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A mutation screen of NR2E1 in patients with aniridia, Peters' anomaly and related eye disorders Borrie, Adrienne E. 2009

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A MUTATION SCREEN OF NR2E1 IN PATIENTS WITH ANIRIDIA, PETERS’ ANOMALY, AND RELATED EYE DISORDERS  by Adrienne E. Borrie B.Sc., McMaster University, 2007  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  in  THE FACULTY OF GRADUATE STUDIES (Medical Genetics)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) December 2009 © Adrienne E. Borrie, 2009  ABSTRACT Aniridia is a rare genetic panocular (whole eye) disorder which, for the majority of cases, is caused by mutations or chromosomal rearrangements involving paired box gene 6 (PAX6). Peters’ anomaly (PA), also a genetic eye disorder, has also been found to be associated with mutations in PAX6, and also in FOXC1, PITX2, and CYP1B1. However, in approximately 20% of patients who have aniridia and 75% of patients with PA, no mutation has been found in PAX6, or other genes involved these eye disorders, and for these patients, their genetic mutation is unknown. This precludes these patients from genetic testing and thus, from gaining the benefits from genetic counseling and early medical interventions. My hypothesis was that patients who have aniridia, Peters’ anomaly or related eye disorders, for which genetic cause is unknown, have mutations in NR2E1. The purpose of this thesis was to study patients with aniridia, Peters’ anomaly, and related eye disorders in order to identify mutations in a novel candidate gene, NR2E1. Here, the first germline amino acid change was identified in the NR2E1 gene in a patient with Peters’ anomaly and his mother, and not found in 392 control subjects. The identification of an amino acid variant in NR2E1 is significant as it supports the hypothesis that NR2E1 is a regulator of eye development in humans and has implications for the gene in the development of eye disorders. If future analysis leads to the identification of NR2E1 mutations in additional patients, it will allow patients with eye disorders of otherwise unknown genetic etiology to receive the benefits of modern genetic medicine and genetic counseling. This future work endeavors to provide the scientific and medical community with a greater depth of knowledge of the role of NR2E1 in genetic eye disorders.  ii  TABLE OF CONTENTS ABSTRACT ....................................................................................................................... ii TABLE OF CONTENTS ................................................................................................. iii LIST OF TABLES ............................................................................................................ v LIST OF FIGURES ......................................................................................................... vi LIST OF ABBREVIATIONS......................................................................................... vii ACKNOWLEDGMENTS ............................................................................................. viii CO-AUTHORSHIP STATEMENT ............................................................................... ix CHAPTER 1: INTRODUCTION .................................................................................... 1 1.1.1 1.1.2  Structure of the human eye.................................................................................. 1 Aniridia................................................................................................................ 2  1.1.3 Peters’ anomaly ................................................................................................... 4 1.1.4 NR2E1 ................................................................................................................. 7 1.1.5 NR2E1 and PAX6 ................................................................................................ 9 1.1.5 NR2E1 and NR2E3 ............................................................................................ 10 1.1.6 NR2E3 and eye disorder .................................................................................... 12 1.1.7 Hypothesis and thesis objectives ....................................................................... 14 1.2 REFERENCES ........................................................................................................ 15 CHAPTER 2: A MUTATION SCREEN OF NR2E1 IN PATIENTS WITH ANIRIDIA, PETERS’ ANOMALY, AND RELATED EYE DISORDERS .............. 26 2.1 INTRODUCTION ................................................................................................... 26 2.2 METHODS .............................................................................................................. 28 2.2.1 2.2.2  Patient information and DNA collection ........................................................... 28 Control DNA ..................................................................................................... 31  2.2.3 Sequencing ....................................................................................................... 32 2.2.4 PAX6 characterization ....................................................................................... 33 2.3 RESULTS................................................................................................................. 33 2.3.1 2.3.2  Sequence variation found in NR2E1 ................................................................. 33 Analysis of linkage disequilibrium in patients and related controls ................. 34  2.3.3 Initially discovered patient specific variants studied further ............................ 35 2.3.4 NR2E1 amino acid altering variant identified in patient with Peters’ anomaly 37 2.4 DISCUSSION .......................................................................................................... 40 2.5 CONCLUSION ........................................................................................................ 42  iii  2.6 REFERENCES ........................................................................................................ 44 CHAPTER 3: GENERAL DISCUSSION ..................................................................... 49 3.1 SUMMARY OF RESULTS .................................................................................... 49 3.2 FUTURE DIRECTIONS ........................................................................................ 51 3.2.1 3.2.2  Expanding collection of patients with eye disorders ......................................... 51 Functional assay to assess the effect of an R274G variant in NR2E1 ............... 54  3.3 CONCLUSION ........................................................................................................ 56 3.4 REFERENCES ........................................................................................................ 57  iv  LIST OF TABLES Table 1.1: Mutations in NR2E3 result in eye disorders ............................................... 13 Table 2.1: Patient data .................................................................................................... 29 Table 2.2: Characteristics of the study population ...................................................... 31 Table 2.3: PCR primers used to amplify NR2E1 sequences ........................................ 33 Table 2.4: Sequence variation of NR2E1 detected in patients with aniridia and related eye disorders ..................................................................................... 34 Table 2.5: Characterization of three NR2E1 patient variants .................................... 37  v  LIST OF FIGURES Figure 1.1: Anatomy of the eye ........................................................................................ 1 Figure 1.2: Similarity between Nr2e1 overexpressor and Pax6+/- mice ..................... 10 Figure 1.3: Alignment of Human NR2E1 and NR2E3 ................................................. 11 Figure 2.1: Visual representation of linkage disequilibrium ...................................... 35 Figure 2.2: Novel NR2E1 variant resulting in amino acid change ............................. 37 Figure 2.3: Amino acid variant R274G found in proband’s mother .......................... 38 Figure 2.4: NR2E1 amino acid variant at residue 274 is highly conserved................ 39 Figure 3.1: R274G variant identified in this study identical to homologous mutation in NR2E3. .................................................................................................... 49 Figure 3.2: Similarity between Nr2e1 null mice and humans with retinitis pigmentosa .................................................................................................. 53  vi  LIST OF ABBREVIATIONS ADRP ASD CGH CYP1B1 DBD ESCS FOXC1 FOXE3 Kb LD LBD NR2E1 NR2E3 PA PAX6 PITX2 Sey  Autosomal dominant retinitis pigmentosa Anterior segment dysgenesis Comparative genomic hybridization Cytochrome P450, family 1, subfamily B, polypeptide 1 DNA binding domain Enhanced S-cone syndrome Forkhead box gene C1 Forkhead box gene E3 Kilobase Linkage disequilibrium Ligand binding domain Nuclear receptor subfamily 2, group E, member 1 Nuclear receptor subfamily 2, group E, member 3 Peters’ anomaly Paired box gene 6 Paired-like homeodomain 2 Small eye  vii  ACKNOWLEDGMENTS First and foremost, I would like to thank my supervisor, Dr. Elizabeth M. Simpson for providing the wonderful opportunity to challenge myself in the scientific field of Medical Genetics. Your supervision and guidance has been instrumental in my development as a scientist. I would also like to thank the entire Simpson Lab, past and present, for their contributions to my scientific training and for their input into my project and thesis. In particular, I would like to thank Bibiana Wong, Russell Bonaguro, and Kathy Banks for patiently training me many different laboratory techniques and instructing me in several essential genetics software programs and websites. To my committee members, Dr. Robert Molday, Dr. Deborah Giaschi and Dr. Angela BrooksWilson, thank you for graciously giving your time to provide guidance in my project. Thank you to Cheryl Bishop, our Medical Genetics graduate secretary, for helping me all throughout my project, and for being so cheerful and kind. I would like to thank Dr. Dave Rollo for all of our conversations over the years, both scientific and philosophical, and for selflessly promoting my professional growth. Thank you to Dr. Angela Brooks-Wilson, for generously collaborating with me and for welcoming me into her laboratory at the BC Cancer Research Center. I would also like to express my gratitude to all the members of Angie’s lab. It was such a joy to work along side of each of you, thank you for all of your insights and encouragement. To So Yamanda, thank you for being so positive, knowledgeable, and patient when training me in sequencing techniques. Johanna Schuetz, you have been an incredible mentor. Thank you for your kindness and patience, for your guidance and problem-solving skills, and for being such a good friend to me. I have an incredible amount of support from my family and friends. My graduate school comrades, Fergil, Khat, Stefano, Lee, and Giorgia, I will never forget our neuroscience adventures and science talk over meals at Vancouver’s finest. To Fergil, thank you for inspiring me, for enthusiastically editing my thesis, and for being my partner in crime. Flora, your silliness, and positive spirit continuously lifted me up. Thank you to Donna, for all of our sushi lunches, for being a wonderful friend, and for believing in my abilities. Nadine, you are the best room mate a person could ask for, and I know that I would have never made it without your daily optimism and confidence in me. Thank you to my two beautiful sisters for your letters, phone calls, visits to Vancouver, and kind thoughts. Most of all, thank you to my amazing parents, Nancy and Michael Borrie, for your unconditional love, encouragement, and support.  viii  CO-AUTHORSHIP STATEMENT Chapter 2: The project described in this chapter was conceived and initiated by Dr. Elizabeth M. Simpson. The project became focused under my direction. Samples were collected from our collaborators: Dr. Veronica van Heyningen, Dr. Michael Walter, Dr. Ian Macdonald, Dr. Karen Gronskøv, Dr. Francesca Pasutto, and Dr. Brian Brooks. Dr. Angela Brooks-Wilson and her student Johanna Schultz provided training and mentorship in sequencing techniques. I generated the results presented, performed all data analysis, wrote the paper, and created all the figures for this manuscript in preparation.  ix  CHAPTER 1: INTRODUCTION 1.1.1  Structure of the human eye Human visual perception relies considerably on proper development of the many  tissues which comprise the eye (Figure 1.1).  Figure 1.1: Anatomy of the eye A cross-section of the adult human eye. Modified from (Kolb et al., 2007). The anterior segment is comprised of the front third of the eye, and includes the iris, cornea, lens, trabecular meshwork, and ciliary body (Chow & Lang, 2001). The iris, the coloured membrane of the eye, is responsible for regulating the amount of light entering the eye, and protects the retina from phototoxicity. The transparent lens and cornea allow for appropriate light refraction. The key role of the ciliary body is production of the aqueous humor, a clear fluid which supplies nutrition to avascular tissues in the anterior segment of the eye, such as the cornea, the lens, and the trabecular 1  meshwork (Llobet et al., 2003). The aqueous humor is secreted into the posterior chamber located between the iris and the lens. It then runs into the anterior chamber between the cornea and iris (Chow & Lang, 2001). The trabecular meshwork is a spongelike tissue which drains the aqueous humor from the anterior segment of the eye and is thus responsible for maintaining intraocular pressure, which creates the shape of the eye and generates constant distances between the retina, the cornea and the lens (Llobet et al., 2003). Abnormal development of the anterior segment of the eye is a symptom of aniridia, Peters’ anomaly (PA), and related ocular disorders which severely reduce visual function (Chow & Lang, 2001, Ciralsky & Colby, 2007, Lee et al., 2008). 1.1.2  Aniridia Aniridia is a rare genetic eye disorder which is characterized by congenital  deficiency of iris tissue and underdevelopment and/or degeneration of the retina, resulting in progressive visual loss (Lee et al., 2008). The most striking feature of aniridia is hypoplasia of the iris. This underdevelopment ranges from a complete deficiency of iris tissue to only mild hypoplasia detectable by slit lamp examination. While aniridia, literally meaning ‘lack of iris’, is named for its most visible symptom, the disorder affects the entire eye, including the retina, cornea, lens, macula and optic nerve (Lee et al., 2008). The incidence rate of aniridia ranges from 1:64,000 to 1:96,000 (Brauner et al., 2008). Two thirds of all aniridia cases are inherited through autosomal dominant transmission, with almost complete penetrance and variable expressivity (Brauner et al., 2008, Ton et al., 1991), and one third of cases occur sporadically. Patients with aniridia often have retinal detachment, as well as underdevelopment of retinal tissue. Retinal dysfunction may be caused by underdevelopment of the fovea, or  2  by phototoxicity as a result of an abnormal iris (Dowler et al., 1995, Lee et al., 2008, Tremblay et al., 1998). Approximately 20% of individuals with aniridia have limbal stem cell deficiency which leads to aniridia-associated keratopathy, a thickening and vascularization of the cornea. Corneal opacities develop early in life and can eventually lead to complete opacification of the cornea which severely impairs vision (Brauner et al., 2008). Cataracts, a gradual clouding of the lens, affect approximately 50–85% of patients with aniridia and have a progressively significant impact on visual acuity as the patient ages. Approximately 10% of patients with aniridia have optic nerve hypoplasia, an underdevelopment of the optic nerve, which can result in a form of involuntary eye movement called nystagmus (Mcculley et al., 2005). Glaucoma, an eye disorder which is characterized by damage to the optic nerve, has been reported in approximately 50% of cases with aniridia, and generally appears in early adulthood (Swanner et al., 2004). PAX6 (paired box gene 6), located on chromosome 11p13, was originally suggested as a candidate gene for aniridia through positional cloning (Ton et al., 1991). It is a paired-box transcription factor and is one of the master control genes found to be central in the development of the eye. Mutations which lead to the loss of function of one copy of PAX6 have been found to cause aniridia, while homozygous mutations in PAX6 are lethal during embryonic development, likely due to the importance of this gene during brain development (Halder et al., 1995). To date, over 290 mutations in PAX6 have been shown to cause aniridia (LOVD-PAX6, 2009). Phenotypic variability has been reported among patients with aniridia, occasionally within a family with identical mutations, however there is a correlation between the severity of the phenotype and the level of PAX6 activity (Brauner et al., 2008, Glaser et al., 1992). During embryonic development,  3  PAX6 is expressed in cells of the retina, lens, cilary body and cornea (Nishina et al., 1999). PAX6 also shows expression in the central nervous system and the pancreas (Dohrmann et al., 2000). The human PAX6 gene is 22 kb and has 14 exons, including exons 1-13, as well as an alternate splicing of exon 5, identified as 5a. The protein is 422 amino acids long and consists of two DNA binding domains, a paired domain and a homeodomain, which are separated by a linker segment, and is followed by a C-terminal domain which is rich in proline, serine and threonine and that possesses transcriptional trans-activation function (Lee et al., 2008, Neethirajan et al., 2009). Pax6 mutations in mice are dominant, and produce the small eye (Sey) phenotype which is characterized by microphthalmia and aniridia-like symptoms such as optic nerve hypoplasia and corneal opacification. As seen in humans, the homozygous Sey mouse dies during embryogenesis, with gross brain and eye abnormalities (Halder et al., 1995). The majority of individuals affected by aniridia have mutations or chromosomal rearrangements involving PAX6, however, in approximately 20% of patients who have aniridia, no mutation has been found in the PAX6 gene, and their genetic mutation is unknown (Crolla & Van Heyningen, 2002, Gronskov et al., 2001, Traboulsi et al., 2008). 1.1.3  Peters’ anomaly Peters’ anomaly is a rare genetic eye disorder characterized primarily by  abnormal cleavage of the anterior chamber of the eye during prenatal development. PA has been reported to occur with aniridia, and may be associated with other abnormalities of the eye, including myopia, corneal opacity, and optic disk hypoplasia. The ocular disorder was first described by Peters in 1906 and has the prevalence estimated at  4  approximately 1:100,000 newborns (Ciralsky & Colby, 2007). PA is grouped under the disease classification of anterior segment dysgenesis (ASD) which comprises a group of eye disorders resulting from abnormal development of the anterior segment of the eye and are often associated with glaucoma (Chavarria-Soley et al., 2006, Ciralsky & Colby, 2007). The major clinical symptoms of PA originate during prenatal development when abnormal cleavage of the anterior segment of the eye occurs. The lens vesicle, which eventually becomes the lens and the anterior chamber, and the surface ectoderm, which develops into the cornea, fail to separate normally from one another. Patients have corneal opacity, as well as adhesions between the cornea and lens, and between the cornea and iris. PA is also associated with thinning of the corneal stroma, the thick middle layer of the cornea which is comprised of collagen fibers and gives the cornea its form, elasticity, and strength. The Descemet’s membrane, which lies between the corneal stroma and the endothelium of the anterior chamber, is responsible for acting as a protective barrier against infection and injuries. Patients with PA often have an abnormally formed or absent Descemet’s membrane (Ciralsky & Colby, 2007, Ozeki et al., 2000). Approximately 50-70% of patients with PA develop glaucoma, and PA may also exist simultaneously with other eye disorders, such as aniridia, coloboma, and microphthalmia (Ciralsky & Colby, 2007, Ozeki et al., 2000), or as a multi-systemic disorder which is characterized by PA in the eyes, combined with mental retardation, cleft lip, cleft palate, facial dysmorphic features, and short stature (Maillette De Buy Wenniger-Prick & Hennekam, 2002).  5  The majority of cases of PA are classified as sporadic; however some PA families have been studied and have been shown to have both autosomal dominant and autosomal recessive inheritance. PA families have shown incomplete penetrance, meaning that in some families, some individuals fail to express the trait, even when they carry the causative mutation. Variable expressivity is also prevalent in families with PA, thus some patients with the disease have severe anterior segment dysgenesis while other patients with the identical mutation have a lesser phenotype, such as cataracts (Edward et al., 2004, Summers et al., 2008). Mutations in the PAX6 gene are the primary known cause of aniridia, and interestingly, some patients with PA have been reported to possess PAX6 mutations as well (Hanson et al., 1994). However subsequent analysis showed that the majority of PA patients had no PAX6 mutations or deletions, suggesting that there are likely other genes involved (Churchill et al., 1998, Dansault et al., 2007, Hanson et al., 1994). Other genes known to be essential in anterior segment development have also been investigated in patients with PA. Forkhead box C1 (FOXC1) is a transcription factor which plays a role in the regulation of ocular development. Mutations in this gene have been associated with primary congenital glaucoma and Axenfeld-Rieger anomaly, an anterior segment dysgenesis which is characterized by abnormal development of the peripheral anterior segment of the eye accompanied by non-ocular abnormalities, such as dental hypoplasia (Kaur et al., 2009). A missense mutation in FOXC1, Phe112Ser, has been linked to a family with Axenfeld-Rieger syndrome, including one family member with PA (Honkanen et al., 2003). Forkhead box E3 (FOXE3), is a transcription factor responsible for regulation of lens development. A family with anterior segment  6  dysgenesis, including an individual with PA, was recently shown to possess a mutation in FOXE3 (Iseri et al., 2009). Paired-like homeodomain transcription factor 2 (PITX2), also known as RIEG1, has been reported to cause anterior segment disorders, specifically Axenfeld-Rieger anomaly. A recent publication has identified a PITX2 mutation in one individual with PA (Arikawa et al., 2009). CYP1B1, a gene which belongs to the cytochrome P450 family, has been identified as one of the major causes of autosomal recessive primary congenital glaucoma and mutations in CYP1B1 have been identified in patients with PA (Ciralsky & Colby, 2007). While mutations in PAX6, FOXC1, FOXE3, PITX2, and CYP1B1 have been found in a number of patients with PA, several recent multi-gene genetic screens of PA patients have unable to identify causative mutations in any of these aforementioned genes. Therefore the etiology of the PA in these patients is unknown (Berker et al., 2009, Chavarria-Soley et al., 2006, Dansault et al., 2007, Edward et al., 2004, Vincent et al., 2006). Taken together, these results suggest that there is likely another eye development gene implicated in PA which has not yet been identified. 1.1.4 NR2E1 Human nuclear receptor 2E1 (NR2E1), also known as TLX, is comprised of 9 exons and spans 21 kb at the 6q21 region. NR2E1 is highly conserved and the 386-amino acid human NR2E1 protein shares 97% and 99% identity with chick and mouse Nr2e1, respectively (Jackson et al., 1998). NR2E1 is classified as an orphan nuclear receptor, meaning that to date no ligand for NR2E1 has been identified. NR2E1 has been identified as a having a key role in the development of the nervous system, including cortical  7  development, ocular development, and neurogenesis (Monaghan et al., 1996, Young et al., 2002). Studies in mice have illustrated the importance of Nr2e1 in brain and eye development. Mice that are null for both copies of Nr2e1 (Nr2e1-/-) have brain development and behavioural abnormalities. These mice have underdevelopment of the cortex, hippocampus, amygdala, corpus callosum, and olfactory bulbs (Monaghan et al., 1997, Young et al., 2002). Mice that lack Nr2e1 also display striking behavioural abnormalities. Nr2e1-/- mice, particularly the males, are extremely aggressive, and will bite and kill their littermates as well as intended mating partners. Additionally, female Nr2e1-/- mice have reduced maternal behaviour and are unable to raise their pups approximately 50% of the time (Young et al., 2002). Mice that are null for Nr2e1 also display an eye phenotype that is characterized by retinal degeneration, optic nerve hypoplasia, abnormal vascularization of the retina, and reduced or flat electroretinogram (Young et al., 2002). At the cellular level, loss of Nr2e1 leads to abnormal development of Muller cells and impaired astrocyte network formation (Miyawaki et al., 2004). Nr2e1 has been shown to be expressed in optic invagination as early as embryonic day E8.5, and is also expressed in the retinas of adult mice (Monaghan et al., 1995). Excitingly, the Simpson laboratory generated multicopy random insertion transgenic mice containing the human NR2E1 and was able to successfully rescue the brain and behavioural phenotype of Nr2e1-null mice (Abrahams et al., 2005). This rescue paradigm provides strong support for the hypothesis of conserved function between the mouse Nr2e1 and human NR2E1. Interestingly, the eye phenotype was not able to be completely rescued (Abrahams et al., 2005). The transgenic mouse which was  8  created has been shown to contain multiple copies of the human NR2E1 transgene (Wong, 2009). It is possible that the mouse eyes are sensitive not only to the presence and absence of the gene, but to changes in gene dosage. Taken together, these findings in mice suggest that mutations of human NR2E1 could potentially lead to eye abnormalities. I hypothesize that NR2E1 may act as a significant regulator of human eye development and could be a promising genetic candidate for several human eye disorders, including aniridia, PA, and other anterior segment dysgenesis disorders. 1.1.5 NR2E1 and PAX6 Further evidence that implicates NR2E1 as a candidate gene for abnormal ocular development in humans can be drawn when comparing it to PAX6, a transcription factor involved in aniridia and PA. Both NR2E1 and PAX6 have been shown to be important for development of the eye in many different species including mouse, Xenopus, and chicken (Cvekl et al., 1995, Hollemann et al., 1998, Stenman et al., 2003, Yu et al., 2000). These two genes have also been shown to cooperate genetically in development of the mouse brain, specifically in creating the pallio-subpallial boundary. Stenman et al., 2003 demonstrated this by comparing Pax6+/+,Nr2e1-/-; Pax6+/-,Nr2e1-/-; and Pax6-/-,Nr2e1-/mice (Stenman et al., 2003). The absence of Pax6 and Nr2e1 leads to a more severe brain phenotype than Pax6+/+;Nr2e1-/- (Stenman et al., 2003). Although this group did not examine the mouse eyes, the interaction between these two genes in the mouse brain suggests that Nr2e1 and Pax6 could have a similar interaction in the developing mouse eye. A compelling and recent finding is that overexpression of Nr2e1 creates an eye phenotype in mice which is almost identical to Pax6+/- (Sey) mice, which are a well-  9  established mouse model for aniridia (Figure 1.2). Mutant mice for these two genes model human aniridia. Like Sey mice, Nr2e1-overexpressor eyes are either underdeveloped or absent, and they show other symptoms present in human patients with aniridia, such as hypoplasia of the optic nerve and corneal opacity (Simpson et al., unpublished, (Schedl et al., 1996).  Figure 1.2: Similarity between Nr2e1 overexpressor and Pax6+/- mice In both strains, mice show phenotypic variability in pathology of the eye. For each strain, the eye on the left is more normal; the eye on the right shows microphthalmia and corneal opacity. 1.1.5 NR2E1 and NR2E3 Human nuclear receptor 2E3 (NR2E3), is the most closely related gene to NR2E1 in the genome. NR2E3 is a transcription factor composed of 8 exons, which are translated into a polypeptide chain of 410 amino acids. It is expressed exclusively in the retina and is postulated to play an important role in photoreceptor development (Haider et al., 2000, Schorderet & Escher, 2009). NR2E3 codes for a nuclear receptor that is specific to photoreceptors in the eye, and has been shown to repress cone-specific genes and activate rod-specific genes (Schorderet & Escher, 2009). NR2E3 has a high level of amino acid  10  similarity and identity with NR2E1, particularly in the DNA binding domains (DBD) and ligand binding domains (LBD) of the two genes (Figure 1.3).  Figure 1.3: Alignment of Human NR2E1 and NR2E3 BLAST (Basic Local Alignment Search Tool, http://genome.ucsc.edu/) results depict alignment between NR2E1 and NR2E3. NR2E1 has 43% amino acid identity and 50% amino acid similarity with NR2E3. Blue highlighting indicates amino acid identity, while grey indicates amino acid similarity. The DNA binding domain is outlined in red; the ligand binding domain is outlined in black.  11  1.1.6 NR2E3 and eye disorder Mutations in the NR2E3 gene have been found to cause the recessive and dominant retinopathies enhanced S-cone syndrome (ESCS), and autosomal dominant Retinitis Pigmentosa (ADRP) (Escher et al., 2008, Sharon et al., 2003). The clinical features of ESCS include night blindness, which presents early in life, sensitivity to blue light, retinal degeneration and vision loss. Patients with ESCS possess hyperfunction of S-cones, known as the ‘blue’ cones, and impaired M-cones and L-cones, as well as impaired rod functions (Haider et al., 2000). This disorder has an autosomal recessive pattern of inheritance and more than 20 different mutations in NR2E3, resulting in amino acid changes, have been associated with ESCS. These mutations are located in the evolutionary-conserved DBD and LBD of NR2E3, with the most common change p.R311Q, present in approximately 15% of patients with ESCS (Bernal et al., 2008, Fradot et al., 2007, Haider et al., 2000, Hayashi et al., 2005, Lam et al., 2007, Nakamura et al., 2004, Pachydaki et al., 2009, Schorderet & Escher, 2009, Sharon et al., 2003). Autosomal dominant retinitis pigmentosa is a type of retinal degeneration in which pathology of the photoreceptors or/and the retinal pigment epithelium lead to progressive vision loss. It is a clinically heterogeneous disorder characterized by night blindness, bone spicule-like pigmentary deposits which mottle the retinal pigment epithelium, constriction of the peripheral visual fields, also known as tunnel vision, and finally, loss of central vision (Gire et al., 2007, Schorderet & Escher, 2009). Twelve genes have been shown to be associated with ADRP, including the rhodopsin gene (RHO), the autosomal dominant retinitis pigmentosa gene (ADRP), the retinitis pigmentosa gene (RP1), retinal degeneration slow gene (RDS), inosine monophosphate  12  dehydrogenase 1(IMPDH1), and NR2E3. Mutations in NR2E3 account for approximately 1-2% of all cases of ADRP (Bernal et al., 2008, Escher et al., 2008, Gire et al., 2007). NR2E3 mutations that are known to cause eye disorders are summarized in Table 1.1.  Table 1.1: Mutations in NR2E3 result in eye disorders Location Mutation Type Result Associated Phenotype Reference Intron1 A to C mutation Splice site acceptor change Retinitis pigmentosa, ECSC Haider et al., 2000 AA 44 Ser to Leu Amino acid change Retinitis Pigmentosa Bernal et al., 2008 AA 56 Gly to Arg Amino acid change Retinitis Pigmentosa Escher et al. 2008 AA 65 Asn to Asn Synonymous change Retinitis Pigmentosa Bernal et al., 2008 AA 67-69 Deletion Three amino acid deletion Enhanced S-cone Syndrome Bernal et al., 2008 AA 71 Deletion Amino acid deletion Goldmann-Favre Syndrome Pachydaki et al. 2009 AA 76 Arg to Trp Amino acid change Enhanced S-cone Syndrome dnSNP AA 76 Arg to Gln Amino acid change Enhanced S-cone Syndrome dnSNP AA 97 Arg to His Amino acid change Enhanced S-cone Syndrome Haider et al. 2000 AA 104 Arg to Gln Amino acid change Enhanced S-cone Syndrome Hayashi et al., 2005 AA 104 Arg to Trp Amino acid change Enhanced S-cone Syndrome Haider et al. 2000 AA 121 Glu to Lys Amino acid change Enhanced S-cone Syndrome Haider et al. 2000 AA 140 Glu to Gly Amino acid change Enhanced S-cone Syndrome Haider et al. 2000 AA 163 Met to Thr Amino acid change Enhanced S-cone Syndrome Haider et al. 2000 AA 234 Trp to Ser Amino acid change Enhanced S-cone Syndrome Fradot et al. 2006 AA 256 Ala to Glu Amino acid change Enhanced S-cone Syndrome Sharon et al., 2003 AA 256 Ala to Val Amino acid change Enhanced S-cone Syndrome Lam et al., 2007 AA 287 Gly to Ser Amino acid change Retinitis Pigmentosa Bernal et al., 2008 AA 309 Arg to Gly Amino acid change Enhanced S-cone Syndrome Haider et al. 2000 AA 311 Arg to Gln Amino acid change Enhanced S-cone Syndrome Haider et al. 2000 AA 324 Lys to Arg Amino acid change Retinitis Pigmentosa Bernal et al., 2008 AA 334 Arg to Gly Amino acid change Enhanced S-cone Syndrome Hayashi et al., 2005 AA 345 Deletion Deletion creating stop codon Retinitis Pigmentosa dbSNP AA 350 Gln to Arg Amino acid change Enhanced S-cone Syndrome Pachydaki et al. 2009 AA 350 Deletion Deletion creating stop codon Enhanced S-cone Syndrome Nakamura, et al. 2004 AA 385 Arg to Pro Amino acid change Enhanced S-cone Syndrome Haider et al. 2000 AA 407 Met to Lys Amino acid change Enhanced S-cone Syndrome Haider et al. 2000 3'UTR Deletion 5 bp deletion Retinitis Pigmentosa Bernal et al., 2008 Mutations in NR2E3 that are known to cause eye disorder. Pink highlighting indicates mutations that lie in the DNA binding domain, yellow highlighting indicates mutations that lie in the ligand binding domain.  Mutations in NR2E3 play a well-established role in eye disorders. Because NR2E1 is highly homologous to NR2E3, and both genes are expressed the retinal cells of the eye, it is possible that mutations in NR2E1 could also result in abnormal development of the eye.  13  1.1.7  Hypothesis and thesis objectives We hypothesized that that patients who had aniridia, PA, or related eye disorders,  not explained by mutations in PAX6, have mutations in NR2E1. The primary objective of this thesis was to sequence NR2E1 in patients of unknown etiology who have aniridia, PA and related eye disorders. This project aimed to identify candidate mutations within the NR2E1 gene that could cause aniridia, PA, or related eye disorders. This research also endeavored to provide the scientific and medical community with a greater depth of knowledge of the role of NR2E1 in eye disorders.  14  1.2  REFERENCES  Abrahams, B.S., Kwok, M.C., Trinh, E., Budaghzadeh, S., Hossain, S.M. & Simpson, E.M. (2005) Pathological aggression in "fierce" mice corrected by human nuclear receptor 2E1. J Neurosci, 25, 6263-6270. Arikawa, A., Yoshida, S., Yoshikawa, H., Ishikawa, K., Yamaji, Y., Arita, R.I., Ueno, A. & Ishibashi, T. (2009) Case of novel PITX2 gene mutation associated with Peters' anomaly and persistent hyperplastic primary vitreous. Eye. Baum, L., Pang, C.P., Fan, D.S., Poon, P.M., Leung, Y.F., Chua, J.K. & Lam, D.S. (1999) Run-on mutation and three novel nonsense mutations identified in the PAX6 gene in patients with aniridia. Hum Mutat, 14, 272-273. Berker, N., Alanay, Y., Elgin, U., Volkan-Salanci, B., Simsek, T., Akarsu, N. & Alikasifoglu, M. (2009) A new autosomal dominant Peters' anomaly phenotype expanding the anterior segment dysgenesis spectrum. Acta Ophthalmol, 87, 52-57. Bernal, S., Solans, T., Gamundi, M.J., Hernan, I., de Jorge, L., Carballo, M., Navarro, R., Tizzano, E., Ayuso, C. & Baiget, M. (2008) Analysis of the involvement of the NR2E3 gene in autosomal recessive retinal dystrophies. Clin Genet, 73, 360-366. Blackshaw, S. & Livesey, R. (2002) Applying genomics technologies to neural development. Curr Opin Neurobiol, 12, 110-114. Brauner, S.C., Walton, D.S. & Chen, T.C. (2008) Aniridia. Int Ophthalmol Clin, 48, 7985. Chauhan, B.K., Yang, Y., Cveklova, K. & Cvekl, A. (2004) Functional properties of natural human PAX6 and PAX6(5a) mutants. Invest Ophthalmol Vis Sci, 45, 385392.  15  Chavarria-Soley, G., Michels-Rautenstrauss, K., Caliebe, A., Kautza, M., Mardin, C. & Rautenstrauss, B. (2006) Novel CYP1B1 and known PAX6 mutations in anterior segment dysgenesis (ASD). J Glaucoma, 15, 499-504. Chow, R.L. & Lang, R.A. (2001) Early eye development in vertebrates. Annu Rev Cell Dev Biol, 17, 255-296. Churchill, A.J., Booth, A.P., Anwar, R. & Markham, A.F. (1998) PAX 6 is normal in most cases of Peters' anomaly. Eye, 12 (Pt 2), 299-303. Ciralsky, J. & Colby, K. (2007) Congenital corneal opacities: a review with a focus on genetics. Semin Ophthalmol, 22, 241-246. Crolla, J.A. & van Heyningen, V. (2002) Frequent chromosome aberrations revealed by molecular cytogenetic studies in patients with aniridia. Am J Hum Genet, 71, 1138-1149. Cvekl, A., Sax, C.M., Li, X., McDermott, J.B. & Piatigorsky, J. (1995) Pax-6 and lensspecific transcription of the chicken delta 1-crystallin gene. Proc Natl Acad Sci U S A, 92, 4681-4685. Dansault, A., David, G., Schwartz, C., Jaliffa, C., Vieira, V., de la Houssaye, G., Bigot, K., Catin, F., Tattu, L., Chopin, C., Halimi, P., Roche, O., Van Regemorter, N., Munier, F., Schorderet, D., Dufier, J.L., Marsac, C., Ricquier, D., Menasche, M., Penfornis, A. & Abitbol, M. (2007) Three new PAX6 mutations including one causing an unusual ophthalmic phenotype associated with neurodevelopmental abnormalities. Mol Vis, 13, 511-523. Davis, J.A. & Reed, R.R. (1996) Role of Olf-1 and Pax-6 transcription factors in neurodevelopment. J Neurosci, 16, 5082-5094.  16  Dohrmann, C., Gruss, P. & Lemaire, L. (2000) Pax genes and the differentiation of hormone-producing endocrine cells in the pancreas. Mech Dev, 92, 47-54. Dowler, J.G., Lyons, C.J. & Cooling, R.J. (1995) Retinal detachment and giant retinal tears in aniridia. Eye, 9 (Pt 3), 268-270. Edward, D., Al Rajhi, A., Lewis, R.A., Curry, S., Wang, Z. & Bejjani, B. (2004) Molecular basis of Peters anomaly in Saudi Arabia. Ophthalmic Genet, 25, 257270. Ellett, M.L. (1996) Gilbert syndrome. Gastroenterol Nurs, 19, 102-104. Escher, P., Gouras, P., Roduit, R., Tiab, L., Bolay, S., Delarive, T., Chen, S., Tsai, C.C., Hayashi, M., Zernant, J., Merriam, J.E., Mermod, N., Allikmets, R., Munier, F.L. & Schorderet, D.F. (2009) Mutations in NR2E3 can cause dominant or recessive retinal degenerations in the same family. Hum Mutat. 30, 342-351. Fradot, M., Lorentz, O., Wurtz, J.M., Sahel, J.A. & Leveillard, T. (2007) The loss of transcriptional inhibition by the photoreceptor-cell specific nuclear receptor (NR2E3) is not a necessary cause of enhanced S-cone syndrome. Mol Vis, 13, 594-601. Gire, A.I., Sullivan, L.S., Bowne, S.J., Birch, D.G., Hughbanks-Wheaton, D., Heckenlively, J.R. & Daiger, S.P. (2007) The Gly56Arg mutation in NR2E3 accounts for 1-2% of autosomal dominant retinitis pigmentosa. Mol Vis, 13, 19701975. Glaser, T., Walton, D.S. & Maas, R.L. (1992) Genomic structure, evolutionary conservation and aniridia mutations in the human PAX6 gene. Nat Genet, 2, 232239.  17  Gronskov, K., Olsen, J.H., Sand, A., Pedersen, W., Carlsen, N., Bak Jylling, A.M., Lyngbye, T., Brondum-Nielsen, K. & Rosenberg, T. (2001) Population-based risk estimates of Wilms tumor in sporadic aniridia. A comprehensive mutation screening procedure of PAX6 identifies 80% of mutations in aniridia. Hum Genet, 109, 11-18. Haider, N.B., Jacobson, S.G., Cideciyan, A.V., Swiderski, R., Streb, L.M., Searby, C., Beck, G., Hockey, R., Hanna, D.B., Gorman, S., Duhl, D., Carmi, R., Bennett, J., Weleber, R.G., Fishman, G.A., Wright, A.F., Stone, E.M. & Sheffield, V.C. (2000) Mutation of a nuclear receptor gene, NR2E3, causes enhanced S cone syndrome, a disorder of retinal cell fate. Nat Genet, 24, 127-131. Halder, G., Callaerts, P. & Gehring, W.J. (1995) New perspectives on eye evolution. Curr Opin Genet Dev, 5, 602-609. Hamel, C. (2006) Retinitis pigmentosa. Orphanet J Rare Dis, 1, 40. Hanson, I.M., Fletcher, J.M., Jordan, T., Brown, A., Taylor, D., Adams, R.J., Punnett, H.H. & van Heyningen, V. (1994) Mutations at the PAX6 locus are found in heterogeneous anterior segment malformations including Peters' anomaly. Nat Genet, 6, 168-173. Hayashi, T., Gekka, T., Goto-Omoto, S., Takeuchi, T., Kubo, A. & Kitahara, K. (2005) Novel NR2E3 mutations (R104Q, R334G) associated with a mild form of enhanced S-cone syndrome demonstrate compound heterozygosity. Ophthalmology, 112, 2115.  18  Hollemann, T., Bellefroid, E. & Pieler, T. (1998) The Xenopus homologue of the Drosophila gene tailless has a function in early eye development. Development, 125, 2425-2432. Honkanen, R.A., Nishimura, D.Y., Swiderski, R.E., Bennett, S.R., Hong, S., Kwon, Y.H., Stone, E.M., Sheffield, V.C. & Alward, W.L. (2003) A family with AxenfeldRieger syndrome and Peters Anomaly caused by a point mutation (Phe112Ser) in the FOXC1 gene. Am J Ophthalmol, 135, 368-375. Iseri, S.U., Osborne, R.J., Farrall, M., Wyatt, A.W., Mirza, G., Nurnberg, G., Kluck, C., Herbert, H., Martin, A., Hussain, M.S., Collin, J.R., Lathrop, M., Nurnberg, P., Ragoussis, J. & Ragge, N.K. (2009) Seeing clearly: the dominant and recessive nature of FOXE3 in eye developmental anomalies. Hum Mutat, 30, 1378-1386. Jackson, A., Panayiotidis, P. & Foroni, L. (1998) The human homologue of the Drosophila tailless gene (TLX): Characterization and mapping to a region of common deletion in human lymphoid leukemia on chromosome 6q21. Genomics, 50, 34-43. Kaur, K., Ragge, N.K. & Ragoussis, J. (2009) Molecular analysis of FOXC1 in subjects presenting with severe developmental eye anomalies. Mol Vis, 15, 1366-1373. Kim, S.K., Haines, J.L., Berson, E.L. & Dryja, T.P. (1994) Nonallelic heterogeneity in autosomal dominant retinitis pigmentosa with incomplete penetrance. Genomics, 22, 659-660. Kitambi, S.S. & Hauptmann, G. (2006) The zebrafish orphan nuclear receptor genes nr2e1 and nr2e3 are expressed in developing eye and forebrain. Gene Expr Patterns. 7, 521-528.  19  Kolb H., Fernandez.E., Nelson R., (2007) Webvision: The Organization of the Retina and Visual System, University of Utah, John Moran Eye Center, Salt Lake City. Kumar, R.A., Leach, S., Bonaguro, R., Chen, J., Yokom, D.W., Abrahams, B.S., Seaver, L., Schwartz, C.E., Dobyns, W., Brooks-Wilson, A. & Simpson, E.M. (2007) Mutation and evolutionary analyses identify NR2E1-candidate-regulatory mutations in humans with severe cortical malformations. Genes Brain Behav, 6, 503-516. Kumar, S. & Hedges, S.B. (1998) A molecular timescale for vertebrate evolution. Nature, 392, 917-920. Lam, B.L., Goldberg, J.L., Hartley, K.L., Stone, E.M. & Liu, M. (2007) Atypical mild enhanced S-cone syndrome with novel compound heterozygosity of the NR2E3 gene. Am J Ophthalmol, 144, 157-159. Lee, H., Khan, R. & O'Keefe, M. (2008) Aniridia: current pathology and management. Acta Ophthalmol, 86, 708-715. Llobet, A., Gasull, X. & Gual, A. (2003) Understanding trabecular meshwork physiology: a key to the control of intraocular pressure? News Physiol Sci, 18, 205-209. Maillette de Buy Wenniger-Prick, L.J. & Hennekam, R.C. (2002) The Peters' plus syndrome: a review. Ann Genet, 45, 97-103. Martinez-De Luna, R.I. & El-Hodiri, H.M. (2007) The Xenopus ortholog of the nuclear hormone receptor Nr2e3 is primarily expressed in developing photoreceptors. Int J Dev Biol, 51, 235-240. McCulley, T.J., Mayer, K., Dahr, S.S., Simpson, J. & Holland, E.J. (2005) Aniridia and optic nerve hypoplasia. Eye, 19, 762-764.  20  Meyer, J.S., Shearer, R.L., Capowski, E.E., Wright, L.S., Wallace, K.A., McMillan, E.L., Zhang, S.C. & Gamm, D.M. (2009) Modeling early retinal development with human embryonic and induced pluripotent stem cells. Proc Natl Acad Sci U S A, 106, 16698-16703. Miyawaki, T., Uemura, A., Dezawa, M., Yu, R.T., Ide, C., Nishikawa, S., Honda, Y., Tanabe, Y. & Tanabe, T. (2004) Tlx, an Orphan Nuclear Receptor, Regulates Cell Numbers and Astrocyte Development in the Developing Retina. J Neurosci, 24, 8124-8134. Monaghan, A.P., Bock, D., Gass, P., Schwager, A., Wolfer, D.P., Lipp, H.P. & Schutz, G. (1997) Defective limbic system in mice lacking the tailless gene. Nature, 390, 515-517. Monaghan, A.P., Gass, P., Lipp, H.-P., Wolfer, D., Lang, R., Stagliar, M. & Schütz, G. (1996) Cerebral hemisphere reduction and sever aggression associated with mice lacking the orphan nuclear receptor. CSH Mouse Molecular Genetics Abstracts, 178. Monaghan, A.P., Grau, E., Bock, D. & Schütz, G. (1995) The mouse homolog of the orphan nuclear receptor tailless is expressed in the developing forebrain. Development, 121, 839-853. Moore, A.T., Fitzke, F., Jay, M., Arden, G.B., Inglehearn, C.F., Keen, T.J., Bhattacharya, S.S. & Bird, A.C. (1993) Autosomal dominant retinitis pigmentosa with apparent incomplete penetrance: a clinical, electrophysiological, psychophysical, and molecular genetic study. Br J Ophthalmol, 77, 473-479.  21  Nakamura, Y., Hayashi, T., Kozaki, K., Kubo, A., Omoto, S., Watanabe, A., Toda, K., Takeuchi, T., Gekka, T. & Kitahara, K. (2004) Enhanced S-cone syndrome in a Japanese family with a nonsense NR2E3 mutation (Q350X). Acta Ophthalmol Scand, 82, 616-622. Neethirajan, G., Solomon, A., Krishnadas, S.R., Vijayalakshmi, P. & Sundaresan, P. (2009) Genotype/phenotype association in Indian congenital aniridia. Indian J Pediatr, 76, 513-517. Nishina, S., Kohsaka, S., Yamaguchi, Y., Handa, H., Kawakami, A., Fujisawa, H. & Azuma, N. (1999) PAX6 expression in the developing human eye. Br J Ophthalmol, 83, 723-727. Ozeki, H., Shirai, S., Nozaki, M., Sakurai, E., Mizuno, S., Ashikari, M., Matsunaga, N. & Ogura, Y. (2000) Ocular and systemic features of Peters' anomaly. Graefes Arch Clin Exp Ophthalmol, 238, 833-839. Pachydaki, S.I., Klaver, C.C., Barbazetto, I.A., Roy, M.S., Gouras, P., Allikmets, R. & Yannuzzi, L.A. (2009) Phenotypic features of patients with NR2E3 mutations. Arch Ophthalmol, 127, 71-75. Parsons, D.W., Jones, S., Zhang, X., Lin, J.C., Leary, R.J., Angenendt, P., Mankoo, P., Carter, H., Siu, I.M., Gallia, G.L., Olivi, A., McLendon, R., Rasheed, B.A., Keir, S., Nikolskaya, T., Nikolsky, Y., Busam, D.A., Tekleab, H., Diaz, L.A., Jr., Hartigan, J., Smith, D.R., Strausberg, R.L., Marie, S.K., Shinjo, S.M., Yan, H., Riggins, G.J., Bigner, D.D., Karchin, R., Papadopoulos, N., Parmigiani, G., Vogelstein, B., Velculescu, V.E. & Kinzler, K.W. (2008) An integrated genomic analysis of human glioblastoma multiforme. Science, 321, 1807-1812.  22  Ramaesh, T., Collinson, J.M., Ramaesh, K., Kaufman, M.H., West, J.D. & Dhillon, B. (2003) Corneal abnormalities in Pax6+/- small eye mice mimic human aniridiarelated keratopathy. Invest Ophthalmol Vis Sci, 44, 1871-1878. Redeker, E.J., de Visser, A.S., Bergen, A.A. & Mannens, M.M. (2008) Multiplex ligation-dependent probe amplification (MLPA) enhances the molecular diagnosis of aniridia and related disorders. Mol Vis, 14, 836-840. Schedl, A., Ross, A., Lee, M., Engelkamp, D., Rashbass, P., van Heyningen, V. & Hastie, N.D. (1996) Influence of PAX6 gene dosage on development: overexpression causes severe eye abnormalities. Cell, 86, 71-82. Schorderet, D.F. & Escher, P. (2009) NR2E3 mutations in enhanced S-cone sensitivity syndrome (ESCS), Goldmann-Favre syndrome (GFS), clumped pigmentary retinal degeneration (CPRD), and retinitis pigmentosa (RP). Hum Mutat. 30, 1475 - 1485. Sharon, D., Sandberg, M.A., Caruso, R.C., Berson, E.L. & Dryja, T.P. (2003) Shared mutations in NR2E3 in enhanced S-cone syndrome, Goldmann-Favre syndrome, and many cases of clumped pigmentary retinal degeneration. Arch Ophthalmol, 121, 1316-1323. Singh, S., Chao, L.Y., Mishra, R., Davies, J. & Saunders, G.F. (2001) Missense mutation at the C-terminus of PAX6 negatively modulates homeodomain function. Hum Mol Genet, 10, 911-918. Stenman, J., Yu, R.T., Evans, R.M. & Campbell, K. (2003) Tlx and Pax6 co-operate genetically to establish the pallio-subpallial boundary in the embryonic mouse telencephalon. Development, 130, 1113-1122.  23  Summers, K.M., Withers, S.J., Gole, G.A., Piras, S. & Taylor, P.J. (2008) Anterior segment mesenchymal dysgenesis in a large Australian family is associated with the recurrent 17 bp duplication in PITX3. Mol Vis, 14, 2010-2015. Swanner, J.C., Walton, D.S. & Chen, T.C. (2004) Prevention of aniridic glaucoma with goniosurgery. Int Ophthalmol Clin, 44, 67-71. Ton, C.C., Hirvonen, H., Miwa, H., Weil, M.M., Monaghan, P., Jordan, T., van Heyningen, V., Hastie, N.D., Meijers-Heijboer, H., Drechsler, M. & et al. (1991) Positional cloning and characterization of a paired box- and homeobox-containing gene from the aniridia region. Cell, 67, 1059-1074. Traboulsi, E.I., Ellison, J., Sears, J., Maumenee, I.H., Avallone, J. & Mohney, B.G. (2008) Aniridia with preserved visual function: a report of four cases with no mutations in PAX6. Am J Ophthalmol, 145, 760-764. Tremblay, F., Gupta, S.K., De Becker, I., Guernsey, D.L. & Neumann, P.E. (1998) Effects of PAX6 mutations on retinal function: an electroretinographic study. Am J Ophthalmol, 126, 211-218. Vincent, A., Billingsley, G., Priston, M., Glaser, T., Oliver, E., Walter, M., Ritch, R., Levin, A. & Heon, E. (2006) Further support of the role of CYP1B1 in patients with Peters anomaly. Mol Vis, 12, 506-510. Wong, B.K.Y. (2009) Evaluating the effects of variable NR2E1 levels on gene expression, behaviour, and neural and ocular development. Medical Genetics. University of British Columbia, Vancouver, p. 183. Wright, A.F., Reddick, A.C., Schwartz, S.B., Ferguson, J.S., Aleman, T.S., Kellner, U., Jurklies, B., Schuster, A., Zrenner, E., Wissinger, B., Lennon, A., Shu, X.,  24  Cideciyan, A.V., Stone, E.M., Jacobson, S.G. & Swaroop, A. (2004) Mutation analysis of NR2E3 and NRL genes in Enhanced S Cone Syndrome. Hum Mutat, 24, 439. Young, K.A., Berry, M.L., Mahaffey, C.L., Saionz, J.R., Hawes, N.L., Chang, B., Zheng, Q.Y., Smith, R.S., Bronson, R.T., Nelson, R.J. & Simpson, E.M. (2002) Fierce: a new mouse deletion of Nr2e1; violent behaviour and ocular abnormalities are background-dependent. Behavioural Brain Research, 132, 145-158. Yu, R.T., Chiang, M.Y., Tanabe, T., Kobayashi, M., Yasuda, K., Evans, R.M. & Umesono, K. (2000) The orphan nuclear receptor Tlx regulates Pax2 and is essential for vision. Proceedings of the National Academy of Sciences USA, 97, 2621-2625. Ziviello, C., Simonelli, F., Testa, F., Anastasi, M., Marzoli, S.B., Falsini, B., Ghiglione, D., Macaluso, C., Manitto, M.P., Garre, C., Ciccodicola, A., Rinaldi, E. & Banfi, S. (2005) Molecular genetics of autosomal dominant retinitis pigmentosa (ADRP): a comprehensive study of 43 Italian families. J Med Genet, 42, e47.  25  1  CHAPTER 2: A MUTATION SCREEN OF NR2E1 IN PATIENTS WITH ANIRIDIA, PETERS’ ANOMALY, AND RELATED EYE DISORDERS  2.1  INTRODUCTION Aniridia is a rare genetic panocular disorder, characterized by the  underdevelopment of the iris in the eye and degeneration of the retina, resulting in progressive visual loss (Lee et al., 2008). The majority of individuals affected by aniridia have mutations or chromosomal rearrangements involving paired box gene 6 (PAX6), a homeobox gene that has been found to be central in the development of the eye (Crolla & Van Heyningen, 2002). Over 290 mutations in the PAX6 gene are used as markers for early diagnosis, which can lead to timely medical interventions that can significantly improve the quality of life of the affected individual (LOVD-PAX6, 2009). Peters’ anomaly (PA), an anterior segment dysgenesis characterized by abnormal cleavage of the anterior chamber and corneal opacity, has been found to be associated with mutations in PAX6, FOXC1, PITX2, and CYP1B1 (Berker et al., 2009). However, in approximately 20% of patients who have aniridia and 75% of patients with PA, no mutation has been found in PAX6, or in the other genes known to be involved in these disorders, and their genetic mutation is unknown (Berker et al., 2009, Chavarria-Soley et al., 2006, Crolla & Van Heyningen, 2002, Dansault et al., 2007, Edward et al., 2004, Gronskov et al., 2001, Vincent et al., 2006). Thus, it is likely that other genes are involved in the pathogenesis of aniridia and PA, and it is important to identify alternative genes that are responsible for these malformations. NR2E1 is a potential candidate.  1  A version of this chapter will be submitted for publication. Borrie A.E., Schuetz J.M., van Heyningen V., Walter M.A., Macdonald I.M., Pasutto F., Brooks B.P., Rosenberg T., Gronskøv K., Brooks-Wilson A.R., Simpson E.M. A mutation screen of NR2E1 in patients with aniridia, Peters’ anomaly, and related eye disorders.  26  PAX6 and NR2E1 are both transcription factors expressed in the brain and eye of multiple species, including human, mouse, frog (Xenopus) and chicken. In mouse brain, Pax6 is known to genetically interact with Nr2e1, and in the mouse eye, Pax6 and Nr2e1 are both expressed in retinal progenitor cells and Müller glia cells (Davis & Reed, 1996, Miyawaki et al., 2004, Stenman et al., 2003). The Nr2e1 null mouse demonstrates a variety of phenotypic abnormalities, including hyperplasia of the cortex, olfactory bulb, and retina, indicating that Nr2e1 regulates eye development in addition to brain development (Monaghan et al., 1995, Young et al., 2002). The mouse mutation lacking Nr2e1 can be completely rescued by the human gene in the brain and behaviour domains, but the rescue is only partial for eye (Abrahams et al., 2005). NR2E3 is the most closely related gene in the human genome to NR2E1. It is a transcription factor that codes for a photoreceptor-specific nuclear receptor which represses cone-specific genes and activates rod-specific genes (Haider et al., 2000). Nr2e1 and Nr2e3 have overlapping patterns of expression in the developing retinas of Xenopus and zebrafish (Kitambi & Hauptmann, 2006, Martinez-De Luna & El-Hodiri, 2007). Mutations in the NR2E3 gene have been shown to cause the dominant and recessive retinopathies Autosomal Dominant Retinitis Pigmentosa (ADRP) and Enhanced S-cone Syndrome (ESCS) (Escher et al., 2008). The high level of similarity between these two genes, and overlapping expression patterns in model organisms, provides evidence that NR2E1 could also play a role in human eye disorders. Together, these findings point to NR2E1 as a regulator of eye development and have implications for the gene in the development of human eye disorders, such as  27  aniridia and PA. We hypothesize that patients who have aniridia or PA, not explained by mutations in PAX6, will have mutations in NR2E1.  2.2  METHODS  2.2.1  Patient information and DNA collection We studied a total of 89 patients; 52 unrelated patients with aniridia, 15 patients  with familial aniridia who belonged to 6 different families, 12 unrelated patients with PA, 5 unrelated patients with Rieger syndrome, and 5 unrelated patients with related eye disorders including Axenfeld-Rieger anomaly, microphthalmia, optic nerve malformation, coloboma, and congenital cataract. Patient demographic and clinical data are reported in Table 2.1 and Table 2.2. Our sample collected included patients whose PAX6 gene had been previously characterized, as well as patients whose PAX6 had not been characterized (Table 2.1 and 2.2). Approval for this study was obtained from the Clinical Research Ethics Board of the University of British Columbia (Certificate of Approval #C99-0524). The research followed Canada’s Tri-Council Statement on ‘Ethical Conduct for Research Involving Humans’. Oragene™ DNA self-collection kits were used to collect saliva, following the manufacturer’s instructions (DNA Genotek, Kanata, Canada) from patients and relatives from the 2007 Aniridia International Medical Conference in Memphis, TN, in July 2007, and via mail. Saliva samples were stored and shipped at room temperature, and DNA purified from saliva was stored at 4°C. Patient DNA which was sent from collaborators had been DNA purified from blood and was shipped and stored at 4°C.  28  Table 2.1: Patient data  29  Table 2.1: Patient data (continued)  (1) Gronskov K et al. 2001, Hum Genet 109:11-18 Samples provided by: Walter (Dr. Michael Walter), Conference (taken at the Aniridia International Medical Conference in Memphis, TN, in July 2007), van Heyningen (Dr. Veronica van Heyningen), Brooks (Dr. Brian P. Brooks), Gronskov (Dr. Karen Gronskov), and Pasutto (Dr. Francesca Pasutto).  30  Table 2.2: Characteristics of the study population  2.2.2  Control DNA The following control subjects were studied: 1) 18 unaffected relatives of patients  with aniridia who appeared to have no eye abnormalities at time of DNA collection, 2) 4 unaffected relatives of patients with PA who appeared to have no eye abnormalities at time of DNA collection, 3) 94 individuals of Caucasian descent obtained from the Coriell Cell Repository, who appeared to be unaffected, though not given eye exams (http://coriell.umdnj.edu/), and 4) 94 “neurologically normal” Caucasians, which had been given eye exams and found to have normal vision and no eye disorders, obtained from the Coriell Cell Repository (catalog no. NDPT096) (http://coriell.umdnj.edu/). DNA from unaffected family members was collected using Oragene™ (DNA Genotek, Kanata, Canada) DNA self-collection kits. Additional sequencing data from control individuals from a previous study was included (Kumar et al., 2007). This included two 94-well plates including 94 Caucasians who appeared to be unaffected, though not given eye  31  exams obtained, from the Coriell Cell Repository (http://coriell.umdnj.edu/) and 94 Caucasian patients diagnosed with Gilbert syndrome. Gilbert syndrome is a genetic disorder in which an enzyme responsible for the disposal of bilirubin, a chemical that results from breakdown of hemoglobin, is abnormal. The main resulting phenotype is jaundice, and while these 94 individuals were not given eye exams, Gilbert syndrome is not co-morbid with any eye abnormalities (Ellett, 1996). 2.2.3 Sequencing We sequenced NR2E1 using DNA amplicons generated from 20 polymerase chain reaction (PCR) assays that covered the coding region, complete 5′- and 3′-untranslated regions (UTRs), exon-flanking regions including consensus splice sites, and six evolutionarily conserved non-coding regions as previously described (Kumar et al., 2007) with adjustments to the primers used for the PCR reactions (Table 2.3). Human genomic NR2E1 sequence AL078596 (http://www.ncbi.nlm.nih.gov/) was used as the reference sequence. Sequences were visually inspected and scored by at least two individuals using the Mutation Surveyor software suite (http://www.softgenetics.com/ms/). Every variant found was confirmed by repeating the PCR and sequencing process.  32  Table 2.3: PCR primers used to amplify NR2E1 sequences  Primers that were redesigned from Kumar et al., 2007 are indicated in grey. 5′-TGTAAAACGACGGCCAGT-3′ sequence (-21M13F) was added to the 5′ end of each forward primer to facilitate sequencing. 5′-CAGGAAACAGCTATGAC-3′ sequence (M13R) was added to the 5′ end of each reverse primer to facilitate sequencing. CE: Conserved element.  2.2.4 PAX6 characterization DNA samples from Patient 1380 and 1325 were analyzed by GeneDx (Gaithersburg, MD). They performed bi-directional sequencing for patients 1325 and 1380 on coding and non-coding sequences, more specifically, exonic regions of PAX6 (exons 1-13), the alternatively spliced exon 5a, and splice junctions. GeneDx (Gaithersburg, MD) also performed targeted array CGH analysis with exon-level resolution on patient 1380 to identify deletions or duplications of one or more exons of PAX6 (Redeker et al., 2008). 2.3  RESULTS  2.3.1  Sequence variation found in NR2E1 To establish whether individuals with aniridia and related eye disorder have  coding or regulatory variation in NR2E1, we amplified and sequenced NR2E1 in 89 patients with aniridia, PA, and other related eye disorders. In this analysis, we identified a non-synonymous (amino acid altering) coding variant in exon 7 of patient 1380 who was 33  effected by PA. No synonymous coding region variants were found. We also observed 16 non-coding variants in our patient group, of which 5 were detected in the 5'-UTR, 5 in the intronic sequence, and 7 within conserved regulatory elements. Transitions accounted for 60%, and transversions accounted for 40%, of all single nucleotide variants. 12% of the variation found in NR2E1 was accounted for by small insertions and deletions. Of the 89 patients sequenced, there were 7 patients that lacked allelic heterozygosity at every locus. There was no sequence variation specific only to control subjects. Among the 17 variants detected, 4 were novel and had not been previously reported in the literature or described by public DNA repositories (http://www.ncbi.nlm.nih.gov/projects/SNP/; Build 129) (Table 2.4). Table 2.4: Sequence variation of NR2E1 detected in patients with aniridia and related eye disorders  *Numbers adjusted to include data from Kumar et al., 2007. Red indicates variant location. Variants were initially found to be patient specific are indicated in grey.  2.3.2  Analysis of linkage disequilibrium in patients and related controls Analysis using Haploview, a bioinformatics software tool used to visualize  patterns of linkage disequilibrium in sequencing data, was conducted on the 89 patients  34  and revealed that variants g.-1492G>A, g.-200G>C, g.-34C>T were in linkage disequilibrium with each other (Figure 2.1).  Figure 2.1: Visual representation of linkage disequilibrium Haploview map represents patterns of linkage disequilibrium in genetic data from this study. Boxes contain pairwise r2 values ranging from 0 to 100, where 0 represents variants that are not in linkage disequilibrium with each other, and 100 represents variants that are in high linkage disequilibrium with each other. Variants observed only once were excluded from this analysis. 2.3.3  Initially discovered patient specific variants studied further Three of the four novel variants (g.3153C>T, g.14122C>G, g.14256C>T) were  initially only found in patients (Table 2.4). However, we expanded our control population, by amplifying and sequencing the corresponding regions in 188 control subjects that were  35  neurologically and ophthalmologically normal. Variant g.3153C>T was detected in 44 of the 376 control chromosomes sequenced, as shown in table 2.5. Variants g.14122C>G, found in patient 1380, and g.14256C>T found in patient 1325, were not detected in any of the 376 control chromosomes, and had not been detected in 376 control chromosomes previously reported (Kumar et al., 2007). As aniridia and PA are known to be caused by mutations in the PAX6 gene, it was essential to rule out the possibility that the eye disorders observed in patients 1380 were caused by mutations in PAX6. We had previously characterized patient 1380’s PAX6 gene using dideoxyfingerprinting and no detectable mutations in PAX6 had been found, and patient 1325 had not previously been characterized. The PAX6 gene in these patients of interest was further analyzed by GeneDx (Gaithersburg, MD). Sequencing of PAX6 was performed on patient 1325, who was found be heterozygous for Stop423Leu mutation in PAX6, a mutation that has been reported previously in association with aniridia (Baum et al., 1999, Singh et al., 2001). Because the PAX6 mutation could explain aniridia in patient 1325, this patient and the NR2E1 variant, g.14256C>T, was no longer of interest. Sequencing analysis revealed that patient 1380 did not possess any mutations in PAX6, and targeted array CGH did not reveal a deletion or duplication of PAX6. Further analyses of these three variants are summarized in Table 2.5.  36  Table 2.5: Characterization of three NR2E1 patient variants  *Numbers adjusted to include data from Kumar et al., 2007. Variant g.14122C>G, which was found to be patient specific, and found to have no detectable PAX6 mutations, is indicated in grey.  2.3.4 NR2E1 amino acid altering variant identified in patient with Peters’ anomaly The g.14122C>G variant, that was identified in patient 1380, results in the replacement of arginine (CGA) with glycine (GGA) at residue 274 (Figure 2.2).  Figure 2.2: Novel NR2E1 variant resulting in amino acid change A chromatogram showing the novel NR2E1 variant which results in an amino acid change. Chromatograms for the normal allele are shown above the mutant allele. Arrow indicates the position of the variant. The region corresponding to the R274G variant was amplified and sequenced in family members of patient 1380, including both parents and a sister. The father and the sister of patient 1380 did not possess the variant; however the mother of patient 1380 harbored the amino acid change (See Figure 2.3 for pedigree).  37  Figure 2.3: Amino acid variant R274G found in proband’s mother Pedigree of extended family of patient 1380 with genotypes for patient and family members. Males and females are represented by squares and circles, respectively, and the affected proband is a darkened. An ophthalmological examination of Patient 1380 revealed that the patient had central corneal opacity, retinal abnormalities, and adhesions between the iris and the cornea. Intraocular pressure was normal, suggesting a low risk for developing glaucoma, and length of the eye was also normal. Patient had reportedly suffered from hydrocephaly and intraventricular bleeding early in life; this was corrected with surgery. Basepair local alignment search tool (BLAST) analysis revealed that residue 274 in NR2E1 is highly conserved from human to zebrafish (Figure 2.4). This amino acid is highly conserved, indicating it is of functional and evolutionary importance. Interestingly, the corresponding amino acid in NR2E3, residue 309, is also conserved to zebrafish (Figure 2.4).  38  Figure 2.4: NR2E1 amino acid variant at residue 274 is highly conserved BLAST homology results depicting the amino acid similarity of human NR2E1 at residue 274 across an array of species in NR2E1. The homologous residue 309 in NR2E3 is also depicted as being highly conserved across an array of species (Sharon et al., 2003). SIFT (Sorting Intolerant From Tolerant) (http://sift.jcvi.org/), which distinguishes functionally neutral from deleterious amino acid changes, was utilized to calculate the probability of the arginine to glycine amino acid change being tolerated at codon 274. It was determined that this missense mutation would not be tolerated, and it is likely that the resulting protein would have altered function. This data was strengthened by data from an additional online protein analysis tool, Polyphen (http://genetics.bwh.harvard.edu/pph/), which determined that the mutation would be possibly damaging.  39  2.4  DISCUSSION This study is the first genetic report of NR2E1 in clinical samples of patients with  eye disorders. The R274G variant is the first germline amino acid change found in human NR2E1 to date. Previously, a heterozygous somatic mutation, g.298C>T; p.R100C, was identified in NR2E1, found in a glioblastoma multiforme tumor sample. However, it was a somatic mutation and was one of 1334 mutations found in that particular tumor (Parsons et al., 2008). There is evidence to support the hypothesis that the R274G variant identified in this study could conceivably underlie a human eye disorder such as PA by altering the protein function of NR2E1. The arginine amino acid at position 274 is conserved from human to zebrafish. Humans and zebrafish diverged from one another approximately 450 million year ago and lack of variability at this residue strongly suggests functional importance (Kumar & Hedges, 1998). Information pertaining to the importance of the amino acid change can be derived from the structure of the amino acids themselves. Arginine is a polar, positively charged amino acid. Thus, a change to a small, non-polar amino acid, such as glycine, would likely not be tolerated. An interesting finding that may point to functional importance of the amino acid change, R274G, is an identical mutation found at the homologous residue in the gene most closely related to NR2E1, NR2E3. Haider et al. (2000), identified an arginine to glycine amino acid change at residue 309 (R309G) in a patient with enhanced S-cone syndrome. This finding lends support to the hypothesis that an amino acid change at this location could be damaging, and is interesting because this amino acid change is known to cause an eye disorder in this closely related gene.  40  The finding of an apparently unaffected parent carrying this variant was unexpected. This would normally indicate that this variant was not de novo unlikely to cause disease. However, as there is evidence to support the hypothesis that the R274G variant could adversely effect eye development, it is prudent to explore alternative explanations. Three possible hypotheses explaining this result are discussed below. First, it is possible that the mother of patient 1380 is a mosaic and possesses two populations of cells with different genotypes (Strachan & Read, 1999). DNA sequenced from patient 1380’s mother was purified from saliva, but perhaps this variant is not found in the cells of the eye. In this case, the mother would be de novo for a variant that was passed down to the proband. A second hypothesis is that the variant was inherited, but there was reduced expressivity in the mother of patient 1380. Families with PA often have phenotypic variability. A recent paper reported that members of an extended family with PA noted that there was phenotypic variability in family members with the same mutation in PITX3 (Summers et al., 2008). Expression of the eye disorder ranged from cataract only to severe anterior segment mesenchymal dysgenesis (ASMD) (Summers et al., 2008). An additional study which examined 10 extended families with PA described reduced expressivity within families who possessed the same mutations in CYP1B1 (Edward et al., 2004). Therefore, an ophthalmological examination of the mother would be warranted to determine whether a less severe phenotype is present. Finally, if the mother was given an eye exam and found to be completely normal, the presence of a variant in her NR2E1 gene could be explained by incomplete penetrance.  41  The study by Edward et al. (2004) reported incomplete penetrance in families with PA caused by mutations in CYP1B1. Linkage disequilibrium (LD) analysis of the variants found in this patient population revealed weak LD in this region. However, there was significant LD between variants g.-1492G>A, g.-200G>C, and g.-34C>T. This is consistent with linkage disequilibrium data from a previous NR2E1 sequencing study (Kumar et al., 2007). Future studies could utilize a variety of experimental strategies to test the functionality of the R274G human variant in the laboratory. One method of testing the effect of the amino acid change is to introduce the NR2E1 R274G variant into the mouse genome using genetic engineering techniques. By generating strains of mice carrying the R274G human variant, we could test to see if they develop an eye phenotype and whether the phenotype is inherited from generation to generation with the variant. The DNA sequencing techniques utilized in this study are unable to detect large deletions, and therefore the possibility that large-scale deletions including NR2E1 may underlie eye development disorders in some patients cannot be excluded. Molecular techniques such as quantitative PCR could be utilized in the future to uncover any large deletions in these patients. Furthermore, the amino acid change was discovered in a patient with PA, and in this study, a total of 12 patients with PA were sequenced. Thus, a limitation of this work is small sample size. Expansion of our patient base, particularly of patients with PA, may yield a better understanding of the role of NR2E1 in eye disorders. 2.5  CONCLUSION This investigation of the human NR2E1 gene provides evidence of a rare genetic  variant found within the coding region of the gene that may contribute to human eye  42  disorder. The variant identified here provides the scientific and medical community with a greater depth of knowledge of the role of NR2E1 in genetic eye disorders and may lead to future research into the molecular basis of eye disorders such as aniridia and Peters’ anomaly.  43  2.6  REFERENCES  Abrahams, B.S., Kwok, M.C., Trinh, E., Budaghzadeh, S., Hossain, S.M. & Simpson, E.M. (2005) Pathological aggression in "fierce" mice corrected by human nuclear receptor 2E1. J Neurosci, 25, 6263-6270. Baum, L., Pang, C.P., Fan, D.S., Poon, P.M., Leung, Y.F., Chua, J.K. & Lam, D.S. (1999) Run-on mutation and three novel nonsense mutations identified in the PAX6 gene in patients with aniridia. Hum Mutat, 14, 272-273. Berker, N., Alanay, Y., Elgin, U., Volkan-Salanci, B., Simsek, T., Akarsu, N. & Alikasifoglu, M. (2009) A new autosomal dominant Peters' anomaly phenotype expanding the anterior segment dysgenesis spectrum. Acta Ophthalmol, 87, 52-57. Chavarria-Soley, G., Michels-Rautenstrauss, K., Caliebe, A., Kautza, M., Mardin, C. & Rautenstrauss, B. (2006) Novel CYP1B1 and known PAX6 mutations in anterior segment dysgenesis (ASD). J Glaucoma, 15, 499-504. Crolla, J.A. & van Heyningen, V. (2002) Frequent chromosome aberrations revealed by molecular cytogenetic studies in patients with aniridia. Am J Hum Genet, 71, 1138-1149. Dansault, A., David, G., Schwartz, C., Jaliffa, C., Vieira, V., de la Houssaye, G., Bigot, K., Catin, F., Tattu, L., Chopin, C., Halimi, P., Roche, O., Van Regemorter, N., Munier, F., Schorderet, D., Dufier, J.L., Marsac, C., Ricquier, D., Menasche, M., Penfornis, A. & Abitbol, M. (2007) Three new PAX6 mutations including one causing an unusual ophthalmic phenotype associated with neurodevelopmental abnormalities. Mol Vis, 13, 511-523.  44  Davis, J.A. & Reed, R.R. (1996) Role of Olf-1 and Pax-6 transcription factors in neurodevelopment. J Neurosci, 16, 5082-5094. Edward, D., Al Rajhi, A., Lewis, R.A., Curry, S., Wang, Z. & Bejjani, B. (2004) Molecular basis of Peters anomaly in Saudi Arabia. Ophthalmic Genet, 25, 257270. Ellett, M.L. (1996) Gilbert syndrome. Gastroenterol Nurs, 19, 102-104. Escher, P., Gouras, P., Roduit, R., Tiab, L., Bolay, S., Delarive, T., Chen, S., Tsai, C.C., Hayashi, M., Zernant, J., Merriam, J.E., Mermod, N., Allikmets, R., Munier, F.L. & Schorderet, D.F. (2008) Mutations in NR2E3 can cause dominant or recessive retinal degenerations in the same family. Hum Mutat. Gronskov, K., Olsen, J.H., Sand, A., Pedersen, W., Carlsen, N., Bak Jylling, A.M., Lyngbye, T., Brondum-Nielsen, K. & Rosenberg, T. (2001) Population-based risk estimates of Wilms tumor in sporadic aniridia. A comprehensive mutation screening procedure of PAX6 identifies 80% of mutations in aniridia. Hum Genet, 109, 11-18. Haider, N.B., Jacobson, S.G., Cideciyan, A.V., Swiderski, R., Streb, L.M., Searby, C., Beck, G., Hockey, R., Hanna, D.B., Gorman, S., Duhl, D., Carmi, R., Bennett, J., Weleber, R.G., Fishman, G.A., Wright, A.F., Stone, E.M. & Sheffield, V.C. (2000) Mutation of a nuclear receptor gene, NR2E3, causes enhanced S cone syndrome, a disorder of retinal cell fate. Nat Genet, 24, 127-131. Hollemann, T., Bellefroid, E. & Pieler, T. (1998) The Xenopus homologue of the Drosophila gene tailless has a function in early eye development. Development, 125, 2425-2432.  45  Kitambi, S.S. & Hauptmann, G. (2006) The zebrafish orphan nuclear receptor genes nr2e1 and nr2e3 are expressed in developing eye and forebrain. Gene Expr Patterns. Kumar, R.A., Leach, S., Bonaguro, R., Chen, J., Yokom, D.W., Abrahams, B.S., Seaver, L., Schwartz, C.E., Dobyns, W., Brooks-Wilson, A. & Simpson, E.M. (2007) Mutation and evolutionary analyses identify NR2E1-candidate-regulatory mutations in humans with severe cortical malformations. Genes Brain Behav, 6, 503-516. Kumar, S. & Hedges, S.B. (1998) A molecular timescale for vertebrate evolution. Nature, 392, 917-920. Lee, H., Khan, R. & O'Keefe, M. (2008) Aniridia: current pathology and management. Acta Ophthalmol, 86, 708-715. Martinez-De Luna, R.I. & El-Hodiri, H.M. (2007) The Xenopus ortholog of the nuclear hormone receptor Nr2e3 is primarily expressed in developing photoreceptors. Int J Dev Biol, 51, 235-240. Miyawaki, T., Uemura, A., Dezawa, M., Yu, R.T., Ide, C., Nishikawa, S., Honda, Y., Tanabe, Y. & Tanabe, T. (2004) Tlx, an Orphan Nuclear Receptor, Regulates Cell Numbers and Astrocyte Development in the Developing Retina. J Neurosci, 24, 8124-8134. Monaghan, A.P., Grau, E., Bock, D. & Schütz, G. (1995) The mouse homolog of the orphan nuclear receptor tailless is expressed in the developing forebrain. Development, 121, 839-853.  46  Parsons, D.W., Jones, S., Zhang, X., Lin, J.C., Leary, R.J., Angenendt, P., Mankoo, P., Carter, H., Siu, I.M., Gallia, G.L., Olivi, A., McLendon, R., Rasheed, B.A., Keir, S., Nikolskaya, T., Nikolsky, Y., Busam, D.A., Tekleab, H., Diaz, L.A., Jr., Hartigan, J., Smith, D.R., Strausberg, R.L., Marie, S.K., Shinjo, S.M., Yan, H., Riggins, G.J., Bigner, D.D., Karchin, R., Papadopoulos, N., Parmigiani, G., Vogelstein, B., Velculescu, V.E. & Kinzler, K.W. (2008) An integrated genomic analysis of human glioblastoma multiforme. Science, 321, 1807-1812. Redeker, E.J., de Visser, A.S., Bergen, A.A. & Mannens, M.M. (2008) Multiplex ligation-dependent probe amplification (MLPA) enhances the molecular diagnosis of aniridia and related disorders. Mol Vis, 14, 836-840. Sharon, D., Sandberg, M.A., Caruso, R.C., Berson, E.L. & Dryja, T.P. (2003) Shared mutations in NR2E3 in enhanced S-cone syndrome, Goldmann-Favre syndrome, and many cases of clumped pigmentary retinal degeneration. Arch Ophthalmol, 121, 1316-1323. Singh, S., Chao, L.Y., Mishra, R., Davies, J. & Saunders, G.F. (2001) Missense mutation at the C-terminus of PAX6 negatively modulates homeodomain function. Hum Mol Genet, 10, 911-918. Stenman, J., Yu, R.T., Evans, R.M. & Campbell, K. (2003) Tlx and Pax6 co-operate genetically to establish the pallio-subpallial boundary in the embryonic mouse telencephalon. Development, 130, 1113-1122. Summers, K.M., Withers, S.J., Gole, G.A., Piras, S. & Taylor, P.J. (2008) Anterior segment mesenchymal dysgenesis in a large Australian family is associated with the recurrent 17 bp duplication in PITX3. Mol Vis, 14, 2010-2015.  47  Vincent, A., Billingsley, G., Priston, M., Glaser, T., Oliver, E., Walter, M., Ritch, R., Levin, A. & Heon, E. (2006) Further support of the role of CYP1B1 in patients with Peters anomaly. Mol Vis, 12, 506-510. Young, K.A., Berry, M.L., Mahaffey, C.L., Saionz, J.R., Hawes, N.L., Chang, B., Zheng, Q.Y., Smith, R.S., Bronson, R.T., Nelson, R.J. & Simpson, E.M. (2002) Fierce: a new mouse deletion of Nr2e1; violent behaviour and ocular abnormalities are background-dependent. Behavioural Brain Research, 132, 145-158.  48  CHAPTER 3: GENERAL DISCUSSION This thesis explored the role of NR2E1 in human eye disorders using human molecular techniques and genomic sequencing strategies. Here, I summarize the major findings of my project and propose experimental strategies to better understand the role of NR2E1 in aniridia, PA, and related eye disorders. 3.1  SUMMARY OF RESULTS The work presented in this thesis is the first analysis of NR2E1 in clinical patients  with eye disorders. We identified the first germline amino acid change in NR2E1, the g.14122C>G; p.R274G variant in patient 1380 and patient 1380’s mother. This amino acid change was detected in a patient with PA, a rare genetic eye disorder in which the genetic mechanism is yet to be determined. The amino acid at residue 274 of NR2E1 is very highly conserved, suggesting evolutionary importance, and could indicate that it is part of a binding site for an NR2E1 cofactor. The location of the amino change is of particular interest because the identical mutation found at the homologous residue in NR2E3, the most closely related gene in the to NR2E1, is known to cause ESCS (Figure 3.1) (Haider et al., 2000, Wright et al., 2004).  Figure 3.1: R274G variant identified in this study identical to homologous mutation in NR2E3. Residue 274 in NR2E1 (top ‘R’ in red) is homologous to residue 309 in NR2E3 (bottom ‘R’ in red). Residue 274 in NR2E1 is also two amino acids away from the homologous residue 311 in NR2E3 (bottom ‘R’ in blue). 49  Furthermore, residue 274 in NR2E1 is in close homologous proximity to R311Q in NR2E3, which is the most common amino acid change in patients with ESCS (Bernal et al., 2008, Fradot et al., 2007, Haider et al., 2000, Hayashi et al., 2005, Lam et al., 2007, Nakamura et al., 2004, Pachydaki et al., 2009, Schorderet & Escher, 2009, Sharon et al., 2003). Because changes to residues 309 and 311 in NR2E3 are known to cause an eye disorder, this implicates the region that the R274G variant was found in as being functionally important, thus changes to residues 274 and 276 would likely result in a protein with altered function. The variant that we identified was also detected in the apparently unaffected mother of patient 1380. This would normally indicate that this variant was unlikely to cause disease. Yet, there is evidence to support the hypothesis that the R274G variant could adversely effect eye development, thus it is imperative that alternative explanations are explored thoroughly. The hypothesis that the mother of patient 1380 is a mosaic could be tested through quantitative pyrosequencing. Pyrosequencing technology is unique among genotyping and sequencing methods in that each allele is measured quantitatively. When assessing whether an individual is a mosaic, it is necessary to perform genetic testing on cells from different parts of the body. By performing pyrosequencing on both DNA samples extracted from saliva and DNA samples purified from blood, we can assess whether there is a difference between either of the two samples, and can provide evidence that the mother is a mosaic. This hypothesis would be supported by analyzing the maternal grandparents. If neither of the maternal grandparents possess the variant, and both are ophthalmologically normal, this would support the hypothesis of mosaicism in the mother,  50  and that some of her cells, including her germ cells, possess a de novo variant, which was passed to patient 1380. If ophthalmological analysis of the mother revealed a normal eye phenotype, the presence of a variant in her NR2E1 gene could also be explained by incomplete penetrance. The maternal grandparents of patient 1380 would have their NR2E1 gene sequenced and be given an eye exam to determine whether either grandparent had PA, and whether the presence of PA coincided with the presence of the R274G variant. However, if one of the maternal grandparents harbored the variant and was unaffected by PA, this would support the hypothesis that the R274G variant is not a causative mutation. 3.2  FUTURE DIRECTIONS  3.2.1  Expanding collection of patients with eye disorders We propose that the collection of aniridia and PA patients should be expanded,  particularly patients whose PAX6 and related eye development genes have been screened and no mutation has been found. The amino acid change found in our study was detected in a patient with PA, and this project sequenced only a small number of patients with PA. Thus, collecting and sequencing additional samples would provide us with a larger sample size and could lead to the detection of additional amino acid changes in NR2E1. A recent study investigated the role of PAX6, PITX2, MYOC, and CYP1B1 in eye disorders by sequencing these genes in 15 patients with PA (Vincent et al., 2006). Three of the patients with PA possessed mutations in CYP1B1. However, the other 12 patients were found to have no mutation in these four genes (Vincent et al., 2006). In 2004, Edwards et al, studied 11 PA patients in 10 families and screened the coding regions of PAX6, FOXC1, FOXE3, PITX2, and CYP1B1. Homozygous CYP1B1 mutations were  51  identified in 6 of the individuals in five families. However, researchers were unable to identify any mutation for the remaining five patients in five extended families. More recently, Berker et al. investigated another large five-generation family with PA. The complete pedigree consisted of 38 members, 19 of whom were affected. However, when a genetic screen was performed on these subjects, no mutations were found in PAX6, CYP1B1, PITX2, or MAF (Berker et al., 2009). Taken together, the results of these studies strongly suggest that there is a gap in our knowledge, an eye development gene which has yet to be identified. These individuals represent an excellent study population for future genetic investigations of NR2E1.(Blackshaw & Livesey, 2002) I would like to suggest an additional group of eye disorder patients that would also be an appropriate candidate for future genetic studies of NR2E1. Autosomal dominant retinitis pigmentosa (ADRP) is a type of retinal degeneration which leads to progressive visual loss. ADRP is characterized by night blindness, mottling of retinal pigment epithelium, attenuation of retinal vessels, and loss of central vision. Patients with ADRP have a reduced or absent electroretinogram (ERG) recording (Hamel, 2006). Nr2e1-/- mice show retinal hypoplasia, mottling of the retinal pigment epithelium, and a flat ERG (Abrahams et al., 2005). Both Nr2e1-/- mice and human with ADRP have disturbances in retinal vessels, though the phenotype is slightly different, as patients with ADRP display attenuation of the retinal vessels, and Nr2e1-/- mice have reduced vessel number (Abrahams et al., 2005, Hamel, 2006). Comparison of fundus photos of the Nr2e1-/- mice and patients with ADRP is compelling evidence that NR2E1 is a potential candidate for this eye disorder (Figure 3.2).  52  Figure 3.2: Similarity between Nr2e1 null mice and humans with retinitis pigmentosa A) Fundus of patient with ADRP. Note that pigment mottling is present in the mid periphery and retinal vessels are attenuated and reduced in number (Hamel, 2006). B) Fundus of Nr2e1-/- (fierce) mouse. Note mottling, indicated with an arrow) and reduced vessel number when compared to a wild-type (Wt) mouse (Abrahams et al., 2005). ADRP has been shown to be both clinically and genetically heterogeneous, and mutations in 20 different genes have been identified in patients with ADRP. Although many different genetic factors have been investigated, the results of a recent mutational screen of 12 genes in 237 affected individuals from 43 different families strongly suggest the presence of unidentified genes that are involved in ADRP. More than 70% of families investigated possessed no mutation in the coding exons of RHO, RDS, RP1, IMPDH1, PRPF31, CRX, NRL, FSCN2, HPRP3, and RP9 (Ziviello et al., 2005). Studies of ADRP in families revealed incomplete penetrance of the disorder, suggesting that there may be genetic or environmental modifiers that have yet to be uncovered (Kim et al., 1994, Moore et al., 1993, Ziviello et al., 2005).  53  As mentioned previously, NR2E3, the closest known relative of NR2E1 has been shown to cause ADRP in approximately 2% of patients with the disorder. Furthermore, Nr2e1 and Nr2e3 have both been shown to be expressed in the developing neural retina of Xenopus (Martinez-De Luna & El-Hodiri, 2007) However, later in development and in adulthood, Nr2e1 and Nr2e3 were expressed in different retinal regions. Nr2e1 was expressed in the retinal ciliary margin, which gives rise to retina and iris, while Nr2e3 expression was restricted to the photoreceptor layer (Martinez-De Luna & El-Hodiri, 2007). Analysis of eye development in zebrafish found that both Nr2e1 and Nr2e3 are expressed in the retina, however Nr2e3 expression is primarily in the photoreceptor layer, while Nr2e1 expression is more widespread, in the forebrain, midbrain and retinal ciliary margin (Kitambi & Hauptmann, 2006). The high level of homology between NR2E1 and NR2E3, and similar patterns of expression in model organisms makes NR2E1 an appealing candidate gene for this disorder. 3.2.2  Functional assay to assess the effect of an R274G variant in NR2E1 There are a variety of different experimental strategies that could be utilized in  future studies to test the functionality of the R274G human variant. Because of the high level of homology between human NR2E1 and mouse Nr2e1, a mouse model with the R274G variant would be useful to assess the effect of this change. PAX6 mutations in human aniridia and the Sey mutation in mice are inherited in an autosomal dominant fashion (Lee et al., 2008, Ramaesh et al., 2003). One hypothesis for the mechanism of this inheritance is that the aniridia or Sey phenotypes are caused by abnormal PAX6 proteins that are produced by the mutant allele. This hypothesis is supported by functional studies which revealed that missense mutations in PAX6  54  produced mutated proteins which could still interact with different PAX6-binding sites, and induced transcriptional responses which differed from normal PAX6 activity (Chauhan et al., 2004). We hypothesize a similar mechanism could exist with the NR2E1 missense R274G variant. Using genetic engineering techniques, mice carrying the R274G human variant could be produced. We could then perform analysis on the gross phenotype of the eye, looking in particular for the hallmark corneal opacity of PA. We could also dissect the eye and perform immunohistochemistry on sections of the eye to detect any abnormalities of the retina or the anterior segment. Finally, we could assess the inheritance pattern of the disease in families of mice, to better understand the effect of this variant on penetrance and expressivity. The amino acid change from a large, polar arginine to a small, non-polar glycine at residue 274 is likely to be a change that is significant and would not be tolerated. However, it is difficult to predict the clinical effect of an amino acid change using a webbased program and this theory would need to be tested using a functional protein assay, possibly in vitro. Human embryonic stem cells (hESC) could be utilized as a comprehensive model that aids in a better understanding of the early stages of human development. A recent study demonstrated the ability of human embryonic stem cells to differentiate into cells of the eye and mimic the sequence and time course of human retinal development (Meyer et al., 2009). The functionality of our R274G variant could be tested by generating a hESC line with our variant using homologous recombination. After allowing the cells to differentiate, we would then compare the number and morphology of the mutant cells to wild type cells. Investigation of which cell types specifically were affected by the R274G variant could illuminate disease pathology and  55  mechanism. This future work aims to investigate functionality and the effects on eye development of the variant identified in this thesis. 3.3  CONCLUSION The work presented in this thesis provides the scientific and medical community  with a greater depth of knowledge of the role of NR2E1 in genetic eye disorders. These results may open new therapeutic strategies, and could lead to future research into the molecular basis of eye disorders such as aniridia and PA.  56  3.4  REFERENCES  Abrahams, B.S., Kwok, M.C., Trinh, E., Budaghzadeh, S., Hossain, S.M. & Simpson, E.M. (2005) Pathological aggression in "fierce" mice corrected by human nuclear receptor 2E1. J Neurosci, 25, 6263-6270. Berker, N., Alanay, Y., Elgin, U., Volkan-Salanci, B., Simsek, T., Akarsu, N. & Alikasifoglu, M. (2009) A new autosomal dominant Peters' anomaly phenotype expanding the anterior segment dysgenesis spectrum. Acta Ophthalmol, 87, 52-57. Bernal, S., Solans, T., Gamundi, M.J., Hernan, I., de Jorge, L., Carballo, M., Navarro, R., Tizzano, E., Ayuso, C. & Baiget, M. (2008) Analysis of the involvement of the NR2E3 gene in autosomal recessive retinal dystrophies. Clin Genet, 73, 360-366. Blackshaw, S. & Livesey, R. (2002) Applying genomics technologies to neural development. Curr Opin Neurobiol, 12, 110-114. Chauhan, B.K., Yang, Y., Cveklova, K. & Cvekl, A. (2004) Functional properties of natural human PAX6 and PAX6(5a) mutants. Invest Ophthalmol Vis Sci, 45, 385392. Fradot, M., Lorentz, O., Wurtz, J.M., Sahel, J.A. & Leveillard, T. (2007) The loss of transcriptional inhibition by the photoreceptor-cell specific nuclear receptor (NR2E3) is not a necessary cause of enhanced S-cone syndrome. Mol Vis, 13, 594-601. Haider, N.B., Jacobson, S.G., Cideciyan, A.V., Swiderski, R., Streb, L.M., Searby, C., Beck, G., Hockey, R., Hanna, D.B., Gorman, S., Duhl, D., Carmi, R., Bennett, J., Weleber, R.G., Fishman, G.A., Wright, A.F., Stone, E.M. & Sheffield, V.C. (2000)  57  Mutation of a nuclear receptor gene, NR2E3, causes enhanced S cone syndrome, a disorder of retinal cell fate. Nat Genet, 24, 127-131. Hamel, C. (2006) Retinitis pigmentosa. Orphanet J Rare Dis, 1, 40. Hayashi, T., Gekka, T., Goto-Omoto, S., Takeuchi, T., Kubo, A. & Kitahara, K. (2005) Novel NR2E3 mutations (R104Q, R334G) associated with a mild form of enhanced S-cone syndrome demonstrate compound heterozygosity. Ophthalmology, 112, 2115. Kim, S.K., Haines, J.L., Berson, E.L. & Dryja, T.P. (1994) Nonallelic heterogeneity in autosomal dominant retinitis pigmentosa with incomplete penetrance. Genomics, 22, 659-660. Kitambi, S.S. & Hauptmann, G. (2006) The zebrafish orphan nuclear receptor genes nr2e1 and nr2e3 are expressed in developing eye and forebrain. Gene Expr Patterns. Lam, B.L., Goldberg, J.L., Hartley, K.L., Stone, E.M. & Liu, M. (2007) Atypical mild enhanced S-cone syndrome with novel compound heterozygosity of the NR2E3 gene. Am J Ophthalmol, 144, 157-159. Lee, H., Khan, R. & O'Keefe, M. (2008) Aniridia: current pathology and management. Acta Ophthalmol, 86, 708-715. Martinez-De Luna, R.I. & El-Hodiri, H.M. (2007) The Xenopus ortholog of the nuclear hormone receptor Nr2e3 is primarily expressed in developing photoreceptors. Int J Dev Biol, 51, 235-240. Meyer, J.S., Shearer, R.L., Capowski, E.E., Wright, L.S., Wallace, K.A., McMillan, E.L., Zhang, S.C. & Gamm, D.M. (2009) Modeling early retinal development with  58  human embryonic and induced pluripotent stem cells. Proc Natl Acad Sci U S A, 106, 16698-16703. Moore, A.T., Fitzke, F., Jay, M., Arden, G.B., Inglehearn, C.F., Keen, T.J., Bhattacharya, S.S. & Bird, A.C. (1993) Autosomal dominant retinitis pigmentosa with apparent incomplete penetrance: a clinical, electrophysiological, psychophysical, and molecular genetic study. Br J Ophthalmol, 77, 473-479. Nakamura, Y., Hayashi, T., Kozaki, K., Kubo, A., Omoto, S., Watanabe, A., Toda, K., Takeuchi, T., Gekka, T. & Kitahara, K. (2004) Enhanced S-cone syndrome in a Japanese family with a nonsense NR2E3 mutation (Q350X). Acta Ophthalmol Scand, 82, 616-622. Pachydaki, S.I., Klaver, C.C., Barbazetto, I.A., Roy, M.S., Gouras, P., Allikmets, R. & Yannuzzi, L.A. (2009) Phenotypic features of patients with NR2E3 mutations. Arch Ophthalmol, 127, 71-75. Ramaesh, T., Collinson, J.M., Ramaesh, K., Kaufman, M.H., West, J.D. & Dhillon, B. (2003) Corneal abnormalities in Pax6+/- small eye mice mimic human aniridiarelated keratopathy. Invest Ophthalmol Vis Sci, 44, 1871-1878. Schorderet, D.F. & Escher, P. (2009) NR2E3 mutations in enhanced S-cone sensitivity syndrome (ESCS), Goldmann-Favre syndrome (GFS), clumped pigmentary retinal degeneration (CPRD), and retinitis pigmentosa (RP). Hum Mutat. Sharon, D., Sandberg, M.A., Caruso, R.C., Berson, E.L. & Dryja, T.P. (2003) Shared Mutations in NR2E3 in Enhanced S-cone Syndrome, Goldmann-Favre Syndrome, and Many Cases of Clumped Pigmentary Retinal Degeneration. Arch Ophthalmol, 121, 1316-1323.  59  Vincent, A., Billingsley, G., Priston, M., Glaser, T., Oliver, E., Walter, M., Ritch, R., Levin, A. & Heon, E. (2006) Further support of the role of CYP1B1 in patients with Peters anomaly. Mol Vis, 12, 506-510. Wright, A.F., Reddick, A.C., Schwartz, S.B., Ferguson, J.S., Aleman, T.S., Kellner, U., Jurklies, B., Schuster, A., Zrenner, E., Wissinger, B., Lennon, A., Shu, X., Cideciyan, A.V., Stone, E.M., Jacobson, S.G. & Swaroop, A. (2004) Mutation analysis of NR2E3 and NRL genes in Enhanced S Cone Syndrome. Hum Mutat, 24, 439. Ziviello, C., Simonelli, F., Testa, F., Anastasi, M., Marzoli, S.B., Falsini, B., Ghiglione, D., Macaluso, C., Manitto, M.P., Garre, C., Ciccodicola, A., Rinaldi, E. & Banfi, S. (2005) Molecular genetics of autosomal dominant retinitis pigmentosa (ADRP): a comprehensive study of 43 Italian families. J Med Genet, 42, e47.  60  

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