Genetic studies and phenotypic expansion of hearing loss (HL) for people living in Africa are greatly needed. We evaluated the clinical phenotypes of three affected siblings presenting non-syndromic (NS) HL and five unaffected members of a consanguineous Ghanaian family. Analysis of exome sequence data was performed for all affected and one unaffected family members. In-depth genetic and cellular characterization studies were performed to investigate biological significance of the implicated variant using bioinformatic tools and cell-based experimentation. Audiological examinations showed severe-to-profound, bilateral, symmetrical, and post-lingual onset. The whole-exome sequencing (WES) identified a homozygous frameshift variant: MARVEL domain containing 2 (MARVELD2):c.1058dup;p.(Val354Serfs*5) in all affected siblings. This frameshift variant leads to an early stop codon insertion and predicted to be targeted by nonsense medicated decay (mutant protein predicted to lack conserved C-terminal domain if translated). Cell immunofluorescence and immunocytochemistry studies exposed the functional impact of the mutant protein's expression, stability, localization, protein-protein binding, barrier function, and actin cytoskeleton architecture. The identified variant segregates with NSHL in the index Ghanaian family. The data support this nonsense variant as pathogenic, likely to impact the homeostasis of ions, solutes, and other molecules, compromising membrane barrier and signaling in the inner ear spaces.

Future VirologyVol. 17, No. 10 CommentaryOpen AccessHIV infection and the risk of cancer: tumorigenicity of HIV-1 auxiliary proteinsShaheen Mowla & Riyaadh AhmedShaheen Mowla *Author for correspondence: Tel.: +27 214 066 560; E-mail Address: shaheen.mowla@uct.ac.za.https://orcid.org/0000-0001-6802-0495Department of Pathology, Division of Haematology, Faculty of Health Sciences, University of Cape Town, Observatory, Cape Town, Western Cape, 7925, South Africa & Riyaadh AhmedDepartment of Pathology, Division of Haematology, Faculty of Health Sciences, University of Cape Town, Observatory, Cape Town, Western Cape, 7925, South AfricaPublished Online:19 Jul 2022https://doi.org/10.2217/fvl-2022-0065AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail Keywords: cancergp120HIV-1NefP17TatThe association of viruses with human cancers is an established phenomenon, supported by clear evidence that shows that these viruses can influence key physiological events, leading to the development and advancement of malignancies. In fact, studies show that globally one in six cancers can be attributable to an infectious etiology, with the majority of the infectious agents being viruses [1,2]. This fraction is likely to be an underestimation in developing countries where the population harbor a significantly larger proportion of virus-associated cancers when compared with elsewhere in the world. While there had been tentative reports of viruses as cancer causing agents in humans from the early 1900s, the first oncogenic virus, Epstein–Barr virus (EBV), was reported in 1964 in cultured Burkitt lymphoma cells [3]. To date, at least seven viruses have been named as having established involvement in human cancers, with a few others having possible causative links [2]. In many instances these viruses are part of common infections, establishing chronic long-term associations with the host, and do not cause cancer in a majority of cases. Additionally, viral infection alone is not sufficient for driving the complex multistep process of oncogenesis, but is dependent on cumulative events, with the state of the host immunity being a major determining factor.Regardless of virus genome type (RNA/DNA), it appears that the induction of tumors is a biological accident rather than an intended outcome of the viral life cycle. Viruses infect cells in order to exploit the host's cellular machinery and establish viral replication, and the long-term establishment of these viruses in the genome of host cells provide ample time for the accumulation of mutagenic events which eventually lead to the development of tumors. Contrary to expectation, the tumor mass is not laden with infectious virions, and in some cases (for example: in the case of EBV or Kaposi sarcoma-associated herpes virus [KSHV]) the viruses are in a latent state in most cancer cells. Eventually, the viral genome could be entirely lost to the tumor cells, without the malignant phenotype being affected [4]. The 'hit-and-run' theory has been proposed to describe the phenomenon where viruses are responsible for the triggering event in cancer formation but are no longer needed over time as mutations accumulate to support cancer progression. This means that there is a potential underestimation of the number of cancers driven by viruses, if the presence of the virus is used to establish etiology. Nevertheless, oncogenic viruses have played an extremely significant role in the discovery of oncogenes and tumor suppressor genes. There is no doubt that studies on cellular transformation by viruses and the association of viral proteins with host proteins have enhanced our understanding of cancer biology and thus has been pivotal in the field of cancer research. The continued efforts in defining the role of viruses in tumorigenesis is therefore essential.Although the HIV virus is not classified as oncogenic, people living with the virus are at a significantly higher risk of developing certain malignancies. While virus-induced immune suppression and dysfunctions of the immune system remain the primary causative link, the persistence of malignancy despite successful antiretroviral therapy (ART) has prompted a deeper look into the mechanisms driving HIV-associated cancers. Accumulating evidence reveals that HIV and its auxiliary proteins can directly promote carcinogenesis in a manner similar to mechanisms described for known oncogenic viruses. The emerging concept of HIV as an oncogenic virus is rapidly gaining credibility as more reports emerge supporting this. Viral protein can be detected in the serum/plasma of patients even when HIV RNA levels are undetectable. These proteins have been shown to affect bystander cells within their microenvironment, in ways which can lead to oncogenesis.HIV-1 TatThe HIV-1 Tat protein is an essential factor in HIV DNA transcription and survival, with several non-viral replication associated functions described, linked to HIV-related diseases including cancer. This small protein (86–102 amino acids) is secreted from both latently infected cells as well as cells with active HIV replication and has been shown to become internalized by bystander cells [5]. The Tat protein contains multiple domains shown to promote a range of cellular events, one of which being co-operation with transcription factors leading to alterations in gene expression known to support oncogenic events. For instance, exposure of healthy B cells to Tat promotes the oncogenic co-localization of the c-MYC and IGH loci, a genetic hallmark of aggressive lymphomas which are over-represented among HIV-infected individuals [6]. Although not host to HIV, Tat can be detected within the tumor cells of Burkitt lymphoma (BL) patients. The prevalence of BL among people infected with HIV is disproportionately high when compared with the immunocompetent population, with some reports showing no to marginal improvement in incidence despite the rollout of ART [7]. Mechanistically, the Tat protein has the ability to directly affect MYC transcription through complexing with the AP-1 factor JunB, a known regulator of the c-MYC promoter [8]. In Kaposi sarcoma (KS), an AIDS-defining cancer, Tat promotes tumorigenesis via induction of viral IL-6 and the PI3K/phosphate and tensin homolog/AKT/GSK-3b pathway, and can act synergistically with Orf-K1, a KSHV protein, to alter expression of cellular miRNAses [9]. Additionally, genome-wide studies demonstrate that Tat has affinity for binding widely within the cellular genome, at specific sites within gene regulatory regions [10]. Thus, the true impact of Tat on HIV-related oncogenesis remains as yet largely unexplored.HIV-1 NefAn oncogenic role for the HIV Nef protein has been demonstrated in several cancer types. This 27-kDa myristoylated protein is abundantly expressed during early stages of infection and is responsible for enhancing the infectivity of progeny virions through mechanisms which are not yet completely understood. Plasma Nef levels correlate with plasma HIV RNA in untreated patients and can be detected in the serum of approximately half of patients on ART, indicating that the source may be from HIV reservoirs [11]. Like Tat, circulating Nef can become internalized by bystander cells where it can alter cellular function. In KS, which remains one of the most common cancers among people living with HIV, Nef has been shown to interact with the KSHV through synergizing with viral IL-6 to enhance angiogenesis and tumorigenesis. The use of in vivo models such as nude mice revealed that mechanistically this is supported via activation of the AKT pathway through induction of cellular miR-718, an inhibitor of phosphate and tensin homolog [12]. Nef has also been linked to the development of lymphoma. Through nanotubule conduits, macrophages shuttle Nef into B lymphocytes and the viral protein has been found to play an active role in the formation of these actin conduits. BL cells expressing Nef has enhanced expression of lymphoma-driving genes, one of which being activation-induced cytidine deaminase, an essential driver of translocations, including the oncogenic MYC/IGH translocation [13]. Despite these reports, the cellular mechanisms of Nef-driven lymphomagenesis remain largely undefined, and lymphoma remains one of the most prevalent cancers among HIV-infected individuals. Non-small-cell lung cancer is one of the most frequent non-AIDS defining malignancies. In a study investigating the role of Nef in HIV-related lung disease, the viral protein was shown to promote changes in metabolism, cell survival and invasion and increase angiogenesis of lung cancer cells [14]. This could be as a result of a global decrease in miRNA and miRNA-biogenesis protein expression brought about by the presence of Nef, thus indicating a direct involvement of Nef in tumorigenesis.HIV-1 P17The structural HIV-1 p17 matrix protein, which plays a regulatory role during various phases of the virus life cycle such as viral entry and replication, gets secreted from HIV-1-infected cells into the extracellular space where it accumulates. As is the case for Nef and Tat, p17 can be detected in serum even in patients on highly active antiretroviral therapy who are virally suppressed [15]. This viral protein has been shown to influence the biological function of many different cell types primarily through interactions between its N-terminal AT20 epitope and cell surface receptors. In particular, p17 has a strong association with angiogenesis. Through high affinity binding to CXCR2, which is related to the IL-8 receptor CXCR1, p17 was shown to promote the formation of capillary-like structures on endothelial cells [16]. In lymphomagenesis, p17 was found to bind and become internalized by B lymphocytes, with higher affinity for EBV-infected cells which preferentially express CXCR2 [17]. A natural variant of p17, S75X, bound more efficiently to these cells and specifically and directly impacted on tumorigenesis – the expression of oncogenic latent membrane protein-1 was enhanced, leading to activation of Akt, ERK1/2 and STAT3 signaling, with associated upregulation of cyclin D2 and D3, and enhanced proliferation. Although not a HIV-associated malignancy, breast cancer in HIV-positive patients is generally more aggressive, with poorer outcomes despite these patients being on ART [18]. When the human breast cancer cell line MDA-MB 231 was exogenously exposed to p17, the migratory abilities and invasiveness of the cancer cells were enhanced. Once again, these effects were mediated via the binding of the viral protein to CXCR2, and this led to the activation of the ERK1/2 signaling pathway. P17 may therefore likely be one of the determining factors of the clinical course of breast cancer in HIV-positive patients.HIV-1 gp120HIV-1 envelope glycoprotein gp120 is yet another viral protein that has demonstrated the ability to drive oncogenic transformation. Glioblastomas are over-represented among people infected with HIV, as is the case for other cancers mentioned above, with patients having an inferior median survival, despite successful viral control [19]. Studies, both primary as well as established glioma cell lines, found that when exposed to gp120, these cells have higher proliferation rates, migratory abilities and survival. Additionally, these cells had enhanced expression and activity of certain glycolytic enzymes, with increased glucose uptake and oxygen consumption. This shift in glycolysis, also known as the Warburg effect, is a typical feature of cancer cells which enhanced proliferation, migration and promotion of DNA, protein and lipid synthesis. Notably, mice expressing the viral protein in their brain developed larger tumors than their control counterparts, with shorter median survival. A study on the integrity of the mucosal epithelial cell layer of the tonsil, the cervix and the foreskin revealed disruptions in the expression of cell adherence junction proteins upon exposure to HIV-1 viral proteins including gp120 [20]. This was accompanied by enhanced epithelial–mesenchymal transition, a critical step in the metastatic cascade of solid tumors.Cancer remains a leading cause of death among HIV-infected persons and the evidence for an oncogenic role for HIV and its auxiliary proteins continues to accumulate. Nevertheless, the actions of established oncogenic viruses including KSHV and EBV, the high-risk HPV and others remain the major causative agents, with some of the evidence demonstrating a collaborative link between HIV and these viruses. It has become clear that, even in patients who are virally suppressed, HIV-encoded proteins remain in tissues and in circulation, and while not inherently oncogenic, these factors potentiate oncogenic risk in non-HIV host bystander cells, compounded by an environment of chronic inflammation and immune dysfunction, among others. These represent carcinogenic characteristics which are unique to HIV-associated malignancies and are not present within the same malignancy that develops in an HIV-negative background. Over time, the deeper understanding of this complex landscape will inform new approaches to diagnosis and treatment of cancers in people who are infected with HIV.Author contributionsS Mowla conceptualized and designed the article, analyzed, collected, interpreted and collated 60% of the content, drafted the manuscript, critically revised it and made the final approval of the submitted version. R Ahmed analyzed, collected, interpreted and collated 40% of the content, and revised a final draft for correctness.Financial & competing interests disclosureThe authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.No writing assistance was utilized in the production of this manuscript.Open accessThis work is licensed under the Attribution-NonCommercial-NoDerivatives 4.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/4.0/References1. Plummer M, De Martel C, Vignat J, Ferlay J, Bray F, Franceschi S. Global burden of cancers attributable to infections in 2012: a synthetic analysis. Lancet Glob. Health 4(9), e609–e616 (2016).Crossref, Medline, Google Scholar2. Ferreira DA, Tayyar Y, Idris A, Mcmillan NA. A "hit-and-run" affair – a possible link for cancer progression in virally driven cancers. Biochim. Biophys. Acta Rev. Cancer 1875(1), 188476 (2021).Crossref, Medline, CAS, Google Scholar3. Ahuja R, Jamal A, Nosrati N et al. Human oncogenic viruses and cancer. Current Sci. 107(5), 768–785 (2014).CAS, Google Scholar4. Chang Y, Moore PS, Weiss RA. Human oncogenic viruses: nature and discovery. Philos. Trans. R. Soc. B Biol. Sci. 372(1732), 20160264 (2017).Crossref, Medline, Google Scholar5. Ajasin D, Eugenin EA. HIV-1 Tat: role in bystander toxicity. Front. Cell. Infect. Microbiol. 10, 61 (2020).Crossref, Medline, CAS, Google Scholar6. Germini D, Tsfasman T, Klibi M et al. HIV Tat induces a prolonged MYC relocalization next to IGH in circulating B-cells. Leukemia 31(11), 2515–2522 (2017).Crossref, Medline, CAS, Google Scholar7. Yarchoan R, Uldrick TS. HIV-associated cancers and related diseases. N. Engl. J. Med. 378(11), 1029–1041 (2018).Crossref, Medline, Google Scholar8. Alves De Souza Rios L, Mapekula L, Mdletshe N, Chetty D, Mowla S. HIV-1 transactivator of transcription (Tat) co-operates with AP-1 factors to enhance c-MYC transcription. Front. Cell Dev. Biol. 9, 693706 (2021).Crossref, Medline, Google Scholar9. Zhou F, Xue M, Qin D et al. HIV-1 Tat promotes Kaposi's sarcoma-associated herpesvirus (KSHV) vIL-6-induced angiogenesis and tumorigenesis by regulating PI3K/PTEN/AKT/GSK-3β signaling pathway. PLOS ONE 8(1), e53145 (2013).Crossref, Medline, CAS, Google Scholar10. Marban C, Su T, Ferrari R et al. Genome-wide binding map of the HIV-1 Tat protein to the human genome. PLOS ONE 6(11), e26894 (2011).Crossref, Medline, CAS, Google Scholar11. Ferdin J, Goričar K, Dolžan V et al. Viral protein Nef is detected in plasma of half of HIV-infected adults with undetectable plasma HIV RNA. PLOS ONE 13(1), e0191613 (2018).Crossref, Medline, Google Scholar12. Xue M, Yao S, Hu M et al. HIV-1 Nef and KSHV oncogene K1 synergistically promote angiogenesis by inducing cellular miR-718 to regulate the PTEN/AKT/mTOR signaling pathway. Nucleic Acids Res. 42(15), 9862–9879 (2014).Crossref, Medline, CAS, Google Scholar13. Mdletshe N, Nel A, Shires K, Mowla S. HIV Nef enhances the expression of oncogenic c-MYC and activation-induced cytidine deaminase in Burkitt lymphoma cells, promoting genomic instability. Infect. Agents Cancer 15(1), 1–10 (2020).Crossref, Medline, Google Scholar14. Santerre M, Chatila W, Wang Y, Mukerjee R, Sawaya BE. HIV-1 Nef promotes cell proliferation and microRNA dysregulation in lung cells. Cell Cycle 18(2), 130–142 (2019).Crossref, Medline, CAS, Google Scholar15. Caccuri F, Marsico S, Fiorentini S, Caruso A, Giagulli C. HIV-1 matrix protein p17 and its receptors. Curr. Drug Targets 17(1), 23–32 (2016).Crossref, Medline, CAS, Google Scholar16. Caccuri F, Giagulli C, Bugatti A et al. HIV-1 matrix protein p17 promotes angiogenesis via chemokine receptors CXCR1 and CXCR2. Proc. Natl Acad. Sci. 109(36), 14580–14585 (2012).Crossref, Medline, CAS, Google Scholar17. Martorelli D, Muraro E, Mastorci K et al. A natural HIV p17 protein variant up‐regulates the LMP‐1 EBV oncoprotein and promotes the growth of EBV‐infected B‐lymphocytes: implications for EBV‐driven lymphomagenesis in the HIV setting. Int. J. Cancer 137(6), 1374–1385 (2015).Crossref, Medline, CAS, Google Scholar18. Caccuri F, Giordano F, Barone I et al. HIV-1 matrix protein p17 and its variants promote human triple negative breast cancer cell aggressiveness. Infect. Agents Cancer 12(1), 1–9 (2017).Crossref, Medline, Google Scholar19. Valentín-Guillama G, López S, Kucheryavykh YV et al. HIV-1 envelope protein gp120 promotes proliferation and the activation of glycolysis in glioma cell. Cancers 10(9), 301 (2018).Crossref, Google Scholar20. Lien K, Mayer W, Herrera R, Rosbe K, Tugizov SM. HIV-1 proteins gp120 and tat induce the epithelial–mesenchymal transition in oral and genital mucosal epithelial cells. PLOS ONE 14(12), e0226343 (2019).Crossref, Medline, CAS, Google ScholarFiguresReferencesRelatedDetails Vol. 17, No. 10 Follow us on social media for the latest updates Metrics History Received 29 March 2022 Accepted 23 June 2022 Published online 19 July 2022 Published in print October 2022 Information© 2022 Shaheen MowlaKeywordscancergp120HIV-1NefP17TatAuthor contributionsS Mowla conceptualized and designed the article, analyzed, collected, interpreted and collated 60% of the content, drafted the manuscript, critically revised it and made the final approval of the submitted version. R Ahmed analyzed, collected, interpreted and collated 40% of the content, and revised a final draft for correctness.Financial & competing interests disclosureThe authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.No writing assistance was utilized in the production of this manuscript.Open accessThis work is licensed under the Attribution-NonCommercial-NoDerivatives 4.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/4.0/PDF download

We have previously reported CLIC5A and SLC12A2 variants in two families from Cameroon and Ghana, segregating non-syndromic hearing impairment (NSHI). In this study, biological assays were performed to further functionally investigate the pathogenicity of CLIC5 [c.224T>C; p.(L75P)] and SCL12A2 [c.2935G>A: p.(E979K)] variants. Ectopic expression of the proteins in a cell model shows that compared to wild-type, both the CLIC5A and SLC12A2 variants were overexpressed. The mutant CLIC5A protein appears as aggregated perinuclear bodies while the wild-type protein was evenly distributed in the cytoplasm. Furthermore, cells transfected with the wild-type CLIC5A formed thin membrane filopodia-like protrusions which were absent in the CLIC5A mutant expressing and control cells. On the other hand, the wild-type SLC12A2 expressing cells had an axon-like morphology which was not observed in the mutant expressing and control cells. A network analysis revealed that CLIC5A can interact with at least eight proteins at the base of the stereocilia. This study has generated novel biological data associated with the pathogenicity of targeted variants in CLIC5A and SLC12A2, found in two African families, and therefore expands our understanding of their pathobiology in hearing impairment.

Congenital hearing impairment (HI) is genetically heterogeneous making its genetic diagnosis challenging. Investigation of novel HI genes and variants will enhance our understanding of the molecular mechanisms and to aid genetic diagnosis. We performed exome sequencing and analysis using DNA samples from affected members of two large families from Ghana and Pakistan, segregating autosomal-dominant (AD) non-syndromic HI (NSHI). Using in silico approaches, we modeled and evaluated the effect of the likely pathogenic variants on protein structure and function. We identified two likely pathogenic variants in SLC12A2 , c.2935G>A:p.(E979K) and c.2939A>T:p.(E980V), which segregate with NSHI in a Ghanaian and Pakistani family, respectively. SLC12A2 encodes an ion transporter crucial in the homeostasis of the inner ear endolymph and has recently been reported to be implicated in syndromic and non-syndromic HI. Both variants were mapped to alternatively spliced exon 21 of the SLC12A2 gene. Exon 21 encodes for 17 residues in the cytoplasmatic tail of SLC12A2, is highly conserved between species, and preferentially expressed in cochlear tissues. A review of previous studies and our current data showed that out of ten families with either AD non-syndromic or syndromic HI, eight (80%) had variants within the 17 amino acid residue region of exon 21 (48 bp), suggesting that this alternate domain is critical to the transporter activity in the inner ear. The genotypic spectrum of SLC12A2 was expanded and the involvement of SLC12A2 in ADNSHI was confirmed. These results also demonstrate the role that SLC12A2 plays in ADNSHI in diverse populations including sub-Saharan Africans.

HIV-1 infection often leads to the development of co-morbidities including cancer. Burkitt lymphoma (BL) is one of the most over-represented non-Hodgkin lymphoma among HIV-infected individuals, and displays a highly aggressive phenotype in this population group, with comparatively poorer outcomes, despite these patients being on anti-retroviral therapy. Accumulating evidence indicates that the molecular pathogenesis of HIV-associated malignancies is unique, with components of the virus playing an active role in driving oncogenesis, and in order to improve patient prognosis and treatment, a better understanding of disease pathobiology and progression is needed. In this study, we found HIV-1 Tat to be localized within the tumor cells of BL patients, and enhanced expression of oncogenic c-MYC in these cells. Using luciferase reporter assays we show that HIV-1 Tat enhances the c-MYC gene promoter activity and that this is partially mediated via two AP-1 binding elements located at positions -1128 and -1375 bp, as revealed by mutagenesis experiments. We further demonstrate, using pull-down assays, that Tat can exist within a protein complex with the AP-1 factor JunB, and that this complex can bind these AP-1 sites within the c-MYC promoter, as shown by in vivo chromatin immunoprecipitation assays. Therefore, these findings show that in HIV-infected individuals, Tat infiltrates B-cells, where it can enhance the expression of oncogenic factors, which contributes toward the more aggressive disease phenotype observed in these patients.

Background/Aim: Activation-induced cytidine deaminase (AID) is a DNA modifying enzyme which has an essential function in promoting antibody diversification. Its overexpression is strongly associated with B-cell derived malignancies including Burkitt lymphoma, where AID is required for the characteristic c-MYC/IGH translocation. This study aimed at defining AID’s oncopathogenic role which is still poorly understood. Materials and Methods: We created over-expressing and knock-down cell culture models of AID, and used cellular assays to provide insight into its contribution to lymphomagenesis. Results: We showed that AID expression is highly specific to, and abundantly expressed in B-cell-derived cancers and that ectopic overexpression of AID leads to rapid cell death. Using a knock-down model, we revealed that AID expression significantly impacts genomic stability, proliferation, migration and drug resistance. Conclusion: AID is an important driver of lymphoma, impacting multiple cellular events, and is potentially a strong candidate for targeted therapy in lymphoma.

Viruses and viral components have been shown to manipulate the expression of host microRNAs (miRNAs) to their advantage, and in some cases to play essential roles in cancer pathogenesis. Burkitt lymphoma (BL), a highly aggressive B-cell derived cancer, is significantly over-represented among people infected with HIV. This study adds to accumulating evidence demonstrating that the virus plays a direct role in promoting oncogenesis. A custom miRNA PCR was used to identify 32 miRNAs that were differently expressed in Burkitt lymphoma cells exposed to HIV-1, with a majority of these being associated with oncogenic processes. Of those, hsa-miR-200c-3p, a miRNA that plays a crucial role in cancer cell migration, was found to be significantly downregulated in both the array and in single-tube validation assays. Using an in vitro transwell system we found that this downregulation correlated with significantly enhanced migration of BL cells exposed to HIV-1. Furthermore, the expression of the ZEB1 and ZEB2 transcription factors, which are promotors of tumour invasion and metastasis, and which are direct targets of hsa-miR-200c-3p, were found to be enhanced in these cells. This study therefore identifies novel miRNAs as role players in the development of HIV-associated BL, with one of these miRNAs, hsa-miR-200c-3p, being a candidate for further clinical studies as a potential biomarker for prognosis in patients with Burkitt lymphoma, who are HIV positive.

Background: Hearing impairment (HI) genes are poorly studied in African populations. Methods: We used whole exome sequencing (WES) to investigate pathogenic and likely pathogenic (PLP) variants in 10 individuals with HI, from four multiplex families from Cameroon, two of which were previously unresolved with a targeted gene enrichment (TGE) panel of 116 genes. In silico protein modelling, western blotting and live imaging of transfected HEK293 cells were performed to study protein structure and functions. Results: All PLP variants previously identified with TGE were replicated. In one previously unresolved family, we found a homozygous frameshift PLP variant in GRXCR2 (OMIM: 615762), NM_001080516.1(GRXCR2):c.251delC p.(Ile85SerfsTer33), in two affected siblings; and additionally, in 1/80 unrelated individuals affected with non-syndromic hearing impairment (NSHI). The GRXCR2-c.251delC variant introduced a premature stop codon, leading to truncation and loss of a zinc-finger domain. Fluorescence confocal microscopy tracked the wild-type GRXCR2 protein to the cellular membrane, unlike the mutated GRXCR2 protein. Conclusion: This study confirms GRXCR2 as a HI-associated gene. GRXCR2 should be included to the currently available TGE panels for HI diagnosis.

Hearing impairment (HI) is a sensory disorder with a prevalence of 0.0055 live births in South Africa. DNA samples from a South African family presenting with progressive, autosomal dominant non-syndromic HI were subjected to whole-exome sequencing, and a novel monoallelic variant in REST [c.1244GC; p.(C415S)], was identified as the putative causative variant. The co-segregation of the variant was confirmed with Sanger Sequencing. The variant is absent from databases, 103 healthy South African controls, and 52 South African probands with isolated HI. In silico analysis indicates that the p.C415S variant in REST substitutes a conserved cysteine and results in changes to the surrounding secondary structure and the disulphide bonds, culminating in alteration of the tertiary structure of REST. Localization studies using ectopically expressed GFP-tagged Wild type (WT) and mutant REST in HEK-293 cells show that WT REST localizes exclusively to the nucleus; however, the mutant protein localizes throughout the cell. Additionally, mutant REST has an impaired ability to repress its known target AF1q. The data demonstrates that the identified mutation compromises the function of REST and support its implication in HI. This study is the second report, worldwide, to implicate REST in HI and suggests that it should be included in diagnostic HI panels.

Abstract Burkitt lymphoma (BL) is one of the most common HIV-associated lymphomas. This cancer represents one of the most frequent causes of mortality among HIV+ people in Southern Africa, which has the highest incidence of HIV/AIDS worldwide. As is the case for Kaposi sarcoma, recent reports associate an oncogenic function with HIV in lymphoma development. This study explores the oncogenic potential of HIV-1 protein Transactivator of transcription (Tat) in BL via its ability to manipulate the expression of c-MYC and AID, two key drivers of disease in BL. The ability of HIV-1 Tat to influence the activity of the full-length (FL) (2861 bp) c-MYC promoter was assessed using Dual Luciferase Reporter (DLR) assays. Using sequential deletions, the minimal promoter region mediating the effect was identified. Site-directed mutagenesis (SDM) was used to identify promoter elements mediating the response. Co-immunoprecipitation assays (Co-IP) were used to assess interactions between Tat and the AP-1 factor JunB. Chromatin Immunoprecipitation (ChIP) assays were done to confirm that JunB bound the promoter in vivo. To assess the ability of Tat to mediate the expression of miRNA-181b-5p, a known repressor of AICDA, Ramos cells were transfected with Tat followed by qRT-PCR. Corresponding changes in AID protein levels were determined using Western blot. To assess whether miRNA-181b-5p targets AICDA in B cells, the FL AICDA 3'UTR was cloned into the pGL3-Promoter vector and DLR assays were performed in the presence of miRNA-181b-5p mimic. Our data reveal that Tat enhances c-MYC promoter activity in a dose-dependent manner, with a maximum activity of 2.6-fold recorded when 500ng of pcDNA-Tat was used on 400ng of pGL3-c-MYC plasmid harboring the FL promoter. A loss of 35% activity was observed [1.7 (±0.05) fold] upon promoter deletion and two AP-1 sites (positions -1128 bp and -1376 bp) within the deleted region were found to mediate the Tat-dependent increase. A loss in activity of 20.6% was recorded when AP-1 site 1 was mutated, and 25% when AP-1 site 2 was mutated. A double mutant decreased the activity by 29%. Co-IP revealed a strong interaction between Tat and AP-1 factor JunB, and ChIP data showed that JunB was strongly recruited to those two AP-1 sites in the presence of Tat. Our result also reveals that the expression of the AICDA specific miRNA181b-5p is significantly reduced in the presence of Tat, which correlates with an increase in AID protein levels. We also confirm that the AICDA FL 3'UTR, which contains putative miRNA-181b-5p binding sites, is partially repressed by a miRNA-181b-5p mimic. Our study reveals that Tat potentiates oncogenesis by enhancing the expression of c-MYC and AID, two key lymphoma drivers. We show that Tat can enhance c-MYC expression by enhancing the recruitment of cellular TFs to the promoter. We also show that the same HIV protein can enhance the expression of AID by modulating the expression of miRNA-181b-5p. Citation Format: Leonardo Alves de Souza Rios, Nontlantla Mdletshe, Shaheen Mowla. HIV-1 transactivator of transcription deregulates key Burkitt lymphoma associated oncogenes at both the transcriptional and post-transcriptional level [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-31.

DNA samples from five members of a multiplex non-consanguineous Cameroonian family, segregating prelingual and progressive autosomal recessive non-syndromic sensorineural hearing impairment, underwent whole exome sequencing. We identified novel bi-allelic compound heterozygous pathogenic variants in CLIC5. The variants identified, i.e., the missense [NM_016929.5:c.224T>C; p.(L75P)] and the splicing (NM_016929.5:c.63+1G>A), were validated using Sanger sequencing in all seven available family members and co-segregated with hearing impairment (HI) in the three hearing impaired family members. The three affected individuals were compound heterozygous for both variants, and all unaffected individuals were heterozygous for one of the two variants. Both variants were absent from the genome aggregation database (gnomAD), the Single Nucleotide Polymorphism Database (dbSNP), and the UK10K and Greater Middle East (GME) databases, as well as from 122 apparently healthy controls from Cameroon. We also did not identify these pathogenic variants in 118 unrelated sporadic cases of non-syndromic hearing impairment (NSHI) from Cameroon. In silico analysis showed that the missense variant CLIC5-p.(L75P) substitutes a highly conserved amino acid residue (leucine), and is expected to alter the stability, the structure, and the function of the CLIC5 protein, while the splicing variant CLIC5-(c.63+1G>A) is predicted to disrupt a consensus donor splice site and alter the splicing of the pre-mRNA. This study is the second report, worldwide, to describe CLIC5 involvement in human hearing impairment, and thus confirms CLIC5 as a novel non-syndromic hearing impairment gene that should be included in targeted diagnostic gene panels.

Background Non-Hodgkin lymphoma is of high prevalence among HIV-infected people. In particular, the incidence of HIV-associated Burkitt lymphoma (BL) remains high despite the advent of Highly Active Anti-Retroviral Therapy. Recent evidence shows that serum-soluble HIV proteins can enhance oncogenesis, particularly in lymphoid tissues. This study sought to define the role of HIV protein Negative regulatory factor (Nef) in BL development by assessing its effect on key lymphoma driver genes. Methods A recombinant Nef protein was used to assess changes in expressions of activation-induced cytidine deaminase ( AICDA /AID) and c-MYC in B lymphocytes exposed extracellularly to the protein. Additionally, changes in the promoter activities of these genes were measured using a Nef-expressing cellular model and reporter assays. Confocal microscopy was used to observe c-MYC and AID expression and localization, and genomic integrity via the recruitment of phosphorylated γ-H2AX, in Nef-exposed cells. Results mRNA transcription of c-MYC and AICDA were significantly enhanced in lymphoma cells, up to 2-fold for c-MYC and up to 4-fold for AICDA , when exposed to varying concentrations of Nef (0–1000 ng/ml) and for different periods of time (3, 6 and 12 h). The protein expressions of AID and c-MYC followed a similar pattern and these effects were specific to BL but not lymphoblastoid cells. While the promoter activity of c-MYC was enhanced in the presence of Nef in a dose-dependent manner, the same was not observed for AICDA . Both AID and c-MYC accumulated within the cytoplasmic and nuclear spaces of Nef-exposed lymphoma cells, with a concomitant increase in DNA double strand breaks within the genome. Conclusions Exposure to HIV Nef leads to significant increases in AID and c-MYC, leading to genomic instability, potentially enhancing the oncogenic potential of Burkitt lymphoma. Our findings align with that of others to show that HIV proteins can directly contribute to the development and pathogenesis of HIV-associated lymphoma and accounts for the elevated incidence of BL observed in the HIV-infected population.

Activation Induced cytidine Deaminase (AID) is an essential enzyme of the adaptive immune system. Its canonical activity is restricted to B lymphocytes, playing an essential role in the diversification of antibodies by enhancing specificity and changing affinity. This is possible through its DNA deaminase function, leading to mutations in DNA. In the last decade, AID has been assigned an additional function: that of a powerful DNA demethylator. Adverse cellular conditions such as chronic inflammation can lead to its deregulation and overexpression. It is an important driver of B-cell lymphoma due to its natural ability to modify DNA through deamination, leading to mutations and epigenetic changes. However, the deregulation of AID is not restricted to lymphoid cells. Recent findings have provided new insights into the role that this protein plays in the development of non-lymphoid cancers, with some research shedding light on novel AID-driven mechanisms of cellular transformation. In this review, we provide an updated narrative of the normal physiological functions of AID. Additionally, we review and discuss the recent research studies that have implicated AID in carcinogenesis in varying tissue types including lymphoid and non-lymphoid cancers. We review the mechanisms, whereby AID promotes carcinogenesis and highlight important areas of future research.

Human cancers attributed to viral infections represent a growing proportion of the global cancer burden, with these types of cancers being the leading cause of morbidity and mortality in some regions. The concept that viruses play a causal role in human cancers is not new, but the mechanism thereof, while well described for some viruses, still remains elusive and complex for others, especially in the case of HIV-associated B-cell derived cancers. In the last decade, compelling evidence has demonstrated that cellular microRNAs are deregulated in cancers, with an increasing number of studies identifying microRNAs as potential biomarkers for human cancer diagnosis, prognosis and therapeutic targets or tools. Recent research demonstrates that viruses and viral components manipulate host microRNA expressions to their advantage, and the emerging picture suggests that the virus/microRNA pathway interaction is defined by a plethora of complex mechanisms. In this review, we highlight the current knowledge on virus/microRNA pathway interactions in the context of cancer and provide new insights on HIV as an oncogenic virus.

Human cancers attributed to viral infections represent a growing proportion of the global cancer burden, with these types of cancers being the leading cause of morbidity and mortality in some regions. The concept that viruses play a causal role in human cancers is not new, but the mechanism thereof, while well described for some viruses, still remains elusive and complex for others, especially in the case of HIV-associated B-cell derived cancers. In the last decade, compelling evidence has demonstrated that cellular microRNAs are deregulated in cancers, with an increasing number of studies identifying microRNAs as potential biomarkers for human cancer diagnosis, prognosis and therapeutic targets or tools. Recent research demonstrates that viruses and viral components manipulate host microRNA expressions to their advantage, and the emerging picture suggests that the virus/microRNA pathway interaction is defined by a plethora of complex mechanisms. In this review, we highlight the current knowledge on virus/microRNA pathway interactions in the context of cancer and provide new insights on HIV as an oncogenic virus.

Background: Environmental pollution such as exposure to pro-carcinogens including benzo-α-pyrene is becoming a major problem globally. Moreover, the effects of benzo-α-pyrene (BaP) on drug pharmacokinetics, pharmacodynamics, and drug resistance warrant further investigation, especially in cancer outpatient chemotherapy where exposure to environmental pollutants might occur. Method: We report here on the effects of benzo-α-pyrene on esophageal cancer cells in vitro, alone, or in combination with chemotherapeutic drugs cisplatin, 5-flurouracil, or paclitaxel. As the study endpoints, we employed expression of proteins involved in cell proliferation, drug metabolism, apoptosis, cell cycle analysis, colony formation, migration, and signaling cascades in the WHCO1 esophageal cancer cell line after 24 h of treatment. Results: Benzo-α-pyrene had no significant effect on WHCO1 cancer cell proliferation but reversed the effect of chemotherapeutic drugs by reducing drug-induced cell death and apoptosis by 30–40% compared to drug-treated cells. The three drugs significantly reduced WHCO1 cell migration by 40–50% compared to control and BaP-treated cells. Combined exposure to drugs was associated with significantly increased apoptosis and reduced colony formation. Evaluation of survival signaling cascades showed that although the MEK-ERK and Akt pathways were activated in the presence of drugs, BaP was a stronger activator of the MEK-ERK and Akt pathways than the drugs. Conclusion: The present study suggest that BaP can reverse the effects of drugs on cancer cells via the activation of survival signaling pathways and upregulation of anti-apoptotic proteins such as Bcl-2 and Bcl-xL. Our data show that BaP contribute to the development of chemoresistant cancer cells.

Southern Africa has the highest HIV incidence worldwide. Thus far there is no cure; however, the lives of individuals infected with the virus can be prolonged through the use of Highly Active AntiRetroviral Therapy (HAART). According to the United Nations Programme on HIV and AIDS (UNAIDS) fact sheet for 2015, 2 out of 3 new HIV infections are in the Sub-Saharan African region, with approximately 25.8 million people living with HIV and 1.4 million new infections in 2014. In 2004, South Africa introduced ART (Anti-retroviral Therapy) free of charge in the public healthcare sector, and today South Africa has the largest treatment programme in the world approximately 2.4 million people have received the life-saving treatment which has significantly increased their life expectancy. Shaheen Mowla* Department of Pathology, Faculty of Health Sciences, University of Cape Town, South Africa Shaheen Mowla Clinics in Oncology General Oncology Remedy Publications LLC., | http://clinicsinoncology.com/ 2017 | Volume 2 | Article 1366 2 novel targeted agents for these two subtypes [8]. Despite undergoing therapy, about 40% of HIV negative DLBCL patients suffer from relapsed or refractory disease under current the chemotherapeutic regimens (R-CHOP-rituximab with cyclophosphamide, doxorubicin, vincristine and prednisone) [9]. The majority of them succumb to their disease. The poor response is largely attributed to how genetically and molecularly heterogeneous the disease is [10]. Among DLBCL patients who are HIV positive, response to standard therapy is even more heterogeneous. Currently, the genetic causes of HIVDLBCL are unknown. Additionally, the contribution of Epstein Barr Virus (EBV) co-infection, which is common in HIV-DLBCLs, is also unknown and current studies in our laboratory are focusing on exploring the genetic and molecular subgroups of HIV-DLBCL patients within our population. While HIV uninfected patients with BL generally respond well to existing therapy, the response in HIV infected patients remains poor, and in some settings, there is report of no improvement at all [1113]. The pathogenesis of BL is strongly related to the overexpression of c-MYC resulting from translocation of the c-MYC gene to the highly active immunoglobulin (IGH) locus in B cells. The common treatment approach for BL is surgical resection if necessary, followed by chemotherapy. Different combinations of chemotherapeutic agents are used such as cyclophosphamidine, Vincristine, doxorubicin and dexamethasone; and in HIV positive patients this is administered after HAART [13,14]. Several case studies have shown that despite combination antiretroviral therapy, the outcome of HIV infected individuals with BL remains inferior compared to HIV-uninfected patients suffering from this cancer. Contrary to what is expected, some reports found that BL incidence among HIV infected individuals has not improved significantly despite the advent of HAART, and that the cancer develops in patients with relatively high CD4 counts [5]. The latter observation points to other enabling factors, aside from an immunocompromised state. Indeed, in recent years, our laboratory as well as others have shown that HIV-encoded proteins can contribute to oncogenesis by activating or enhancing oncogenic pathways in B cells. This is already shown in Kaposi sarcoma where the HIV Tat protein was found to cooperate with the Kaposi sarcoma herpes virus (KSHV) to enhance the development of the cancer [15].

Introduction: The pathogenesis HIV-associated lymphomas involve a complex interplay of biological factors, including the immerging role of HIV encoded proteins. These proteins have been shown to have oncogenic potential and to enhance the pathogenesis of HIV-associated malignancies. However, very little is known about the molecular mechanisms involved in this interaction. Here, we investigate the effect of HIV protein Negative Factor (Nef) on the expression of c-MYC and activation-induced cytidine deaminase (AID) in lymphoblasts and lymphoma cells. Methods: Native histidine-tagged HIV Nef was produced in a bacterial system and used to treat (1) an EBV-immortalized B lymphoblastoid cell line (L1439A) and (2) the established lymphoma cell line Ramos, at various concentrations and for various time points. Changes in the expressions of c-MYC and AID were measured at both the mRNA and proteins levels using qPCR and western blotting respectively. The ability of Nef to regulate the promoter of these two genes was determined using luciferase reporter assays. Furthermore, confocal microscopy was used to investigate the downstream effects of Nef exposure. Results: In L1439A cells, a general decrease in the expression of c-MYC and AID was observed. After 1 and 3 hours of exposure to HIV-Nef both c-MYC and AID mRNA levels reduced by approximately 0.5-fold. In contrast, the expressions of both genes increased significantly upon these treatments in the lymphoma cell line Ramos. After 3 hours of exposure, c-MYC expression increased by ~1.9-fold, while AID expression increased up to 4.6-fold. The protein expression followed a similar pattern, as indicated by western blotting. Furthermore, luciferase reporter assays showed that the effect on c-MYC expression is likely to be direct since the activity of the c-MYC promoter increased in a dose-dependent manner in the presence of Nef. c-MYC translocated to the nucleus, which is indicative of an active protein. Furthermore, an increase in gamma H2AX foci on DNA, indicative of double strand breaks, shows that the increase in AID lead to genomic instability. Conclusion: This study demonstrates a differential effect of HIV Nef on normal immortalized lymphoblasts versus lymphoma cells. More importantly, it shows for the first time that HIV Nef may enhance the pathogenesis of lymphomas by triggering pathways and mechanisms which increase the expression of c-MYC and AID. Citation Format: Shaheen Mowla, Nontlantla Mdletshe. The role of HIV protein Nef in the development of HIV/AIDS-associated lymphomas [abstract]. In: Proceedings of the AACR International Conference: New Frontiers in Cancer Research; 2017 Jan 18-22; Cape Town, South Africa. Philadelphia (PA): AACR; Cancer Res 2017;77(22 Suppl):Abstract nr B31.

BACKGROUNDAn exponential increase in the number of sickle cell disease (SCD) patients in paediatric services in Cape Town, South Africa, has been reported. The trend in adult/adolescent services has not been investigated.OBJECTIVESTo evaluate epidemiological trends of SCD and the profile of patients affected by SCD attending the Haematology Clinic at Groote Schuur Hospital (GSH), Cape Town.METHODS(i) A retrospective review of the number of SCD patients over the past 20 years; (ii) a cross-sectional analysis of clinical and haematological characteristics of SCD patients; and (iii) molecular analysis of the haemoglobin S mutation, the haplotype in the β-globin-like genes cluster, the 3.7 kb α-thalassaemia gene deletion and 19 selected single-nucleotide polymorphisms (SNPs) associated with fetal haemoglobin (HbF) levels.RESULTSFrom 1995 to 2016, 81 adolescent/adult patients with SCD were registered, mostly originating from other African countries (n=61, 75.3%). There was an increase of over 200% in new cases (n=47) during the last quarter of the two decades investigated. Data from 34 of 58 regular attendees (58.6%) were analysed. The mean age of the patients was 26.1 years (standard deviation (SD) 9.8), and 70.6% were male. With the exception of four patients with sickle/β-thalassaemia, all the patients had SCD (haemoglobin SS). The co-inheritance of a single 3.7 kb α-globin deletion was found in 42.3% of cases (n=11). The Bantu haplotype was the most observed (65.4% of chromosomes). Most HbF-promoting SNPs were not associated with variable levels of haematological indices.CONCLUSIONSThere is an increasing burden of adult SCD patients at GSH. National health and academic institutions need to adapt policies and healthcare professional training accordingly.

The major therapeutic benefit of hydroxyurea, the only FDA-approved pharmacologic treatment for sickle cell disease (SCD), is directly related to fetal hemoglobin (HbF) production that leads to significant reduction of morbidity and mortality. However, potential adverse effects such as infertility, susceptibility to infections, or teratogenic effect have been subject of concerns. Therefore, understanding HU molecular mechanisms of action, could lead to alternative therapeutic agents to increase HbF with less toxicity. This paper investigated whether HU-induced HbF could operate through post-transcriptional miRNAs regulation of BCL11A, KLF-1 and MYB, potent negative regulators of HbF. Both ex vivo differentiated primary erythroid cells from seven unrelated individuals, and K562 cells were treated with hydroxyurea (100 μM) and changes in BCL11A, KLF-1, GATA-1, MYB, β- and γ-globin gene expression were investigated. To explore potential mechanisms of post-transcriptional regulation, changes in expression of seven targeted miRNAs, previously associated with basal γ-globin expression were examined using miScript primer assays. In addition, K562 cells were transfected with miScript miRNA inhibitors/anti-miRNAs followed by Western Blot analysis to assess the effect on HbF protein levels. Direct interaction between miRNAs and the MYB 3′-untranslated region (UTR) was also investigated by a dual-luciferase reporter assays.