Jacqueline A.C. Goos

Mutations in TCF12 , encoding a basic helix-loop-helix partner of TWIST1, are a frequent cause of coronal craniosynostosis

Craniosynostosis, the premature fusion of the cranial sutures, is a heterogeneous disorder with a prevalence of ∼1 in 2,200 (refs. 1,2). A specific genetic etiology can be identified in ∼21% of cases, including mutations of TWIST1, which encodes a class II basic helix-loop-helix (bHLH) transcription factor, and causes Saethre-Chotzen syndrome, typically associated with coronal synostosis. Using exome sequencing, we identified 38 heterozygous TCF12 mutations in 347 samples from unrelated individuals with craniosynostosis. The mutations predominantly occurred in individuals with coronal synostosis and accounted for 32% and 10% of subjects with bilateral and unilateral pathology, respectively. TCF12 encodes one of three class I E proteins that heterodimerize with class II bHLH proteins such as TWIST1. We show that TCF12 and TWIST1 act synergistically in a transactivation assay and that mice doubly heterozygous for loss-of-function mutations in Tcf12 and Twist1 have severe coronal synostosis. Hence, the dosage of TCF12-TWIST1 heterodimers is critical for normal coronal suture development.

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.

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.

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

Craniofrontonasal 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.

A novel mutation in FGFR2

Craniosynostosis is a congenital anomaly that can occur as an isolated condition or as part of a syndrome. Although several genes are known to cause syndromic craniosynostosis, only 24% can be attributed to known genes. Therefore, it is likely that more mutations and other genes are involved. We present the identification of a novel point mutation in fibroblast growth factor receptor 2 (FGFR2), c.812G>T, p.(Gly271Val) or c.1851G>C, p.(Leu617Phe). Furthermore, we describe a mutation that has been identified just recently, c.812G>T, (p.Gly271Val) or c.1851G>C, (p.Leu617Phe). In addition, we describe findings from a sequence analysis of all coding exons and exon/intron boundaries of FGFR2 performed on 124 patients with syndromic craniosynostosis.

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

BackgroundMutations 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 presentationHere 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.ConclusionsThese 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.

Genetic Causes of Craniosynostosis: An Update

In 1993, Jabs et al. were the first to describe a genetic origin of craniosynostosis. Since this discovery, the genetic causes of the most common syndromes have been described. In 2015, a total of 57 human genes were reported for which there had been evidence that mutations were causally related to craniosynostosis. Facilitated by rapid technological developments, many others have been identified since then. Reviewing the literature, we characterize the most common craniosynostosis syndromes followed by a description of the novel causes that were identified between January 2015 and December 2017.

De Novo and Inherited Loss-of-Function Variants in TLK2: Clinical and Genotype-Phenotype Evaluation of a Distinct Neurodevelopmental Disorder

Next-generation sequencing is a powerful tool for the discovery of genes related to neurodevelopmental disorders (NDDs). Here, we report the identification of a distinct syndrome due to de novo or inherited heterozygous mutations in Tousled-like kinase 2 (TLK2) in 38 unrelated individuals and two affected mothers, using whole-exome and whole-genome sequencing technologies, matchmaker databases, and international collaborations. Affected individuals had a consistent phenotype, characterized by mild-borderline neurodevelopmental delay (86%), behavioral disorders (68%), severe gastro-intestinal problems (63%), and facial dysmorphism including blepharophimosis (82%), telecanthus (74%), prominent nasal bridge (68%), broad nasal tip (66%), thin vermilion of the upper lip (62%), and upslanting palpebral fissures (55%). Analysis of cell lines from three affected individuals showed that mutations act through a loss-of-function mechanism in at least two case subjects. Genotype-phenotype analysis and comparison of computationally modeled faces showed that phenotypes of these and other individuals with loss-of-function variants significantly overlapped with phenotypes of individuals with other variant types (missense and C-terminal truncating). This suggests that haploinsufficiency of TLK2 is the most likely underlying disease mechanism, leading to a consistent neurodevelopmental phenotype. This work illustrates the power of international data sharing, by the identification of 40 individuals from 26 different centers in 7 different countries, allowing the identification, clinical delineation, and genotype-phenotype evaluation of a distinct NDD caused by mutations in TLK2.

Identification of causative variants in TXNL4A in Burn-McKeown syndrome and isolated choanal atresia

Burn-McKeown syndrome (BMKS) is a rare syndrome characterized by choanal atresia, prominent ears, abnormalities of the outer third of the lower eyelid, structural cardiac abnormalities, conductive and sensorineural hearing loss, and cleft lip. Recently, causative compound heterozygous variants were identified in TXNL4A. We analyzed an individual with clinical features of BMKS and her parents by whole-genome sequencing and identified compound heterozygous variants in TXNL4A (a novel splice site variant (c.258-2A>G, (p.?)) and a 34 bp promoter deletion (hg19 chr18:g.77748581_77748614del (type 1Δ) in the proband). Subsequently, we tested a cohort of 19 individuals with (mild) features of BMKS and 17 individuals with isolated choanal atresia for causative variants in TXNL4A by dideoxy-sequence analysis. In one individual with BMKS unrelated to the first family, we identified the identical compound heterozygous variants. In an individual with isolated choanal atresia, we found homozygosity for the same type 1Δ promoter deletion, whilst in two cousins from a family with choanal atresia and other minor anomalies we found homozygosity for a different deletion within the promoter (hg19 chr18: g.77748604_77748637del (type 2Δ)). Hence, we identified causative recessive variants in TXNL4A in two individuals with BMKS as well as in three individuals (from two families) with isolated choanal atresia.

Mutations of TCF12, encoding a basic-helix-loop-helix partner of TWIST1, are a frequent cause of coronal craniosynostosis

Abstract Background Craniosynostosis, the premature fusion of the cranial sutures, is the second most common craniofacial malformation. A genetic aetiology can be identified in about 21% of cases, including mutations of TWIST1 that cause Saethre-Chotzen syndrome and are associated with coronal synostosis. By contrast, the cause of non-syndromic craniosynostosis is largely unknown. Methods We undertook exome sequencing of seven individuals with bilateral coronal synostosis, identifying mutations of TCF12 in three samples. We sequenced TCF12 in a further 347 patients with craniosynostosis. We performed mutation testing in extended families and determined the effects of mutations on mRNA expression. We examined the genetic interaction between loss-of-function mutations of Tcf12 and Twist1 in mice. Findings Heterozygous TCF12 mutations were present in 38 of the 347 unrelated patients with craniosynostosis. These included 22 (32%) of 69 with isolated bilateral coronal synostosis, and 14 (10%) of 141 with unilateral coronal synostosis, but none with premature fusion of only the metopic, sagittal, or lambdoid sutures (p −6 ). An additional two patients had either bilateral or right coronal synostosis in addition to sagittal synostosis. 14 cases arose de novo, but vertical transmission was demonstrated in 23 families. 16 mutation-positive individuals (47%) had craniosynostosis or suspicious clinical features, but 19 (53%) of 35 mutation-positive relatives were non-penetrant for craniosynostosis. TCF12 mutations were associated with diminished mRNA expression, indicating haploinsufficiency. With appropriate surgical correction the clinical outcome was usually good; six of 66 individuals had learning disability. Mice doubly heterozygous for Twist1 and Tcf12 mutations had severe bilateral coronal synostosis. Interpretation Mutations of TCF12 are a frequent and specific cause of coronal craniosynostosis; mutation testing is indicated in the assessment of these patients. Genetic interaction of Tcf12 and Twist1 is crucial for coronal suture development. Funding National Institute for Health Research Biomedical Research Centre, Oxford (BRC)/Oxford University Clinical Academic Graduate School (OUCAGS), and Oxfordshire Health Services Research Committee (OHSRC), Oxford Craniofacial Unit Charitable Fund; Thames Valley Comprehensive Local Research Network; The Dutch Center for Translational Molecular Medicine; Carolien Bijl Foundation; US National Institutes for Health; Wellcome Trust.