There has been an increase in rates of chromosomal abnormalities in newborns as a result of reproductive aging. For the past decades, a lot of effort has been placed on identifying pregnancies at risk of genetic defects. Conventional prenatal genetic diagnosis is achieved by invasive procedures that have been associated with an increased risk of pregnancy loss. This has led the researchers to explore the use of non-/minimally invasive techniques for prenatal diagnosis. Trophoblasts are known to be shed from regressing chorionic villi into the lower uterine pole of pregnant women during the first trimester. These cells are trapped within cervical mucus, which can be retrieved with a cytobrush. By using human leukocyte antigen G (HLA-G) and cytokeratin-7 (CK7) as trophoblast markers, this study aims to investigate the possibility of isolating individual fetal trophoblast from transcervical samples for genetic diagnosis. 195 healthy pregnant women requesting for legal termination of pregnancy (TOP) were recruited in this study. Transcervical cells were collected from them with the use of a cytobrush before TOP. HLA-G+ or CK7+ cells were then isolated by a combination of mucolytic action, fluorescent immunohistochemistry, and micromanipulation. The origin of these cells was subsequently investigated by either fluorescent in situ hybridization (FISH) or allelic profiling by quantitative fluorescent polymerase chain reaction (QF-PCR) based on chromosome 16, chromosome X, amelogenin gene and sex determining region Y (SRY) gene. This study first demonstrated the presence of fetal cells in transcervical samples based on the detection of chromosome Y signal by ordinary PCR. Cells expressing HLA-G and CK7 were also identified among transcervical cells. Immunopositive cells were isolated by micromanipulation under fluorescent microscopy. One isolated cell expressing CK7 was shown to inherit paternal allele at a locus on chromosome 16, suggesting the possible fetal origin of this cell. However, this study was still hampered by a number of technical factors. Further optimization of the protocol is required before transcervical trophoblasts can be retrieved in a reliable manner. / published_or_final_version / Obstetrics and Gynaecology / Master / Master of Philosophy

Deficiency of the cytochrome P-450 steroid 21-hydroxylase (21-OHase) which causes Congenital Adrenal Hyperplasia (CAH) is a monogenic autosomal recessive disorder which is linked to HLA. There are two 21-OHase genes in man, A and B, and they are mapped to the HLA class III region ~ 3 kb 3' to the complement genes C4A and C4B, respectively. Two genes encoding 21-OHase were isolated, characterized and sequenced. Both 21-OHase genes are ~ 3.3 kb in length and are split into 10 exons by nine introns. Comparison of the two genes showed that although they are highly conserved, there are three deleterious mutations in the 21-OHase A gene which cause frameshifts and introduce in phase premature termination codons. Thus the 21-OHase A gene is a pseudogene. Comparison of the 21-OHase B gene to the other cytochrome P-450 sequences revealed that although the cysteine-429 was conserved in 21-OHase, there is very little homology with other cytochrome P-450, indicating it belongs to a separate family of genes within the superfamily. Clear evidence of polymorphism in 21-OHase is apparent on comparison with other 21-OHase B sequences. There is a size polymorphism of 494 and 495 amino acids. The differing severities of 21-OHase deficiency in CAH may be due to allelic variants of the 21-OHase B gene, since in most cases the defect is not due to gene deletion (Rumsby et al., 1986). A 21-OHase B gene from a patient with CAH was characterized and sequenced. There were 13 nucleotide alterations in his single 21-OHase B gene, one of which at codon 269 caused a serine to change to a threonine residue. The G → C transversion in the 21-OHase B gene from the patient at codon 269 introduced a new NcoI restriction site into the gene. This restriction fragment length polymorphism (RFLP) was used to study other patients with CAH and normal individuals. The NcoI RFLP was found not to be confined to the 21-OHase B gene but was also present in some 21-OHase A genes. It is likely therefore that the mutation occurred in the pseudogene first and then transferred to some 21-OHase B genes.

Bibliography : leaves 182-203. / 203 leaves : ill, map ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Describes the molecular analysis of the HLA class I and class II genes in the Australian Aboriginal population and also provides a comparison of serological and molecular tissue typing methods in view of genetic mutations at the HLA loci and the expression of serological HLA "blanks". / Thesis (Ph.D.)--University of Adelaide, Dept. of Medicine, 1996?

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 5 uppsatser.

Thesis (Ph. D.)--Cornell University, 1989. / Vita. Includes bibliographical references.

Thesis (Ph. D.)--University of Missouri--Columbia, 1996. / Typescript. Vita. Includes bibliographical references (leaves: [167]-204). Also available on the Internet.

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves [140]-144).

Thesis (Ph. D.)--University of Texas at El Paso, 2009. / Title from title screen. Vita. CD-ROM. Includes bibliographical references. Also available online.

Thesis (Ph. D.)--University of Hong Kong, 1993. / Cover title.

Thesis (doctoral)--Uppsala University, 1983. / Includes bibliographical references (p. 47-57).