Transferase galactosemia is an autosomal recessively inherited disorder caused by a block in the conversion of galactose to glucose. Manifestations include jaundice, vomiting, cataracts, mental retardation, speech abnormalities and poor growth. This disorder is due to a deficiency of galactose- 1-phosphate uridyl transferase (GAIT), an enzyme which catalyses the conversion of uridyldisphosphoglucose (UDPG) and galactose-l-phosphate to uridyldisphosphogalactose (UDPGal) and glucose-1-phosphate. The GALT gene has been mapped and 50 mutations have been reported in this; gene to date. The aims of this project were to identity and characterise galactosemia-causing mutations in the South African negroid population and to determine the frequency of these mutations in order to estimate the incidence of transferase galactosemia in the South African negroid population. Twentysix negroid patients and one obligate carrier were investigated and the SI35L mutation was found to account for approximately 91% (48/53) of galactosemia alleles in this group. The estimated S135L allele carrier frequency in 600 healthy unrelated negroid individuals (1/75) was used in conjunction with the proportion of non-S135L galactosemia alleles present in galactosemics to arrive at an estimated galactosemia incidence of 1 per 18 455 births. This is over three times the estimated world average of one per 70 000. Three caucasoid galactosemia patients, one galactosemic patient of mixed ancestry and two obligate carriers (one caucasoid and one of mixed ancestry) were screened for mutations in the GALT gene. The Q188R mutation was found to account for 57% (4/7) of the galactosemia mutations in the South African caucasoid galactosemics which is similar to the overall frequency detected in other caucasoid populations. Both the SDSL and the Q188R mutations were detected in the individuals of mixed ancestry. - .......... Several populations from western, central and southern Africa were screened for the SDSL mutation. This mutation was found at low frequencies (± ISE) in western and central Africa, 0.003 (+2.5 x 10'3) and 0.003 (±1.96 x 10""), respectively and was detected at a higher frequency (+ 1SE) of 0.006 (±2.25 x lO"3) in the southeastern Bantu, but was not detected in the San populations screened. This mutation also accounts for approximately 48% of the galactosemia mutations in African American galactosemics. Those results suggest an African origin of the S135L mutation. The South African negroid galactosemics and 202 randomly ascertained negroid individuals were screened for the QI88R mutation and &c/RFLP in the GALT gene. The Q188R mutation was not detected in these groups; thus indicating that this mutation was not a major cause of transferase galactosemia in South African negroids. No Sad" alleles were detected in these individuals suggesting that this allele was not associated with the galactosemia mutations in negroids and was less frequent in this population than in caucasoids. Seventy South African Indians were screened for the N314D mutation which results in the Duarte electrophoretic phenotype (both Duarte and Los Angeles alleles result in this phenotype) is associated with the Duarte variants of galactosemia. The N314D mutation was found at the high frequency (± 1 SE) of 0.20 (± 0.033) in this group and this mutation was detected in cis with the Sad" allele (Duarte) or in cis with the L218L mutation (Los Angeles). A previously undescribed mutation, G to A transition at bp 997 in exon 4 of the GALT gene, was discovered in a randomly ascertained individual from the Central African Rephiic. This mutation is predicted to result in the substitution of arginine by glutamine at amino acid IZi, sad did not appear to affect the level of GALT activity in red blood cells.

Duarte galactosemia is a variant form of galactosemia that on average results in a reduction of the galactose-1-phosphate uridyltransferase enzyme to 25% activity. This enzyme is involved in the metabolism of galactose in the body. On the contrary, patients diagnosed with the classic form of galactosemia have a galactose-1-phosphate uridyltransferase enzyme activity of zero or near-zero. As a result, classic galactosemics are placed on galactose-restricted diets to prevent acute neonatal signs of disease that can ultimately lead to death. These diets are instituted for the rest of the patients' lives. However, even with dietary treatment, classic galactosemia patients go on to experience long-term neurodevelopmental outcomes, most notably cognitive defects and speech and language delay. Duarte galactosemia patients, as a result of their residual enzyme activity, experience much milder disease symptoms. Many specialists agree that these patients have a benign disease and therefore treatment is not consistently agreed upon nor prescribed. Most Duarte patients follow an unrestricted diet and if a diet is prescribed, it is only for the first year of life. While these patients have enough enzyme activity to prevent acute neonatal signs of disease, there is still limited information regarding any long-term neurodevelopmental outcomes in the Duarte galactosemia population. This study examined developmental outcomes and need for special services of a sample of Duarte galactosemia patients. The outcome data were compared to the general population as well as to a classic galactosemia group. A convenience cohort of clinical charts for patients seen for neuropsychological evaluations from 1978 to 2013 was reviewed. Developmental scores, neuropsychological outcomes, and need for special services for patients diagnosed with a form of galactosemia were entered into an electronic database. Recorded developmental information on twenty-two Duarte galactosemia patients were found. All of the 22 Duarte patients were found to have developmental test scores within normal range. However, 38.9% of Duarte patients containing information regarding special services were found to participate in early intervention, 71.4% of which received speech therapy. Furthermore, 22.2% of Duarte patients containing information regarding special services were found to participate in special education, and 100% of these children received speech therapy. In conclusion, despite Duarte galactosemia patients not exhibiting lower learning test scores, there was a large proportion of them participating in special services, particularly in speech therapy. This indicates some speech and language difficulties in children with Duarte galactosemia.

A galactosemia clássica ou tipo I (GC) é um erro inato do metabolismo da galactose causada pela deficiência da enzima galactose-1-fosfato uridiltransferase (GALT). É transmitida como uma doença autossômica recessiva e é tipicamente caracterizada pela intolerância neonatal a galactose, com complicações que vão desde icterícia, para os casos mais leves, à insuficiência hepática nos mais graves, e às complicações tardias, como disfunções motoras e reprodutivas. A galctosemia também é heterogénea do ponto de vista molecular, com 266 mutações diferentes descritas no gene GALT, algumas específicas para certas populações, refletindo o que se espera de alguns eventos de efeito fundador. O objetivo deste trabalho foi avaliar o perfil de mutações no gene GALT, dos pacientes brasileiros com galactosemia clássica e fazer um estudo da correlação do genótipo com o fenótipo, uma vez que se sabe que parte da variação observada na evolução clínica está relacionada com o nível de atividade residual da enzima e do genótipo. Para tanto, foram incluídos no estudo 31 pacientes com o diagnóstico bioquímico de galactosemia de diversas regiões do Brasil, que tiveram seus dados clínicos obtidos a partir de revisão de prontuários médicos e preenchimento de ficha clínica. Foi realizado o sequenciamento genético direto bidirecional do gene GALT e também estudos adicionais, como genotipagem do gene GALK1 de um paciente, estudo de ancestralidade de sete pacientes, além de simulações de patogenicidade in silico das novas mutações identificadas. Os principais achados clínicos dos pacientes que participaram deste estudo foram hepatomegalia, icterícia, baixo ganho pondero-estatural, vômito recorrente, anemia e catarata. As principais mutações que causam GC descritas na literatura foram identificadas neste estudo, como por exemplo, a p.Q188R, p.S135L e p.K285N, bem como o alelo Duarte 2 e seis mutações novas, p.M1T, p.R33S, p.P73S, IVS3+1G>A, IVS4+4A>C e p.Q169P. Este resultado era esperado, dada a elevada miscigenação da população brasileira. Alguns indivíduos foram diagnosticados através do teste de triagem neonatal expandido, que não está disponível rotineiramente a todos os recém-nascidos, portanto, começaram o tratamento dietético antes de desenvolverem os sinais e sintomas da doença. Para estes indivíduos não foi possível fazer uma análise da relação genótipo-fenótipo. Para os demais indivíduos esta relação foi consistente com o que é descrito na literatura, com os indivíduos homozigotos para a mutação p.Q188R com uma evolução mais grave do que os indivíduos que tinham pelo menos uma mutação p.S135L. Para os indivíduos com as mutações novas, foi observado um amplo espectro de fenótipos, como de pacientes que foi a óbito por insuficiência hepática e sepse à um caso assintomático. Este estudo amplia o espectro de mutação no gene GALT descrito na literatura e reforça a importância tanto do diagnóstico precoce quanto da introdução do tratamento dietético; também acrescenta mais evidências para a discussão sobre a introdução da galactosemia no programa de triagem neonatal do Brasil, onde a incidência da doença é estimada em cerca de 1:20.000. / Classical galactosemia (CG) or type I galactosemia is an inborn error of galactose metabolism caused by the deficiency of the galactose-1-phosphate uridyltransferase enzyme (GALT). It is transmitted as an autosomal recessive disease and is typically characterized by neonatal galactose intolerance, with complications ranging from neonatal jaundice and liver failure to late complications, such as motor and reproductive dysfunctions. Galactosemia is also heterogeneous from a molecular standpoint, with 266 mutations described to date in the GALT gene, some of them specific to certain populations, reflecting what is expected as some events of founder effect. The objective of this study was to evaluate the profile of mutations in the GALT gene of Brazilian patients with classical galactosemia and perform a genotype-phenotype correlation study, since it is known that part of the observed variation in clinical outcome is related to the level of residual enzyme activity and genotype. Therefore, this study included 31 patients with biochemical diagnosis of galactosemia from different regions of Brazil, who had their clinical data obtained from review of medical records and from a standardized case report form. We conducted a direct bidirectional sequencing of the GALT gene and also additional studies, as GALK1 genotyping for a patient, ancestrality study of seven patients and in silico simulation of pathogenicity for the new mutations identified. The main clinical features of the patients in this study were hepatomegaly, jaundice, low weight and height gain, recurrent vomiting, anemia and cataract. The major CG causing mutations described in the literature have been identified in this study, for example, p.Q188R, p.S135L and p.K285N, as well as the Duarte 2 allele and six novel mutations: p.M1T; p.R33S; p.P73S; IVS3+1G>A; IVS4+4A>C and p.Q169P. This result was expected, given the high miscegenation of the Brazilian population. Some individuals were diagnosed through expanded newborn screening test, which is not available routinely to all newborns, and so began dietary treatment before they develop signs and symptoms of the disease. For these individuals, was not possible to analyze the genotype-phenotype correlation. For other individuals this relationship was consistent with what is described in the literature, with the homozygous for p.Q188R mutation presenting a more severe phenotype than individuals who had at least one p.S135L mutation. For individuals with new mutations, was observed a wide range of phenotypes, from patients who died due to liver failure and sepsis to an asymptomatic case. This study expands the spectrum of mutations in the GALT gene described in the literature and reinforces the importance of early diagnosis and the introduction of dietary treatment; also adds more evidence to the discussion on the introduction of galactosemia in the neonatal screening program of Brazil, where the incidence of the disease is estimated at about 1:20,000.

A galactosemia clássica ou tipo I (GC) é um erro inato do metabolismo da galactose causada pela deficiência da enzima galactose-1-fosfato uridiltransferase (GALT). É transmitida como uma doença autossômica recessiva e é tipicamente caracterizada pela intolerância neonatal a galactose, com complicações que vão desde icterícia, para os casos mais leves, à insuficiência hepática nos mais graves, e às complicações tardias, como disfunções motoras e reprodutivas. A galctosemia também é heterogénea do ponto de vista molecular, com 266 mutações diferentes descritas no gene GALT, algumas específicas para certas populações, refletindo o que se espera de alguns eventos de efeito fundador. O objetivo deste trabalho foi avaliar o perfil de mutações no gene GALT, dos pacientes brasileiros com galactosemia clássica e fazer um estudo da correlação do genótipo com o fenótipo, uma vez que se sabe que parte da variação observada na evolução clínica está relacionada com o nível de atividade residual da enzima e do genótipo. Para tanto, foram incluídos no estudo 31 pacientes com o diagnóstico bioquímico de galactosemia de diversas regiões do Brasil, que tiveram seus dados clínicos obtidos a partir de revisão de prontuários médicos e preenchimento de ficha clínica. Foi realizado o sequenciamento genético direto bidirecional do gene GALT e também estudos adicionais, como genotipagem do gene GALK1 de um paciente, estudo de ancestralidade de sete pacientes, além de simulações de patogenicidade in silico das novas mutações identificadas. Os principais achados clínicos dos pacientes que participaram deste estudo foram hepatomegalia, icterícia, baixo ganho pondero-estatural, vômito recorrente, anemia e catarata. As principais mutações que causam GC descritas na literatura foram identificadas neste estudo, como por exemplo, a p.Q188R, p.S135L e p.K285N, bem como o alelo Duarte 2 e seis mutações novas, p.M1T, p.R33S, p.P73S, IVS3+1G>A, IVS4+4A>C e p.Q169P. Este resultado era esperado, dada a elevada miscigenação da população brasileira. Alguns indivíduos foram diagnosticados através do teste de triagem neonatal expandido, que não está disponível rotineiramente a todos os recém-nascidos, portanto, começaram o tratamento dietético antes de desenvolverem os sinais e sintomas da doença. Para estes indivíduos não foi possível fazer uma análise da relação genótipo-fenótipo. Para os demais indivíduos esta relação foi consistente com o que é descrito na literatura, com os indivíduos homozigotos para a mutação p.Q188R com uma evolução mais grave do que os indivíduos que tinham pelo menos uma mutação p.S135L. Para os indivíduos com as mutações novas, foi observado um amplo espectro de fenótipos, como de pacientes que foi a óbito por insuficiência hepática e sepse à um caso assintomático. Este estudo amplia o espectro de mutação no gene GALT descrito na literatura e reforça a importância tanto do diagnóstico precoce quanto da introdução do tratamento dietético; também acrescenta mais evidências para a discussão sobre a introdução da galactosemia no programa de triagem neonatal do Brasil, onde a incidência da doença é estimada em cerca de 1:20.000. / Classical galactosemia (CG) or type I galactosemia is an inborn error of galactose metabolism caused by the deficiency of the galactose-1-phosphate uridyltransferase enzyme (GALT). It is transmitted as an autosomal recessive disease and is typically characterized by neonatal galactose intolerance, with complications ranging from neonatal jaundice and liver failure to late complications, such as motor and reproductive dysfunctions. Galactosemia is also heterogeneous from a molecular standpoint, with 266 mutations described to date in the GALT gene, some of them specific to certain populations, reflecting what is expected as some events of founder effect. The objective of this study was to evaluate the profile of mutations in the GALT gene of Brazilian patients with classical galactosemia and perform a genotype-phenotype correlation study, since it is known that part of the observed variation in clinical outcome is related to the level of residual enzyme activity and genotype. Therefore, this study included 31 patients with biochemical diagnosis of galactosemia from different regions of Brazil, who had their clinical data obtained from review of medical records and from a standardized case report form. We conducted a direct bidirectional sequencing of the GALT gene and also additional studies, as GALK1 genotyping for a patient, ancestrality study of seven patients and in silico simulation of pathogenicity for the new mutations identified. The main clinical features of the patients in this study were hepatomegaly, jaundice, low weight and height gain, recurrent vomiting, anemia and cataract. The major CG causing mutations described in the literature have been identified in this study, for example, p.Q188R, p.S135L and p.K285N, as well as the Duarte 2 allele and six novel mutations: p.M1T; p.R33S; p.P73S; IVS3+1G>A; IVS4+4A>C and p.Q169P. This result was expected, given the high miscegenation of the Brazilian population. Some individuals were diagnosed through expanded newborn screening test, which is not available routinely to all newborns, and so began dietary treatment before they develop signs and symptoms of the disease. For these individuals, was not possible to analyze the genotype-phenotype correlation. For other individuals this relationship was consistent with what is described in the literature, with the homozygous for p.Q188R mutation presenting a more severe phenotype than individuals who had at least one p.S135L mutation. For individuals with new mutations, was observed a wide range of phenotypes, from patients who died due to liver failure and sepsis to an asymptomatic case. This study expands the spectrum of mutations in the GALT gene described in the literature and reinforces the importance of early diagnosis and the introduction of dietary treatment; also adds more evidence to the discussion on the introduction of galactosemia in the neonatal screening program of Brazil, where the incidence of the disease is estimated at about 1:20,000.

Dissertação de Mestrado apresentada ao Programa de Pós Graduação em Ciências da Saúde, da Universidade do Extremo Sul Catarinense – UNESC, para obtenção do título de Mestre em Ciências da Saúde. / A galactosemia é uma condição causada pela deficiência de uma das quatro enzimas envolvidas no metabolismo da galactose: galactose mutarotase, galactocinase, galactose-1-fosfato uridiltransferase e UDP galactose - 4 - epimerase. Como consequência deste bloqueio, os indivíduos afetados pela galactosemia não podem efetivamente converter galactose em glicose, apresentando acúmulo de galactose e seus metabólitos em seus tecidos, especialmente nas células do tecido nervoso central, fígado e rins. Dentre os principais achados clínicos, destaca-se disfunção cerebral com atraso de desenvolvimento e deficiência de aprendizado e de memória, cuja fisiopatologia ainda não está esclarecida. Considerando-se o parco conhecimento a respeito dos mecanismos fisiopatológicos do dano cerebral apresentado por pacientes galactosêmicos, o presente trabalho avaliou o efeito da administração aguda de galactose sobre vias apoptóticas em cérebros de ratos jovens. Para tanto, vinte e quatro ratos machos da linhagem Wistar de 30 dias foram divididos em dois grupos: controle e galactosemia. Os animais do grupo galactosemia receberam uma única administração subcutânea de 5 μmol/g de peso corporal de galactose. Os animais do grupo controle receberam solução salina nos mesmos volumes por via subcutânea. Uma hora após a administração deste carboidrato, os animais sofreram eutanásia por decapitação com guilhotina e o córtex cerebral e o cerebelo foram isolados e limpos. Foram então determinados os imunoconteúdo de Bax, Bcl-2, AIF e p53, proteínas que fazem parte de vias de morte celular por apoptose, e a razão Bax/Bcl-2 nestes tecidos por western blotting. Não foram observadas diferenças significativas entre os grupos nos conteúdos das proteínas avaliadas, bem como na razão entre Bax/Bcl-2 em nenhuma das estruturas cerebrais estudadas. Os resultados do presente trabalho sugerem que a administração aguda de galactose não altera os processos de morte celular por apoptose via proteínas Bax, Bcl-2, AIF e p53 em cérebro de ratos jovens.

Addendum pasted inside the back end-paper. / x, 179 leaves : / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / This thesis presents a hypothesis that there are abnormalities of N-glycosylation in classical galactosaemia and that these abnormalities could contribute to the long-term complications. The aim of the thesis is to characterise and model N-glycosylation in skin fibroblasts from patients with galactosaemia. The study identifies a disturbance in the synthesis and processing of dolichol-linked oligosaccharides. It is anticipated that the serum glycoproteins in untreated galactosaemia may contain N-glycans that are partly absent or truncated. / Thesis (M.D.)--University of Adelaide, Dept. of Paediatrics, 1997

Classic Galactosemia (CG) is an autosomal recessive disorder caused by deleterious mutations of galactose-1-phosphate uridyltransferase (E.C. 2.7.7.12) (GALT) gene, which results in the inability to metabolize galactose and the accumulation of galactose-1-phosphate (gal-1-p) in patients' cells. Newborn screening has resulted in presymptomatic diagnosis and treatment. Although a galactose-restricted diet prevents the neonatal lethality of this disorder, many well-treated patients continue to develop debilitating complications such as premature ovarian insufficiency (POI), dyspraxic speech, ataxia and other neurological defects. The causes of these unsatisfactory outcomes remain unclear, but accumulation of gal-1-p is regarded as the major factor responsible for these chronic complications. In previous studies we found that gal-1-p was a competitive inhibitor of the UTP dependent, glucose-1-phosphate pyrophosphorylase (EC 2.7.7.9) and inositol monophosphatase (EC 3.1.3.25). As a result there were deficiencies in cellular UDP-glucose and UDP-galactose as well as impaired G-protein-stimulated inositol responses of Ca++ release, respectively. In this study, we found that when we challenged GALT-deficient yeast and GALT-deficient human diploid fibroblasts with galactose, these cells exhibited environmental stress and endoplasmic reticulum (ER) stress, which was characterized by the up-regulation of the gene encoding the master regulator of ER stress, GRP78/BiP. In separate studies using GALT-deficient diploid fibroblasts and comparative expression arrays, we found that the expression level of a tumor suppressor gene called aplysia ras homolog I (ARHI) was significantly higher in patient cells under galactose challenge. This ARHI gene was lost in rodents during evolution and GALT knockout mice did not express the human phenotype for galactosemia whereas over-expression of this gene in transgenic mice resulted in phenotypes characteristic of those seen in patients with galactosemia. We therefore propose here that ARHI could be an important target of galactose toxicity in Classic Galactosemia, and also explain the absence of patient phenotypes in GALT knock-out mice. In order to prevent accumulation of gal-1-p caused by GALT-deficiency, we experimentally screened over 300,000 chemical compounds against human galactokinase (GALK) in vitro. To date, we obtained from two high-throughput screenings (HTS), 200 GALK inhibitors with IC50s ranging from 700nM to 35μM. We subsequently established selectivity and toxicity profiles of 34 selected GALK inhibitors. Based on these results, we selected four compounds for further characterization, which included kinetic studies, site-directed mutagenesis and molecular docking experiments. From these experiments, we learned how these compounds interacted with the GALK enzyme and built detailed binding models for each of them. We demonstrated that three of the in vitro inhibitors of GALK could lower intracellular gal-1-p accumulation in GALT-deficient cells. Of considerable interest to us was that one of the compounds, cluster 25-1, not only reduced gal-1-p accumulation, but also corrected the level of GRP78/BiP back to background in the galactose-challenged GALT-deficient cells. These results were the first to demonstrate a direct link between GALT-deficiency and ER stress and provided proof of concept that we could prevent both gal-1-p accumulation and ER stress with GALK inhibitors in GALT deficient human cells. Lastly, we developed a new, virtual method of identifying novel GALK inhibitors by combining software-based, high-throughput virtual screening (HTVS) and fragment-based linkage using docking software. The initial HTVS validation experiments with compounds from the "ZINC" database identified four active GALK inhibitors with IC50s ranging from 70μM to 400μM. We then used HTVS to identify chemical fragments which bind to the active site of the human GALK enzyme. Using fragment-linking software, we identified chemical fragments which could potentially result in high-affinity inhibitors when chemically joined.

Thesis (M. Phil.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 86-111).

published_or_final_version / abstract / toc / Anatomy / Master / Master of Philosophy

published_or_final_version / abstract / toc / Anatomy / Doctoral / Doctor of Philosophy