A pilot evaluation of whole blood finger-prick sampling for point-of-care HIV viral load measurement: the UNICORN study.

There is a global need for HIV viral load point-of-care (PoC) assays to monitor patients receiving antiretroviral therapy. UNICORN was the first study of an off-label protocol using whole blood finger-prick samples tested with and without a simple three minute spin using a clinic-room microcentrifuge. Two PoC assays were evaluated in 40 HIV-positive participants, 20 with detectable and 20 with undetectable plasma viral load (pVL) (<20 copies/ml). Using 100 µl finger-prick blood samples, the Cepheid Xpert HIV-1 Viral Load and HIV-1 Qual cartridges were compared with laboratory pVL assessment (TaqMan, Roche). For participants with undetectable viraemia by TaqMan, there was poor concordance without centrifugation with the TaqMan platform with only 40% 'undetectable' using Xpert VL and 25% 'not detected' using the Qual assay. After a 3 minute spin, 100% of samples were undetectable using either assay, showing full concordance with the TaqMan assay. Defining a lower limit of detection of 1000 copies/ml when including a spin, there was 100% concordance with the TaqMan platform with strong correlation (rho 0.95 and 0.94; p < 0.0001 for both assays). When including a simple microcentrifugation step, finger-prick PoC testing was a quick and accurate approach for assessing HIV viraemia, with excellent concordance with validated laboratory approaches.

High level expression of N-acetylgluosamine-6-O-sulfotransferase is characteristic of a subgroup of paediatric precursor-B acute lymphoblastic leukaemia.

Microarray analysis is a powerful technology, but its impact on routine diagnosis for the near future maybe in revealing individual genes, which are useful diagnostic markers. Recently microarray analysis has identified a novel subgroup of childhood precursor-B acute lymphoblastic leukaemia (ALL) from a unique gene expression profile of over 30 genes. We have evaluated the four most highly expressed genes from this profile, by quantitative real time RT-PCR, to determine whether any of these genes by itself could be useful as a diagnostic indicator. The levels of expression of N-acetylglucosamine-6-O-sulfotransferase (GN6ST), protein tyrosine phosphatase receptor M (PTPRmu), G protein-coupled receptor 49 (HG38) and KIAA1099 protein were determined in childhood precursor-B ALL samples from a cohort of 116 Indian patients. In nine cases, three or four of these genes exhibited very high expression levels, but only GN6ST was consistently over-expressed. We suggest that very high level expression of GN6ST is a useful diagnostic marker for a subgroup of previously unclassified ALL.

Factor V G1691A (Leiden) and prothrombin G20210A single-nucleotide polymorphisms in type 2 diabetes mellitus.

The association of the single nucleotide polymorphisms (SNPs) G1691A in coagulation factor V (FV)-Leiden and G20210A in prothrombin (PRT) genes with type 2 diabetes mellitus (T2DM) were analyzed in 112 T2DM patients (58 males, 54 females; mean age 55.24 +/- 13.5 years) and 249 healthy control subjects (118 males, 131 females; mean age 53.03 +/- 13.8 years). No association was found for FV-Leiden with T2DM, as the frequency of the G/G (82.1% vs. 85.5%), G/A (17.0% vs. 14.1%), and A/A (0.9% vs. 0.4%) genotypes was not different between patients and controls, respectively (P = 0.644). Similarly, lack of association of PRT G20210A with T2DM was seen among the population studied, and the frequency of the G/G (92.9% vs. 97.2%), G/A (6.3% vs. 2.8%), and A/A (0.9% vs. 0.0%) genotypes was similar among patients and controls, respectively (P = 0.094). Neither FV-Leiden nor PRT G20210A was associated with, and no evidence for interactions between these mutations was seen in, T2DM.

A case control study on the contribution of factor V-Leiden, prothrombin G20210A, and MTHFR C677T mutations to the genetic susceptibility of deep venous thrombosis.

BACKGROUND: Insofar as the inherited prothrombotic single nucleotide polymorphisms (SNPs) factor V G1691A (FV-Leiden), prothrombin (PRT) G20210A, and methylenetetrahydrofolate reductase (MTHFR), C677T are inherited risk factors of venous thromboembolism (VTE), the aim of this study was to determine the prevalence of single and combined SNPs in 198 patients with documented deep venous thrombosis (DVT), and 697 control subjects, and to estimate the associated risks. METHODS: Factor V-Leiden, PRT G20210A, and MTHFR C677T were analyzed by PCR and restriction fragment length polymorphism (RFLP). RESULTS: The prevalence of the heterozygote and homozygous variants for FV-Leiden (52.02 vs. 14.78%, RR 6.28), PRT G20210A (19.2 vs. 3.6%; RR 6.38), and to a lesser extent the T/T genotype of MTHFR C677T (20.71 vs. 11.0%; RR 1.49) were higher among DVT patients vs. controls, respectively. Two or more SNPs were detected in 90 of 198 patients (45.5%) and in 60 of 697 controls (8.6%), with odds ratios of 16.754 for joint occurrence of FV-Leiden and PRT G20210A, 10.471 for FV-Leiden and MTHFR C677T, and 6.283 for PRT G20210A SNPs and MTHFR 677T/T. Logistic regression analysis showed a further increased odds for FV-Leiden in combination with PRT G20210A (85.198) or homozygous MTHFR C677T (81.133), and to a lesser extent for PRT G20210A in combination with homozygous MTHFR C677T (20.812). CONCLUSIONS: This indicates that FV-Leiden and PRT G20210A, more than MTHFR C677T, are important risk factors for DVT, and that the presence of more than one prothrombotic SNPs was associated with a significant risk of DVT.

Single monochrome real-time RT-PCR assay for identification, quantification, and breakpoint cluster region determination of t(9;22) transcripts.

t(9;22) generates the BCR-ABL fusion gene, the hallmark of chronic myeloid leukemia (CML) but also found in acute lymphoblastic leukemia (ALL). Multiple chimeric transcripts translate to proteins of 190 or 210 kd and, rarely, 230 kd. CML typically carries p210 BCR-ABL while ALL is most often associated with p190. Detection and quantification of these fusion transcripts is useful in clinical management. We have exploited the unique melting profiles of these transcripts to design a new, simple, and cost-effective assay based on monochrome multiplex real-time RT-PCR for identification and quantification of each of these transcripts (b3-a2, b2-a2, and e1-a2) without further manipulation. The sensitivity of this assay was 10(-4) for e1-a2 and 10(-5) for b3-a2/b2-a2, which is appropriate for detection of minimal residual disease (MRD). Inter- and intra-assay variation was minimal. We applied this assay to assess the distribution of p190 and p210 in 260 childhood ALL samples from India. BCR-ABL was detected in 19 (7.3%), including one T-ALL. Eight patients (3.1%) demonstrated mBCR-ABL (p190) and 11 (4.2%) had MBCR-ABL (p210). Transcript levels varied markedly (up to 3000-fold) but e1-a2 were generally expressed at higher levels than b3/b2-a2 (P = 0.05). This simple real-time multiplex assay can thus be easily applied to monitor patients with ALL as well as CML.

A rapid RT-PCR screening assay incorporating multiplexed validated control genes for CBF rearrangements at diagnosis in AML.

AIMS: Our objective was to establish a multiplexed assay using the Biomed 1 primers to detect AML1-ETO transcripts and 10 different CBFB-MYH11 transcripts, using BCR and ABL transcripts as controls. METHODS: Control genes were systematically tested for characteristics of optimal controls. The final assay was validated on 50 AML patient samples. RESULTS: Testing confirmed that the designated control gene criteria were fulfilled. Of 50 patient samples tested, four RT-PCR results were discordant with the cytogenetic result. In three cytogenetically negative cases, RT-PCR detected cryptic CBF rearrangements (one AML1-ETO and two CBFB-MYH11). The fourth case was inv(16) positive but negative by RT-PCR; however, the control gene result revealed suboptimal RNA quality. CONCLUSIONS: We have described a robust multiplex RT-PCR assay that incorporates experimentally validated control genes that are important for accurate interpretation. The assay is more sensitive than cytogenetics in the detection of CBF AML. Application to large patient cohorts will determine the prognostic significance of cryptic CBF rearrangements compared with their cytogenetic counterparts.

Frequent silencing of fragile histidine triad gene (FHIT) in Burkitt's lymphoma is associated with aberrant hypermethylation.

The fragile histidine triad (FHIT) gene, a potential tumor-suppressor gene, is frequently inactivated in multiple human cancers. However, the FHIT gene remains largely unexplored in Burkitt's lymphoma (BL). Hence, we assessed whether loss of FHIT expression occurs in BL, and, if so, what is the mechanism of such loss. Lack of protein expression was observed in 50% of BL cell lines. Methylation-specific polymerase chain reaction (MSP) showed that 45% of BL cell lines carried aberrantly methylated FHIT alleles. Sequencing of bisulfite-treated DNA confirmed these data and indicated a very high density of methylation in all methylated alleles. Real-time, quantitative reverse-transcription PCR analysis indicated that attenuation of full-length FHIT transcription was correlated with methylation. Sequencing of transcripts illustrated that aberrant transcription resulting in loss of FHIT exons occurred more commonly in BL containing unmethylated FHIT genes. However, such transcripts often coexisted with full-length FHIT transcripts. Not surprisingly, therefore, loss of FHIT protein in BL correlated with CpG island methylation, rather than with aberrant transcription. FHIT methylation also was detected in 31% (16 of 51) of the primary BLs examined, including 2 samples whose derived cell lines also manifested FHIT hypermethylation. Aberrant methylation can thus occur in vivo. In summary, this report provides evidence that epigenetic modification frequently results in loss of FHIT expression in BL.

Preclinical validation of a monochrome real-time multiplex assay for translocations in childhood acute lymphoblastic leukemia.

PURPOSE: The purpose is to develop a real-time multiplex reverse transcription-PCR assay for detection and quantification of leukemia-specific chimeric transcripts that identify the genetic subgroups of acute lymphoblastic leukemias (ALLs) proposed by the WHO classification. EXPERIMENTAL DESIGN: Real-time multiplex assay for t(12;21), t(4;11), and t(1;19) with hypoxanthine phosphoribosyltransferase as internal standard used in tandem with a new real time quantitative-RT-PCR assay for the t(9;22). This new strategy was designed to yield an amplicon from each translocation with a distinct melting peak allowing dependable identification using only Sybr green I, without any need for expensive hybridization probes. RESULTS: We validated this method with 92 primary ALLs and identified 4 E2A-PBX1, 4 mBCR-ABL and 10 TEL-AML1. When compared with conventional RT-PCRs and Southern blot analyses, 100% concordance was obtained. During the course of these studies, we found marked variations in the levels of the TEL-AML1 transcripts in individual patients. We, therefore, extended the study to accurately and reproducibly determine TEL-AML1 mRNA levels in 47 additional patients with t(12;21). The results indicated that the level of expression of TEL-AML1 varied among individual patients, and it was independent of the WBC count. CONCLUSIONS: Our new real-time multiplex assay can be used for rapid, simple, and reliable classification of pediatric ALL. Its reproducible quantification results should also facilitate studies on minimal residual disease. The observed variation in TEL-AML1 transcript levels is of interest because it could reflect biological and/or clinical heterogeneity in the behavior of these leukemias.