Application of Direct cDNA Sequencing for the Characterisation of Molecular Pathology in Acute Intermittent Porphyria

Mgone, Charles S (1991) Application of Direct cDNA Sequencing for the Characterisation of Molecular Pathology in Acute Intermittent Porphyria. PhD thesis, University of Glasgow.

Abstract

Acute intermittent porphyria (AIP) is an autosomal dominant disorder caused by partial deficiency of the enzyme porphobilinogen deaminase (PBG-D). It is heterogeneous and commonly gene carriers of the disorder remain asymptomatic and may not always be diagnosed by conventional biochemical methods. Since detection of gene carriers is of central importance to the management of this condition, an alternative method of diagnosis is essential. Mutations causing acute intermittent porphyria have not however, been fully characterised. In the current study, direct cDNA sequencing of polymerase chain reaction (PCR) amplified templates has been developed and applied for the characterisation of mutations associated with this disorder. The procedure was developed by amplifying RNA from various sources including human placenta, chorion, lymphocytes and lymphoblastoid cells. The amplification was performed by a technique referred to as reverse-transcriptase polymerase chain reaction (R-T PCR) in which both the first strand cDNA synthesis and the subsequent amplification are performed in the same reaction mixture. Two approaches to the R-T PCR amplification were employed and compared. In the first approach, the first strand cDNA synthesis was carried out with one of the primers complementary to the non-erythroid PBG-D mRNA and in the second, by using oligo(dT)12-18. Both methods were successful and comparable, but the later was preferred because it could be modified in asymmetric PCR to directly produce either the sense or the anti-sense strand. The PBG-D cDNA synthesised and amplified by the R-T PCR was either directly sequenced as double-stranded (ds) templates or eluted and reamplified by asymmetric PCR to produce singlestranded templates. Alternatively, single-stranded templates were produced directly by 'asymmetric' R-T PCR. Prior to sequencing, the PCR amplified templates were concentrated, desalted and purified to remove excess deoxyribonucleoside triphosphates (dNTPs) and amplification primers. Several purification methods were employed and their efficacy compared. These included, spun-column chromatography, nucleic acid chromatography system (NACS) purification, centrifuge-driven dialysis, geneclean TM purification, gel fractionation and selective precipitation in ammonium acetate and propan-2-ol. Selective precipitation with ammonium acetate and propan-2-ol was found to be the simplest and most effective method of template purification. In addition it was also inexpensive, reliable and convenient. Dideoxy sequencing of both double-stranded and single-stranded (ss) templates was performed with either Sequenase T7 DNA polymerase or Taq DNA polymerase. Sequencing of the singlestranded templates, especially when produced by asymmetric reamplification of cDNA gave the most consistent and reliable results. For routine sequencing, there was no difference in the performance of the two sequencing enzymes used, although Taq DNA polymerase was better than Sequenase T7 DNA polymerase in handling templates with complex secondary structures. The procedure of direct sequencing was applied on asymetrically amplified templates of thirty patients with acute intermittent porphyria (AIP) and ten normal controls. The diagnosis of acute intermittent porphyria was based on increased excretion of aminolevulinic acid (ALA) and porphobilinogen (PBG) in urine and decreased activity of erythrocyte porphobilinogen deaminase (PBG-D) coupled with a clinical history of one or more acute attacks. The mean erythrocyte porphobilinogen activity in the acute intermittent porphyria patients was 22. 3 nmol/h/ml erythrocytes. The normal adult activity range for the enzyme is 25-42 nmol/h/ml erythrocytes in the females and 30-48 in males. After optimisation of the R-T PCR, correct sized products were obtained from the amplification of all samples, indicating absence of any major deletions, Sequencing of these products revealed seven point mutations in twelve patients with acute intermittent porphyria and none in the control subjects. All mutations were due to single base substitutions, four of which were associated with amino acid substitutions and are likely to be the cause of AIP in these individuals. The remaining three were silent mutations without change of amino acid and are therefore regarded as neutral polymorphisms. The detected mutations were Q34K (C100→A) seen in two related individuals, L177R (T530→G) also observed in two unrelated individuals, R167Q (G500→A) and H256N (C766→A) each seen separately in single subjects. The silent mutation L42L (G117→A) was seen in one individual whereas S45S (G135→A) and V202V (G606→T) were seen in two and four individuals respectively. With the exception of the mutation R167Q which has been previously reported in four other individuals, the rest of the mutations were novel, emphasising the heterogeneity of this condition. (Abstract shortened by ProQuest.).

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Keywords: Medicine, Genetics, Bioinformatics
• Date of Award: 1991
• Depositing User: Enlighten Team
• Unique ID: glathesis:1991-78387
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 30 Jan 2020 15:29
• Last Modified: 30 Jan 2020 15:29
• URI: https://theses.gla.ac.uk/id/eprint/78387