Mutation Screening of Candidate Genes in Patients with Nonsyndromic Sagittal Craniosynostosis

Abstract

Background: Craniosynostosis is a condition that includes the premature fusion of one or multiple cranial sutures. Among various craniosynostosis forms, sagittal nonsyndromic craniosynostosis is the most prevalent. Although different gene mutations have been identified in some craniosynostosis syndromes, the cause of sagittal nonsyndromic craniosynostosis remains largely unknown.

Methods: To screen for candidate genes for sagittal nonsyndromic craniosynostosis, the authors sequenced DNA of 93 sagittal nonsyndromic craniosynostosis patients from a population-based study conducted in Iowa and New York states. FGFR1-3 mutational hotspots and the entire TWIST1, RAB23, and BMP2 coding regions were screened because of their known roles in human nonsyndromic or syndromic sagittal craniosynostosis, expression patterns, and/or animal model studies.

Results: The authors identified two rare variants in their cohort. A FGFR1 insertion c.730_731insG, which led to a premature stop codon, was predicted to abolish the entire immunoglobulin-like III domain, including the ligand-binding region. A c.439C>G variant was observed in TWIST1 at its highly conserved loop domain in another patient. The patient's mother harbored the same variant and was reported with jaw abnormalities. These two variants were not detected in 116 alleles from unaffected controls or seen in the several databases; however, TWIST1 variant was found in a low frequency of 0.000831 percent in Exome Aggregation Consortium database.

Conclusions: The low mutation detection rate indicates that these genes account for only a small proportion of sagittal nonsyndromic craniosynostosis patients. The authors' results add to the perception that sagittal nonsyndromic craniosynostosis is a complex developmental defect with considerable genetic heterogeneity.

Clinical question/level of evidence: Risk, II.

Fig. 1

Sequencing chromatogram of novel variants identified in this study. The arrows indicate the positions of the variants. (Left) TWIST1 c.439C>G; (Right) FGFR1 c.730_731insG.

Fig. 2

Assessment of the functional impact of the variant p.Q147E (c.439 C>G) in TWIST1. A reduced presentation of the multiple sequence alignment of the protein region with mutated residue produced by Mutation Assessor. The whole alignment is composed of 393 sequence homologs of TWIST1 (both human and non-human) and divided into 29 subfamilies; only 50 sequences and 4 subfamilies are shown. The positions in the alignment are assessed by evolutionary conservation within entire protein family and by specificity, i.e. evolutionary conservation within subfamilies; the lengths of the bars above the alignment represent the relative strength of conservation and specificity in positions of the multiple alignment. The residues, which are conserved within subfamilies, i.e. specific to the subfamilies, are typically responsible for the actual binding specificity of proteins. The mutated residue is located in one of the top specificity positions, both the original glutamine and the mutated glutamic acid residues are represented by subfamilies; however the dominant residue in this position is a glutamic acid.

Fig. 3

The 3D view of the complex of the neurogenic differentiation factor 1 (mouse homolog of TWIST1) and the transcription factor E2-alpha bound to DNA (PDB code 2QL2). The conserved and the specificity residues derived from multiple sequence alignment of TWIST1 homologs are mapped on the 3D structure of the neurogenic differentiation factor 1 and shown in blue and orange, respectively; DNA fragment is shown in violet; an alpha-helix fragment of E2-alpha is shown in green. The glutamic acid E147 in the position of the mutated residue (shown in dark red) contributes to the complex formation by interacting with the charged residues of the arginine 600 and the main chain of the glutamic acid 601 of E2-alpha protein; it also interacts with the hydroxyl group of serine 138 of TWIST1. The mutation of the glutamine Q147 to glutamic acid in TWIST1 is predicted to affect the stability, the complex and the regulation of transcription performed by TWIST1 molecule.