The perception is that inborn errors of metabolism (IEM) are rare, but the reality is that more than 600 lEMs are now recognized. The organic aciduria, 3-methylcrotonyl-CoA carboxylase (MCC) deficiency arises when 3-methylcrotonyl-Coenzyme A (CoA) carboxylase that participates in the fourth step of the leucine catabolism is defective. Tandem mass spectrometry (MS/MS) based screening programmes in North America, Europe and Australia, showed that MCC deficiency is the most frequent organic aciduria detected, with an average frequency of 1:50 000. Therefore MCC deficiency is considered an emerging disease in these regions. The incidence of MCC deficiency in the Republic of South Africa (RSA) is not yet known. However, one 48 year old male Caucasian individual (HGS) was diagnosed suffering from mild MCC deficiency, since elevated levels of 3-hydroxyisovaleric acid, 3- hydroxyisovalerylcarnitine, 3-methylcrotonylglycine was present in his urine. Several groups are currently working on various aspects of this emerging disease with the focus on the molecular characterisation of MCC deficiency. In the RSA no molecular based diagnostic method which complements MS/MS screening programmes have yet been implemented. Therefore, the aim of this study was to implement the necessary techniques for the molecular characterisation of MCC deficiency, the determination of the sequence of the open reading frame (ORF) of mccA and mccB subunits to determine which mutation(s) are present in the South African MCC deficient patient. For the implementation of the molecular characterisation, a two-pronged approached was used to characterize MCC of a MCC non-deficient individual (CFC). This approach included the reverse transcriptase polymerase chain reaction (RT-PCR) amplification of the ORFs of the associated genes [mccA (19 exons) and mccB (17 exons] and the PCR amplification of selected (genomic deoxyribonucleic acid (gDNA) regions (exons mccA8, mccA11 , mccB5, mccB6 and mccB5-intron 5-6 exon 6 (mccB5-6) which have been found to have mutations associated with MCC deficiency in Caucasians. The sequence analyses produced surprising results of the amplified ORFs (CFCmccA and CFCmccB) of the MCC non-deficient individual CFC. A non-synonymous single nucleotide polymorphism (SNP) (1391C→A, H464P) associated with MCC deficiency (Gallardo et al., 2001) was identified in the CFCmccA subunit. Another SNP (1368G→A, A456A) recently listed in GenBank was observed in the amplified CFCmccB ORF. No significant novel variations or described mutations were identified in the amplified genomic regions mccA8, mccA11 ,mccB5, mccB6 and mccB5-6. The implemented molecular approach was used to characterise MCC of our MCC deficient patient (HGS). The patient did not have any mutation in the four selected exons mccA8, mccA11, mccB5, mccB6 or the genomic region mccB5-6. The RT-PCR amplification of both ORFs (HGSmccA and HGSmccB) resulted in multiple amplicons. Gel extracted amplicons of the expected size were sequenced. Of the 36 exons, 34 exons were sequenced. This includes all 19 exons of HGSmccA and 15 of 17 exons of HGSmccB (exons 1-6 and exons 9-17). The non-synonymous SNP (1391C→A, H464P) detected in CFCmccA (MCC non-deficient individual), seems to be present in the HGSmccA subunit of the MCC deficient individual, HGS. The HGSmccB amplicons could not be entirely sequenced. However, the region exon 1-6 and 9-17 was sequenced but no described or novel mutations were identified. The lack of sequence data of region exon 7-8 led to an incomplete molecular characterisation of the MCC deficiency in HGS. In conclusion, the basic methods and techniques for the molecular characterisation of MCC deficient patients have been implemented locally. A few additional sequencing primers need to be designed to cover mccB7 and mccB8 as well as the entire coding and non-coding strands of each MCC gene (mccA and mccB). The primers for RT-PCR of both mccA and mccB need to be further refined to ensure better specificity. / Thesis (M.Sc. (Biochemistry))--North-West University, Potchefstroom Campus, 2007.

Introdução: A enzima 3-hidroxiacil CoA desidrogenase de cadeia longa (LCHAD) é uma das enzimas envolvidas na beta-oxidação mitocondrial de ácidos graxos e faz parte do complexo enzimático chamado proteína trifuncional. Sua deficiência segue um modelo de herança autossômica recessiva com uma mortalidade maior que 70%, porém um tratamento dietoterápico adequado reduz substancialmente a morbimortalidade. Estudos recentes descreveram que gestantes de fetos homozigóticos para defeitos de LCHAD apresentam grandes chances de desenvolver síndrome HELLP e doença hepática aguda da gestação, com risco de morte materna, fetal e do recém-nascido. A síndrome HELLP é caracterizada por plaquetopenia, enzimas hepáticas elevadas e hemólise, podendo se apresentar na forma completa ou parcial. Estudos mostram que investigar a relação entre síndrome HELLP e defeitos de LCHAD, possibilita a prevenção de futuras gestações de risco por meio de aconselhamento genético e o diagnóstico precoce deste erro inato do metabolismo. Objetivos: Verificar a associação entre gestantes com síndrome HELLP e lactentes com defeitos LCHAD e identificar problemas de saúde gerados pela doença nesses conceptos. Metodologia: Análise de prontuários, necropsias e desfecho atual dos conceptos de 42 gestantes com síndrome HELLP e 84 controles. Resultados: Entre as gestantes que compunham os casos, a maioria apresentou proteinúria; além de sintomas como vômitos e epigastralgia. O parto cesárea foi realizado em 93% das gestantes. Quase metade das puérperas apresentou algum tipo de complicação materna. Quanto ao desfecho perinatal, 90% dos conceptos apresentaram baixo peso ao nascer e 23,8% evoluíram para óbito. Entre esses 10 óbitos, resgatamos 7 imagens histopatológicas com esteatose hepática. Inferimos doença metabólica nesses casos, que levou a uma associação de 11% com a síndrome HELLP. Entre o grupo controle, 46,2% das mulheres já haviam sofrido pelo menos um aborto. Na atual gestação 6,4% desenvolveram pré-eclampsia; entre outras complicações. Encontramos gravidezes subsequentes das gestantes do grupo Caso com recorrência de HELLP e óbito. Conclusão: Os resultados reforçam a importância do diagnóstico precoce de síndrome HELLP, além da investigação da associação do defeito de LCHAD e HELLP mesmo post morten afim de evitar futuras gestações de risco e diminuir a morbimortalidade materna e neonatal. / Long-chain 3-hydroxyacyl CoA dehydrogenase (LCHAD) is one of the enzymes involved in the mitochondrial fatty acids beta-oxidation and part of the enzymatic complex called trifunctional protein. Its deficiency follows an autosomal recessive model with a higher mortality, but an adequate dietary treatment reduces its morbimortality. Recent studies reported that mothers of fetuses homozygous for LCHAD deficiency have higher chances of developing HELLP and acute fatty liver of pregnancy with risk of maternal, infant and fetal death. Thrombocytopenia, elevated liver enzymes and hemolysis characterize HELLP syndrome, which can be diagnosed as complete or partial. Studies demonstrate that investigate the association between HELLP syndrome and LCHAD defects can prevent future risk pregnancies through genetic counseling and early diagnosis of this inborn error of metabolism. Objectives: To investigate the association between pregnant women and concepts with LCHAD deficiency and identify health problems caused by the disease in these fetuses. Methodology: Analysis of medical records, autopsy reports and current outcome of fetuses of 42 pregnant women with HELLP syndrome and 84 controls. Results: In case group, most patients presented proteinuria; as well as symptoms as vomiting and epigastric pain. The cesarean delivery was performed in 93% of pregnant women. Almost half of women presented maternal complications. In perinatal outcome, 90% of fetuses has low weight at birth and 23.8% died. Among these 10 deaths, we rescued 7 hystopathological images with hepatic steatosys. We could infer metabolic disease in these cases, which led to an association of 11% to the HELLP syndrome. Among the control group, 46.2% of women had at least one abortion before this pregnancy. During the pregnancy 6.4% developed pre-eclampsia among other complications. In control group, we find HELLP syndrome recurrence and death in subsequent pregnancy. Conclusion: The results reinforce the importance of early diagnosis of HELLP syndrome, as well as research LCHAD and HELLP association even post mortem to avoid future risk pregnancies and reduce maternal and neonatal morbidity and mortality.

Introdução: A enzima 3-hidroxiacil CoA desidrogenase de cadeia longa (LCHAD) é uma das enzimas envolvidas na beta-oxidação mitocondrial de ácidos graxos e faz parte do complexo enzimático chamado proteína trifuncional. Sua deficiência segue um modelo de herança autossômica recessiva com uma mortalidade maior que 70%, porém um tratamento dietoterápico adequado reduz substancialmente a morbimortalidade. Estudos recentes descreveram que gestantes de fetos homozigóticos para defeitos de LCHAD apresentam grandes chances de desenvolver síndrome HELLP e doença hepática aguda da gestação, com risco de morte materna, fetal e do recém-nascido. A síndrome HELLP é caracterizada por plaquetopenia, enzimas hepáticas elevadas e hemólise, podendo se apresentar na forma completa ou parcial. Estudos mostram que investigar a relação entre síndrome HELLP e defeitos de LCHAD, possibilita a prevenção de futuras gestações de risco por meio de aconselhamento genético e o diagnóstico precoce deste erro inato do metabolismo. Objetivos: Verificar a associação entre gestantes com síndrome HELLP e lactentes com defeitos LCHAD e identificar problemas de saúde gerados pela doença nesses conceptos. Metodologia: Análise de prontuários, necropsias e desfecho atual dos conceptos de 42 gestantes com síndrome HELLP e 84 controles. Resultados: Entre as gestantes que compunham os casos, a maioria apresentou proteinúria; além de sintomas como vômitos e epigastralgia. O parto cesárea foi realizado em 93% das gestantes. Quase metade das puérperas apresentou algum tipo de complicação materna. Quanto ao desfecho perinatal, 90% dos conceptos apresentaram baixo peso ao nascer e 23,8% evoluíram para óbito. Entre esses 10 óbitos, resgatamos 7 imagens histopatológicas com esteatose hepática. Inferimos doença metabólica nesses casos, que levou a uma associação de 11% com a síndrome HELLP. Entre o grupo controle, 46,2% das mulheres já haviam sofrido pelo menos um aborto. Na atual gestação 6,4% desenvolveram pré-eclampsia; entre outras complicações. Encontramos gravidezes subsequentes das gestantes do grupo Caso com recorrência de HELLP e óbito. Conclusão: Os resultados reforçam a importância do diagnóstico precoce de síndrome HELLP, além da investigação da associação do defeito de LCHAD e HELLP mesmo post morten afim de evitar futuras gestações de risco e diminuir a morbimortalidade materna e neonatal. / Long-chain 3-hydroxyacyl CoA dehydrogenase (LCHAD) is one of the enzymes involved in the mitochondrial fatty acids beta-oxidation and part of the enzymatic complex called trifunctional protein. Its deficiency follows an autosomal recessive model with a higher mortality, but an adequate dietary treatment reduces its morbimortality. Recent studies reported that mothers of fetuses homozygous for LCHAD deficiency have higher chances of developing HELLP and acute fatty liver of pregnancy with risk of maternal, infant and fetal death. Thrombocytopenia, elevated liver enzymes and hemolysis characterize HELLP syndrome, which can be diagnosed as complete or partial. Studies demonstrate that investigate the association between HELLP syndrome and LCHAD defects can prevent future risk pregnancies through genetic counseling and early diagnosis of this inborn error of metabolism. Objectives: To investigate the association between pregnant women and concepts with LCHAD deficiency and identify health problems caused by the disease in these fetuses. Methodology: Analysis of medical records, autopsy reports and current outcome of fetuses of 42 pregnant women with HELLP syndrome and 84 controls. Results: In case group, most patients presented proteinuria; as well as symptoms as vomiting and epigastric pain. The cesarean delivery was performed in 93% of pregnant women. Almost half of women presented maternal complications. In perinatal outcome, 90% of fetuses has low weight at birth and 23.8% died. Among these 10 deaths, we rescued 7 hystopathological images with hepatic steatosys. We could infer metabolic disease in these cases, which led to an association of 11% to the HELLP syndrome. Among the control group, 46.2% of women had at least one abortion before this pregnancy. During the pregnancy 6.4% developed pre-eclampsia among other complications. In control group, we find HELLP syndrome recurrence and death in subsequent pregnancy. Conclusion: The results reinforce the importance of early diagnosis of HELLP syndrome, as well as research LCHAD and HELLP association even post mortem to avoid future risk pregnancies and reduce maternal and neonatal morbidity and mortality.

La chromatographie en phase gazeuse couplée à la spectrométrie de masse simple quadripolaire (GC-qMS) est la technique la plus utilisée dans l’exploration des acides organiques urinaires dans le cadre du diagnostic des aciduries organiques (AO). Cependant, après analyse par GC-MS de plus de 1000 échantillons d'urines recueillis auprès d'enfants atteints de troubles neurologiques, moins de 0,5% d'entre eux ont reçu un diagnostic positif. Ceci témoigne de l’urgence de rechercher de nouveaux biomarqueurs dans le domaine des troubles neurologiques associés à des erreurs innées du métabolisme (EIM).L'objectif principal de cette thèse a été de développer une nouvelle méthode d’analyse des échantillons urinaires par chromatographie bidimensionnelle couplée à la spectrométrie de masse (GCxGC-qMS) et d'évaluer son apport dans le profilage métabolique de ces échantillons. Par ailleurs, dans le cadre du diagnostic des AO, dans un objectif de simplification de la procédure de préparation de l’échantillon, nous avons évalué une autre approche d’analyse directe en temps réel couplée à la spectrométrie de masse « DART-MS » des acides organiques urinaires. La première étape de notre travail a été de développer un protocole de préparation de l’échantillon compatible avec une méthode de séparation bidimensionnelle. Puis, grâce à l’étude systématique des différentes conditions de séparation, nous avons développé une méthode par CGxCG-qMS parfaitement adaptée à la séparation des composés extraits.Après validation analytique, la méthode proposée a été ensuite évaluée par le profilage des composés organiques présents dans des échantillons urinaires prélevés chez des volontaires sains. Par rapport aux profils obtenus par une méthode par GC-qMS classique, optimisée, les profils chromatographiques obtenus par la méthode proposée présentent une sensibilité et une résolution nettement plus élevées. Ceci se traduit par la détection en GCxGC-qMS de nombreux composés supplémentaires, jusqu’à 92, selon les échantillons. Parmi les composés supplémentaires détectés et caractérisés, certains sont d’un intérêt diagnostic reconnu.Malgré les difficultés liées à l’étape de préparation de l’échantillon et au traitement des données, l’ensemble des résultats obtenus par la méthode proposée sur de nombreux échantillons urinaires a confirmé les potentialités et la nécessité d’utiliser la chromatographie bidimensionnelle dans le domaine de la découverte de candidats biomarqueurs. Il s’agit maintenant de poursuivre l’identification des composés supplémentaires détectés dans les échantillons urinaires étudiés et d’appliquer la méthode proposée à un plus grand nombre d’échantillons témoins et pathologiques afin d’essayer d’identifier des candidats biomarqueurs.Enfin, les essais préliminaires par la technique DART-MS, effectués dans un laboratoire de la Direction des Applications Militaires (DAM) du CEA, ont été axés sur la détection d’un panel d’acides organiques représentatifs des différentes anomalies rencontrées lors des AO. Malgré les effets de suppression ionique qui restent à éliminer, les résultats obtenus montrent que cette approche mérite d’être approfondie. / Among inherited metabolic diseases, organic acidemia (OA) or organic aciduria is characterized by urinary excretion of abnormal amounts or types of organic acids. OA is mostly associated with genetic conditions resulting in a specific step of amino acid catabolism dysfunction. Such alterations can produce disease states that range from mild to lethal neurological involvement. Gas chromatography coupled to mass spectrometry (GC-MS) remains the most used analytical technique for detecting specific urinary organic metabolites related to OA. However, after analyzing more than 1,000 urine samples collected from children with neurological disorders, by this technique less than 0.5% of them were positive. Thus, the discovery of new biomarker candidates for other metabolic diseases is urgently needed. The main objective of this thesis was to evaluate a new method using two-dimensional gas chromatography coupled to mass spectrometry (GCxGC-MS) for the both qualitative and quantitative metabolic profiling of children’s urine. As compared to GC-MS, GCxGC-MS shows great resolution power and high peak capacity. For this purpose, we first developed a GCxGC-MS method with an appropriate sample preparation protocol for urinary organic acids profiling. Applied to urine samples of healthy children and children with neurological disorders, the proposed method showed high sensitivity and peak capacity thus opening new possibilities for the characterization of new biomarker candidates. For instance, by this technique, we were able to detect in urines samples more than ninety additional compounds, which are not detected by a conventional GC-MS method. However, taken together the obtained results show that while the GCxGC technique has unmatched power to separate compounds in a complex mixture, the sample preparation protocol remains a limiting step for the precise quantification of the detected compounds. On another hand, in order to reduce the sample preparation step, we evaluated the direct analysis in real time (DART) method for the urinary organic acids screening. The obtained results are very promising.

目前廣泛應用于胎兒醫學的唐氏綜合症篩查法，即結合早孕期胎兒頸項透明層的超聲檢查，及母體血清生化指標的綜合篩查法。頸項透明層是指在早孕期利用超聲檢測到的胎兒頸后的皮下積水，其作為預測胎兒異常的一項重要“軟指標，其臨床意義，尤其是與胎兒染色體異常及器官結構異常之間的關係，逐漸得到深入的認識，但其形成機制尚未明確。現在已知有一百餘種畸形及遺傳綜合征與胎兒頸項透明層增厚相關，但其染色體異常譜系，尤其是亞顯微的染色體異常仍有待明確。大部分頸項透明層增厚但核型正常的胎兒預後良好，但約3-10%的這部分胎兒會伴有畸形或出生后的神經智力發育缺陷。而傳統核型分析無法檢測到亞顯微的染色體異常，從而無法判斷這部分核型正常卻伴有缺陷的胎兒是否因為這類染色體異常而致病。 / 微陣列比較基因組雜交芯片作為檢測兒童發育遲緩者及器官結構異常原因的重要手段已廣泛應用于臨床。在染色體核型正常的胎兒中，若伴有器官結構異常的胎兒，5-12%被檢出與該畸形相關的微缺失及微重複；若僅伴有孕婦高齡或唐氏篩查高危，則微缺失及微重複檢出率約1%。 / 該課題旨在研究頸項透明層增厚但核型正常的胎兒中，染色體拷貝數變異發生的頻率及頻譜；評估微陣列比較基因組雜交芯片在協助臨床判斷胎兒預後中的作用。因此，我們開展該多中心隊列研究，通過納入449例頸項透明層厚度≧3.5 mm但正常核型胎兒的，檢測其染色體拷貝數變異，監測并記錄其圍產、產後及新生兒期情況。微陣列比較基因組雜交芯片總共檢出2.8%的異常拷貝數變異，其大小範圍為0.1 kb至18Mb。在伴有器官結構異常的胎兒組中，異常拷貝數變異檢出率達7.8%。對於頸項透明層厚度≧4.0 mm的胎兒，異常拷貝數變異檢出率可達7.3%。 / 對於頸項透明層增厚的胎兒，致病拷貝數變異暫未發現特定的頻譜。但，該研究中發現重複的致病拷貝數變異，如22號染色體長臂1區1帶的微重複或微缺失，2號染色體長臂2區2帶的微缺失。未在3號、7號、12號、13號、18號、20號、21號或Y染色體上發現與胎兒頸項透明層增厚相關的致病拷貝數變異。 / 頸項透明層增厚的胎兒79.3%預後良好；若經微陣列比較基因組雜交芯片未檢出致病拷貝數變異，則81.2%預後良好。如果僅頸項透明層增厚不伴有結構異常的胎兒，經微陣列比較基因組雜交芯片未檢出致病拷貝數變異，則93.5%預後良好。 / 綜上所述，微陣列比較基因組雜交芯片顯著提高了致病拷貝數變異的檢出率。可考慮將微陣列比較基因組雜交芯片作為頸項透明層厚度≧4.0 mm的胎兒染色體異常檢查的首要方法。對於僅頸項透明層增厚不伴有結構異常的胎兒，且經微陣列比較基因組雜交芯片未檢出致病拷貝數變異，絶大部分預後良好。對於頸項透明層增厚的胎兒，致病拷貝數變異暫未發現特定的頻譜，但發現重複出現的致病拷貝數變異。通過初步的基因本體分析及基因通路分析，神經嵴細胞的分化遷徙功能異常可作為今後研究頸項透明層增厚的病理生理機制的方向。 / Measurement of nuchal translucency (NT) has been recognized as a sensitive marker for fetal chromosomal disorders for more than a decade, and is presently used as a routine first-trimester screening test. Although over 100 abnormalities and genetic syndromes have been reported to be associated with increased NT, these associations have not been fully explored and the relevant spectrum of associated submicroscopic chromosomal abnormalities has not been sufficiently investigated. The majority of euploid fetuses with increased NT have a good outcome, but around 3-10% of fetuses present with structural or neurodevelopmental abnormalities postnatally. A range of genetic syndromes has been reported, many of which are linked to submicroscopic chromosomal abnormalities that are typically missed by conventional karyotyping. / Microarray-based comparative genomic hybridization (arrayCGH) has been applied as the first-tier diagnostic tool for the evaluation of developmental delay and structural malformations in children. In fetuses with a normal karyotype, microarray analysis revealed clinically relevant deletions or duplications in 5-12% with a structural anomaly and in about 1% of those whose indications were advanced maternal age or positive screening results. / The objectives of this study were to delineate the frequency and spectrum of pathogenic chromosome copy number variants (CNVs) among fetuses with increased NT and normal karyotype; to evaluate the role of arrayCGH to predict the prognosis of the high NT fetuses; to explore the genotype-phenotype correlations of increased NT. Therefore, a multi-centre cohort of 449 fetuses with NT ≧3.5 mm and normal karyotype were further investigated by arrayCGH. Antenatal surveillance, pregnancy outcome and paediatric follow up were documented. ArrayCGH detected abnormal CNVs in 2.8% (14 of 449) of the fetuses with high NT; the size of CNVs ranged from 0.1 kb to 18Mb. Among fetuses with major congenital abnormalities the incidence of abnormal CNV reached 7.8% (4 of 51). By adjusting the NT to ≧4.0 mm as the referral indication, 7.3% (14 of 192) of the fetuses would have abnormal arrayCGH results. The spectrum of pathogenic CNVs found associated with increased NT was diverse. However, there were recurrent ones such as the deletions or duplications at chromosomal region 22q11, and deletions in ZEB2. There was no pathogenic CNV related with increased NT found in chromosomes 3, 7, 12, 13, 18, 20, 21, or Y. The total normal outcome rate of euploid fetuses with an increased NT was 79.3%; for fetuses with normal arrayCGH results 81.2% had a normal outcome. In fetuses with isolated increased NT, normal arrayCGH results predict a favorable prognosis of 93.5%. / In conclusion, arrayCGH significantly increased the diagnostic yield of pathogenic CNVs. In clinical practice arrayCGH may be considered as the first tier investigation in fetuses with an increased NT more than 4.0 mm. In cases with an isolated increased NT with normal arrayCGH results the pregnancy outcome is likely to be favorable. The spectrum of abnormal CNVs found by arrayCGH is diverse but there are recurrent cases such as del/dup 22q11 and del ZEB2. Our preliminary gene ontology and pathway analysis showed that gene pathways related to neural crest cells may be considered as a future study for physiopathologic mechanisms of NT. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Huang, Jin. / Thesis (Ph.D.) Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 106-120). / Abstracts also in Chinese.

The perception is that inborn errors of metabolism (IEM) are rare, but the reality is that more than 600 lEMs are now recognized. The organic aciduria, 3-methylcrotonyl-CoA carboxylase (MCC) deficiency arises when 3-methylcrotonyl-Coenzyme A (CoA) carboxylase that participates in the fourth step of the leucine catabolism is defective. Tandem mass spectrometry (MS/MS) based screening programmes in North America, Europe and Australia, showed that MCC deficiency is the most frequent organic aciduria detected, with an average frequency of 1:50 000. Therefore MCC deficiency is considered an emerging disease in these regions. The incidence of MCC deficiency in the Republic of South Africa (RSA) is not yet known. However, one 48 year old male Caucasian individual (HGS) was diagnosed suffering from mild MCC deficiency, since elevated levels of 3-hydroxyisovaleric acid, 3- hydroxyisovalerylcarnitine, 3-methylcrotonylglycine was present in his urine. Several groups are currently working on various aspects of this emerging disease with the focus on the molecular characterisation of MCC deficiency. In the RSA no molecular based diagnostic method which complements MS/MS screening programmes have yet been implemented. Therefore, the aim of this study was to implement the necessary techniques for the molecular characterisation of MCC deficiency, the determination of the sequence of the open reading frame (ORF) of mccA and mccB subunits to determine which mutation(s) are present in the South African MCC deficient patient. For the implementation of the molecular characterisation, a two-pronged approached was used to characterize MCC of a MCC non-deficient individual (CFC). This approach included the reverse transcriptase polymerase chain reaction (RT-PCR) amplification of the ORFs of the associated genes [mccA (19 exons) and mccB (17 exons] and the PCR amplification of selected (genomic deoxyribonucleic acid (gDNA) regions (exons mccA8, mccA11 , mccB5, mccB6 and mccB5-intron 5-6 exon 6 (mccB5-6) which have been found to have mutations associated with MCC deficiency in Caucasians. The sequence analyses produced surprising results of the amplified ORFs (CFCmccA and CFCmccB) of the MCC non-deficient individual CFC. A non-synonymous single nucleotide polymorphism (SNP) (1391C→A, H464P) associated with MCC deficiency (Gallardo et al., 2001) was identified in the CFCmccA subunit. Another SNP (1368G→A, A456A) recently listed in GenBank was observed in the amplified CFCmccB ORF. No significant novel variations or described mutations were identified in the amplified genomic regions mccA8, mccA11 ,mccB5, mccB6 and mccB5-6. The implemented molecular approach was used to characterise MCC of our MCC deficient patient (HGS). The patient did not have any mutation in the four selected exons mccA8, mccA11, mccB5, mccB6 or the genomic region mccB5-6. The RT-PCR amplification of both ORFs (HGSmccA and HGSmccB) resulted in multiple amplicons. Gel extracted amplicons of the expected size were sequenced. Of the 36 exons, 34 exons were sequenced. This includes all 19 exons of HGSmccA and 15 of 17 exons of HGSmccB (exons 1-6 and exons 9-17). The non-synonymous SNP (1391C→A, H464P) detected in CFCmccA (MCC non-deficient individual), seems to be present in the HGSmccA subunit of the MCC deficient individual, HGS. The HGSmccB amplicons could not be entirely sequenced. However, the region exon 1-6 and 9-17 was sequenced but no described or novel mutations were identified. The lack of sequence data of region exon 7-8 led to an incomplete molecular characterisation of the MCC deficiency in HGS. In conclusion, the basic methods and techniques for the molecular characterisation of MCC deficient patients have been implemented locally. A few additional sequencing primers need to be designed to cover mccB7 and mccB8 as well as the entire coding and non-coding strands of each MCC gene (mccA and mccB). The primers for RT-PCR of both mccA and mccB need to be further refined to ensure better specificity. / Thesis (M.Sc. (Biochemistry))--North-West University, Potchefstroom Campus, 2007.

Copies of author's previously published articles inserted / Includes bibliographic references / 2 v. (various foliations) : ill ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Summary: Studies the enzymology of some of the nine lysosomal exo-enzyme activities which act together to degrade the more highly sulphated regions of the glycosaminoglycans heparin and heparan sulphate. A deficiency of any one of these enzyme activities can result in one of the lysosomal storage disorders collectively known as the Mucopolysaccharidoses (MPS) / Thesis (Ph.D.)--University of Adelaide, Dept. of Paediatrics, 1991

Introdução: A Doença da Urina do Xarope do Bordo (DXB) é um Erro Inato do Metabolsimo (EIM), causada pela deficiência da atividade do complexo enzimático desidrogenase dos L-cetoácidos de cadeia ramificada (CACR). O programa público brasileiro de triagem neonatal não inclui o diagnóstico e o tratamento da DXB. Objetivo: Caracterizar aspectos relacionados ao diagnóstico e ao tratamento de pacientes brasileiros com DXB. Metodologia: Estudo retrospectivo. Os pacientes foram identificados a partir dos registros de laboratório considerado referência nacional para o diagnóstico de DXB, e de contato com demais serviços nacionais de referência em genética médica. O diagnóstico foi realizado entre 1992-2011. Os dados analisados foram obtidos a partir da revisão de prontuários. Resultados: Oitenta e três pacientes, oriundos de 75 famílias, foram incluídos no estudo (mediana de idade= 3 anos; IQ25- 75= 0,57-7). A mediana da idade de início dos sintomas foi de 10 dias (IQ= 5-30), enquanto que a mediana da idade ao diagnóstico foi de 60 dias (IQ= 29-240, p= 0,001). Apenas três (3,6%) pacientes foram diagnosticados antes de desenvolverem manifestações clínicas. A comparação entre os pacientes com (n=12) e sem (n=71) diagnóstico precoce mostrou que o mesmo associa-se com a presença de história familiar positiva e diminuição da prevalência de manifestações clínicas ao diagnóstico, mas não com melhor resultado; além disso, a maioria desses diagnósticos foi realizada entre 2002-2011 (n= 10/12). Considerando a amostra total, 98,8% dos pacientes apresentam atraso de desenvolvimento neuropsicomotor. Conclusão: Os pacientes com DXB são diagnosticados tardiamente no Brasil, de forma que as complicações associadas a esta condição não são prevenidas. Entretanto, existem indícios de que está havendo um melhora gradual desse panorama desde a última década. Os nossos dados indicam que o diagnóstico precoce dessa condição, mesmo que em fase sintomática, associa-se a um melhor prognóstico do paciente. Sugere-se a elaboração de políticas públicas específicas para doenças raras no país. / Introduction: Maple syrup urine disease (MSUD) is an Inborn Errors of Metabolism, is caused by a deficiency in activity of the branched-chain L-keto acid dehydrogenase complex. The Brazilian neonatal screening program does not include the diagnosis and treatment of MSUD. Objective: To draw a profile of aspects related to the diagnosis and treatment of Brazilian patients who received. Methods: In this retrospective study, patients were identified through a search of records from a national reference laboratory for the diagnosis of MSUD and through contact with other medical genetics services across Brazil. A diagnosis of MSUD between 1992 and 2011. Data were collected by means of a chart review. Results: Eighty-three patients from 75 families were enrolled in the study (median age 3 years; interquartile range, 0.57–7). Median age at onset of symptoms was 10 days (IQR 5–30), whereas median age at diagnosis was 60 days (IQR 29–240, p=0.001). Only three (3.6%) patients were diagnosed before the onset of clinical manifestations. A comparison between patients with (n=12) and without (n=71) an early diagnosis show that early diagnosis is associated with the presence of positive familial history and decreased prevalence of clinical manifestations at the time of diagnosis, but not with a better outcome. Overall, 98.8% of patients have some psychomotor or neurodevelopmental delay. Conclusion: In Brazil, patients with MSUD receive a late diagnosis and show neurological compromise and poor survival even with an early diagnosis. We suggest that specific public policies for rare diseases should be developed and implemented in the country.

Introdução: A Doença da Urina do Xarope do Bordo (DXB) é um Erro Inato do Metabolsimo (EIM), causada pela deficiência da atividade do complexo enzimático desidrogenase dos L-cetoácidos de cadeia ramificada (CACR). O programa público brasileiro de triagem neonatal não inclui o diagnóstico e o tratamento da DXB. Objetivo: Caracterizar aspectos relacionados ao diagnóstico e ao tratamento de pacientes brasileiros com DXB. Metodologia: Estudo retrospectivo. Os pacientes foram identificados a partir dos registros de laboratório considerado referência nacional para o diagnóstico de DXB, e de contato com demais serviços nacionais de referência em genética médica. O diagnóstico foi realizado entre 1992-2011. Os dados analisados foram obtidos a partir da revisão de prontuários. Resultados: Oitenta e três pacientes, oriundos de 75 famílias, foram incluídos no estudo (mediana de idade= 3 anos; IQ25- 75= 0,57-7). A mediana da idade de início dos sintomas foi de 10 dias (IQ= 5-30), enquanto que a mediana da idade ao diagnóstico foi de 60 dias (IQ= 29-240, p= 0,001). Apenas três (3,6%) pacientes foram diagnosticados antes de desenvolverem manifestações clínicas. A comparação entre os pacientes com (n=12) e sem (n=71) diagnóstico precoce mostrou que o mesmo associa-se com a presença de história familiar positiva e diminuição da prevalência de manifestações clínicas ao diagnóstico, mas não com melhor resultado; além disso, a maioria desses diagnósticos foi realizada entre 2002-2011 (n= 10/12). Considerando a amostra total, 98,8% dos pacientes apresentam atraso de desenvolvimento neuropsicomotor. Conclusão: Os pacientes com DXB são diagnosticados tardiamente no Brasil, de forma que as complicações associadas a esta condição não são prevenidas. Entretanto, existem indícios de que está havendo um melhora gradual desse panorama desde a última década. Os nossos dados indicam que o diagnóstico precoce dessa condição, mesmo que em fase sintomática, associa-se a um melhor prognóstico do paciente. Sugere-se a elaboração de políticas públicas específicas para doenças raras no país. / Introduction: Maple syrup urine disease (MSUD) is an Inborn Errors of Metabolism, is caused by a deficiency in activity of the branched-chain L-keto acid dehydrogenase complex. The Brazilian neonatal screening program does not include the diagnosis and treatment of MSUD. Objective: To draw a profile of aspects related to the diagnosis and treatment of Brazilian patients who received. Methods: In this retrospective study, patients were identified through a search of records from a national reference laboratory for the diagnosis of MSUD and through contact with other medical genetics services across Brazil. A diagnosis of MSUD between 1992 and 2011. Data were collected by means of a chart review. Results: Eighty-three patients from 75 families were enrolled in the study (median age 3 years; interquartile range, 0.57–7). Median age at onset of symptoms was 10 days (IQR 5–30), whereas median age at diagnosis was 60 days (IQR 29–240, p=0.001). Only three (3.6%) patients were diagnosed before the onset of clinical manifestations. A comparison between patients with (n=12) and without (n=71) an early diagnosis show that early diagnosis is associated with the presence of positive familial history and decreased prevalence of clinical manifestations at the time of diagnosis, but not with a better outcome. Overall, 98.8% of patients have some psychomotor or neurodevelopmental delay. Conclusion: In Brazil, patients with MSUD receive a late diagnosis and show neurological compromise and poor survival even with an early diagnosis. We suggest that specific public policies for rare diseases should be developed and implemented in the country.

Introdução: A Doença da Urina do Xarope do Bordo (DXB) é um Erro Inato do Metabolsimo (EIM), causada pela deficiência da atividade do complexo enzimático desidrogenase dos L-cetoácidos de cadeia ramificada (CACR). O programa público brasileiro de triagem neonatal não inclui o diagnóstico e o tratamento da DXB. Objetivo: Caracterizar aspectos relacionados ao diagnóstico e ao tratamento de pacientes brasileiros com DXB. Metodologia: Estudo retrospectivo. Os pacientes foram identificados a partir dos registros de laboratório considerado referência nacional para o diagnóstico de DXB, e de contato com demais serviços nacionais de referência em genética médica. O diagnóstico foi realizado entre 1992-2011. Os dados analisados foram obtidos a partir da revisão de prontuários. Resultados: Oitenta e três pacientes, oriundos de 75 famílias, foram incluídos no estudo (mediana de idade= 3 anos; IQ25- 75= 0,57-7). A mediana da idade de início dos sintomas foi de 10 dias (IQ= 5-30), enquanto que a mediana da idade ao diagnóstico foi de 60 dias (IQ= 29-240, p= 0,001). Apenas três (3,6%) pacientes foram diagnosticados antes de desenvolverem manifestações clínicas. A comparação entre os pacientes com (n=12) e sem (n=71) diagnóstico precoce mostrou que o mesmo associa-se com a presença de história familiar positiva e diminuição da prevalência de manifestações clínicas ao diagnóstico, mas não com melhor resultado; além disso, a maioria desses diagnósticos foi realizada entre 2002-2011 (n= 10/12). Considerando a amostra total, 98,8% dos pacientes apresentam atraso de desenvolvimento neuropsicomotor. Conclusão: Os pacientes com DXB são diagnosticados tardiamente no Brasil, de forma que as complicações associadas a esta condição não são prevenidas. Entretanto, existem indícios de que está havendo um melhora gradual desse panorama desde a última década. Os nossos dados indicam que o diagnóstico precoce dessa condição, mesmo que em fase sintomática, associa-se a um melhor prognóstico do paciente. Sugere-se a elaboração de políticas públicas específicas para doenças raras no país. / Introduction: Maple syrup urine disease (MSUD) is an Inborn Errors of Metabolism, is caused by a deficiency in activity of the branched-chain L-keto acid dehydrogenase complex. The Brazilian neonatal screening program does not include the diagnosis and treatment of MSUD. Objective: To draw a profile of aspects related to the diagnosis and treatment of Brazilian patients who received. Methods: In this retrospective study, patients were identified through a search of records from a national reference laboratory for the diagnosis of MSUD and through contact with other medical genetics services across Brazil. A diagnosis of MSUD between 1992 and 2011. Data were collected by means of a chart review. Results: Eighty-three patients from 75 families were enrolled in the study (median age 3 years; interquartile range, 0.57–7). Median age at onset of symptoms was 10 days (IQR 5–30), whereas median age at diagnosis was 60 days (IQR 29–240, p=0.001). Only three (3.6%) patients were diagnosed before the onset of clinical manifestations. A comparison between patients with (n=12) and without (n=71) an early diagnosis show that early diagnosis is associated with the presence of positive familial history and decreased prevalence of clinical manifestations at the time of diagnosis, but not with a better outcome. Overall, 98.8% of patients have some psychomotor or neurodevelopmental delay. Conclusion: In Brazil, patients with MSUD receive a late diagnosis and show neurological compromise and poor survival even with an early diagnosis. We suggest that specific public policies for rare diseases should be developed and implemented in the country.