The causes of retinal dystrophy and the development of more comprehensive screening approach

Inherited retinal diseases (IRDs) are a group of genetically and phenotypically heterogenous disorders caused by variants in around 280 genes. Additional loci have also been localised to chromosomal regions, though the causative genes remain unknown. Recent improvements in screening technologies have increased the detection of pathogenic variants in IRD. This thesis describes the use of next generation sequencing (second (short-read) and third (long-read) generation sequencing) to find missing or hard to find pathogenic variants in IRD patients. The first results chapter describes use of whole exome sequencing to screen 24 individuals with syndromic and non-syndromic IRDs. This identified pathogenic variants in known genes in eight cases; CDHR1 (c.1527T>G, p.Y509*), RHO (c.284T>C, p.L95P), PRPF31 (c.797delC, p.S266*), CNGA3 (c.1088T>C, p.L363P), BBS10 (c.728-731delAAGA, p. K243Ifs*15), USH2A (c.252T>G, p.C84W), ABCA4 (c.2588G>C, p.G863A and c.6089G>A, p.R2030Q), and SLC25A46 (c.670A>G, p.T224A). In addition, several candidate variants were highlighted for further investigation. In the second results chapter, seven patients with late onset macular dystrophy and one with age related macular degeneration were found to carry the same heterozygous ~126 kb deletion encompassing CRX, TPRX1 and SULT2A1. This phenotype has already been documented in patients with heterozygous variants in the gene encoding retinal transcription factor CRX, while there is no known functional or phenotypic link with variants in TPRX1 or SULT2A1. This therefore confirms that CRX haploinsufficiency is pathogenic, a finding that had previously been debated in the ophthalmic literature. The deletion was characterized using a PCR assay followed by cloning and Sanger sequencing or direct Sanger sequencing. Haplotype analysis was done by microsatellite genotyping. The third results chapter describes use of SMRT PacBio and nanopore long-read sequencing to screen the hard-to-sequence mutation hotspot RPGR-ORF15. Both approaches were effective in reading throughout ORF15 and allowed sequencing indexed pooled samples, and 218 IRD patients were screened, detecting known and new variants. Nanopore sequencing on the smaller Flongle flowcell allowed low-cost optimisation, but pores rapidly blocked, probably due to ORF15 secondary structures. Repeated DNase I washes reopened the pores but required use of the more expensive MinION flowcells. Ultimately, the PacBio sequencer proved simpler to use, cheaper, and more scalable

Determining the causes of recessive retinal dystrophy

Inherited retinal dystrophies (RDs) are a clinically heterogeneous group of eye diseases that result from mutations in more than 250 genes. Genetic diagnosis of these diseases has, until recently, been hampered by the lack of suitable technologies to perform high throughput screening. This thesis describes two different strategies for using next generation sequencing (NGS) in RD patients to find the pathogenic mutation(s) involved. In the first results chapter, a customised capture reagent (called Retinome) designed against the known retinal dystrophy genes (RetNet, June 2010) was used in NGS analysis of 20 RD families. The disease-causing mutations were identified in 12 of 20 cases (60%). These included previously reported mutations in ABCA4 (c.6088C>T, p.R2030*; c.5882G>A, p.G1961E), RDH12 (c.601T>C, p.C201R; c.506G>A, p.R169Q), PROM1 (c.1117C>T, p.R373C), GUCY2D (c.2512C>T, p.R838C), RPGRIP1 (c.3565C>T, p.R1189*), BBS2 (c.1895G>C, p.R632P) and SPATA7 (c.253C>T, p.R85*) and new mutations in CRB1 (c.2832_2842+23del), USH2A (c.12874A>G, p.N4292D), RP2 (c.884-1G>T) and ABCA4 (c.3328+1G>C). In eight cases the causative mutation could not be unambiguously identified. In the second results chapter, whole-exome NGS was performed on five RD families that had been pre-screened with the Retinome reagent. This identified mutations in three known RD genes, MFSD8 (c.1006G>C, p.E336Q; c.1394G>A, p.R465Q), C8orf37 (c.555G>A, p.W185*) and TTLL5 (c.1627G>A, p.E543K), and mutations in two potentially new RD genes, LARGE (c.2089G>T, p.V697L) and FDFT1 (c.930C>G, p.F310L). In the third results chapter, whole-exome NGS was performed, without pre-screening of known genes, in a family with atypical adult-onset RD with early macular involvement. NGS identified a mutation in a novel RD gene, DRAM2 (c.140delG, p.G47Vfs*3). Further DRAM2 screening in DNA panels identified a compound heterozygote case (c.494G>A, p.W165*; c.131G>A, p.S44N). DRAM2 was localised to the photoreceptor inner segment and retinal pigment epithelium. The relative merits of each approach are discussed. Identifying the pathogenic mutation facilitates counselling, carrier testing and may lead to a clearer prognosis. It may also influence future prospects for these families as new treatments become available

The role of common genetic variation in model polygenic and monogenic traits

The aim of this thesis is to explore the role of common genetic variation, identified through genome-wide association (GWA) studies, in human traits and diseases, using height as a model polygenic trait, type 2 diabetes as a model common polygenic disease, and maturity onset diabetes of the young (MODY) as a model monogenic disease. The wave of the initial GWA studies, such as the Wellcome Trust Case-Control Consortium (WTCCC) study of seven common diseases, substantially increased the number of common variants associated with a range of different multifactorial traits and diseases. The initial excitement, however, seems to have been followed by some disappointment that the identified variants explain a relatively small proportion of the genetic variance of the studied trait, and that only few large effect or causal variants have been identified. Inevitably, this has led to criticism of the GWA studies, mainly that the findings are of limited clinical, or indeed scientific, benefit. Using height as a model, Chapter 2 explores the utility of GWA studies in terms of identifying regions that contain relevant genes, and in answering some general questions about the genetic architecture of highly polygenic traits. Chapter 3 takes this further into a large collaborative study and the largest sample size in a GWA study to date, mainly focusing on demonstrating the biological relevance of the identified variants, even when a large number of associated regions throughout the genome is implicated by these associations. Furthermore, it shows examples of different features of the genetic architecture, such as allelic heterogeneity and pleiotropy. Chapter 4 looks at the predictive value and, therefore, clinical utility, of variants found to associate with type 2 diabetes, a common multifactorial disease that is increasing in prevalence despite known environmental risk factors. This is a disease where knowledge of the genetic risk has potentially substantial clinical relevance. Finally, Chapter 5 approaches the monogenic-polygenic disease bridge in the direction opposite to that approached in the past: most studies have investigated genes mutated in monogenic diseases as candidates for harboring common variants predisposing to related polygenic diseases. This chapter looks at the common type 2 diabetes variants as modifiers of disease onset in patients with a monogenic but clinically heterogeneous disease, maturity onset diabetes of the young (MODY)

Antioxidant effect of Linalool on testicular-injury induced by carbon tetrachloride in male rats

Background: The oxidative stress and generation of free radicals plays an important role in testicular impairment. The aim of this study was to investigate the protective effect of linalool on carbon tetrachloride (CCl4)-induced male reproductive system damage. Methods: In this study, 24 male rats were divided into four groups. Two of the groups were normal control group andCCl4 damage control group which received a daily dose of distilled water for 14 days. Two of the other groups were pretreatment groups; the rats in one of them received a daily dose of 25 mg/kg linalool and those in the other were administered with a daily dose of 100 mg/kg silymarin for 14 days. On the 14th day, the damage control group as well as the pretreatment groups was intraperitoneally injected with 1 ml/kg of the mixture of CCl4and olive oil (1:1). The rats in the normal control group were only administered with olive oil. 48 hours after the injection of CCl4, a part of the testis tissue was separated for conducting antioxidant and malondialdehyde (MDA) tests. Results: The injection of CCl4 into the rats caused a significant increase in the concentration of MDA and insignificant decrease in the level of antioxidants in the testicular lysate as compared to the normal control group (P<0.01). Treatment with linalool improved the level of MDA and enhanced antioxidant as compared to the damage control group (P<0.01). Conclusion: The results of this study indicated that linalool has antioxidant properties and can have a therapeutic effect against CCl4-induced testicular injuries