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

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.)

Strengthening of the clinical research capacity for malaria: a shared responsibility

Lack of adequate human resource capacity, good governance, sound physical infrastructure and well-functioning systems impede economic growth in low- and middle-income countries. The heavy burden from disease compounds this. To overcome these setbacks a concerted effort needs to be taken. This requires collective effort of all including the public and private sectors from development partners and from low- and medium-income countries themselves. Specific research capacity gaps, such as lack of expertise and infrastructure to engage in upstream research and development of new products, need to be addressed. Special attention should also be given to those with more acute capacity needs and high disease burden, such as communities in conflict-affected regions. Capacity building approaches need to be innovative and responsive to needs and the ever changing scientific landscape. Therefore, for example, as the global community aims to eliminate and eventually eradicate malaria, there should be an appropriately matched effort to strengthen the capacity to meet these challenges

Sequence diversity and molecular evolution of the merozoite surface antigen 2 of plasmodium falciparum

Eleven new alleles of the Plasmodium falciparum merozoite surface antigen 2 (MSA2) from Papua New Guinea were analyzed by direct sequencing of polymerase chain reaction (PCR) products. We have used the sequence information to trace the molecular evolution of MSA2. The repeats of ten alleles belonging to the 3D7 allelic family differed considerably in size, nucleotide sequence, and repeat copy number. In the repeat region of these new alleles, codon usage was extremely biased with an exclusive use of NNT codons. Another new allele sequenced belonged to the FC27 family and confirmed the family-specific conserved structure of 96 and 36 bp repeats. In order to assess sequence microheterogeneity within samples defined as the same genotype by restriction fragment length polymorphism (RFLP), we have analyzed single-strand conformation polymorphism (SSCP) of different samples of the most frequent allele (D10 of the FC27 family) in the study population. No sequence heterogeneity could be detected within the repeat region. Based on analysis of the repeat regions in both allelic families, we discuss the hypothesis of a different evolutionary strategy being represented by each of the allelic familie

Host erythrocyte polymorphisms and exposure to Plasmodium falciparum in Papua New Guinea

Contains fulltext : 69991.pdf (publisher's version ) (Open Access)BACKGROUND: The protection afforded by human erythrocyte polymorphisms against the malaria parasite, Plasmodium falciparum, has been proposed to be due to reduced ability of the parasite to invade or develop in erythrocytes. If this were the case, variable levels of parasitaemia and rates of seroconversion to infected-erythrocyte variant surface antigens (VSA) should be seen in different host genotypes. METHODS: To test this hypothesis, P. falciparum parasitaemia and anti-VSA antibody levels were measured in a cohort of 555 asymptomatic children from an area of intense malaria transmission in Papua New Guinea. Linear mixed models were used to investigate the effect of alpha+-thalassaemia, complement receptor-1 and south-east Asian ovalocytosis, as well as glucose-6-phosphate dehydrogenase deficiency and ABO blood group on parasitaemia and age-specific seroconversion to VSA. RESULTS: No host polymorphism showed a significant association with both parasite prevalence/density and age-specific seroconversion to VSA. CONCLUSION: Host erythrocyte polymorphisms commonly found in Papua New Guinea do not effect exposure to blood stage P. falciparum infection. This contrasts with data for sickle cell trait and highlights that the above-mentioned polymorphisms may confer protection against malaria via distinct mechanisms

Mitochondrial Genetic Diversity and its Determinants in Island Melanesia

For a long time, many physical anthropologists and human geneticists considered Island Melanesian populations to be genetically impoverished, dominated by the effects of random genetic drift because of their small sizes, internally very homogeneous, and therefore of little relevance in reconstructing past human migrations. This view is changing. Here we present the developing detailed picture of mitochondrial DNA (mtDNA) variation in eastern New Guinea and Island Melanesia that reflects linguistic distinctions within the region as well as considerable island-by-island isolation. It also appears that the patterns of variation reflect marital migration distinctions between bush and beach populations. We have identified a number of regionally specific mtDNA variants. We also question the widely accepted hypothesis that the mtDNA variant referred to as the ‘Polynesian Motif’ (or alternatively the ‘Austronesian Motif’) developed outside this region somewhere to the west. It may well have first appeared among certain non-Austronesian speaking groups in eastern New Guinea or the Bismarcks. Overall, the developing mtDNA pattern appears to be more easily reconciled with that of other genetic and biometric variables

Translational research for tuberculosis elimination: priorities, challenges, and actions

Christian Lienhardt and colleagues describe the research efforts needed to end the global tuberculosis epidemic by 2035

Reduced Plasmodium vivax Erythrocyte Infection in PNG Duffy-Negative Heterozygotes

BACKGROUND: Erythrocyte Duffy blood group negativity reaches fixation in African populations where Plasmodium vivax (Pv) is uncommon. While it is known that Duffy-negative individuals are highly resistant to Pv erythrocyte infection, little is known regarding Pv susceptibility among heterozygous carriers of a Duffy-negative allele (+/−). Our limited knowledge of the selective advantages or disadvantages associated with this genotype constrains our understanding of the effect that interventions against Pv may have on the health of people living in malaria-endemic regions. METHODS AND FINDINGS: We conducted cross-sectional malaria prevalence surveys in Papua New Guinea (PNG), where we have previously identified a new Duffy-negative allele among individuals living in a region endemic for all four human malaria parasite species. We evaluated infection status by conventional blood smear light microscopy and semi-quantitative PCR-based strategies. Analysis of a longitudinal cohort constructed from our surveys showed that Duffy heterozygous (+/−) individuals were protected from Pv erythrocyte infection compared to those homozygous for wild-type alleles (+/+) (log-rank tests: LM, p = 0.049; PCR, p = 0.065). Evaluation of Pv parasitemia, determined by semi-quantitative PCR-based methods, was significantly lower in Duffy +/− vs. +/+ individuals (Mann-Whitney U: p = 0.023). Overall, we observed no association between susceptibility to P. falciparum erythrocyte infection and Duffy genotype. CONCLUSIONS: Our findings provide the first evidence that Duffy-negative heterozygosity reduces erythrocyte susceptibility to Pv infection. As this reduction was not associated with greater susceptibility to Pf malaria, our in vivo observations provide evidence that Pv-targeted control measures can be developed safely

Nitric Oxide Production and Nitric Oxide Synthase Activity in Malaria-Exposed Papua New Guinean Children and Adults Show Longitudinal Stability and No Association with Parasitemia

Individuals in areas of intense malaria transmission exhibit resistance (or tolerance) to levels of parasitemia in their blood that would normally be associated with febrile illness in malaria-naïve subjects. The resulting level of parasitemia associated with illness (the pyrogenic threshold) is highest in childhood and lowest in adulthood. Clinical parallels between malarial and bacterial endotoxin tolerance have led to the supposition that both share common physiological processes, with nitric oxide (NO) proposed as a candidate mediator. The hypotheses that NO mediates tolerance and blood stage parasite killing in vivo were tested by determining its relationship to age and parasitemia cross-sectionally and longitudinally in a population of 195 children and adults from Papua New Guinea encountering intense malaria exposure. Despite pharmacological clearance of asymptomatic parasitemia, NO production and mononuclear cell NO synthase (NOS) activity were remarkably stable within individuals over time, were not influenced by parasitemia, and varied little with age. These results contrast with previous smaller cross-sectional studies. Baseline NO production and NOS activity did not protect against recurrent parasitemia, consistent with previous data suggesting that NO does not have antiparasitic effects against blood stage infection in vivo. The NO indices studied were markedly higher in specimens from study subjects than in samples from Australian controls, and NOS activity was significantly associated with plasma immunoglobulin E levels, consistent with induction of NO by chronic exposure to other infections and/or host genetic factors. These results suggest that NO is unlikely to mediate killing of blood stage parasites in this setting and is unlikely to be the primary mediator in the acquisition or maintenance of malarial tolerance