4 and RAB23 was captured with OncoCap Enrichment System (MyGenostics, Beijing, China) based on their establish procedures.

After enrichment, libraries were sequenced on an Illumina Solexa HiSeq 2000 sequencer for paired reads of 100 bp followed by data retrieval using Solexa QA package and a cutadapt program. Expected coverage was 99% of the targeted genomic regions of interest, which achieved an average alignment performance of 98% across all samples. The obtained sequences were aligned to the reference genome (GRCh37/hg19 ) to detect discrepancies. PCR and Sanger sequencing were performed to confirm the candidate mutation using an ABI 3130XL automatic genetic analyzer (Applied Biosystems, Foster City, CA).

The History of this Chinese family was illustrated by the pedigree (Figure 1).

Figure 1: The pedigree of this present Chinese family.

Clinical features and measurements of interest were noted and the data were tabulated as shown in Table 1.

Table 1: Clinical features of 4 patients in the present pedigree.

The proband was 30 years old with chief complaint of being different in appearance from his peers. He has a flat face, hypertelorism and nose deviation. Head examination showed skull deformity as plagiocephaly, flat occiput, and head circumference of 55cm. Broad thumbs and toes with partially bifid end phanlanx were seen Figure 2.

Figure 2: Phenotypic features of the proband.

He was otherwise well built, well nourished, and has normal intelligence. The mother (Figure 3), sister, and nephew of the proband (Figure 4) had the similar phenotypes as the proband including plagiocephaly, flat face, flat occiput, ptosis, proptosis and hypertelorism. Except from the other affected individuals with deviated nose, clinodactyly, broad thumbs and toes, the 2 years old nephew of the proband has normal nose and thumbs.

Figure 3: Phenotypic features of the mother of the proband.

Figure 4: Phenotypic features of the nephew of the proband.

In this pedigree, a heterozygous missense mutation c.395G>C of TWIST1 gene leading to the substitution of amino acid proline by arginine (p.P132R) was identified by NGS and confirmed by Sanger sequencing in all of the 4 patients of three generations Figure 5.

Figure 5: Sanger sequencing analysis of TWIST1 gene: c.395G>C was seen in the patients of this family (a), normal alleles were seen in normal controls (b).

More than 150 syndromes have been found to be associated with craniosynostosis. However, it is not easy to give an accurate diagnosis without genetic confirmation since they share similar symptoms. A multiple genes have been found to be associated with syndromic craniosynostosis. One single gene may be involved in various syndromic craniosynostosis, such as mutation of FGFR2 may present diverse clinical syndromes including Pfeiffer syndrome [14], and Crouzon syndrome [7]. Also, one syndrome could be caused by different genes. Mutations of FGFR2 and POR have been identified to cause Antley-Bixler syndrome [10] and both FGFR2 and FGFR1 mutation could cause Pfeiffer syndrome [16]. Eight known genes including FGFR1, FGFR2, FGFR3, POR, MSX2, TWIST1, RECQL4 and RAB23 have been found as the most common causative genes involved in craniosynostosis syndromes [11]. Genetic test may be an optimal method to give an accurate diagnosis, which may not have a direct impact on the management of the patients in many cases, but could provide accurate prenatal diagnosis [5]. NGS is a very efficient method to identify the etiology of patients with syndromic craniosynostosis.

In the present study, NGS involving 8 genes most frequently associated with craniosynostosis was performed in a Chinese pedigree with four affected members with obvious craniofacial dysmorphisms. This pedigree was diagnosed as Robinow-Sorauf Syndrome (RSS) through their typically craniofacial and digital features and a heterozygous missense mutation c.395G>C (p.R132P) of TWIST1 gene identified by NGS. This mutation is within the DNA binding helix I domain, which has been reported in one family previously [12].

The original case of RSS was reported by Robinow and Sorauf in 1975 [13] then more cases were reported worldwide, but it has never been reported in Chinese populations. The clinical features of RSS include craniosynostosis, plagiocephaly, flat face, hypertelorism, shallow orbits, strabismus, and broad great toes/thumbs with bifid or partially duplicated distal phalanx. Clinically, even with these typical digital anomalies, diagnosing RSS is almost impossible due to overlapping features of other craniosynostosis disorders including Apert syndrome, Crouzon syndrome, Saethre-Chotzen syndrome and Pfeiffer syndrome. Confirmation of RSS could be made not only after exclusion of other craniosynostosis syndrome clinically but based on the genes involved. Next Generation Sequencing is an optimal method to identify the possible causative gene mutation and give differential diagnosis. TWIST1 gene encodes a basic helix-loop-helix (bHLH) transcription factor that was reported to be involved in mesenchymal cell development of cranium. TWIST protein negatively regulates skeletogenesis by interacting with RUNX2 , which is a key transcription factor associated with the development of head and limbs. A loss-offunction of TWIST1 will result in premature closure of sutures while a loss-of-function of RUNX2 will cause a delay of closure. TWIST1 also has been thought to be interacted with FGFR during fetal development [6]. There is no single pathway that caused craniosynostosis, and several independent mechanisms involving extracellular matrixmediated focal adhesion and Ffg/Wnt/Igf signaling pathways may attribute its pathogenesis [15]. Different mutations including missense mutation, nonsense mutation, insertion and whole gene deletion occurring within the exon1 of TWIST1 gene may result in loss-offunction of TWIST 1 [4], leading to skeletal dysplasia including craniosynostosis and limb deformities. TWIST1 is the causing gene for both RSS and Saethre-Chotzen syndrome. However, it seems no apparent correlation between the physical manifestations of the syndromes and the location of TWIST1 mutations [3]. In summary, the affected patients of this Chinese family in the present study were diagnosed as Robinow-Sorauf Syndrome by typical clinical findings and NGS with a heterozygous mutation of TWIST1 gene (c.395G>C, p.P132R), which has not been reported in Chinese population. Craniosynostosis is an etiologically heterogeneous disorder resulting from environmental factors as well as genetics causes, and NGS can be an essential and efficient workup to clarify the genetic etiology.

The authors declare that they have no conflict of interest.