Molecular Genetics of Epilepsy and Mental Retardation Limited to Females (EFMR) A thesis submitted for the degree of Doctor of Philosophy to the University of Adelaide By Kim Hynes School of Molecular and Biomedical Science, Division of Genetics, University of Adelaide December 2009 Chapter 1 – Introduction ! ! ! " $ ' $! $" # # $ & !% $ $ % ! 1.1 Intellectual disability Intellectual disability (ID), also termed mental retardation (MR) (Salvador-Carulla and Bertelli, 2008), is a common disorder affecting an estimated 1.5-2% of the population in Westernised countries (reviewed in Leonard and Wen, 2002). ID is defined by the American Association on Mental Retardation (AAMR) as a disability characterized by significant limitations both in intellectual functioning and in adaptive behaviour that originates before age 18 (Luckasson et al., 2002). ID is subdivided into five categories based on a patient’s intelligence quotient (IQ). Class Intelligence quotient Profound <20 Severe 20-34 Moderate 35-49 Mild 50-69 Borderline 70-85 Table 1.1 The IQ criterion used to classify the severity of ID based on WHO guidelines (WHO, 1996) The most common forms of ID within the population are mild to moderate (Toniolo, 2000, Vasconcelos, 2004). The least common form is severe ID which only affects 0.3-0.5% of the population (Toniolo, 2000, Vasconcelos, 2004). ID can present on its own or more commonly as one of the comorbidities of a more complex syndrome such as Down syndrome (Toniolo, 2000). ID can present in infancy and early childhood but it is commonly diagnosed during school years, as IQ tests are more objective and only reliable in children over five (Reviewed in Toniolo, 2000, Vasconcelos, 2004, Shevell et al., 2003). The underlying causes of ID are highly heterogeneous and include both genetic and non- genetic factors. Non-genetic factors occur pre-natal or during early infancy and result in brain injury. Examples of these include infectious diseases, very premature birth, perinatal anoxia Chapter 1- 1 and foetal alcohol syndrome (Chelly and Mandel, 2001). Developmental disorders that affect the precise pattern of organization and connections in the brain can result in ID (Toniolo, 2000). Metabolic and mitochondrial disorders have also been associated with ID through inducing defects in neurons that alter brain housekeeping functions (Toniolo, 2000). This introduction will focus exclusively on genetic causes of ID and in particular X-linked intellectual disability (XLID). 1.2 X-Linked Intellectual Disability XLID is defined by the occurrence of ID (IQ<70) where the causative gene has been mapped to the X-chromosome (Gecz et al., 2009). Historically the categories of XLID have be further subdivided into syndromic (S-XLID) and non-syndromic (NS-XLID) forms, depending on whether further consistent abnormalities (in addition to ID) are found on physical examination, laboratory investigation or brain imaging (Mulley et al., 1992). However with increased understanding of the molecular basis of XLID a proportion of NS-XLID are turning out to be S-XLID. Once a causative gene has been identified in a multiple NS-XLID patients the specific phenotypes of these individuals with mutations in this gene can be compared as a group against normal individuals. Specific syndromal features can then be indentified for the group. For example, Fragile(X) (Fra(X)) syndrome which was initially characterised as NS- XLID, but is now the most widely recognised example of S-XLID (Ropers and Hamel, 2005). Although some syndromologists can pick some cases of Fra(X) by clinical observation alone, many Fra(X) cases are not so easily diagnosed and require laboratory testing for a definitive diagnosis. A further complexity to the S-XLID / NS-XLID subdivision is that mutations in a gene can result in patients with S-XLID and patients with NS-XLID. For example, mutations in ribosomal protein S6 kinase (RSK2) cause Coffin-Lowry syndrome (CLS) (Trivier et al., 1996) a disorder characterised by severe psychomotor retardation, facial and digital Chapter 1- 2 dysmorphisms and progressive skeletal deformations (Young, 1988). Mutations in RSK2 have also been identified in patients with NS-XLID who do not exhibit any of the facial, digital or skeletal features typical of CLS (Merienne et al., 1999). Genes on the X-chromosome have long been implicated in ID due to the ~25-30% excess of males with ID compared to females (Turner, 1996). More realistic recent estimates predict a contribution from the X-chromosome of between 8-15% (Ropers, 2008, Ropers and Hamel, 2005). This is 2-4 times higher than expected based on the ~4% of the protein coding genes in the human genome being coded for on the X-chromosome (Ropers, 2008). The X- chromosome is a rich source of causative genes for ID. The human X-chromosome contains ~818 known protein-coding genes (Gecz et al., 2009). Roughly 40% of X-chromosome genes are expressed in the brain and are therefore candidate genes for XLID (Ropers and Hamel, 2005). Out of these a large number of genes have been screened for mutations in XLID patients and so far more than 90 genes have been implicated in XLID (Chiurazzi et al., 2008, Tarpey et al., 2009). However mutations in these genes only explain ~50% of families with suspected XLID (reviewed in Gecz et al., 2009). The absence of a definitive diagnosis leaves affected families without accurate genetic counselling and reproductive options such as prenatal diagnosis. ID has a high burden on society, not just on those families directly affected, as it also ranks highly in terms of health care expenditure in developed countries. ID is therefore one of the most important health issues in medicine. Whilst considerable effort has been put into the discovery of genes responsible for ID the landscape is far from complete and requires much more investigation to complete the picture. Chapter 1- 3 1.3 Strategies for identifying causative genes for XLID The identification of causative genes for XLID is not only important at a clinical level to aid those families and individuals affected, but it will also increase our understanding of normal brain function. For example, investigations into the function of FMR1, the gene involved in Fra(X) syndrome, have added to the knowledge of fundamental aspects of normal neuronal function (Willemsen et al., 2004). Gene discovery is considered easier in the case of S-XLID, compared to NS-XLID, as affected families and individuals can be classified and sub-grouped on the basis of physical, metabolic and/or behavioural characteristics, in addition to ID. Linkage data can therefore be combined for families and individuals in a particular sub-group, which greatly enhances the power of linkage analysis and has the potential to significantly reduce the linkage interval to be assessed for candidate genes. The task of gene discovery in monogenic disease has become easier with the availability of the human genome sequence and increased knowledge of gene function (Ropers and Hamel, 2005). However, on the other hand the unexpected degree of genetic heterogeneity and mutation pleiotropy observed for some disorders represents a considerable challenge, as seen for disorders caused by ARX (Aristaless-related homeobox) (Gecz et al., 2006) and ATRX (Gibbons and Higgs, 2000) mutations. Gene discovery for NS-XLID cases remains extremely difficult. Families with NS-XLID are impossible to separate in terms of their phenotype and need to be dealt with family by family because as a group they are clinically homogeneous. Progress in NS-XLID has been slow with less that 50% of causative genes identified due to extreme genetic heterogeneity which prevents the pooling of linkage information from unrelated families (Ropers and Hamel, 2005). Chapter 1- 4 In recent years the strategies used to identify causative genes in XLID have changed significantly. Previous success in gene identification has arisen through the use of breakpoint mapping in patients with rearrangements involving the X-chromosome, positional candidate screening of genes associated with fragile sites and linkage mapping with candidate gene screening (reviewed in Ropers and Hamel, 2005). These were primarily conducted on a patient/patient basis. More recent approaches to gene identification have seen a shift to semi- automated high throughput mutation-detection screening in large cohorts of families. Analysing linkage intervals from 125 families with NS-XLID revealed a potential mutation hotspot with clustering of the underlying mutations to the Xp11.2-11.3 which was predicted to carry 30% of causative genes (Ropers et al., 2003). Subsequent mutation screening of 50 genes expressed in the brain from the Xp11.2-11.3 region identified 5 novel XLMR genes (Ropers and Hamel, 2005, Freude et al., 2004, Jensen et al., 2005, Kalscheuer et al., 2003a, Shoichet et al., 2003, Tarpey et al., 2004). A further large-scale screen for causative point mutations in 90 known and candidate XLID genes in 600 suspected XLID families identified 73 mutations in 21 genes (de Brouwer et al., 2007). From this analysis they identified a causative mutation in 42% of families that contained an obligate female carrier (de Brouwer et al., 2007). It is likely that some of the 600 families analysed in this screen may not have ID due to X-linked genes, this is particularly relevant for the 191 families which only had affected individuals in a single generation with 2-5 affected brothers, there was still a significant proportion of XLID families with no apparent mutation in any of the previously identified and suspected XLID genes. To identify additional causative genes for XLID our laboratory in collaboration with others from the USA and UK embarked on a large scale, comprehensive screen, via high-through- put re-sequencing, of 718 X-chromosome genes in probands from 208 XLID or suspected XLID families (Tarpey et al., 2009). This effort led to the identification of at least 12 novel Chapter 1- 5 XLID genes and contributed to the identification of 4 additional XLID genes (Tarpey et al., 2009).