Facing the Future of Craniofacial Genetics

“It is the common wonder of all men, how among so many millions of faces, there should be none alike,” wrote Sir Thomas Browne in Religio Medici published in 1643. Ever since, people have been intrigued by the development of the human head and face and its biodiversity. Nowadays, we know that both nurture and nature, like cellular and molecular pathways, play a key role in this craniofacial development. If the cellular and/or molecular pathways are disturbed by gene mutations a malformation can occur. The aim of this thesis is to explore the genetic alterations that cause craniofacial malformations using next generation sequencing

Distribution of glycine/GABA neurons in the ventromedial medulla with descending spinal projections and evidence for an ascending glycine/GABA projection

The ventromedial medulla (VM), subdivided in a rostral (RVM) and a caudal (CVM) part, has a powerful influence on the spinal cord. In this study, we have identified the distribution of glycine and GABA containing neurons in the VM with projections to the cervical spinal cord, the lumbar dorsal horn, and the lumbar ventral horn. For this purpose, we have combined retrograde tracing using fluorescent microspheres with fluorescent in situ hybridization (FISH) for glycine transporter 2 (GlyT2) and GAD67 mRNAs to identify glycinergic and/or GABAergic (Gly/GABA) neurons. Since the results obtained with FISH for GlyT2, GAD67, or GlyT2+GAD67 mRNAs were not significantly different, we concluded that glycine and GAB

Apparently synonymous substitutions in FGFR2 affect splicing and result in mild Crouzon syndrome

Background: Mutations of fibroblast growth factor receptor 2 (FGFR2) account for a higher proportion of genetic cases of craniosynostosis than any other gene, and are associated with a wide spectrum of severity of clinical problems. Many of these mutations are highly recurrent and their associated features well documented. Crouzon syndrome is typically caused by heterozygous missense mutations in the third immunoglobulin domain of FGFR2.Case presentation: Here we describe two families, each segregating a different, previously unreported FGFR2 mutation of the same nucleotide, c.1083A>G and c.1083A>T, both of which encode an apparently synonymous change at the Pro361 codon. We provide experimental evidence that these mutations affect normal FGFR2 splicing and document the clinical consequences, which include a mild Crouzon syndrome phenotype and reduced penetrance of craniosynostosis.Conclusions: These observations add to a growing list of FGFR2 mutations that affect splicing and provide important clinical information for genetic counselling of families affected by these specific mutations

Gain-of-Function Mutations in ZIC1 Are Associated with Coronal Craniosynostosis and Learning Disability

Human ZIC1 (zinc finger protein of cerebellum 1), one of five homologs of the Drosophila pair-rule gene odd-paired, encodes a transcription factor previously implicated in vertebrate brain development. Heterozygous deletions of ZIC1 and its nearby paralog ZIC4 on chromosome 3q25.1 are associated with Dandy-Walker malformation of the cerebellum, and loss of the orthologous Zic1 gene in the mouse causes cerebellar hypoplasia and vertebral defects. We describe individuals from five families with heterozygous mutations located in the final (third) exon of ZIC1 (encoding four nonsense and one missense change) who have a distinct phenotype in which severe craniosynostosis, specifically involving the coronal sutures, and variable learning disability are the most characteristic features. The location of the nonsense mutations predicts escape of mutant ZIC1 transcripts from nonsense-mediated decay, which was confirmed in a cell line from an affected individual. Both nonsense and missense mutations are associated with altered and/or enhanced expression of a target gene, engrailed-2, in a Xenopus embryo assay. Analysis of mouse embryos revealed a localized domain of Zic1 expression at embryonic days 11.5-12.5 in a region overlapping the supraorbital regulatory center, which patterns the coronal suture. We conclude that the human mutations uncover a previously unsuspected role for Zic1 in early cranial suture development, potentially by regulating engrailed 1, which was previously shown to be critical for positioning of the murine coronal suture. The diagnosis of a ZIC1 mutation has significant implications for prognosis and we recommend genetic testing when common causes of coronal synostosis have been excluded

Gain-of-Function Mutations in ZIC1 Are Associated with Coronal Craniosynostosis and Learning Disability

Human ZIC1 (zinc finger protein of cerebellum 1), one of five homologs of the Drosophila pair-rule gene odd-paired, encodes a transcription factor previously implicated in vertebrate brain development. Heterozygous deletions of ZIC1 and its nearby paralog ZIC4 on chromosome 3q25.1 are associated with Dandy-Walker malformation of the cerebellum, and loss of the orthologous Zic1 gene in the mouse causes cerebellar hypoplasia and vertebral defects. We describe individuals from five families with heterozygous mutations located in the final (third) exon of ZIC1 (encoding four nonsense and one missense change) who have a distinct phenotype in which severe craniosynostosis, specifically involving the coronal sutures, and variable learning disability are the most characteristic features. The location of the nonsense mutations predicts escape of mutant ZIC1 transcripts from nonsense-mediated decay, which was confirmed in a cell line from an affected individual. Both nonsense and missense mutations are associated with altered and/or enhanced expression of a target gene, engrailed-2, in a Xenopus embryo assay. Analysis of mouse embryos revealed a localized domain of Zic1 expression at embryonic days 11.5-12.5 in a region overlapping the supraorbital regulatory center, which patterns the coronal suture. We conclude that the human mutations uncover a previously unsuspected role for Zic1 in early cranial suture development, potentially by regulating engrailed 1, which was previously shown to be critical for positioning of the murine coronal suture. The diagnosis of a ZIC1 mutation has significant implications for prognosis and we recommend genetic testing when common causes of coronal synostosis have been excluded

A de novo substitution in BCL11B leads to loss of interaction with transcriptional complexes and craniosynostosis

Craniosynostosis, the premature ossification of cranial sutures, is a developmental disorder of the skull vault, occurring in approximately 1 in 2250 births. The causes are heterogeneous, with a monogenic basis identified in ~25% of patients. Using whole-genome sequencing, we identified a novel, de novo variant in BCL11B, c.7C>A, encoding an R3S substitution (p.R3S), in a male patient with coronal suture synostosis. BCL11B is a transcription factor that interacts directly with the nucleosome remodelling and deacetylation complex (NuRD) and polycomb-related complex 2 (PRC2) through the invariant proteins RBBP4 and RBBP7. The p.R3S substitution occurs within a conserved amino-terminal motif (RRKQxxP) of BCL11B and reduces interaction with both transcriptional complexes. Equilibrium binding studies and molecular dynamics simulations show that the p.R3S substitution disrupts ionic coordination between BCL11B and the RBBP4-MTA1 complex, a subassembly of the NuRD complex, and increases the conformational flexibility of Arg-4, Lys-5 and Gln-6 of BCL11B. These alterations collectively reduce the affinity of BCL11B p.R3S for the RBBP4-MTA1 complex by nearly an order of magnitude. We generated a mouse model of the BCL11B p.R3S substitution using a CRISPR-Cas9-based approach, and we report herein that these mice exhibit craniosynostosis of the coronal suture, as well as other cranial sutures. This finding provides strong evidence that the BCL11B p.R3S substitution is causally associated with craniosynostosis and confirms an important role for BCL11B in the maintenance of cranial suture patency

Rapid Low-Cost Microarray-Based Genotyping for Genetic Screening in Primary Immunodeficiency

Background: Genetic tests for primary immunodeficiency disorders (PIDs) are expensive, time-consuming, and not easily accessible in developing countries. Therefore, we studied the feasibility of a customized single nucleotide variant (SNV) microarray that we developed to detect disease-causing variants and copy number variation (CNV) in patients with PIDs for only 40 Euros. Methods: Probes were custom-designed to genotype 9,415 variants of 277 PID-related genes, and were added to the genome-wide Illumina Global Screening Array (GSA). Data analysis of GSA was performed using Illumina GenomeStudio 2.0, Biodiscovery Nexus 10.0, and R-3.4.4 software. Validation of genotype calling was performed by comparing the GSA with whole-genome sequencing (WGS) data of 56 non-PID controls. DNA samples of 95 clinically diagnosed PID patients, of which 60 patients (63%) had a genetically established diagnosis (by Next-Generation Sequencing (NGS) PID panels or Sanger sequencing), w

Diagnostic value of exome and whole genome sequencing in craniosynostosis

Background Craniosynostosis, the premature fusion of one or more cranial sutures, occurs in ~1 in 2250 births, either in isolation or as part of a syndrome. Mutations in at least 57 genes have been associated with craniosynostosis, but only a minority of these are included in routine laboratory genetic testing. Methods We used exome or whole genome sequencing to seek a genetic cause in a cohort of 40 subjects with craniosynostosis, selected by clinical or molecular geneticists as being high-priority cases, and in whom prior clinically driven genetic testing had been negative. Results We identified likely associated mutations in 15 patients (37.5%), involving 14 different genes. All genes were mutated in single families, except for IL11RA (two families). We classified the other positive diagnoses as follows: commonly mutated craniosynostosis genes with atypical presentation (EFNB1, TWIST1); other core craniosynostosis genes (CDC45, MSX2, ZIC1); genes for which mutations are only rarely associated with craniosynostosis (FBN1, HUWE1, KRAS, STAT3); and known disease genes for which a causal relationship with craniosynostosis is currently unknown (AHDC1, NTRK2). In two further families, likely novel disease genes are currently undergoing functional validation. In 5 of the 15 positive cases, the (previously unanticipated) molecular diagnosis had immediate, actionable consequences for either genetic or medical management (mutations in EFNB1, FBN1, KRAS, NTRK2, STAT3). Conclusions This substantial genetic heterogeneity, and the multiple actionable mutations identified, emphasises the benefits of exome/whole genome sequencing to identify causal mutations in craniosynostosis cases for which routine clinical testing has yielded negative results

Assessment of volumetric changes with a best-fit method in three-dimensional stereophotograms

Objective: Different three-dimensional stereophotogrammetry systems and analyzing methods exist that often use landmarks for comparison. Measurement errors in landmark or surface comparison are mostly within 1 mm, which seems clinically acceptable. The aim of this study was to validate a three-dimensional stereophotogrammetric best-fit method of assessing volumetric changes and to compare three devices. Methods: The validation of the best-fit method was at first done on a life-size dummy head. Scans were made in the ideal position, as well as in four additional positions, and a scan was made in which a soft putty specimen was added to the dummy head. The comparison was executed with a best-fit method using triangulation. Student's t tests were used to detect statistically significant differences. Second, comparisons were made among scans of a white man in the ideal position and with volume changes added. Results: The different positions tested for the dummy head showed no significant volume differences within each system or among systems. The differences found when adding a soft putty specimen fell into the same range as the differences between various positions. The differences within a live situation were 10 times greater compared with the dummy-head situation. Conclusions: In a dummy-head situation, the different systems gave similar results when tested with a best-fit method. However, in live situations the differences may become 10 times greater, possibly due to different facial expressions. These differences may become clinically relevant and, therefore, further research in volumetric changes is needed

Phenotypes of craniofrontonasal syndrome in patients with a pathogenic mutation in EFNB1

raniofrontonasal syndrome (CFNS) is an X-linked developmental malformation, caused by mutations in the EFNB1 gene, which have only been described since 2004. A genotype–phenotype correlation seems not to be present. As it is of major importance to adequately counsel patients with EFNB1 mutations and their parents, and to improve diagnosis of new patients, more information about the phenotypic features is needed. This study included 23 patients (2 male, 21 female) with confirmed EFNB1 mutations. All patients underwent a thorough physical examination and photographs were taken. If available, radiological images were also consulted. Hypertelorism, longitudinal ridging and/or splitting of nails, a (mild) webbed neck and a clinodactyly of one or more toes were the only consistent features observed in all patients. Frequently observed phenotypic features were bifid tip of the nose (91%), columellar indentation (91%) and low implantation of breasts (90%). In comparison with anthropometric data of facial proportions, patients with CFNS had a significantly different face in multiple respects. An overview of all phenotypic features is shown. Patients with EFNB1 mutations have a clear phenotype. This study will facilitate genetic counseling of parents and patients, and contribute to the diagnostic and screening process of patients with suspected CFNS