Prof Andrew OM Wilkie FRS FMedSci FRCP
|Research Area:||Genetics and Genomics|
|Scientific Themes:||Genes, Genetics, Epigenetics & Genomics and Bioinformatics, Statistics & Computational Biology|
|Keywords:||Genetics, Craniofacial and Sperm|
About 1 in 40 babies is born with a serious congenital abnormality: in many cases, this is caused by alterations (mutations) in genes involved in the normal process of embryonic development. Many important malformations affect both the skull and limbs, suggesting that similar developmental processes are used to build these distinct structures. Our lab is investigating these processes by identifying the causative mutations, particularly in patients affected with skull malformations. Such individuals frequently require corrective surgery and the group collaborates with the Department of Plastic Surgery in Oxford in the investigation of their patients. The work is principally funded by the Wellcome Trust, with contributions from the NIHR Oxford Biomedical Research Centre and US National Institutes of Health.
In 1995 our group discovered the cause of Apert syndrome, a severe condition characterised by craniosynostosis (early closure of the cranial sutures) and syndactyly (fusion between the digits) of the hands and feet. We identified two specific mutations within the gene for fibroblast growth factor receptor type 2 (FGFR2), one or other of which is present in ~99% of affected individuals. Other FGFR2 mutations are associated with different congenital syndromes and complete mutation screens show that the mutations are non-random, with some being highly recurrent. It is now apparent that a similar spectrum of mutations occur somatically in specific cancers.
In cases where the mutation has arisen de novo, it always originates from the unaffected father, who tends to be older than average (paternal age effect). We have have extended this observation by developing a method to measure the level of the most common Apert mutation in sperm. This has led us to propose that these mutations confer a paradoxical growth advantage to the testis cells in which they arise (Goriely et al 2003, 2005). We next demonstrated a direct link between the occurrence in sperm of a specific mutation in a related gene, FGFR3, and a rare type of testicular tumour (spermatocytic seminoma). We propose that paternal age-effect mutations arise through a shared mechanism involving activation of Ras signalling within the spermatogonial cell (Goriely et al 2009; Giannoulatou et al 2013). We are now exploring further the broader consequences of this novel mechanism (which we term selfish spermatogonial selection) for disease (reviewed by Goriely & Wilkie 2012), and developing immunohistochemical and genetic methods to identify the abnormal clonal events directly in human testes (Lim et al 2012). Most recently we have developed a method to identify these clonal mutational events directly in the seminiferous tubules of normal human testes (Maher al 2016). See our video on: https://youtu.be/NRj4IjkEvws
We have extended the work on Apert syndrome to study the molecular basis of many other conditions with craniofacial and/or limb malformations. Discoveries in the past few years include the identification of mutations of ROR2 in brachydactyly type B and recessive Robinow syndrome, of MSX2 and ALX4 in parietal foramina, of FLNA in the otopalatodigital spectrum disorders, of EFNB1 in craniofrontonasal syndrome (Twigg et al 2004, 2006), RAB23 in Carpenter syndrome (Jenkins et al 2007), the ZRS of SHH in triphalangeal thumb (Furniss et al 2008) and ALX3 in a newly recognised disorder, frontorhiny (Twigg et al 2009). Using whole exome and whole genome sequencing we have recently identified 4 important new genes that are mutated in craniosynostosis. These are MEGF8 (Twigg et al 2012), ERF (Twigg et al 2013), TCF12 (Sharma et al 2013) and ZIC1 (Twigg et al 2015). Recently we comprehensively reviewed the molecular genetic basis of craniosynostosis (Twigg and Wilkie 2015).
We contributed to the first large scale application of whole genome sequencing in a clinical setting (Taylor et al 2016), with one of our patients (see the CT scan on the previous web page) gaining publicity on the front page of the Times (3 August 2011) as possibly the first within the UK to have a clinically applied whole genome sequence. We are continuing to use next generation sequencing technologies to help give better, more accurate genetic diagnoses for our patients.
|Prof Gil McVean||Wellcome Trust Centre for Human Genetics||The University of Oxford||United Kingdom|
|Prof George Mavrothalassitis||IMBB/FORTH||Greece|
|Dr Robert Maxson||USC Keck School of Medicine||United States|
|Prof Tudor A Fulga||Nuffield Division of Clinical Laboratory Sciences||University of Oxford||United Kingdom|
|Dr Wojciech Niedzwiedz||Weatherall Institute of Molecular Medicine||University of Oxford||United Kingdom|
|Prof Irene Mathijssen||Department of Plastic Surgery||Erasmus MC, Rotterdam||Netherlands|
|Dr Simeon Boyadjiev||Dept of Pediatrics||University of California Davis||United States|
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. Hide abstract
Craniosynostosis, the premature fusion of one or more cranial sutures of the skull, provides a paradigm for investigating the interplay of genetic and environmental factors leading to malformation. Over the past 20 years molecular genetic techniques have provided a new approach to dissect the underlying causes; success has mostly come from investigation of clinical samples, and recent advances in high-throughput DNA sequencing have dramatically enhanced the study of the human as the preferred "model organism." In parallel, however, we need a pathogenetic classification to describe the pathways and processes that lead to cranial suture fusion. Given the prenatal onset of most craniosynostosis, investigation of mechanisms requires more conventional model organisms; principally the mouse, because of similarities in cranial suture development. We present a framework for classifying genetic causes of craniosynostosis based on current understanding of cranial suture biology and molecular and developmental pathogenesis. Of note, few pathologies result from complete loss of gene function. Instead, biochemical mechanisms involving haploinsufficiency, dominant gain-of-function and recessive hypomorphic mutations, and an unusual X-linked cellular interference process have all been implicated. Although few of the genes involved could have been predicted based on expression patterns alone (because the genes play much wider roles in embryonic development or cellular homeostasis), we argue that they fit into a limited number of functional modules active at different stages of cranial suture development. This provides a useful approach both when defining the potential role of new candidate genes in craniosynostosis and, potentially, for devising pharmacological approaches to therapy. Hide abstract
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. Hide abstract
The RAS proto-oncogene Harvey rat sarcoma viral oncogene homolog (HRAS) encodes a small GTPase that transduces signals from cell surface receptors to intracellular effectors to control cellular behavior. Although somatic HRAS mutations have been described in many cancers, germline mutations cause Costello syndrome (CS), a congenital disorder associated with predisposition to malignancy. Based on the epidemiology of CS and the occurrence of HRAS mutations in spermatocytic seminoma, we proposed that activating HRAS mutations become enriched in sperm through a process akin to tumorigenesis, termed selfish spermatogonial selection. To test this hypothesis, we quantified the levels, in blood and sperm samples, of HRAS mutations at the p.G12 codon and compared the results to changes at the p.A11 codon, at which activating mutations do not occur. The data strongly support the role of selection in determining HRAS mutation levels in sperm, and hence the occurrence of CS, but we also found differences from the mutation pattern in tumorigenesis. First, the relative prevalence of mutations in sperm correlates weakly with their in vitro activating properties and occurrence in cancers. Second, specific tandem base substitutions (predominantly GC>TT/AA) occur in sperm but not in cancers; genomewide analysis showed that this same mutation is also overrepresented in constitutional pathogenic and polymorphic variants, suggesting a heightened vulnerability to these mutations in the germline. We developed a statistical model to show how both intrinsic mutation rate and selfish selection contribute to the mutational burden borne by the paternal germline. Hide abstract
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. Hide abstract
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. Hide abstract
Advanced paternal age has been associated with an increased risk for spontaneous congenital disorders and common complex diseases (such as some cancers, schizophrenia, and autism), but the mechanisms that mediate this effect have been poorly understood. A small group of disorders, including Apert syndrome (caused by FGFR2 mutations), achondroplasia, and thanatophoric dysplasia (FGFR3), and Costello syndrome (HRAS), which we collectively term "paternal age effect" (PAE) disorders, provides a good model to study the biological and molecular basis of this phenomenon. Recent evidence from direct quantification of PAE mutations in sperm and testes suggests that the common factor in the paternal age effect lies in the dysregulation of spermatogonial cell behavior, an effect mediated molecularly through the growth factor receptor-RAS signal transduction pathway. The data show that PAE mutations, although arising rarely, are positively selected and expand clonally in normal testes through a process akin to oncogenesis. This clonal expansion, which is likely to take place in the testes of all men, leads to the relative enrichment of mutant sperm over time-explaining the observed paternal age effect associated with these disorders-and in rare cases to the formation of testicular tumors. As regulation of RAS and other mediators of cellular proliferation and survival is important in many different biological contexts, for example during tumorigenesis, organ homeostasis and neurogenesis, the consequences of selfish mutations that hijack this process within the testis are likely to extend far beyond congenital skeletal disorders to include complex diseases, such as neurocognitive disorders and cancer predisposition. Hide abstract
Carpenter syndrome is an autosomal-recessive multiple-congenital- malformation disorder characterized by multisuture craniosynostosis and polysyndactyly of the hands and feet; many other clinical features occur, and the most frequent include obesity, umbilical hernia, cryptorchidism, and congenital heart disease. Mutations of RAB23, encoding a small GTPase that regulates vesicular transport, are present in the majority of cases. Here, we describe a disorder caused by mutations in multiple epidermal-growth-factor- like-domains 8 (MEGF8), which exhibits substantial clinical overlap with Carpenter syndrome but is frequently associated with abnormal left-right patterning. We describe five affected individuals with similar dysmorphic facies, and three of them had either complete situs inversus, dextrocardia, or transposition of the great arteries; similar cardiac abnormalities were previously identified in a mouse mutant for the orthologous Megf8. The mutant alleles comprise one nonsense, three missense, and two splice-site mutations; we demonstrate in zebrafish that, in contrast to the wild-type protein, the proteins containing all three missense alterations provide only weak rescue of an early gastrulation phenotype induced by Megf8 knockdown. We conclude that mutations in MEGF8 cause a Carpenter syndrome subtype frequently associated with defective left-right patterning, probably through perturbation of signaling by hedgehog and nodal family members. We did not observe any subject with biallelic loss-of function mutations, suggesting that some residual MEGF8 function might be necessary for survival and might influence the phenotypes observed. © 2012 The American Society of Human Genetics. Hide abstract
The dominant congenital disorders Apert syndrome, achondroplasia and multiple endocrine neoplasia-caused by specific missense mutations in the FGFR2, FGFR3 and RET proteins respectively-represent classical examples of paternal age-effect mutation, a class that arises at particularly high frequencies in the sperm of older men. Previous analyses of DNA from randomly selected cadaveric testes showed that the levels of the corresponding FGFR2, FGFR3 and RET mutations exhibit very uneven spatial distributions, with localised hotspots surrounded by large mutation-negative areas. These studies imply that normal testes are mosaic for clusters of mutant cells: these clusters are predicted to have altered growth and signalling properties leading to their clonal expansion (selfish spermatogonial selection), but DNA extraction eliminates the possibility to study such processes at a tissue level. Using a panel of antibodies optimised for the detection of spermatocytic seminoma, a rare tumour of spermatogonial origin, we demonstrate that putative clonal events are frequent within normal testes of elderly men (mean age: 73.3 yrs) and can be classed into two broad categories. We found numerous small (less than 200 cells) cellular aggregations with distinct immunohistochemical characteristics, localised to a portion of the seminiferous tubule, which are of uncertain significance. However more infrequently we identified additional regions where entire seminiferous tubules had a circumferentially altered immunohistochemical appearance that extended through multiple serial sections that were physically contiguous (up to 1 mm in length), and exhibited enhanced staining for antibodies both to FGFR3 and a marker of downstream signal activation, pAKT. These findings support the concept that populations of spermatogonia in individual seminiferous tubules in the testes of older men are clonal mosaics with regard to their signalling properties and activation, thus fulfilling one of the specific predictions of selfish spermatogonial selection. Hide abstract
Craniosynostosis, defined as the premature fusion of the cranial sutures, presents many challenges in classification and treatment. At least 20% of cases are caused by specific single gene mutations or chromosome abnormalities. This article maps out approaches to clinical assessment of a child presenting with an unusual head shape, and illustrates how genetic analysis can contribute to diagnosis and management. © 2011 Macmillan Publishers Limited All rights reserved. Hide abstract
OBJECTIVES: We describe the first cohort-based analysis of the impact of genetic disorders in craniosynostosis. We aimed to refine the understanding of prognoses and pathogenesis and to provide rational criteria for clinical genetic testing. METHODS: We undertook targeted molecular genetic and cytogenetic testing for 326 children who required surgery because of craniosynostosis, were born in 1993-2002, presented to a single craniofacial unit, and were monitored until the end of 2007. RESULTS: Eighty-four children (and 64 relatives) had pathologic genetic alterations (86% single-gene mutations and 14% chromosomal abnormalities). The FGFR3 P250R mutation was the single largest contributor (24%) to the genetic group. Genetic diagnoses accounted for 21% of all craniosynostosis cases and were associated with increased rates of many complications. Children with an initial clinical diagnosis of nonsyndromic craniosynostosis were more likely to have a causative mutation if the synostoses were unicoronal or bicoronal (10 of 48 cases) than if they were sagittal or metopic (0 of 55 cases; P = .0003). Repeat craniofacial surgery was required for 58% of children with single-gene mutations but only 17% of those with chromosomal abnormalities (P = .01). CONCLUSIONS: Clinical genetic assessment is critical for the treatment of children with craniosynostosis. Genetic testing of nonsyndromic cases (at least for FGFR3 P250R and FGFR2 exons IIIa/c) should be targeted to patients with coronal or multisuture synostoses. Single-gene disorders that disrupt physiologic signaling in the cranial sutures often require reoperation, whereas chromosomal abnormalities follow a more-indolent course, which suggests a different, secondary origin of the associated craniosynostosis. Hide abstract
Genes mutated in congenital malformation syndromes are frequently implicated in oncogenesis, but the causative germline and somatic mutations occur in separate cells at different times of an organism's life. Here we unify these processes to a single cellular event for mutations arising in male germ cells that show a paternal age effect. Screening of 30 spermatocytic seminomas for oncogenic mutations in 17 genes identified 2 mutations in FGFR3 (both 1948A>G, encoding K650E, which causes thanatophoric dysplasia in the germline) and 5 mutations in HRAS. Massively parallel sequencing of sperm DNA showed that levels of the FGFR3 mutation increase with paternal age and that the mutation spectrum at the Lys650 codon is similar to that observed in bladder cancer. Most spermatocytic seminomas show increased immunoreactivity for FGFR3 and/or HRAS. We propose that paternal age-effect mutations activate a common 'selfish' pathway supporting proliferation in the testis, leading to diverse phenotypes in the next generation including fetal lethality, congenital syndromes and cancer predisposition. Hide abstract
The role of Zic1 in cranial suture development
This project will use mouse models to study the role of Zic1 (a highly conserved zinc-finger transcription factor) in cranial suture development, with the aim of understanding how gain-of-function mutations of Zic1 cause craniosynostosis (premature fusion of the cranial sutures of the skull). As Zic1 appears to be expressed in an organising centre for coronal suture formation, the project will also involve identification of Zic1 interaction partners and investigate the relationship of Zic1 to o ...
Selfish Selection in the Human Testis
Work in our lab has revealed a novel mechanism causing the enrichment of specific genetic diseases, whereby the causative “selfish” mutations confer a selective advantage to spermatogonial stem cells or progenitors in the adult testis. We are currently extending the principles underlying this phenomenon from the original exemplars (which involve the FGFR2, FGFR3, HRAS, RET, PTPN11 genes), to a wider range of targets. Current data indicate that mutations affecting one or more pathways involved in ...