High Risk Janus Kinase 2 V617F Allele Burden in a Seven-Year Cohort of Patients with Myeloproliferative Neoplasms.

BACKGROUND: JAK2 V617F is the most common somatic mutation associated with the classical Philadelphia (Ph) chromosome negative myeloproliferative neoplasms (MPNs), which include essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis (PMF). JAK2 V617F allele burden may be used for establishing the diagnosis, determining prognosis, and monitoring progression in these diseases. Limited data is available regarding the epidemiology of MPNs in Africa, and there is paucity of data on demographic, laboratory, and clinical features of MPNs in South Africa. This study determined the JAK2 V617F allele burden in a seven-year retrospective cohort of patients diagnosed with MPNs and described the characteristics of these diseases in a South African setting. METHODS: A laboratory database search was performed to identify patients diagnosed with ET, PV or PMF and a positive JAK2 V617F mutation, diagnosed qualitatively on Fluorescence Resonance Energy Transfer (FRET) real-time PCR and melting curve analysis. The allele burden for these patients was measured on archived residual DNA samples using a quantitative allele specific amplification (QUASA) assay. Demographic data and relevant laboratory results at presentation were analyzed. RESULTS: The search identified 87 patients who tested positive for JAK2 V617F mutation and fulfilled the diagnostic criteria for ET, PV or PMF from 2012 to 2018. Median age at diagnosis was 64 years with a male: female ratio of 1.2:1. ET, PV and PMF accounted for 11.5%, 44.8%, and 43.7% of the MPNs, respectively. Median allele burden for ET, PV, and PMF was 24.9%, 71.1%, and 55.8%, respectively. Allele burden was significantly lower in ET compared to PV (p = 0.0003) and PMF (p = 0.0023) and correlated with leukocytosis, neutrophilia, eosinophilia, and low erythrocyte mean cell volume (p < 0.05). CONCLUSIONS: JAK2 V617F-positive MPNs occurred predominantly in older patients with approximately equal gender ratio. ET was the least common MPN and there was a higher proportion of PMF cases than described in studies in Europe and America. Allele burden was also relatively high for all three subtypes of MPNs when compared to other published data, which may predispose to poorer prognosis.

A Rare Prothrombin Gene Mutation C20209T in a South African Patient with Pulmonary Embolism in Pregnancy: a Case Study and Systematic Review.

BACKGROUND: The G202010A prothrombin gene mutation is a documented prothrombotic risk factor in Caucasian patients. Several other mutations have been described within the prothrombin gene, predominantly in non-Caucasians, including the C20209T mutation. The clinical significance of this mutation is uncertain, but it has been associated with thrombotic events and pregnancy complications. METHODS: We describe a 28-year-old black South African woman who presented with pulmonary embolism during pregnancy. She was investigated for underlying prothrombotic biomarkers. RESULTS: Genetic screening for the prothrombin G202010A mutation by real-time polymerase chain reaction and melting curve analysis demonstrated an atypical mutant peak. Sequencing confirmed a variant C20209T prothrombin mutation. CONCLUSIONS: This is the first report of the C20209T mutation in the Southern African population. It remains uncertain whether genetic testing should be offered routinely to non-Caucasian patients in a resource-limited setting.

Mutational Correlates of Virological Failure in Individuals Receiving a WHO-Recommended Tenofovir-Containing First-Line Regimen: An International Collaboration.

Tenofovir disoproxil fumarate (TDF) genotypic resistance defined by K65R/N and/or K70E/Q/G occurs in 20% to 60% of individuals with virological failure (VF) on a WHO-recommended TDF-containing first-line regimen. However, the full spectrum of reverse transcriptase (RT) mutations selected in individuals with VF on such a regimen is not known. To identify TDF regimen-associated mutations (TRAMs), we compared the proportion of each RT mutation in 2873 individuals with VF on a WHO-recommended first-line TDF-containing regimen to its proportion in a cohort of 50,803 antiretroviral-naïve individuals. To identify TRAMs specifically associated with TDF-selection pressure, we compared the proportion of each TRAM to its proportion in a cohort of 5805 individuals with VF on a first-line thymidine analog-containing regimen. We identified 83 TRAMs including 33 NRTI-associated, 40 NNRTI-associated, and 10 uncommon mutations of uncertain provenance. Of the 33 NRTI-associated TRAMs, 12 - A62V, K65R/N, S68G/N/D, K70E/Q/T, L74I, V75L, and Y115F - were more common among individuals receiving a first-line TDF-containing compared to a first-line thymidine analog-containing regimen. These 12 TDF-selected TRAMs will be important for monitoring TDF-associated transmitted drug-resistance and for determining the extent of reduced TDF susceptibility in individuals with VF on a TDF-containing regimen.

High prevalence of the K65R mutation in HIV-1 subtype C infected patients failing tenofovir-based first-line regimens in South Africa.

BACKGROUND: Tenofovir (TDF) has replaced stavudine (d4T) as the preferred nucleoside reverse transcriptase inhibitor (NRTI) in first-line regimens in South Africa, but limited information is available on the resistance patterns that develop after the introduction of TDF. This study investigated the antiretroviral drug resistance patterns in South African HIV-1 subtype C-infected patients failing stavudine- (d4T) and tenofovir- (TDF) based first-line regimens and assess the suitability of TDF as the preferred first-line nucleotide reverse transcriptase inhibitor (NRTI). METHODS: Resistance patterns of HIV-1 from 160 adult patients virologically failing TDF- (n = 80) and d4T- (n = 80) based first-line regimens were retrospectively analyzed. The pol gene was sequenced using an in-house protocol and mutations were analysed using the IAS-USA 2014 Drug Resistance Mutation list. RESULTS: Compared to d4T-exposed patients (n = 7), patients failing on a TDF-containing regimen (n = 43) were almost 5 times more likely to present with a K65R mutation (aRR 4.86 95% CI 2.29 - 10.34). Y115F was absent in the d4T group, and detected in 13.8% (n = 11) of TDF-exposed patients, p = 0.0007. Virus from 9 of the 11 patients (82.0%) who developed the Y115F mutation also developed K65R. Intermediate or high-level resistance to most NRTIs was common in the TDF-treatment group, but these patients twice more likely to remain susceptible to AZT as compared to those exposed to d4T (aRR 2.09 95% CI 1.13 - 3.90). CONCLUSION: The frequency of the TDF induced K65R mutation was higher in our setting compared to non-subtype C dominated countries. However, despite the higher frequency of cross-resistance to NRTIs, most patients remained susceptible to AZT, which is reflected in the South African treatment guidelines that recommend AZT as an essential component of second-line regimens.