Clinical utility of next generation sequencing based HLA typing for disease association and pharmacogenetic testing.

HLA associations have been linked to many diseases and are important for risk assessment of drug hypersensitivity reactions. The increasing number of HLA alleles discovered generated a list of ambiguities that cannot be resolved with the current clinical assays, which commonly include sequence-specific oligonucleotide probe (SSOP) genotyping, and real-time PCR with melting curve analysis. HLA typing by next-generation sequencing (NGS) has recently been adopted by clinical laboratories for transplantation testing, as it provides unambiguous and cost-effective HLA typing. The goal of this study was to evaluate the feasibility of using NGS-based HLA-B and DQ genotyping for clinical HLA disease association testing, and provide direct comparison with the currently used clinical tests, including SSOP genotyping, and real-time PCR with melting curve analysis. While the real-time PCR method is easy and inexpensive to perform, ambiguities are rapidly increasing as more and more HLA alleles are discovered. SSOP genotyping identifies the alleles present but limitations include ambiguities and underreporting less common alleles. Our data show that HLA typing by NGS is superior to the existing clinical methods for identifying HLA alleles associated with disease or drug hypersensitivity, and offers a viable approach for high volume clinical diagnostic laboratories.

Myristicyclins A and B: antimalarial procyanidins from Horsfieldia spicata from Papua New Guinea.

An antimalarial screen for plants collected from Papua New Guinea identified an extract of Horsfieldia spicata as having activity. Isolation of the active constituents led to the identification of two new compounds: myristicyclins A (1) and B (2). Both compounds are procyanidin-like congeners of myristinins lacking a pendant aromatic ring. Myristicyclin A was found to inhibit the ring, trophozoite, and schizont stages of Plasmodium falciparum at similar concentrations in the mid-µM range.

Malaria severity and human nitric oxide synthase type 2 (NOS2) promoter haplotypes.

Nitric oxide (NO) mediates host resistance to severe malaria and other infectious diseases. NO production and mononuclear cell expression of the NO producing enzyme-inducible nitric oxide synthase (NOS2) have been associated with protection from severe falciparum malaria. The purpose of this study was to identify single nucleotide polymorphisms (SNPs) and haplotypes in the NOS2 promoter, to identify associations of these haplotypes with malaria severity and to test the effects of these polymorphisms on promoter activity. We identified 34 SNPs in the proximal 7.3 kb region of the NOS2 promoter and inferred NOS2 promoter haplotypes based on genotyping 24 of these SNPs in a population of Tanzanian children with and without cerebral malaria. We identified 71 haplotypes; 24 of these haplotypes comprised 82% of the alleles. We determined whether NOS2 promoter haplotypes were associated with malaria severity in two groups of subjects from Dar es Salaam (N = 185 and N = 250) and in an inception cohort of children from Muheza-Tanga, Tanzania (N = 883). We did not find consistent associations of NOS2 promoter haplotypes with malaria severity or malarial anemia, although interpretation of these results was potentially limited by the sample size of each group. Furthermore, cytokine-induced NOS2 promoter activity determined using luciferase reporter constructs containing the proximal 7.3 kb region of the NOS2 promoter and the G-954C or C-1173T SNPs did not differ from NOS2 promoter constructs that lacked these polymorphisms. Taken together, these studies suggest that the relationship between NOS2 promoter polymorphisms and malaria severity is more complex than previously described.

Biologically active components of a Papua New Guinea analgesic and anti-inflammatory lichen preparation.

A traditional preparation of Parmotrema saccatilobum (Taylor) Hale (Family: Parmeliaceae) is being considered for inclusion into the PNG national drug formulary by the Ministry of Health Taskforce on Traditional Medicines. The lichen preparation is traditionally used in the Milne Bay province of Papua New Guinea for analgesic and anti-inflammatory activities. A hexane extract of P. saccatilobum yielded the principle components atranorin and chloroatranorin. Atranorin and chloroatranorin were tested in a COX-1 and -2 enzyme inhibition assay, which showed that atranorin inhibited COX-1 in a dose dependent manner and suggests partial inhibition by atranorin and chloroatranorin of COX-2 and COX-1, respectively.

A new NOS2 promoter polymorphism associated with increased nitric oxide production and protection from severe malaria in Tanzanian and Kenyan children.

BACKGROUND: Nitric oxide (NO) is a mediator of immunity to malaria, and genetic polymorphisms in the promoter of the inducible NO synthase gene (NOS2) could modulate production of NO. We postulated that NOS2 promoter polymorphisms would affect resistance to severe malaria. METHODS: We assessed genomic DNA from healthy children and from those diagnosed with malaria from Tanzania (n=47 and n=138, respectively) and Kenya (n=1106) for polymorphisms by single-stranded conformational polymorphism (SSCP) analysis and sequencing. We also measured in-vivo NO production in Tanzanian children. FINDINGS: We identified a novel single nucleotide polymorphism, -1173 C-->T, in the NOS2 promoter that was significantly associated with protection from symptomatic malaria (odds ratio 0.12, 95% CI 0.03-0.48, p=0.0006) in 179 Tanzanian children, and significantly associated with protection from severe malarial anaemia (adjusted relative risk 0.25, 95% CI 0.09-0.66, p=0.0005) in 1106 Kenyan children studied over 5 years. The risk of parasitaemia was not significantly different in wild-type or -1173 C-->T individuals. -1173 C-->T protection in Tanzanians was independent of the previously recognised NOS2-954 G-->C polymorphism. The (CCTTT)(n) NOS2 polymorphism (Tanzania and Kenya) was not associated with severe malaria outcomes. -1173 C-->T was associated with increased fasting urine and plasma NO metabolite concentrations in Tanzanian children, suggesting that the polymorphism was functional in vivo. Interpretation The NOS2 promoter -1173 C-->T single nucleotide polymorphism is associated with protection against cerebral malaria and severe malarial anaemia. Increased NO production in individuals with the -1173 C-->T polymorphism lends support to a protective role for NO against these syndromes. Targeted interventions to increase NO delivery or production could provide novel preventive and therapeutic strategies against these major causes of mortality in African children.