Development of Erf-Mediated Craniosynostosis and Pharmacological Amelioration.

ETS2 repressor factor (ERF) insufficiency causes craniosynostosis (CRS4) in humans and mice. ERF is an ETS domain transcriptional repressor regulated by Erk1/2 phosphorylation via nucleo-cytoplasmic shuttling. Here, we analyze the onset and development of the craniosynostosis phenotype in an Erf-insufficient mouse model and evaluate the potential of the residual Erf activity augmented by pharmacological compounds to ameliorate the disease. Erf insufficiency appears to cause an initially compromised frontal bone formation and subsequent multisuture synostosis, reflecting distinct roles of Erf on the cells that give rise to skull and facial bones. We treated animals with Mek1/2 and nuclear export inhibitors, U0126 and KPT-330, respectively, to increase Erf activity by two independent pathways. We implemented both a low dosage locally over the calvaria and a systemic drug administration scheme to evaluate the possible indirect effects from other systems and minimize toxicity. The treatment of mice with either the inhibitors or the administration scheme alleviated the synostosis phenotype with minimal adverse effects. Our data suggest that the ERF level is an important regulator of cranial bone development and that pharmacological modulation of its activity may represent a valid intervention approach both in CRS4 and in other syndromic forms of craniosynostosis mediated by the FGFR-RAS-ERK-ERF pathway.

Localized TWIST1 and TWIST2 basic domain substitutions cause four distinct human diseases that can be modeled in Caenorhabditis elegans.

Twist transcription factors, members of the basic helix-loop-helix family, play crucial roles in mesoderm development in all animals. Humans have two paralogous genes, TWIST1 and TWIST2, and mutations in each gene have been identified in specific craniofacial disorders. Here, we describe a new clinical entity, Sweeney-Cox syndrome, associated with distinct de novo amino acid substitutions (p.Glu117Val and p.Glu117Gly) at a highly conserved glutamic acid residue located in the basic DNA binding domain of TWIST1, in two subjects with frontonasal dysplasia and additional malformations. Although about one hundred different TWIST1 mutations have been reported in patients with the dominant haploinsufficiency Saethre-Chotzen syndrome (typically associated with craniosynostosis), substitutions uniquely affecting the Glu117 codon were not observed previously. Recently, subjects with Barber-Say and Ablepharon-Macrostomia syndromes were found to harbor heterozygous missense substitutions in the paralogous glutamic acid residue in TWIST2 (p.Glu75Ala, p.Glu75Gln and p.Glu75Lys). To study systematically the effects of these substitutions in individual cells of the developing mesoderm, we engineered all five disease-associated alleles into the equivalent Glu29 residue encoded by hlh-8, the single Twist homolog present in Caenorhabditis elegans. This allelic series revealed that different substitutions exhibit graded severity, in terms of both gene expression and cellular phenotype, which we incorporate into a model explaining the various human disease phenotypes. The genetic analysis favors a predominantly dominant-negative mechanism for the action of amino acid substitutions at this highly conserved glutamic acid residue and illustrates the value of systematic mutagenesis of C. elegans for focused investigation of human disease processes.

Mutations in CDC45, Encoding an Essential Component of the Pre-initiation Complex, Cause Meier-Gorlin Syndrome and Craniosynostosis.

DNA replication precisely duplicates the genome to ensure stable inheritance of genetic information. Impaired licensing of origins of replication during the G1 phase of the cell cycle has been implicated in Meier-Gorlin syndrome (MGS), a disorder defined by the triad of short stature, microtia, and a/hypoplastic patellae. Biallelic partial loss-of-function mutations in multiple components of the pre-replication complex (preRC; ORC1, ORC4, ORC6, CDT1, or CDC6) as well as de novo stabilizing mutations in the licensing inhibitor, GMNN, cause MGS. Here we report the identification of mutations in CDC45 in 15 affected individuals from 12 families with MGS and/or craniosynostosis. CDC45 encodes a component of both the pre-initiation (preIC) and CMG helicase complexes, required for initiation of DNA replication origin firing and ongoing DNA synthesis during S-phase itself, respectively, and hence is functionally distinct from previously identified MGS-associated genes. The phenotypes of affected individuals range from syndromic coronal craniosynostosis to severe growth restriction, fulfilling diagnostic criteria for Meier-Gorlin syndrome. All mutations identified were biallelic and included synonymous mutations altering splicing of physiological CDC45 transcripts, as well as amino acid substitutions expected to result in partial loss of function. Functionally, mutations reduce levels of full-length transcripts and protein in subject cells, consistent with partial loss of CDC45 function and a predicted limited rate of DNA replication and cell proliferation. Our findings therefore implicate the preIC as an additional protein complex involved in the etiology of MGS and connect the core cellular machinery of genome replication with growth, chondrogenesis, and cranial suture homeostasis.

ERF-related craniosynostosis: The phenotypic and developmental profile of a new craniosynostosis syndrome.

Mutations in the ERF gene, coding for ETS2 repressor factor, a member of the ETS family of transcription factors cause a recently recognized syndromic form of craniosynostosis (CRS4) with facial dysmorphism, Chiari-1 malformation, speech and language delay, and learning difficulties and/or behavioral problems. The overall prevalence of ERF mutations in patients with syndromic craniosynostosis is around 2%, and 0.7% in clinically nonsyndromic craniosynostosis. Here, we present findings from 16 unrelated probands with ERF-related craniosynostosis, with additional data from 20 family members sharing the mutations. Most of the probands exhibited multisutural (including pan-) synostosis but a pattern involving the sagittal and lambdoid sutures (Mercedes-Benz pattern) predominated. Importantly the craniosynostosis was often postnatal in onset, insidious and progressive with subtle effects on head morphology resulting in a median age at presentation of 42 months among the probands and, in some instances, permanent visual impairment due to unsuspected raised intracranial pressure (ICP). Facial dysmorphism (exhibited by all of the probands and many of the affected relatives) took the form of orbital hypertelorism, mild exorbitism and malar hypoplasia resembling Crouzon syndrome but, importantly, a Class I occlusal relationship. Speech delay, poor gross and/or fine motor control, hyperactivity and poor concentration were common. Cranial vault surgery for raised ICP and/or Chiari-1 malformation was expected when multisutural synostosis was observed. Variable expressivity and nonpenetrance among genetically affected relatives was encountered. These observations form the most complete phenotypic and developmental profile of this recently identified craniosynostosis syndrome yet described and have important implications for surgical intervention and follow-up.

Disruption of TWIST1 translation by 5' UTR variants in Saethre-Chotzen syndrome.

Saethre-Chotzen syndrome (SCS), one of the most common forms of syndromic craniosynostosis (premature fusion of the cranial sutures), results from haploinsufficiency of TWIST1, caused by deletions of the entire gene or loss-of-function variants within the coding region. To determine whether non-coding variants also contribute to SCS, we screened 14 genetically undiagnosed SCS patients using targeted capture sequencing, and identified novel single nucleotide variants (SNVs) in the 5' untranslated region (UTR) of TWIST1 in two unrelated SCS cases. We show experimentally that these variants, which create translation start sites in the TWIST1 leader sequence, reduce translation from the main open reading frame (mORF). This is the first demonstration that non-coding SNVs of TWIST1 can cause SCS, and highlights the importance of screening the 5' UTR in clinically diagnosed SCS patients without a coding mutation. Similar 5' UTR variants, particularly of haploinsufficient genes, may represent an under-ascertained cause of monogenic disease.

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.

Identification of Intragenic Exon Deletions and Duplication of TCF12 by Whole Genome or Targeted Sequencing as a Cause of TCF12-Related Craniosynostosis.

TCF12-related craniosynostosis can be caused by small heterozygous loss-of-function mutations in TCF12. Large intragenic rearrangements, however, have not been described yet. Here, we present the identification of four large rearrangements in TCF12 causing TCF12-related craniosynostosis. Whole-genome sequencing was applied on the DNA of 18 index cases with coronal synostosis and their family members (43 samples in total). The data were analyzed using an autosomal-dominant disease model. Structural variant analysis reported intragenic exon deletions (of sizes 84.9, 8.6, and 5.4 kb) in TCF12 in three different families. The results were confirmed by deletion-specific PCR and dideoxy-sequence analysis. Separately, targeted sequencing of the TCF12 genomic region in a patient with coronal synostosis identified a tandem duplication of 11.3 kb. The pathogenic effect of this duplication was confirmed by cDNA analysis. These findings indicate the importance of screening for larger rearrangements in patients suspected to have TCF12-related craniosynostosis.

TCF12 microdeletion in a 72-year-old woman with intellectual disability.

Heterozygous mutations in TCF12 were recently identified as an important cause of craniosynostosis. In the original series, 14% of patients with a mutation in TCF12 had significant developmental delay or learning disability. We report on the first case of TCF12 microdeletion, detected by array-comparative genomic hybridization, in a 72-year-old patient presenting with intellectual deficiency and dysmorphism. Multiplex ligation-dependent probe amplification analysis indicated that exon 19, encoding the functionally important basic helix-loop-helix domain, was included in the deleted segment in addition to exon 20. We postulate that the TCF12 microdeletion is responsible for this patient's intellectual deficiency and facial phenotype.

Inactivation of IL11 signaling causes craniosynostosis, delayed tooth eruption, and supernumerary teeth.

Craniosynostosis and supernumerary teeth most often occur as isolated developmental anomalies, but they are also separately manifested in several malformation syndromes. Here, we describe a human syndrome featuring craniosynostosis, maxillary hypoplasia, delayed tooth eruption, and supernumerary teeth. We performed homozygosity mapping in three unrelated consanguineous Pakistani families and localized the syndrome to a region in chromosome 9. Mutational analysis of candidate genes in the region revealed that all affected children harbored homozygous missense mutations (c.662C>G [p.Pro221Arg], c.734C>G [p.Ser245Cys], or c.886C>T [p.Arg296Trp]) in IL11RA (encoding interleukin 11 receptor, alpha) on chromosome 9p13.3. In addition, a homozygous nonsense mutation, c.475C>T (p.Gln159X), and a homozygous duplication, c.916_924dup (p.Thr306_Ser308dup), were observed in two north European families. In cell-transfection experiments, the p.Arg296Trp mutation rendered the receptor unable to mediate the IL11 signal, indicating that the mutation causes loss of IL11RA function. We also observed disturbed cranial growth and suture activity in the Il11ra null mutant mice, in which reduced size and remodeling of limb bones has been previously described. We conclude that IL11 signaling is essential for the normal development of craniofacial bones and teeth and that its function is to restrict suture fusion and tooth number. The results open up the possibility of modulation of IL11 signaling for the treatment of craniosynostosis.

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.

Homozygous SALL1 mutation causes a novel multiple congenital anomaly-mental retardation syndrome.

OBJECTIVE: To delineate a novel autosomal recessive multiple congenital anomaly-mental retardation (MCA-MR) syndrome in 2 female siblings of a consanguineous pedigree and to identify the disease-causing mutation. STUDY DESIGN: Both siblings were clinically characterized and homozygosity mapping and sequencing of candidate genes were applied. The contribution of nonsense-mediated messenger RNA (mRNA) decay to the expression of mutant mRNA in fibroblasts of a healthy carrier and a control was studied by pyrosequencing. RESULTS: We identified the first homozygous SALL1 mutation, c.3160C > T (p.R1054*), in 2 female siblings presenting with multiple congenital anomalies, central nervous system defects, cortical blindness, and absence of psychomotor development (ie, a novel recognizable, autosomal recessive MCA-MR). The mutant SALL1 transcript partially undergoes nonsense-mediated mRNA decay and is present at 43% of the normal transcript level in the fibroblasts of a healthy carrier. CONCLUSION: Previously heterozygous SALL1 mutations and deletions have been associated with dominantly inherited anal-renal-radial-ear developmental anomalies. We identified an allelic recessive SALL1-related MCA-MR. Our findings imply that quantity and quality of SALL1 transcript are important for SALL1 function and determine phenotype, and mode of inheritance, of allelic SALL1-related disorders. This novel MCA-MR emphasizes SALL1 function as critical for normal central nervous system development and warrants a detailed neurologic investigation in all individuals with SALL1 mutations.

Frank-ter Haar syndrome associated with sagittal craniosynostosis and raised intracranial pressure.

BACKGROUND: Frank-ter Haar syndrome is a rare disorder associated with skeletal, cardiac, ocular and craniofacial features including hypertelorism and brachycephaly. The most common underlying genetic defect in Frank-ter Haar syndrome appears to be a mutation in the SH3PXD2B gene on chromosome 5q35.1. Craniosynostosis, or premature fusion of the calvarial sutures, has not previously been described in Frank-ter Haar syndrome. CASE PRESENTATION: We present a family of three affected siblings born to consanguineous parents with clinical features in keeping with a diagnosis of Frank-ter Haar syndrome. All three siblings have a novel mutation caused by the deletion of exon 13 of the SH3PXD2B gene. Two of the three siblings also have non-scaphocephalic sagittal synostosis associated with raised intracranial pressure. CONCLUSION: The clinical features of craniosynostosis and raised intracranial pressure in this family with a confirmed diagnosis of Frank-ter Haar syndrome expand the clinical spectrum of the disease. The abnormal cranial proportions in a mouse model of the disease suggests that the association is not coincidental. The possibility of craniosynostosis should be considered in individuals with a suspected diagnosis of Frank-ter Haar syndrome.

A deletion of FGFR2 creating a chimeric IIIb/IIIc exon in a child with Apert syndrome.

BACKGROUND: Signalling by fibroblast growth factor receptor type 2 (FGFR2) normally involves a tissue-specific alternative splice choice between two exons (IIIb and IIIc), which generates two receptor isoforms (FGFR2b and FGFR2c respectively) with differing repertoires of FGF-binding specificity. Here we describe a unique chimeric IIIb/c exon in a patient with Apert syndrome, generated by a non-allelic homologous recombination event. CASE PRESENTATION: We present a child with Apert syndrome in whom routine genetic testing had excluded the FGFR2 missense mutations commonly associated with this disorder. The patient was found to harbour a heterozygous 1372 bp deletion between FGFR2 exons IIIb and IIIc, apparently originating from recombination between 13 bp of identical DNA sequence present in both exons. The rearrangement was not present in the unaffected parents. CONCLUSIONS: Based on the known pathogenesis of Apert syndrome, the chimeric FGFR2 protein is predicted to act in a dominant gain-of-function manner. This is likely to result from its expression in mesenchymal tissues, where retention of most of the residues essential for FGFR2b binding activity would result in autocrine activation. This report adds to the repertoire of rare cases of Apert syndrome for which a pathogenesis based on atypical FGFR2 rearrangements can be demonstrated.

Factors influencing success of clinical genome sequencing across a broad spectrum of disorders.

To assess factors influencing the success of whole-genome sequencing for mainstream clinical diagnosis, we sequenced 217 individuals from 156 independent cases or families across a broad spectrum of disorders in whom previous screening had identified no pathogenic variants. We quantified the number of candidate variants identified using different strategies for variant calling, filtering, annotation and prioritization. We found that jointly calling variants across samples, filtering against both local and external databases, deploying multiple annotation tools and using familial transmission above biological plausibility contributed to accuracy. Overall, we identified disease-causing variants in 21% of cases, with the proportion increasing to 34% (23/68) for mendelian disorders and 57% (8/14) in family trios. We also discovered 32 potentially clinically actionable variants in 18 genes unrelated to the referral disorder, although only 4 were ultimately considered reportable. Our results demonstrate the value of genome sequencing for routine clinical diagnosis but also highlight many outstanding challenges.

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.

Reduced dosage of ERF causes complex craniosynostosis in humans and mice and links ERK1/2 signaling to regulation of osteogenesis.

The extracellular signal-related kinases 1 and 2 (ERK1/2) are key proteins mediating mitogen-activated protein kinase signaling downstream of RAS: phosphorylation of ERK1/2 leads to nuclear uptake and modulation of multiple targets. Here, we show that reduced dosage of ERF, which encodes an inhibitory ETS transcription factor directly bound by ERK1/2 (refs. 2,3,4,5,6,7), causes complex craniosynostosis (premature fusion of the cranial sutures) in humans and mice. Features of this newly recognized clinical disorder include multiple-suture synostosis, craniofacial dysmorphism, Chiari malformation and language delay. Mice with functional Erf levels reduced to ∼30% of normal exhibit postnatal multiple-suture synostosis; by contrast, embryonic calvarial development appears mildly delayed. Using chromatin immunoprecipitation in mouse embryonic fibroblasts and high-throughput sequencing, we find that ERF binds preferentially to elements away from promoters that contain RUNX or AP-1 motifs. This work identifies ERF as a novel regulator of osteogenic stimulation by RAS-ERK signaling, potentially by competing with activating ETS factors in multifactor transcriptional complexes.