Browsing by Author "Arowolo, Afolake"

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    Open Access
    A histopathological and genomics study of the mutated human FAM111B gene related POIKTMP disease
    (2023) Tambwe, Nadine; Arowolo, Afolake
    Fibrosis is a pathological feature of many chronic inflammatory diseases, eventually leading to organ failure and death. POIKTMP is a rare, multi-organ fibrosing disease which is associated with mutations of the human FAM111B gene. FAM111B gene codes for a protein whose function is not well characterized. Therefore, elucidating the mechanism of FAM111B or its mutations in POIKTMP is beneficial to understanding the complexities surrounding this multisystemic fibrosing disease. The study sought to understand the pathogenesis of fibrosis, its role in POIKTMP and its causative gene mutation: FAM111B Y621D. First, Sanger sequencing was used to confirm the presence of the FAM111B Y621D mutation using DNA isolated and amplified from post-mortem FFPE tissues of a POIKTMP patient first described with the disease in South African Following that, qRT-PCR was employed to assess gene expression changes between the patient and the familial control. The RT2 Profiler Human fibrosis PCR Array was then used to associate POIKTMP and 84 known fibrotic markers to propose a possible fibrotic pathway associated with POIKTMP disease using mRNA from the lung and skin POIKTMP patient tissues. Gene-set enrichment analysis (GSEA) using Enrichr, a computational GSEA tool, was used to predict enrichment analysis between the identified upregulated fibrosis markers and the FAM111B gene. Finally, Immunohistochemistry was used to identify cellular and sub-cellular protein distribution of FAM111B and other fibroproliferative markers of interest to annotate pathological changes. The results from this study validated the FAM111B Y621D mutation in the affected tissues. Next, FAM111B mRNA was shown to be downregulated in the lungs and skeletal muscle tissues of the POIKTMP patient. The human fibrosis PCR array experiments identified eight upregulated fibrotic markers: MMP3, MMP13, PDGFA, ITGB-1, THBS-2, COL3A1, TGFβ3, and CCN2 in the patient lungs and skin tissues, which were validated by qRT-PCR. Furthermore, these genes with FAM111B form a gene-list that was used in interrogating various gene-set libraries in the gene-set enrichment analysis. FAM111B was enriched in some gene-set libraries within the Diseases/Drugs and Cell type categories. The GSEA terms enriched within these libraries are the pathways associated with SARS-COVID-19 perturbations and cell/tissue types related to the small intestine, breast, oesophagus, thyroid, smooth muscle and stromal cells of some of these organs. Lastly, immunohistochemistry results corroborated this study's mRNA expression analysis by showing that FAM111B was more highly expressed in the skin than in the lung patient. TGF-β1 and Ki-67 markers were assessed from protein expression, which resulted in higher expression in the POIKTMP patient skin tissue than in the lungs. Altogether, our data suggest that FAM111B and mutations in this gene play a pivotal role in POIKTMP and other fibrosing organ diseases, representing a potential disease biomarker and possible therapeutic target in POIKTMP and other fibrotic disorders.
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    Open Access
    A histopathological and genomics study of the mutated human FAM111B gene related POIKTMP disease
    (2023) Tambwe, Nadine; Arowolo, Afolake
    Fibrosis is a pathological feature of many chronic inflammatory diseases, eventually leading to organ failure and death. POIKTMP is a rare, multi-organ fibrosing disease which is associated with mutations of the human FAM111B gene. FAM111B gene codes for a protein whose function is not well characterized. Therefore, elucidating the mechanism of FAM111B or its mutations in POIKTMP is beneficial to understanding the complexities surrounding this multisystemic fibrosing disease. The study sought to understand the pathogenesis of fibrosis, its role in POIKTMP and its causative gene mutation: FAM111B Y621D. First, Sanger sequencing was used to confirm the presence of the FAM111B Y621D mutation using DNA isolated and amplified from post-mortem FFPE tissues of a POIKTMP patient first described with the disease in South African Following that, qRT-PCR was employed to assess gene expression changes between the patient and the familial control. The RT2 Profiler Human fibrosis PCR Array was then used to associate POIKTMP and 84 known fibrotic markers to propose a possible fibrotic pathway associated with POIKTMP disease using mRNA from the lung and skin POIKTMP patient tissues. Gene-set enrichment analysis (GSEA) using Enrichr, a computational GSEA tool, was used to predict enrichment analysis between the identified upregulated fibrosis markers and the FAM111B gene. Finally, Immunohistochemistry was used to identify cellular and sub-cellular protein distribution of FAM111B and other fibroproliferative markers of interest to annotate pathological changes. The results from this study validated the FAM111B Y621D mutation in the affected tissues. Next, FAM111B mRNA was shown to be downregulated in the lungs and skeletal muscle tissues of the POIKTMP patient. The human fibrosis PCR array experiments identified eight upregulated fibrotic markers: MMP3, MMP13, PDGFA, ITGB-1, THBS-2, COL3A1, TGFβ3, and CCN2 in the patient lungs and skin tissues, which were validated by qRT-PCR. Furthermore, these genes with FAM111B form a gene-list that was used in interrogating various gene-set libraries in the gene-set enrichment analysis. FAM111B was enriched in some gene-set libraries within the Diseases/Drugs and Cell type categories. The GSEA terms enriched within these libraries are the pathways associated with SARS-COVID-19 perturbations and cell/tissue types related to the small intestine, breast, oesophagus, thyroid, smooth muscle and stromal cells of some of these organs. Lastly, immunohistochemistry results corroborated this study's mRNA expression analysis by showing that FAM111B was more highly expressed in the skin than in the lung patient. TGF-β1 and Ki-67 markers were 12 assessed from protein expression, which resulted in higher expression in the POIKTMP patient skin tissue than in the lungs. Altogether, our data suggest that FAM111B and mutations in this gene play a pivotal role in POIKTMP and other fibrosing organ diseases, representing a potential disease biomarker and possible therapeutic target in POIKTMP and other fibrotic disorders.
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    Open Access
    A study of the expression and cellular function of the human FAM111B gene
    (2021) Rhoda, Cenza; Arowolo, Afolake
    POIKTMP, a multi-systemic fibrosing disease, results from mutations in the human FAM111B gene. Studies have also suggested high expression of this gene in cancers. Despite rising interest in the pathological effects of FAM111B mutations and overexpression of FAM111B, knowledge of the physiological role of this gene remains limited. Therefore, this study sought out to provide insights into the cellular function of FAM111B and to investigate the pathological effect of the FAM111B Y621D mutation. First, bioinformatics studies coupled with quantitative PCR and Western blots analysis were employed to assess FAM111B gene and protein expression in cancerous and non-cancerous cell lines. Subsequently, FAM111B gene expression was downregulated and upregulated in the human fibrosarcoma (HT1080) cell line by RNA-interference mediated gene silencing and recombinant gene expression technologies. The effect of these FAM111B dysregulations was studied using cellbased functional assays: proliferation, apoptosis, migration, and invasion assays. Furthermore, the functional pathways and interacting proteins of the FAM111B protein was determined using mass spectroscopy proteomics. Finally, preliminary studies in a POIKTMP patient-derived fibroblasts were attempted to recapitulate the results obtained using the HT1080 cell line. The results from this study indicated that FAM111B gene and protein overexpression occurs in cancer cells. Second, the depletion of FAM111B suggests a decelerated rate of cell proliferation and migration (14%), and increased apoptosis (1.4-fold). Conversely, overexpression of FAM111B resulted in a marked reduction in apoptosis (3-fold) and increased cell migration by 27 %, howbeit, no evidence of increased proliferation. Furthermore, Y621D FAM111B mutant cells showed reduced expression of FAM111B, decreased apoptosis (1.1-fold), cellular invasion (24%), and indicates an increase in cell proliferation and migration (18 %). The proteomics data suggested wild-type FAM111B interacts with HSP7C, a molecular chaperone, which alongside BAG3 and BCL2 to minimise apoptosis. Similarly, Y621D's interaction with G3V3W4, a component of the 20S proteasome complex involved in the proteolytic degradation of damaged proteins, may suggest the rapid clearance of this mutant protein.