RESUMEN
We examined baseline HIV-1 protease and reverse transcriptase sequences and HIV clinical parameters from 1,021 consecutive patients (814 male, 207 female) through the Royal Perth Hospital HIV service to investigate HIV-1 subtype diversity and local phylogenetic networks from 2000 to 2014. HIV-1 subtype B virus sequences were demonstrated in 619 (61%) of cases, with increasing non-B HIV-1 subtypes from 23.2% (2000-2003) to 48% (2008-2011) and 43% (2012-2014) (p < 0.001), including the CRF01_AE subtype [6.6% (2000-2003) to 21.5% (2008-2011)] and HIV-1 C subtype [9.5% (2000-2003) to 20.2% (2008-2011)]. More HIV-1 B subtypes were assigned to phylogenetic clusters compared to non-B subtypes (34% vs. 18%; p < 0.001), with larger clusters identified (cluster size >2: 135/211; 64% vs. 13/69; 19%; p = 0.001), including one cluster of 53 HIV-1 B subtype sequences that evolved from 2008 to 2014. Non-B subtype HIV-1 was associated with lower baseline CD4 T cell count (p = 0.005) but not plasma HIV-1 RNA levels (p = 0.31), suggesting relatively delayed diagnosis. Baseline viral load was strongly associated with calendar time [mean 18,620 copies/ml in 2000-2003; 75,858 copies/ml in 2012-2014 (p < 0.001)], and was also associated with larger phylogenetic clusters (size >2) in adjusted analyses (p = 0.03). This study identifies a number of temporal trends over the past 15 years, including an increasing prevalence of non-B subtype HIV-1 that highlights the growing influence of migration and travel on the Australian HIV-1 epidemic and the associated increased role of heterosexual HIV-1 transmission in this context. At the same time, these data indicate that local transmission within predominantly male networks remains a challenging issue for HIV-1 prevention.
Asunto(s)
Transmisión de Enfermedad Infecciosa , Variación Genética , Genotipo , Infecciones por VIH/transmisión , Infecciones por VIH/virología , VIH-1/clasificación , Adolescente , Adulto , Anciano , Recuento de Linfocito CD4 , Niño , Preescolar , Femenino , Infecciones por VIH/epidemiología , Infecciones por VIH/patología , Proteasa del VIH/genética , Transcriptasa Inversa del VIH/genética , VIH-1/genética , VIH-1/aislamiento & purificación , Humanos , Lactante , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Epidemiología Molecular , Filogenia , Prevalencia , ARN Viral/sangre , Análisis de Secuencia de ADN , Carga Viral , Australia Occidental/epidemiología , Adulto JovenRESUMEN
OBJECTIVE: To establish gene copy number (GCN)-specific normal ranges for serum C4 genes and to determine their utility with respect to the interpretation of chronically low serum C4 concentrations in patients with clinically quiescent systemic lupus erythematosus (SLE). METHODS: C4 serum concentrations were estimated by automated turbidimetry, and C4 GCNs were determined using the TaqMan real-time polymerase chain reaction (PCR) analysis in 184 unselected individuals and in 10 patients with type 1 diabetes mellitus (DM) who were selected for the presence of only 2 copies of the C4 gene. C4 GCNs were also determined in 11 patients with clinically quiescent SLE who had chronically low serum C4 concentrations. RESULTS: A total of 33% of the variation in serum C4 concentrations could be accounted for by both C4A and C4B GCNs (R(2) = 0.30, P ≤ 0.0001). There was a median of 2 gene copies at the C4A locus (53.8%) and 2 at the C4B locus (58.7%). The median total number of C4 genes was 4 (55.4%). C4 GCN-specific normal ranges were established. A chronically low serum C4 concentration was explained by a low C4 GCN in 3 of 11 patients tested. CONCLUSION: This study establishes the feasibility of establishing C4 GCN-specific normal ranges using the TaqMan real-time PCR assay. Chronically low serum C4 concentrations in SLE patients are sometimes explained by low C4 GCNs.
Asunto(s)
Complemento C4/genética , Dosificación de Gen , Predisposición Genética a la Enfermedad , Lupus Eritematoso Sistémico/genética , Adulto , Anciano , Alelos , Complemento C4/metabolismo , Femenino , Genotipo , Humanos , Lupus Eritematoso Sistémico/sangre , Masculino , Persona de Mediana Edad , Valores de ReferenciaRESUMEN
Classical, alternative, or lectin pathways may activate the complement system cascade. The classical pathway includes the C4 protein and functions in the prevention of immune complex precipitation and in clearance of immune complexes.Two isotypes of C4-C4A and C4B-are coded by genes located at two loci within the major histocompatibility complex (MHC) on chromosome 6. While these isotypes share over 99% amino acid sequence homology, five nucleotide differences located in exon 26 are responsible for major structural and functional differences between the C4 isotypes.C4A and C4B are highly polymorphic with over 40 alleles, gene duplications, and "null alleles". C4 genes may be short (14.6 kb) or long (21 kb), due to the absence or presence of an endogenous retroviral sequence-HERV-K(C4)-in intron 9, respectively. The C4 gene copy number (GCN) can vary from 1-3 per haplotype or 2-6 per diploid genome. The variation in GCN leads to a range of C4 plasma protein concentrations among healthy subjects. In subjects with equal numbers of C4 genes, subjects with short genes have C4 plasma levels relatively higher than subjects with long genes.Variation of the C4 GCN, the gene size (long or short) and the C4 isotypes (C4A and C4B) may also lead to susceptibility to autoimmune disease. Therefore, in subjects with autoimmune disease, a low serum C4 level may be due to ongoing disease activity associated with complement activation and consumption or it may be due to genetic factors. Distinguishing between these will have clinical implications.Exact determination of GCN can be difficult, at least in part due to the high degree of homology between C4A and C4B and a variety of techniques has been described. This chapter describes a quantitative TaqMan real-time PCR (qPCR) copy number assay, based on our laboratory experience using this assay.
Asunto(s)
Complemento C4/genética , Dosificación de Gen/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Variación Genética/genética , HumanosRESUMEN
This chapter describes the application of diagnostic HLA typing for disease association and five methods used for specific HLA genotypes. The methods utilise a combination of polymerase chain reaction (PCR) amplification to detect sequence polymorphism by the presence or absence of amplification, nucleotide sequencing of the PCR product, and hybridisation of the PCR product with labelled probes. The probes are specific for sequence polymorphism associated with the genotype and are attached to either a Micro Bead or a Solid Phase. In addition, the detection of single nucleotide polymorphism(s) which "tag" for the genotype using a real-time PCR is described.