Errata inserted on back end paper. / Bibliography: leaves 226-254. / xviii, 261 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Describes the development of techniques to genotype and simultaneously assess allele dosage at the ABO locus using PCR and allele-specific restriction enzyme digestion. / Thesis (Ph.D.)--University of Adelaide, Dept. of Medicine, 1996

Blood group glycolipids are important tools in the study of microbial receptor interactions and other biological phenomena. Presently blood group glycolipids of interest are isolated from biological samples. However, all glycolipids are not readily available due to the low frequency of some phenotypes in the general population. The ability to acquire the rare glycolipids from the degradation of common glycolipids would be a useful alternative to trying to obtain the molecules from biological sources.This research set out to establish the ability of blood group glycolipids to be degraded into useful glycolipids in a controlled manner by acid hydrolysis and possibly metal catalysis. The initial experiments investigated the degradation/hydrolysis of the more readily available glycolipid globoside with a range of salts and acids to establish degradation concepts such as; temperature, type of acid, acid concentration, and the role of metal ions in glycolipid degradation. These concepts then led to a series of degradation experiments with the blood group glycolipids Leb and ALeb. These glycolipids were incubated with a range of acid concentrations and varying temperatures. Thin layer chromatography separation and chemical and immunochemical staining were the main methods used to identify the products of degradation.It was established that metal ions were not directly involved in the catalysis of glycolipids in the short-term, however some metal ions were indirectly implicated in their degradation due to their ability to form acid solutions. Acid hydrolysis was established as the principle mechanism for glycan chain degradation. In general it was found that the glycan chain primarily lost its fucose groups (in no particular order) and was then followed by sequential degradation of the remaining glycan chain. The glycan chain also appeared to have a protective function on the ceramide moiety. Degradation of globoside established a simple sequential pathway of glycan chain reduction from the non-reducing end. Blood group glycolipids ALeb and Leb first lost their fucose side groups followed by sequential reduction of the glycan chain. Although not fully controllable, degradation of Leb was able to produce Lea, Led and Lec. In contrast degradation of ALeb did not produce any Lea or Led. Instead A-type 1 and two novel A-like structures, 'linear A' and 'GalNAc-Lea' were generated. Lec was only produced from ALeb in extremely acidic conditions. This research established the ability to generate, by acid hydrolysis, a range of rare and "unnatural" novel glycolipids from more commonly available structures. It is of interest that the so-called unnatural glycolipids obtained from the acid hydrolysis of ALeb may, in theory, occur naturally in the acid environment of the stomach, and as such could have the potential to be implicated in disease. It is probable that by applying the principles learned here, a range of novel and natural structures suitable for use in the study of biological interactions can be obtained.

The knowledge of the variability of antigens of blood groups is essential in the transfusional practice, mainly to avoid grave alloimmunizations. This study had as objective to evaluate the prevalence of the phenotypes from the blood donors from Santa Maria´s Blood Center. These donors were evaluated for the mainly antigens of ABO, Rh and Kell systems. About the phenotyped samples with Rh system, 1274 samples (54.18%) were phenotyped as positives Rh and 1077 samples (45.82%) phenotyped as negative Rh. From the phenotyped donors as negatives Rh, 103 samples (9.5%) were positives for the ―C‖ or ―E‖ antigen. Relating the percentage of the Kell system positive in donors with negative Rh, was gotten 8.3%. We concluded that the negative Rh donor must be analyzed for other antigens of Rh system and for the Kell antigen, exactly being less immunogenics, these antigens are able to cause Grave Hemolytic Disease and Alloimmunizations. The D antigen Phenotypic expression may vary due to changes qualitative/quantitative: weak D, partial D. This study analyzed a sample of donors by genotyping to identify the most common D variants in this region was found (44%) weak D, (3%) and partial D. Strengthens thus the importance of established protocols for use of rare blood and ensure the proper use of blood products, as well as the safety of blood transfusion. / O conhecimento da variabilidade dos antígenos de grupos sanguíneos é essencial na prática transfusional, principalmente para evitar aloimunizações graves. Assim, este estudo teve como objetivo avaliar a prevalência dos fenótipos dos doadores de sangue do Hemocentro de Santa Maria, os quais foram avaliados para os principais antígenos dos sistemas ABO, Rh e Kell. Das amostras fenotipadas quanto ao sistema Rh, 1274 amostras (54.18%) foram fenotipadas como Rh positivos e 1077 amostras (45,82%) fenotipadas como Rh negativo. Dos doadores fenotipados como Rh negativos, 103 amostras (9,5%) foram positivas para o antígeno ―C‖ e/ou ―E‖. Relacionando o percentual do sistema Kell positivo em doadores Rh negativos foi de 8,3%. Conclui-se, então, que o doador Rh negativo deve ser analisado para os demais antígenos do sistema Rh e para o antígeno Kell, pois, mesmo sendo menos imunogênicos, estes antígenos são capazes de causar doença hemolítica graves e aloimunizações. O antígeno D pode variar de expressão fenotípica, devido a alterações qualitativas/quantitativas: D fraco, D parcial. Este trabalho analisou uma amostragem de doadores, através de genotipagem para identificar quais as variantes de D mais freqüentes nesta região, foi encontrado (44%) D fraco, (3%) D parcial. Reforça, dessa forma, a importância de ser estabelecidos protocolos para utilização destes sangues raros e garantir o uso correto destes hemocomponentes, assim como a segurança transfusional.

Les antigènes de groupes sanguins érythrocytaires peuvent être responsables d’une allo-immunisation anti-érythrocytaire et d’accidents immuno-hémolytiques lors de transfusion ou de grossesse. La transfusion des populations d’Afrique sub-saharienne est complexifiée par l’absence d’expression d’antigènes publics, l’expression d’antigènes privés et l’expression d’antigènes partiels en particulier pour le système RH. L’étude des systèmes de groupes sanguins d’intérêt transfusionnel chez les donneurs de Bamako a confirmé l’efficacité de la stratégie du génotypage multiplex incluant des polymorphismes d’appels pour identifier des donneurs rares, qui permet d’accéder aux phénotypes déduits des prélèvements. L’exploration du système RH réalisée par séquençage chez les Dogons et les Peulhs de Mopti met clairement en évidence que la diversité allélique et la fréquence de certains allèles RH sont fonction de l’ethnicité. Un nouvel haplotype associant un allèle RHD*DIVa codant pour un antigène D partiel, des antigènes ce potentiellement partiels, et une réactivité partielle anti-C, a été identifié chez les Dogons. L’exploration des allèles codant pour les antigènes de haute et basse fréquence en Afrique subsaharienne d’Est en Ouest constitue un exemple d’étude qui distingue clairement les populations d’Afrique subsaharienne de celles d’Europe par des différences de fréquences des allèles définissant la diversité génétique d’une population par rapport à une autre. Des orientations stratégiques en fonction du contexte local ont été identifiées pour l’évolution de la transfusion au Mali dans les prochaines années. / Blood group antigens may be responsible for alloimmunization and immuno-hemolytic accidents during transfusion or pregnancy. The transfusion of of sub-Saharan Africa populations is complex due to absence of high antigens expression, low antigens expression and partial antigens expression particularly for RH system.The study of blood group for transfusion of interest among donors in Bamako confirmed the effectiveness of multiplex genotyping strategy including polymorphisms calls to identify the rare donors, which permit access to phenotypes derived samples. In a second phase, the exploration of RH blood group system by sequencing among Dogon and Fulani in Mopti clearly shows that the allelic diversity and the frequency of some alleles RH depend on the ethnicity. A new haplotype RHD*DIVa/RHCE*ceTI(D2) combining an RHD*DIVa allele encoding a partial D antigen, potentially partial ce antigens, and a partial reactivity with anti-C, was identified among Dogon. In a third phase, the exploration of alleles encoding of the high and low frequency antigens in sub-Saharan Africa from East to West is an example of a study that clearly makes a difference between the populations of sub-Saharan African and those of Europe in terms of frequencies of alleles that define genetic diversity of one population compared to another. Thus, knowledge of ethnicity is more relevant than knowing the geographical origin in order to optimize transfusion in Saharan Africa and in European countries where some of these populations live. Guidelines strategic in relation with the local context have been identified for development of transfusion for next years in Mali.

This dissertation reconstructs how blood groups were made into pre-eminent objects of human genetic research and powerful markers for producing human biological difference. By tracing the ways in which three British laboratories became international centres for blood-group genetic research, it also offers an expanded history of postwar human genetics. In early 1930s Britain a community of geneticists, including R.A. Fisher and B.S. Haldane, promoted blood groups as having the potential to give the study of human heredity 'a solidly objective foundation, under strict statistical control'. Fisher and colleagues at the Cambridge Galton Serum Unit- especially Robert Race and Arthur Mourant- implemented this vision, the dissertation shows, using the arrangements for large-scale blood transfusion set up early in the Second World War. In 1946, Mourant became director of the Blood Group Reference Laboratory and Race of the Blood Group Research Unit, both at London's Lister Institute. As well as standardising blood-grouping reagents and investigating serological problems for the World Health Organization, these laboratories collected, analysed and published vast quantities of genetic data, making the Lister the global centre for blood-group genetics. During this period, human genetics changed from a marginal research field to an established discipline, partly, the dissertation argues, as a result of this blood-group research. By the 1950s a third of all human genetics publications were on blood groups: as one of the few human traits with simple Mendelian inheritance, they formed the basis for linkage studies and association surveys, and underpinned innovation in theoretical population genetics. Against a backdrop of intense international discussion about the meaning and scope of race science, blood groups were also made into tools for a supposedly 'obj ective' and 'unprejudiced' anthropology. This first history of how blood groups became scientific objects follows their collection in Britain and overseas, the grouping of samples, their transformation into data, and their presentation as credible genetic knowledge. It also offers the first sustained analysis of the functions of genetic nomenclatures. I argue that mid-century human genetics was profoundly influenced by the questions and practices of physical anthropology, by clinical practice, and by international infrastructures for medical research.

Lectin affinity chromatography has proven to be a powerful method to separate oligosaccharides based on their stereochemical structures. This technique has not been used for the separation of glycolipids since mixtures of these compounds form micelles in aqueous solution. Since N-acetylgalactosamine (GalNAc) is commonly found in glycolipids, three GalNAc-specific lectins were selected to develop a lectin affinity chromatographic method for glycolipids. To circumvent the difficulty of working with micelles, the autoradiographic detection of ¹²⁵l-labeled lectins binding to glycolipids on thin-layer chromatograms was used to study the glycolipid-binding specificity of the lectins from Helix pomatia, Wisteria floribunda and Dolichos biflorus. All three lectins detected the Forssman glycolipid which has a terminal GalNAcα1-3 residue. The Helix pomatia and Wisteria floribunda lectins are also bound to glycolipids with GalNAcβ-linked residues. The interactions of these lectins with glycolipid derived, ³H-labeled oligosaccharides were also analyzed by affinity chromatography on agarose-immobilized lectins. Only the immobilized Helix pomatia lectin was able to specifically bind oligosaccharides with α-linked GalNAc residues. The Helix pomatia lectin was selected to develop an affinity chromatography system for the purification of intact glycolipids having terminal GalNAcα1-3 residues. This technique relies on the ability of the immobilized lectin to bind its oligosaccharide ligands in aqueous solutions of tetrahydrofuran (THF) which inhibits micelle formation and permits the separation of non-specifically bound glycolipids. Forssman glycolipid and a human blood group A-active hexaosylceramide were bound to the Helix pomatia column equilibrated in water/THF (5:95). After applying a step gradient of increasing water content (to 50% water), the specifically bound glycolipids were eluted when GalANc was included in the mobile phase. Using these chromatographic conditions, the Forssman glycolipid from the neutral lipid fraction of sheep erythrocyte stroma and the A-active glycolipids from a total extract of type A human erythrocytes were purified in the Helix pomatia column. The ability to purify human A-active glycolipids from total lipid extracts in a single chromatographic step with the Helix pomatia column was used to isolate A-active glycolipids present in erythrocytes from donors from a rare blood group B(A). The erythrocytes from B(A) subgroup of blood group B individuals, are weakly hemagglutinated by a murine monoclonal anti-A antibody although these erythrocytes should not express blood group A antigens. The Helix pomatia lectin was used to determine the presence and isolate A-active glycolipids from the neutral lipid fraction of erythrocytes from two blood group B(A) donors. However, A-active glycolipids were absent in the glycolipid extracts from erythrocytes from a third B(A) donor and plasma of all three B(A) donors as well as erythrocytes of blood group B and O donors. Based on the fact that only glycolipids and oligosaccharides with GalNAcα1-3 residues specifically bind to the Helix pomatia column, this lectin column was used to isolate the 'terminal products' of the biosynthetic pathway of the human blood group A glycolipids and glycopeptides from the human epidermoid carcinoma cell line A-431. The metabolically active A-431 cells were grown in the presence of ³H-labeled monosaccharide precursors and the Helix pomatia column was used to determine and compare the rate of incorporation of labeled precursors in the A-active glycoconjugates from these cells. / Ph. D.

Serological discrepancies in matching blood group antigens between donors and patients for blood transfusion may lead to alloimmunisation, especially in multiply transfused patients. Blood group genotyping (BGG) has contributed in reducing this issue. ABO, Fy and Jk antigens are among those to be causative for alloimmunisation through transfusion or pregnancy. The number of alleles of these clinically significant blood groups is ever increasing. Currently, all commercially available high-throughput BGG platforms are only based on pre-defined polymorphisms. Consequently, novel or rare alleles that might have clinical significance are not identified. Next generation sequencing (NGS) circumvents this issue by providing high-throughput comprehensive genotyping of blood group genes in discovery mode to find all existing and novel mutations. Accordingly, a large number of individuals can be genotyped in a single run. Here, we describe an NGS-based method coupled with long-range polymerase chain reaction (LR-PCR) for high-throughput, rapid and extensive genotyping of FY, JK and ABO blood group genes. The Ion Torrent Personal Genome Machine (PGMTM) was used for sequencing the entire FY, JK and ABO blood group genes including flanking regions. Accordingly, high resolution genotyping was obtained. 53 genomic DNA samples were sequenced and genotyped for FY, 67 for JK and 47 for ABO. Sequencing data were aligned to the gene reference sequence derived from the human genome (hg19) to analyse variants. Analysis was accomplished by software packages, such as Ion Torrent SuiteTM plugins. Sanger sequencing of cDNA and cDNA clones was used to confirm findings in the JK gene. The sequencing data had a coverage depth of more than 5000x for FY, 700x for JK and 600x for ABO. NGS data matched with the serological phenotypes of FY alleles FY*A, FY*B and FY*02 Null main polymorphisms, such as FY*A/FY*B (125G > A) in exon 2 and (-67 T > C) in the promotor region. JK variant analysis revealed that the JK*01W.01 allele (130G > A) is common (10/67 samples) with normal antigenicity. The previously described silencing polymorphism (810G > A), leading to a purported JK*B null allele, restores a splice site and does not correlate with loss of Jkb antigenicity (10/67 samples). JK intron analysis revealed several new JK alleles described in this thesis. All 7 exons, introns and the flanking regions of the ABO gene were covered by only four amplicons. Several rare O alleles were found, such as O73 and O75, while one suggested novel O allele was characterised by a missense SNP 482G > A (Arg161His) in exon 7. The ABO reference sequence from hg19 appeared to resemble (O01 and O02) alleles. The intronic SNPs might be used to distinguish between alleles more accurately as a correlation of the intronic SNPs with the alleles was noted for the homozygous O alleles. It is predicted that NGS-based genotyping will replace not only microarray-based genotyping but also serology in the blood group typing of individuals, with great advancements in technology and molecular knowledge being expected in the near future.

Os métodos sorológicos para determinar o fenótipo Vel- requerem o uso de antissoros humanos raros e não permitem o rastreamento de muitas amostras ao mesmo tempo, limitando sua aplicação como ferramenta para busca de doadores raros. Neste estudo, desenvolvemos uma estratégia de triagem molecular de baixo custo, usando PCR em tempo real e reciclagem do DNA extraído de pools de plasma da rotina viral NAT (Teste de Ácido Nucleico) para identificar doadores Vel- e Vel+W. Um total de 25.322 doadores de sangue do Sudeste Brasileiro foram genotipados por PCR em tempo real para detectar a deleção de 17nt (c.64_80del17) no gene SMIM1, que determina o fenótipo Vel-, utilizando DNA extraído de pools de plasma de seis doadores rotineiramente descartados após a liberação dos resultados NAT para HBV, HCV e HIV. Cento e oitenta e oito (188) de 4.220 pools foram reativos para deleção. A deleção SMIM1*64_80del17 estava presente em 210 doadores sendo em 208 (0,4%) em heterozigose e em apenas dois (0,008%), em homozigose. A estratégia de genotipagem de pools de DNA, usando o ensaio de PCR em tempo real, desenhado para a identificação da deleção de 17 nucleotídeos no gene SMIM1, mostrou-se eficaz e acurada na identificação de doadores com fenótipo Vel- e Vel+W. A reciclagem do DNA da rotina NAT a torna uma técnica economicamente atrativa e definitivamente superior às técnicas sorológicas para o rastreamento deste fenótipo raro. / Serologic methods to determine the Vel- phenotype require the use of rare human antisera and do not allow for many samples to be tested simultaneously, which limits their application as a tool to search for rare donors. This study developed a low-cost molecular screening strategy using the real-time polymerase chain reaction (PCR) with DNA extracted from plasma pools used for viral nucleic acid test (NAT) screening, to identify Vel- and Vel+W donors. A total of 25,322 blood donors from the Brazilian southeast region were genotyped through real-time PCR targeting the 17-nucleotide (c.64_80del17) deletion in the SMIM1 gene, which determines the Vel- phenotype, by using leftover nucleic acid from plasma pools of six donors, routinely discarded after the release of viral NAT results. One hundred and eighty eight from 4,220 pools tested were reactive. The SMIM1*64_80del17 deletion was present in 210 donors, 208 (0.4%) in heterozygosity and only 2 in homozygosis (0.008%). The DNA pool genotyping strategy using real-time PCR designed to detect the deletion in the SMIM1 gene proved effective and accurate in identifying donors with the Vel- and Vel+W phenotypes. The fact that residual nucleic acid from routine viral NAT screening was successfully employed renders this technique economically attractive and definitely superior to the serologic techniques available to search for this rare phenotype

1. For the first time a wide range of biochemical genetic markers and different typing techniques were used to access levels of genetic variability in Lithuanian horse breeds; 2. DNA based methods were used to access levels of genetic variation in Lithuanian horse breeds; 3. Genetic variation in Lithuanian horses was investigated using mitochondrial DNA (mtDNA) sequencing; 4. Genetic relationship and genetic distances between the breeds were estimated using a wide range of different genetic markers.

At present, cardiovascular diseases cause ca. 30 of deaths worldwide, and are the most common cause of death and disability (The World Health Report 2002; Pearson 1999). Coronary artery disease (CAD) accounts for nearly 50 of all deaths caused by cardiovascular diseases. In 2002, 7.2 million people died of CAD worldwide, and 5.8 million new cases were diagnosed. In 2000, the number of people with CAD around the world amounted to ca. 40 millions (Mackay 2004). The modern understanding of the pathophysiology of atherosclerosis and the concept of “cardiovascular risk factors” started forming in 1950s, when the first findings of the Framingham study were published (Wilson et al. 1998, D’Agostino et al. 2000). Information accumulated during scientific research on atherosclerosis allowed for a significant reduction of CAD-related mortality in the developed countries during the last 20 years, but a more profound analysis showed that the mortality mostly decreased in males, whereas in females it continues to grow. Nearly two-thirds of suddenly deceased women previously showed no clinical symptoms of CAD (AHA 2002). This most probably was influenced by a still predominant erroneous opinion that women, especially of younger age, very rarely have CAD and atherosclerosis of peripheral arteries. Epidemiological studies showed that cardiovascular diseases induced by atherosclerosis are equally frequent cause of death in both males and females. Of all patients who in 2000 in the U.S. died... [to full text]