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.

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.

Thesis (Ph. D.)--University of New South Wales, 1998. / Also available online.

Estudamos uma família com cinco homens (dois falecidos) afetados por deficiência mental (DM) não-sindrômica em duas gerações, num padrão de herança ligada ao cromossomo X. A análise do padrão de inativação do cromossomo X, com base na metilação do gene AR, evidenciou que a mulher portadora obrigatória tinha desvio completo de inativação nos leucócitos, uma característica freqüente em portadoras de mutações do cromossomo X relacionadas com DM. Para o mapeamento da DM, genotipamos 28 locos de microssatélites ao longo do cromossomo X e delimitamos um segmento de cerca de 32 Mb, entre os marcadores DXS986 e DXS8067, compartilhado pelos afetados e pela portadora obrigatória, mas não pelo homem normal ou pelas possíveis portadoras que não tinham desvio do padrão de inativação do cromossomo X. Na busca do gene mutado, analisamos, por seqüenciamento direto, genes mapeados no intervalo compartilhado e já relacionados a DM ou que tivessem expressão em cérebro e leucócitos. Nos afetados e na portadora obrigatória, encontramos a mutação c.845C→T no gene ACSL4, que resulta na substituição do aminoácido histidina, conservado na família de sintetases de acil-CoA humanas e em diversos outros organismos, por tirosina (p.H323Y da isoforma cérebro-específica). Tratando-se de mutação que altera um aminoácido evolutivamente conservado em gene já relacionado com DM, que segregava com a DM na família, não tendo sido encontrada em amostra controle de 160 indivíduos do sexo masculino, concluímos que era a causa da DM na família. Mutações de ponto no gene ACSL4 foram relacionadas com a DM não sindrômica em três famílias descritas na literatura. O gene ACSL4 codifica a acil-coA sintetase 4 da família das sintetases de cadeia longa, que catalisa a formação de ésteres acil-coA a partir de ácidos graxos de cadeia longa. Sua expressão já foi documentada em vários tecidos, incluindo o cérebro e dados recentes mostraram que a proteína é essencial para a formação normal de espinhos dendríticos. A nova mutação do gene ACSL4 que descrevemos como causa de DM vem reforçar a relação alterações desse gene e a DM de herança ligada ao X. O padrão de inativação do X totalmente desviado foi mais uma vez observado em mulher portadora da mutação, indicando a importância da expressão desse gene em leucócitos. A presença de dificuldades de aprendizado na portadora da mutação concorda com o observado nas três famílias da literatura em que o estudo das portadoras foi relatado, indicando o efeito de mutações do gene ACSL4 sobre a função intelectual mesmo em heterozigose. A ausência de correlação entre o padrão de inativação do cromossomo X em células do sangue periférico e o comprometimento intelectual foi confirmada. Na família estudada, a identificação da mutação permitiu o aconselhamento genético. / We studied a family with five men (two of them deceased) affected by nonsyndromic mental retardation in two generations, in a pattern of X-linked inheritance (MRX). The study aimed at identifying the causative mutation. The obligate female carrier showed completely skewed inactivation of the X chromosome, based on the methylation status of the AR gene in peripheral blood in leukocytes, a common feature in carriers of X-linked mutations that cause mental retardation. We genotyped 28 microsatellite loci mapped throughout the X chromosome and delimited a 32 Mb segment, between markers DXS986 and DXS8067, that was shared by the affected males and obligate carrier, but was not present in a normal man or in two women who did not show skewed X-inactivation. We searched for the causative mutation by sequencing genes mapped to this candidate interval that had been associated with MR and/or were expressed in brain and leukocytes. In the affected men and obligate carrier, we found a c.845C→T mutation in the ACSL4 gene, resulting in the amino acid tyrosine substituting for a histidine (p.H323Y in brain isoform), which is conserved in the acyl-CoA synthetase family in humans and others organisms. This mutation was not found in a control sample of 160 men. Previously, point mutations in the ACSL4 gene had been identified as the cause of MRX in three families. ACSL4 encodes the acyl-CoA synthetase long-chain family member 4, which catalyzes the formation of acyl-CoA esters from long-chain fatty acids. It is expressed in several tissues, and in brain it is essential for the normal formation of dendritic spines. The novel mutation here described confirmed the causal association of ACSL4 mutations with non-syndromic mental retardation. The completely skewed Xinactivation, also observed in the previously described carriers, supported a functional role for this gene in peripheral blood leukocytes. The intellectual impairment present in the carrier in the family here reported is in accordance with previous findings pointing to the effect on intellectual abilities of ACSL4 mutations in heterozygosis. The absence of correlation between the pattern of X-inactivation in leukocytes and mental status was confirmed.

Estudamos uma família com cinco homens (dois falecidos) afetados por deficiência mental (DM) não-sindrômica em duas gerações, num padrão de herança ligada ao cromossomo X. A análise do padrão de inativação do cromossomo X, com base na metilação do gene AR, evidenciou que a mulher portadora obrigatória tinha desvio completo de inativação nos leucócitos, uma característica freqüente em portadoras de mutações do cromossomo X relacionadas com DM. Para o mapeamento da DM, genotipamos 28 locos de microssatélites ao longo do cromossomo X e delimitamos um segmento de cerca de 32 Mb, entre os marcadores DXS986 e DXS8067, compartilhado pelos afetados e pela portadora obrigatória, mas não pelo homem normal ou pelas possíveis portadoras que não tinham desvio do padrão de inativação do cromossomo X. Na busca do gene mutado, analisamos, por seqüenciamento direto, genes mapeados no intervalo compartilhado e já relacionados a DM ou que tivessem expressão em cérebro e leucócitos. Nos afetados e na portadora obrigatória, encontramos a mutação c.845C→T no gene ACSL4, que resulta na substituição do aminoácido histidina, conservado na família de sintetases de acil-CoA humanas e em diversos outros organismos, por tirosina (p.H323Y da isoforma cérebro-específica). Tratando-se de mutação que altera um aminoácido evolutivamente conservado em gene já relacionado com DM, que segregava com a DM na família, não tendo sido encontrada em amostra controle de 160 indivíduos do sexo masculino, concluímos que era a causa da DM na família. Mutações de ponto no gene ACSL4 foram relacionadas com a DM não sindrômica em três famílias descritas na literatura. O gene ACSL4 codifica a acil-coA sintetase 4 da família das sintetases de cadeia longa, que catalisa a formação de ésteres acil-coA a partir de ácidos graxos de cadeia longa. Sua expressão já foi documentada em vários tecidos, incluindo o cérebro e dados recentes mostraram que a proteína é essencial para a formação normal de espinhos dendríticos. A nova mutação do gene ACSL4 que descrevemos como causa de DM vem reforçar a relação alterações desse gene e a DM de herança ligada ao X. O padrão de inativação do X totalmente desviado foi mais uma vez observado em mulher portadora da mutação, indicando a importância da expressão desse gene em leucócitos. A presença de dificuldades de aprendizado na portadora da mutação concorda com o observado nas três famílias da literatura em que o estudo das portadoras foi relatado, indicando o efeito de mutações do gene ACSL4 sobre a função intelectual mesmo em heterozigose. A ausência de correlação entre o padrão de inativação do cromossomo X em células do sangue periférico e o comprometimento intelectual foi confirmada. Na família estudada, a identificação da mutação permitiu o aconselhamento genético. / We studied a family with five men (two of them deceased) affected by nonsyndromic mental retardation in two generations, in a pattern of X-linked inheritance (MRX). The study aimed at identifying the causative mutation. The obligate female carrier showed completely skewed inactivation of the X chromosome, based on the methylation status of the AR gene in peripheral blood in leukocytes, a common feature in carriers of X-linked mutations that cause mental retardation. We genotyped 28 microsatellite loci mapped throughout the X chromosome and delimited a 32 Mb segment, between markers DXS986 and DXS8067, that was shared by the affected males and obligate carrier, but was not present in a normal man or in two women who did not show skewed X-inactivation. We searched for the causative mutation by sequencing genes mapped to this candidate interval that had been associated with MR and/or were expressed in brain and leukocytes. In the affected men and obligate carrier, we found a c.845C→T mutation in the ACSL4 gene, resulting in the amino acid tyrosine substituting for a histidine (p.H323Y in brain isoform), which is conserved in the acyl-CoA synthetase family in humans and others organisms. This mutation was not found in a control sample of 160 men. Previously, point mutations in the ACSL4 gene had been identified as the cause of MRX in three families. ACSL4 encodes the acyl-CoA synthetase long-chain family member 4, which catalyzes the formation of acyl-CoA esters from long-chain fatty acids. It is expressed in several tissues, and in brain it is essential for the normal formation of dendritic spines. The novel mutation here described confirmed the causal association of ACSL4 mutations with non-syndromic mental retardation. The completely skewed Xinactivation, also observed in the previously described carriers, supported a functional role for this gene in peripheral blood leukocytes. The intellectual impairment present in the carrier in the family here reported is in accordance with previous findings pointing to the effect on intellectual abilities of ACSL4 mutations in heterozygosis. The absence of correlation between the pattern of X-inactivation in leukocytes and mental status was confirmed.

Neste trabalho utilizamos diferentes abordagens para o estudo de genes associados com desenvolvimento e funcionamento do SNC assim como com doenças neurológicas: 1) uma das abordagens consistiu na identificação do alelo associado a uma forma de retardo mental sindrômico com herança recessiva ligada ao cromossomo X, síndrome de Snyder-Robinson, em uma família Brasileira. Utilizando as estratégias de estudo de ligação genética e análise de genes candidatos, identificamos a segunda mutação patogênica no gene SMS (que codifica a enzima espermina sintase) associada à síndrome de Snyder-Robinson. A identificação dessa mutação contribuiu para: delinear e expandir o espectro clínico da síndrome, evidenciar domínios importantes para o funcionamento da proteína espermina sintase, comprovar a importância dessa proteína nos processos cognitivos, e também possibilitar um aconselhamento genético preciso para membros da família; 2) outra abordagem consistiu em analisar (triar mutação) o gene codificador da proteína colibistina (ARHGEF9), a qual está envolvida em sinaptogênese inibitória, em pacientes Brasileiros portadores de hiperecplexia (6 pacientes) e em pacientes portadores de retardo mental associado com epilepsia (22 pacientes). Não identificamos nenhuma alteração patogênica no gene ARHGEF nos 28 pacientes estudados; contudo, o número de pacientes analisados foi muito pequeno. Julgamos que a análise de um número maior de pacientes com essas doenças neurológicas pode vir a revelar novas mutações deletérias em ARHGEF9; 3) a última abordagem consistiu no estudo funcional da proteína colibistina. Com o objetivo de identificar outras proteínas que interagem com a colibistina humana utilizamos o sistema de duplo-híbrido em leveduras e experimentos de co-imunoprecipitação in vitro e in vivo. Identificamos a proteína eIF3-p40 interagindo com a proteína colibistina e também com a proteína gefirina (a qual, por sua vez, também interage com colibistina e está envolvida com funcionamento de sinapses inibitórias). A proteína eIF3-p40 é uma das subunidades do complexo do fator 3 de iniciação de tradução protéica em eucariotos (eIF3). Essas interações ligam as proteínas colibistina e a gefirina à maquinaria de tradução protéica, revelando uma provável nova função dessas proteínas no controle da tradução em sítios pós-sinápticos inibitórios. / In this work we have used different approaches to the study of genes associated with CNS development and function as well as with neurological diseases: 1) one study involved the identification of the allele associated with an X-linked recessive sindromic form of mental retardation, Snyder-Robinson syndrome, in a Brazilian family. Using genetic linkage analysis and candidate gene strategy, we identified the second pathogenic mutation in the SMS gene (that encodes the spermine synthase enzyme) associated with the Snyder-Robinson syndrome. The identification of this mutation contributed to: the delineation and expansion of the clinical spectrum of the syndrome, highlight important domains for spermine synthase protein functioning, demonstrate the importance of this protein in cognitive processes, and also a precise genetic counseling for family members; 2) a second study involved the mutation screening of ARHGEF9, gene encoding the collybistin protein, which is involved in inhibitory synaptogenesis, in Brazilian patients with hyperekplexia (6 patients) and in patients with mental retardation associated with epilepsy (22 patients). We did not identify any pathogenic alteration in the ARHGEF9, gene in the 28 studied patients, but the number of patients analysed was very small. However, the possibility remains that additional mutations in ARHGEF9, may contribute to other cases of hyperekplexia and mental retardation associated with epilepsy; 3) the last study involved the functional analysis of collybistin protein. In order to identify other proteins that interact with human collybistin, we used the yeast two-hybrid system and in vitro e in vivo co-immunoprecipitation experiments. We identified the eIF3-p40 protein as collybistin and gephyrin (another protein involved in the function of inhibitory synapses that also interacts with collybistin) binding partner. The eIF3-p40 protein is one of the subunits of the eukaryotic initiation factor 3 complex (eIF3). These interactions link the collybistin and gephyrin proteins to the protein translation machinery, revealing a putative new role of these proteins in the translation control at inhibitory postsynaptic sites.

Neste trabalho utilizamos diferentes abordagens para o estudo de genes associados com desenvolvimento e funcionamento do SNC assim como com doenças neurológicas: 1) uma das abordagens consistiu na identificação do alelo associado a uma forma de retardo mental sindrômico com herança recessiva ligada ao cromossomo X, síndrome de Snyder-Robinson, em uma família Brasileira. Utilizando as estratégias de estudo de ligação genética e análise de genes candidatos, identificamos a segunda mutação patogênica no gene SMS (que codifica a enzima espermina sintase) associada à síndrome de Snyder-Robinson. A identificação dessa mutação contribuiu para: delinear e expandir o espectro clínico da síndrome, evidenciar domínios importantes para o funcionamento da proteína espermina sintase, comprovar a importância dessa proteína nos processos cognitivos, e também possibilitar um aconselhamento genético preciso para membros da família; 2) outra abordagem consistiu em analisar (triar mutação) o gene codificador da proteína colibistina (ARHGEF9), a qual está envolvida em sinaptogênese inibitória, em pacientes Brasileiros portadores de hiperecplexia (6 pacientes) e em pacientes portadores de retardo mental associado com epilepsia (22 pacientes). Não identificamos nenhuma alteração patogênica no gene ARHGEF nos 28 pacientes estudados; contudo, o número de pacientes analisados foi muito pequeno. Julgamos que a análise de um número maior de pacientes com essas doenças neurológicas pode vir a revelar novas mutações deletérias em ARHGEF9; 3) a última abordagem consistiu no estudo funcional da proteína colibistina. Com o objetivo de identificar outras proteínas que interagem com a colibistina humana utilizamos o sistema de duplo-híbrido em leveduras e experimentos de co-imunoprecipitação in vitro e in vivo. Identificamos a proteína eIF3-p40 interagindo com a proteína colibistina e também com a proteína gefirina (a qual, por sua vez, também interage com colibistina e está envolvida com funcionamento de sinapses inibitórias). A proteína eIF3-p40 é uma das subunidades do complexo do fator 3 de iniciação de tradução protéica em eucariotos (eIF3). Essas interações ligam as proteínas colibistina e a gefirina à maquinaria de tradução protéica, revelando uma provável nova função dessas proteínas no controle da tradução em sítios pós-sinápticos inibitórios. / In this work we have used different approaches to the study of genes associated with CNS development and function as well as with neurological diseases: 1) one study involved the identification of the allele associated with an X-linked recessive sindromic form of mental retardation, Snyder-Robinson syndrome, in a Brazilian family. Using genetic linkage analysis and candidate gene strategy, we identified the second pathogenic mutation in the SMS gene (that encodes the spermine synthase enzyme) associated with the Snyder-Robinson syndrome. The identification of this mutation contributed to: the delineation and expansion of the clinical spectrum of the syndrome, highlight important domains for spermine synthase protein functioning, demonstrate the importance of this protein in cognitive processes, and also a precise genetic counseling for family members; 2) a second study involved the mutation screening of ARHGEF9, gene encoding the collybistin protein, which is involved in inhibitory synaptogenesis, in Brazilian patients with hyperekplexia (6 patients) and in patients with mental retardation associated with epilepsy (22 patients). We did not identify any pathogenic alteration in the ARHGEF9, gene in the 28 studied patients, but the number of patients analysed was very small. However, the possibility remains that additional mutations in ARHGEF9, may contribute to other cases of hyperekplexia and mental retardation associated with epilepsy; 3) the last study involved the functional analysis of collybistin protein. In order to identify other proteins that interact with human collybistin, we used the yeast two-hybrid system and in vitro e in vivo co-immunoprecipitation experiments. We identified the eIF3-p40 protein as collybistin and gephyrin (another protein involved in the function of inhibitory synapses that also interacts with collybistin) binding partner. The eIF3-p40 protein is one of the subunits of the eukaryotic initiation factor 3 complex (eIF3). These interactions link the collybistin and gephyrin proteins to the protein translation machinery, revealing a putative new role of these proteins in the translation control at inhibitory postsynaptic sites.

The first part of the thesis describes a systematic function analysis that identified in vitro E2 partners for ten different HECT E3 ligase proteins. Using mass spectrometry, the linkage composition for the resulting autoubiquitylation products of a number of functional E2-HECT pairs was determined. HECT domains from different subfamilies catalyze the formation of very different types of Ub chains, largely independent of the E2 in the reaction. The second part of the thesis describes the characterization of the RAD6-interactome. Using affinity purification coupled with mass spectrometry, I identified a novel RAD6-interacting E3 ligase, KCMF1, which binds to a different surface on RAD6 than the other RAD6-associated E3 ligases. KCMF1 also recruits additional proteins to RAD6, and this new complex points to novel RAD6 functions. Interestingly, the RAD6A R11Q mutant polypeptide, found in X-linked mental retardation patients specifically loses the interaction with KCMF1, but not with other RAD6-associated E3 ligases.