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Retinal degenerative disease, which includes age-related macular degeneration and retinitis pigmentosa, is the main cause of blindness in developed countries. Degeneration of the retinal pigment epithelium (RPE) and photoreceptor cells through apoptosis is believed to be the main mechanism of cell death. X-linked inhibitor of apoptosis (XIAP) is an endogenous anti-apoptotic protein that mediates its effects through the inhibition of caspases - the proteins regulating the final stages of apoptosis. The neuroprotection of XIAP has been demonstrated in various neurodegenerative models. Retinal gene therapy based on adeno-associated virus (AAV) has recently been proven safe and effective in clinical trials of Leber's congenital amaurosis. However, studies are very limited so far about AAV-mediated XIAP effect on degeneration of the RPE and photoreceptor cells. In this thesis, a comprehensive study of AAV-mediated XIAP was performed in the RPE and photoreceptor degenerative models. First, an oxidative stress model was investigated using H2O2 in a human RPE cell line. Second, AAV2-mediated XIAP conferred marked protection on the RPE cells against H2O2 induced apoptosis. Third, an in vivo analysis using confocal scanning laser ophthalmoscope was applied to the NaIO3 induced retinopathy in two transgenic mice (NRL-GFP and B6TGOPN1LW-EGFP). However, subretinal injection of AAV2-XIAP did not rescue photoreceptor cells in the NaIO3-treated animals. Finally, AAV8-XIAP was tested in a rhodopsin mutant mouse line with retinal degeneration (the Rho-/- B6TGOPN1LW-EGFP mouse) but did not reveal any protection on cone photoreceptors. Overall the work in this thesis indicates a limited protection of AAV-mediated XIAP on the RPE and photoreceptor cells in the degenerative models used. XIAP based gene therapy may be helpful for RPE preservation in atrophic AMD, but it needs further research.

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MSc (Med), Faculty of Health Sciences, University of the Witwatersrand / Mental retardation (MR) is estimated to affect ~2-3% of the general population and may result from genetic causes and/or environmental causes. X-linked mental retardation (XLMR) is a heterogeneous group of disorders with a broad range of phenotypes and can be classified into syndromic XLMR (S-XLMR) and nonsyndromic XLMR (NS-XLMR) types. A number of X-linked genes have been identified that are associated with the different forms of XLMR. In an attempt to refine the diagnostic service to patients with XLMR, the Division of Human Genetics, Molecular Laboratory at the National Health Laboratory Service (NHLS) has investigated a number of associated X-linked genes. The main objective of this project was to investigate three genes, FMR2, XNP and ARX, associated with NS-XLMR and their contribution to XLMR in the South African (SA) population of MR males. Patients from different ethnic groups, referred to the Division of Human Genetics for fragile X MR syndrome that tested negative for the FMR1 expansion mutation were investigated for mutations in these genes. In addition, a cohort of Black institutionalized males was also investigated. The FMR2 expansion mutation responsible for fragile X E syndrome was not identified in 1194 FMR1 expansion negative MR male patients. FMR2 allele distribution analysis showed that a GCC repeat size of 15 was common in the MR cohort, accounting for 42% of alleles identified. From a total of 210 FMR1 expansion negative MR male subjects screened for mutations in a hotspot region (exons 7, 8 and 9) of the XNP gene, none was found to have a mutation in this region. Two patients from a cohort of 868 FMR1 expansion negative MR males were found to have a mutation in the ARX gene – one patient tested positive for the common 24 bp duplication mutation and a second patient appeared to have a deletion in the region amplified. These results indicate that the FMR2, XNP and ARX genes do not contribute significantly to MR in the SA population. As a result of this study, routine DNA testing for the FMR2 expansion, mutation screening in the hotspot region of the XNP gene and screening for the common 24 bp duplication mutation in the ARX gene in FMR1 expansion negative MR male subjects will not be implemented. A retrospective analysis was also done on 1862 probands referred to the Molecular Genetics Diagnostic service from 1992 to 2009 for fragile X MR syndrome testing. The FMR1 full expansion mutation was detected in 6.2% of probands, higher than reported worldwide figures. FMR1 allele distribution analysis in a cohort of 1184 FMR1 expansion negative MR males showed that 29 CGG repeats was the most frequent repeat size observed, accounting for 32.6% of all alleles in the cohort. The analysis of FMR1 alleles in MR males shows a similar distribution between different ethnic groups and compares well with other reported studies. This study reinforces the presence of fragile X MR syndrome in the SA Black population. Molecular investigations were also undertaken on 3 patients clinically suspected to have X-linked -thalassaemia mental retardation syndrome (ATR-X) and extended family members. Mutations were identified in each of the patients – two patients were found to have a novel mutation in the XNP gene and the third patient had a common XNP mutation. As a result, carrier testing and prenatal diagnosis was made possible in these families. A large family positive for the FMR1 expansion causing fragile X A MR syndrome was investigated. As an incidental finding, 2 females were found to be compound heterozygotes for 2 FMR1 alleles. Extended family members were tested and their FMR1 status was determined. Haplotype analysis was used to track the high-risk X chromosome in the family. As a result of this investigation, females at risk for premature ovarian failure and fragile X tremor ataxia syndrome have been identified. The approach to testing genes implicated in NS-XLMR has to be refined to allow for a cheaper and more efficient alternative. The use of newer techniques such as CGH microarray and MLPA has allowed for better detection of mutations. Delineating the causes of MR and their molecular and cellular consequences will assist families but also provide insight into the mechanisms that are required for the normal development of cognitive functions in humans.

The Indonesian archipelago comprises more than 17,000 islands, inhabited by ~200 million people constituting more than 350 recognizable ethnic and tribal groups which can be classified into two broad ethno-linguistic groups [the Austronesian (AN) and non-Austronesian (NAN) speaking peoples] and 3 physical anthropology groups (Deutero Malay, Proto Malay and Papuan). The origins of these groups are of considerable anthropological interest. The anthropology of Indonesia is extremely complex and still controversial. The present populations of Indonesia show very great diversity. The data presented below result from an investigation of the Fragile X A syndrome and the size and distribution of alleles at fragile sites on the X chromosome among Javanese males with developmental disability (DD) and unselected males from 10 major Indonesian ethnic groups. The Fragile X syndrome is caused by expansion of a CGG trinucleotide repeat array in the 5' untranslated region of the FMR-1 gene at Xq27.3. Normal X chromosomes have between 6-54 CGG trinucleotide repeats, whereas premutation alleles have 55-230 and full mutation alleles more than 230 repeats. In a study of predominantly Caucasian males with intellectual disability, the prevalence of Fragile X syndrome is estimated to be approximately 1:4,000. FRAXE mental retardation syndrome is caused by an expansion of a GCC trinucleotide repeat in the 5'UTR of FMR2 gene located 600 kb telomeric to FMR1. The prevalence of FMR2 is 1-2 per 100,000 live births. FMR2 common alleles consist of 11-30 GGC repeats; intermediate alleles between 31-60 GCC repeats; premutation alleles with 61-200 repeats and full mutation alleles have over 200 repeats with attendant methylation of the repeat array The first Indonesian screening program aimed at determining the presence and prevalence of fragile XA syndrome among individuals with mild DD (IQ above 50) from special schools (N=205) and isolated areas (N=50) of Java was undertaken in 1994-1996 by cytogenetic and molecular studies. In this first study 4 fragile X positive children were found among 255 males with DD. The estimated prevalence of fragile-X in males with mild DD from special schools was 1.95% (5/205) and the overall prevalence was 1.57% (4/255). The number of trinucleotide repeats in the 5' untranslated regions of the FMR1 and FMR2 genes were determined by PCR in 254 Fragile XA-negative Javanese male children with DD. The distribution of FMR1 and FMR2 trinucleotide repeat alleles was found to be significantly different in the Indonesian population with DD compared to that in equivalent Caucasian populations. The trimodal distribution of Indonesian FMR1 alleles (29, 30 and 36 repeats) is largely in agreement with findings from other Asian populations). This provides supportive evidence that the origin of Indonesians could be the same as that of the Chinese and Japanese. Sequence analysis was performed on the trinucleotide repeat arrays of the 27 individuals' FMR1 alleles in the 'grey zone' (35-52 repeats). The identification of 16 unrelated individuals with a (CGG)36 allele that also contains a (CGG)6 segment [(CGG)9AGG(CGG)9AGG(CGG)6 AGG(CGG)9 or 9A9A6A9 pattern] is in agreement with earlier observations in the Japanese population. It is proposed that this FMR1 array pattern may be specific for Asian populations and that Javanese and Japanese populations may have arisen from a single progenitor population. The presence of pure 25, 33 and 34 CGGs in FMR1 alleles with 36, 44 and 45 repeats respectively, suggests that these may represent alleles at high risk for instability and may therefore be at early stages of expansion to a premutation. The lack of the characteristic (CGG)6 in all three alleles with ?? 25 pure CGG arrays suggests that the most common Asian 36 repeat allele is not predisposed to slippage expansion. Seven of the 8 alleles with 36 CGG repeats could be sequenced. Seven of 36 CGG repeats FMR1 alleles from the Hiri population has been sequenced and 4 alleles indicated 9A9A6A9 pattern, 1 sample with 10A25 pattern Two of the remaining alleles showed 12A6A6A9 structure, which consisted of a tandem duplication of the (CGG)6 segment. The presence of a tandem duplication of (CGG)6 segments has never been reported in any other population. The other major findings of this study are that FRAXE syndrome is a rare cause of developmental disability in this predominantly-Javanese population. The most common FMR2 (GCC)20 allele in this selected Asian population is significantly longer than that previously reported for Caucasian populations. There was a weak correlation between the overall length of the FMR1 and FMR2 repeat arrays within the normal range (Spearman's Rank Correlation = 0.130, p-value=0.042) in the Indonesian population, which have been no previous associations reported for alleles within the normal range. One approach to studying the origins of the human populations is to study the genetic structure of polymorphic alleles such as those at the FMR1 locus and its linked microsatellite markers DXS548 and FRAXAC1. Length polymorphisms of the FMR1 gene (CGG)n repeat array, DXS548 and FRAXAC1 were studied in a total of 1,008 unselected males from 10 different Indonesian ethnic groups. FMR1 alleles were identified ranging from 8 to 57 CGG repeats. The most common CGG repeat allele was 29 (45.6%) followed by 30 (27.4%) and 36 repeats (8.0%). One hundred and forty four grey zone (3-52 CGG) alleles were found in the study population. Four people of the same ethnic group from an isolated island in Eastern Indonesia (Hiri, Ternate), a representative of the NAN ethnolinguistic group, had CGG repeat lengths of 55-57. The prevalence of these alleles is estimated to be 3.3% (4/120) in the population of Hiri or 0.4% (4/1008) of whole Indonesian population. Thirteen different alleles were found at the DXS548 locus, of which allele numbers 7 [194 bp] (44.1%), 6.5 [195bp] (43.5%) and 6 [196bp] (7.5%) are the most common. Seven rare alleles, some of which have not been previously found in Asian peoples were also identified (190, 191,192, 193, 197,198, 199, 202, 204 and 206) and accounted for 3.9% of the total. The odd number alleles were dominantly found in this study whereas almost none found in Caucasian. The finding of many "odd numbered" alleles DXS548 has never been found in other Asian population and has only been documented extremely rarely in Caucasians and Africans. Five different alleles of FRAXAC1 identified with alleles D [106 bp] (62.2%) and C [108bp] (35.6%) accounting for 97.8% of FRAXAC1 alleles in the population. Three rare alleles (104, 110, 112 bp = 2.2%) were identified that have not been previously found in other Asian populations (1-3). There is a striking linkage disequilibrium of FMR1 alleles with FRAXAC1 (p=0.0001), 88% of 29 (CGG)n repeats alleles associated with FRAXAC1 allele D (106bp) versus only 17% with the 30 (CGG)n repeat alleles, which is in agreement with other studies. The value of D' was calculated to be 0.7. The longer alleles of both DXS548 and FRAXAC1 were found mostly in the NAN ethnolinguistic group. Moreover the Irian Jaya people also showed a higher percentage of people with 30 CGG repeats and the 108 bp FRAXAC1. The Eastern Indonesian NAN groups demonstrate a different genetic background probably due to the contribution of Melanesian peoples. The Analysis of Molecular Variance (AMOVA) identified that the vast majority of genetic diversity occurs within, rather than between, ethnic groups. These data are consistent with a model where there is sufficient migration (~20 per generation) between populations to minimise differentiation of population through genetic drift. The results obtained are consistent with three clusters of populations that share similar allele frequencies at the fragile X locus. The most clearly defined cluster is based in the east of Indonesia and includes the two Irian populations, Minahasans and Hiri. A surprising finding was that the Minahasan who are Deutero-Malay in origin and physical appearance are genetically closer to the Irianese. This may reflect the admixture of Melanesian alleles or other eastern Indonesian alleles as a result of their geographic location in that part of Indonesia. The second major cluster is largely based in the west of the country and is composed of the following Deutero-Malay populations; Javanese, Balinese, Acehnese but which also includes people from Ternate (not including those from Hiri). Using Delta Mu and Nei's genetic distance for FMR1 locus in this study the Javanese were shown to have the closest distance to Balinese which is consistent with anthropological data and with published data. The third group is a "western and central" group composed of Bimanese, Dayak and Sundanese who share some features of the western and eastern clusters but mostly resemble the western Indonesian populations. Bima is located in the lesser Sunda in between west Indonesia and east Indonesia. The Bimanese are of mixed Deutero & Proto Malay origin that is consistent with their geographic location. The Bataks are distinctive and sit somewhat apart in this scheme. In this study, Bataks were found not to resemble the other Proto-Malay group studied (the Dayak). The Dayaks were found to have fewer alleles than the Bataks at FRAXAC1 and DXS548. In all four methods of calculating genetic distance Bataks showed a large genetic distance to almost all other ethnic groups. There are differences in allele frequency between east and west Indonesia as well as other Asian nations, but the genetic similarities between these groups are also very impressive. The findings from this study are consistent with other genetic anthropological evidence that the people of Indonesia have the same origin as North-east Asian groups. This model is referred to as the "express train from Taiwan" in which the Austronesian speakers are proposed to have radiated from Taiwan bringing the Malayo-Polynesian language group to the Philippines, Borneo and Sulawesi around 5000-4500 B.P.E. However Richards et al.(1998) have used the diversity in the mtDNA D Loop to propose an alternative to the "express train" model. The "two train7quot; model proposes that the Austronesian languages originated within eastern Indonesia during the Pleistocene era and spread through Melanesia and into the remote Pacific within the past 6,000 years. Unfortunately the high migration rates between population groups that were demonstrated in this thesis and the known migration patterns of populations through Indonesia preclude determining whether the observed allelic heterogeneity is a function of the original population or due to the admixture of several gene pools in more recent times.

The Indonesian archipelago comprises more than 17,000 islands, inhabited by ~200 million people constituting more than 350 recognizable ethnic and tribal groups which can be classified into two broad ethno-linguistic groups [the Austronesian (AN) and non-Austronesian (NAN) speaking peoples] and 3 physical anthropology groups (Deutero Malay, Proto Malay and Papuan). The origins of these groups are of considerable anthropological interest. The anthropology of Indonesia is extremely complex and still controversial. The present populations of Indonesia show very great diversity. The data presented below result from an investigation of the Fragile X A syndrome and the size and distribution of alleles at fragile sites on the X chromosome among Javanese males with developmental disability (DD) and unselected males from 10 major Indonesian ethnic groups. The Fragile X syndrome is caused by expansion of a CGG trinucleotide repeat array in the 5' untranslated region of the FMR-1 gene at Xq27.3. Normal X chromosomes have between 6-54 CGG trinucleotide repeats, whereas premutation alleles have 55-230 and full mutation alleles more than 230 repeats. In a study of predominantly Caucasian males with intellectual disability, the prevalence of Fragile X syndrome is estimated to be approximately 1:4,000. FRAXE mental retardation syndrome is caused by an expansion of a GCC trinucleotide repeat in the 5'UTR of FMR2 gene located 600 kb telomeric to FMR1. The prevalence of FMR2 is 1-2 per 100,000 live births. FMR2 common alleles consist of 11-30 GGC repeats; intermediate alleles between 31-60 GCC repeats; premutation alleles with 61-200 repeats and full mutation alleles have over 200 repeats with attendant methylation of the repeat array The first Indonesian screening program aimed at determining the presence and prevalence of fragile XA syndrome among individuals with mild DD (IQ above 50) from special schools (N=205) and isolated areas (N=50) of Java was undertaken in 1994-1996 by cytogenetic and molecular studies. In this first study 4 fragile X positive children were found among 255 males with DD. The estimated prevalence of fragile-X in males with mild DD from special schools was 1.95% (5/205) and the overall prevalence was 1.57% (4/255). The number of trinucleotide repeats in the 5' untranslated regions of the FMR1 and FMR2 genes were determined by PCR in 254 Fragile XA-negative Javanese male children with DD. The distribution of FMR1 and FMR2 trinucleotide repeat alleles was found to be significantly different in the Indonesian population with DD compared to that in equivalent Caucasian populations. The trimodal distribution of Indonesian FMR1 alleles (29, 30 and 36 repeats) is largely in agreement with findings from other Asian populations). This provides supportive evidence that the origin of Indonesians could be the same as that of the Chinese and Japanese. Sequence analysis was performed on the trinucleotide repeat arrays of the 27 individuals' FMR1 alleles in the 'grey zone' (35-52 repeats). The identification of 16 unrelated individuals with a (CGG)36 allele that also contains a (CGG)6 segment [(CGG)9AGG(CGG)9AGG(CGG)6 AGG(CGG)9 or 9A9A6A9 pattern] is in agreement with earlier observations in the Japanese population. It is proposed that this FMR1 array pattern may be specific for Asian populations and that Javanese and Japanese populations may have arisen from a single progenitor population. The presence of pure 25, 33 and 34 CGGs in FMR1 alleles with 36, 44 and 45 repeats respectively, suggests that these may represent alleles at high risk for instability and may therefore be at early stages of expansion to a premutation. The lack of the characteristic (CGG)6 in all three alleles with ?? 25 pure CGG arrays suggests that the most common Asian 36 repeat allele is not predisposed to slippage expansion. Seven of the 8 alleles with 36 CGG repeats could be sequenced. Seven of 36 CGG repeats FMR1 alleles from the Hiri population has been sequenced and 4 alleles indicated 9A9A6A9 pattern, 1 sample with 10A25 pattern Two of the remaining alleles showed 12A6A6A9 structure, which consisted of a tandem duplication of the (CGG)6 segment. The presence of a tandem duplication of (CGG)6 segments has never been reported in any other population. The other major findings of this study are that FRAXE syndrome is a rare cause of developmental disability in this predominantly-Javanese population. The most common FMR2 (GCC)20 allele in this selected Asian population is significantly longer than that previously reported for Caucasian populations. There was a weak correlation between the overall length of the FMR1 and FMR2 repeat arrays within the normal range (Spearman's Rank Correlation = 0.130, p-value=0.042) in the Indonesian population, which have been no previous associations reported for alleles within the normal range. One approach to studying the origins of the human populations is to study the genetic structure of polymorphic alleles such as those at the FMR1 locus and its linked microsatellite markers DXS548 and FRAXAC1. Length polymorphisms of the FMR1 gene (CGG)n repeat array, DXS548 and FRAXAC1 were studied in a total of 1,008 unselected males from 10 different Indonesian ethnic groups. FMR1 alleles were identified ranging from 8 to 57 CGG repeats. The most common CGG repeat allele was 29 (45.6%) followed by 30 (27.4%) and 36 repeats (8.0%). One hundred and forty four grey zone (3-52 CGG) alleles were found in the study population. Four people of the same ethnic group from an isolated island in Eastern Indonesia (Hiri, Ternate), a representative of the NAN ethnolinguistic group, had CGG repeat lengths of 55-57. The prevalence of these alleles is estimated to be 3.3% (4/120) in the population of Hiri or 0.4% (4/1008) of whole Indonesian population. Thirteen different alleles were found at the DXS548 locus, of which allele numbers 7 [194 bp] (44.1%), 6.5 [195bp] (43.5%) and 6 [196bp] (7.5%) are the most common. Seven rare alleles, some of which have not been previously found in Asian peoples were also identified (190, 191,192, 193, 197,198, 199, 202, 204 and 206) and accounted for 3.9% of the total. The odd number alleles were dominantly found in this study whereas almost none found in Caucasian. The finding of many "odd numbered" alleles DXS548 has never been found in other Asian population and has only been documented extremely rarely in Caucasians and Africans. Five different alleles of FRAXAC1 identified with alleles D [106 bp] (62.2%) and C [108bp] (35.6%) accounting for 97.8% of FRAXAC1 alleles in the population. Three rare alleles (104, 110, 112 bp = 2.2%) were identified that have not been previously found in other Asian populations (1-3). There is a striking linkage disequilibrium of FMR1 alleles with FRAXAC1 (p=0.0001), 88% of 29 (CGG)n repeats alleles associated with FRAXAC1 allele D (106bp) versus only 17% with the 30 (CGG)n repeat alleles, which is in agreement with other studies. The value of D' was calculated to be 0.7. The longer alleles of both DXS548 and FRAXAC1 were found mostly in the NAN ethnolinguistic group. Moreover the Irian Jaya people also showed a higher percentage of people with 30 CGG repeats and the 108 bp FRAXAC1. The Eastern Indonesian NAN groups demonstrate a different genetic background probably due to the contribution of Melanesian peoples. The Analysis of Molecular Variance (AMOVA) identified that the vast majority of genetic diversity occurs within, rather than between, ethnic groups. These data are consistent with a model where there is sufficient migration (~20 per generation) between populations to minimise differentiation of population through genetic drift. The results obtained are consistent with three clusters of populations that share similar allele frequencies at the fragile X locus. The most clearly defined cluster is based in the east of Indonesia and includes the two Irian populations, Minahasans and Hiri. A surprising finding was that the Minahasan who are Deutero-Malay in origin and physical appearance are genetically closer to the Irianese. This may reflect the admixture of Melanesian alleles or other eastern Indonesian alleles as a result of their geographic location in that part of Indonesia. The second major cluster is largely based in the west of the country and is composed of the following Deutero-Malay populations; Javanese, Balinese, Acehnese but which also includes people from Ternate (not including those from Hiri). Using Delta Mu and Nei's genetic distance for FMR1 locus in this study the Javanese were shown to have the closest distance to Balinese which is consistent with anthropological data and with published data. The third group is a "western and central" group composed of Bimanese, Dayak and Sundanese who share some features of the western and eastern clusters but mostly resemble the western Indonesian populations. Bima is located in the lesser Sunda in between west Indonesia and east Indonesia. The Bimanese are of mixed Deutero & Proto Malay origin that is consistent with their geographic location. The Bataks are distinctive and sit somewhat apart in this scheme. In this study, Bataks were found not to resemble the other Proto-Malay group studied (the Dayak). The Dayaks were found to have fewer alleles than the Bataks at FRAXAC1 and DXS548. In all four methods of calculating genetic distance Bataks showed a large genetic distance to almost all other ethnic groups. There are differences in allele frequency between east and west Indonesia as well as other Asian nations, but the genetic similarities between these groups are also very impressive. The findings from this study are consistent with other genetic anthropological evidence that the people of Indonesia have the same origin as North-east Asian groups. This model is referred to as the "express train from Taiwan" in which the Austronesian speakers are proposed to have radiated from Taiwan bringing the Malayo-Polynesian language group to the Philippines, Borneo and Sulawesi around 5000-4500 B.P.E. However Richards et al.(1998) have used the diversity in the mtDNA D Loop to propose an alternative to the "express train" model. The "two train7quot; model proposes that the Austronesian languages originated within eastern Indonesia during the Pleistocene era and spread through Melanesia and into the remote Pacific within the past 6,000 years. Unfortunately the high migration rates between population groups that were demonstrated in this thesis and the known migration patterns of populations through Indonesia preclude determining whether the observed allelic heterogeneity is a function of the original population or due to the admixture of several gene pools in more recent times.

No description available.