Human DNA Damage Response Disorders Group Site




Understanding the molecular basis of human Microcephalic Primordial Dwarfism.

            This family of conditions is characterised by profound short stature associated with a severely reduced head circumference denoting an extreme impairment of normal brain development (microcephaly: see Alcantara D & O'Driscoll M Am J Med Genet Part C 2014). Very often, the skeletal system is also affected in these disorders. The archetypal example of such conditions is Seckel syndrome, first described as a distinct clinical disorder in 1960 by Dr. Helmut Seckel in a monograph entitled “Bird-Headed Dwarfs. Studies in developmental anthropology including human proportions”. This condition is characterised by a dramatic proportionate dwarfism evident even in utero, a characteristic facial appearance (small chin, protruding nose, receding forehead), severe microcephaly (reduced head circumference) and isolated skeletal abnormalities. We characterised the first identified genetic defect in this disorder as a hypomorphic mutation in gene encoding ATR (ataxia telangiectasia and Rad3-related) , a protein kinase that plays a central role in the DNA damage response (O’Driscoll et al Nat Genet 2003). A ‘humanised’ mouse model of this defect was created by Murga and colleagues (Murga et al, Nat Genet 2009) underscoring the importance of this DNA damage response pathway for normal development and maintenance of tissue homeostasis (O’Driscoll M, Nat Genet 2009, O’Driscoll M, DNA Repair 2009). Recently, we have been involved in a study describing novel ATR mutations in additional cases of Seckel syndrome as well as a novel defect in ATRIP, the ATR interacting protein (Ogi et al PLoS Genet 2012). This work helps consolidate the clinical presentation of impaired ATR-ATRIP function in humans.

            We have also been involved in other studies that have identified novel defects in related conditions including Pericentrin (PCNT) and Origin Recognition Complex 1 (ORC1) in Microcephalic Primordial Dwarfism Type II and Meier-Gorlin syndrome respectively (Griffith et al Nat Genet 2008 and Bicknell et al Nat Genet 2011). These studies highlight the previously hitherto under-appreciated significance of defects in centrosome function and DNA replication licensing to normal human development. Interestingly, we have also identified specific ATR-pathway defects in cells from these conditions and are currently pursuing the characterisation of novel defects in these genetically heterogeneous disorders. Our work on human ORC-defects has also uncovered a pathomechanistic link between these DNA replication controlling factors and cilia formation and function (Stiff T et al PLoS Genet 2013). We have also been involved in the description of a novel kinetochore-based genetic defect (CENPE) causative of Microcephalic Primordial Dwarfism (Mirzaa GM et al Hum Genet 2014). These findings further expand the growing landscape of complex cellular pathomechanisms underlying these conditions.

Defective ATR-pathway function and skeletal development.

            Several isolated skeletal abnormalities are associated, to varying degrees, with Microcephalic Primordial Dwarfism syndromes. Specific examples include, fifth finger clinodactyly, thoracic kyphosis, joint dislocation, ivory epiphysis, delayed ossification, absent patella and dental malocclusion. The molecular aetiology of these clinical features is currently unclear. Using model cell-based systems for osteogenesis and chrondrogenesis we have been investigating how, at the molecular level, defects in genes associated with these conditions can impact upon these processes. Specifically with regard to chondrogenesis and chondroinduction, we have developed a differentiation based system that employs patient-derived material, thereby carrying the clinically relevant pathogenic defects (Stiff T et al PLoS Genet 2013).

Understanding the interplay between mTOR pathway and the DNA damage response.

            Components of the mTOR pathway, specifically PKB/AKT are often over-expressed/hyperactive in malignancy and represent important drug targets for cancer treatment. In fact, mis-regulated mTOR function underlies several cancer predisposition syndromes including Cowden syndrome (PTEN mutations), Tuberous sclerosis (TSC1/2 mutations) and Peutz-Jeghers syndrome (LKB1 mutations). We have been involved in studies identifying novel congenital defects in components of the mTOR signal transduction network in close collaboration with colleagues in the US and Canada. We recently functionally characterised cell lines from patients with complex developmental conditions ('Megalencephaly-capillary malformation' and 'Megalencephaly-polymicrogyria-polydactyly-hydrocephalus' syndromes), with novel defects in AKT3, PIK3R2 and PIK3CA, showing that hyperactive signal transduction through the mTOR network underlay these disorders (Riviere et al Nat Genet 2012). We have also been involed in functional and genetic characterisation of SHORT syndrome. In this case we found hypoactivation of the mTOR network (Dyment DA et al Am J Hum Genet 2013). Furthermore, we also played an active role in the genetic delineation of 'Microcephaly Capillary Malformation syndrome', a devastating developmental condition associated with progressive neurodegeneration and cutaneous vascular abnormalities (McDonell L et al Nat Genet 2013).

Interestingly, various components of the insulin-IGF (insulin growth factor) axis appear to be substrates of ATM/ATR following DNA damage, although the physiological relevance of this remains unclear (Matsouka et al Science 2007). The Insulin-IGF axis plays a fundamental role in cell and organism growth. Interestingly, defective ATR-pathway function in humans is characterised by profound growth retardation. We are currently studying the functional interplay between these pathways using cells from individuals with Microcephalic Primordial Dwarfism and those with congenital defects in the Receptor tyrosine kinase-mTOR pathway.

Genomic Disorders.

            We run several projects characterising the functional significance of copy number variation (CNV; deletion and/or duplication) of genes that are known to function in various aspects of cell cycle checkpoint control, DNA repair and DNA damage signalling, using material derived from individuals with diverse human Genomic Disorders (see Colnaghi et al Sem Cell & Dev Biol 2011 for an overview). To date, in association with colleagues in the US, Canada and Europe we have characterised novel and specific DNA damage response defects in cells from well-known Genomic Disorders including Miller-Dieker Lissencephaly and Williams-Beuren syndromes (O’Driscoll et al Am J Hum Genet ), 1q21.1 CNV syndrome (Harvard C et al Orphanet J Rare Diseases 2011), DiGeorge/Veleocardiofacial syndrome and rec(3) syndrome (Colnaghi R et al Sem Cell & Dev Biol 2011), as well as other well-known syndromes such as 17p13.3 duplication syndrome (Outwin E et al PLoS Genetics 2011) and Wolf-Hirschhorn syndrome (Hart L et al Dis Models & Mech 2014 and Kerzendorfer et al Hum Mol Genet 2012). This findings offer new insight into the distinct pathomechanisms underlying the clinical presentation and progression of these conditions.

Defective ubiquitin pathway function and syndromal mental retardation.

            In this diverse group of disorders, we are interested in understanding how primary genetic defects in ubiquitin pathway components can impact on various aspects of the DNA damage response and how these combine to present clinically. For example, we have found that cells derived from patients with mutations in the E3-ubiquitinin ligase Cullin 4B are highly sensitive the topoisomerase I (Topo I) poison camptothecin (CPT) and that this sensitivity is likely a result of impaired CPT-mediated Topo I degradation suggesting Topo I to be a CUL4B-dependent substrate (Kerzendorfer et al Hum Mol Genet 2010). We are currently working on understanding other impacts of defective CUL4B function, on how these may contribute to the human disorder or could be exploited to sensitise cells to certain DNA damaging agents (Kerzendorfer et al Mech Ageing & Dev 2011). We are also working on understanding the pathophysiological impact of clinically relevant deficits of other syndromal mental retardation genes that encode ubiquitin pathway components with specific relevance to the DDR and genome stability networks. Our work on Microcephaly Capillary Malformation Syndrome (MIC-CAP) characterised the functional consequences of pathogenic defects in STAMBP/AMSH, which encodes a deubiquitinating isopeptidase (McDonell L et al Nat Genet 2013).

‘Novel’ DNA damage response-defective disorders.

            Many human DNA damage response/repair defective disorders exhibit the specific clinical combination of microcephaly and pre- and post-natal growth retardation. Provocatively, the medical literature abounds with clinical descriptions of genetically uncharacterised syndromes exhibiting similar features, many of which also appear to be cancer prone. Whether any of these disorders are caused by a defective response to DNA damage is unknown. Recently, we were involved in a study identify a pathogenic defect in ATR in an autosomal dominant oropharyngeal cancer syndrome family (Tanaka et al Am J Hum Genet 2012). This unexpected clinical presentation of an ATR-pathway defect illustrates how much we have yet to learn concerning the intricate biological connections between the DDR, genomic instability and normal human development.

           Another aspect of our work concerns the identification and characterisation of novel DNA damage response disorders. This evolving aspect of our research has been greatly facilitated by the recent technological revolution in genomic array and next generation sequencing technologies, which can generate target candidate lists from which we can prioritise characterisation studies (e.g. see Raffan et al, Frontiers in Genomic Endocrinology 2011). Together with our collaborators from the UK, mainland Europe, North America and Canada, we are currently working on several novel congenital defects associated with fascinating human disorders concerning various aspects of the DDR, DNA replication and cell cycle control. For example, together with colleagues in the UK, we described and characterised novel defects in the SMC5-6-associated SUMO Ligase, NSE2 (mms21/NSMCE2), in individuals exhibiting primordial dwafism and extreme insulin resistance (Payne, Colnaghi, Rocha & Seth et al, J Clin Invest 2014). This further highlights the functional cross-talk between genome stability pathways and those that control metabolic homeostasis.

Immunosuppression, DNA damage and synthetic lethality.

            We also have a more ‘translational’ project based on our previous demonstration that cyclosporine A (CSA), a cyclic peptide routinely clinically used as a prophylaxis against Graft-versus-Host disease, an important contributor to bone marrow and solid organ transplantation morbidity, can induce DNA double strand breaks (DSBs) in human cells (O’Driscoll et al, Bone Marrow Transplantation 2008). Interestingly, prolonged CSA treatment is associated with cancer development, assumed to be a consequence of a lack of tumour immune-surveillance, although any impact of CSA-induced genomic instability has not been fully considered. We are currently investigating the mechanism of CSA-induced DSB formation and how we can exploit this feature in synthetic lethal approaches for the treatment of certain cancers with specific genetic backgrounds.