Associação entre polimorfismos do gene FCN3 da via das lectinas do complemento com o pênfigo foliáceo

Orientador: Angelica Beate Winter BoldtCo-orientadora: Fabiana Antunes de AndradeMonografia (Bacharelado) - Universidade Federal do Paraná. Setor de Ciências Biológicas. Curso de Graduação em Ciências Biológica

Ensino de Genética : validação de um modelo didático sobre formação, pareamento, recombinação e segregação de cromossomos rearranjados

Orientadora: Araci Asinelli-Luz.Coorientadora: Angelica Beate Winter Boldt.Trabalho de Conclusão de Curso (Licenciatura) - Universidade Federal do Paraná. Setor de Ciências Biológicas. Curso de Graduação em Ciências Biológicas

Investigação integrativa das vias de morte celular no pênfigo

Orientadora: Profa. Dra. Angelica Beate Winter BoldtCoorientadores: Profa. Dra. Jennifer Elisabeth Hundt e Prof. Dr. Gabriel Adelman CippolaTese (doutorado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Genética. Defesa : Curitiba, 31/03/2022Inclui referênciasResumo:Abstract

Genetic Associations and Differential mRNA Expression Levels of Host Genes Suggest a Viral Trigger for Endemic Pemphigus Foliaceus

The long search for the environmental trigger of the endemic pemphigus foliaceus (EPF, fogo selvagem) has not yet resulted in any tangible findings. Here, we searched for genetic asso ciations and the differential expression of host genes involved in early viral infections and innate antiviral defense. Genetic variants could alter the structure, expression sites, or levels of the gene products, impacting their functions. By analyzing 3063 variants of 166 candidate genes in 227 EPF patients and 194 controls, we found 12 variants within 11 genes associated with differential suscepti bility (p < 0.005) to EPF. The products of genes TRIM5, TPCN2, EIF4E, EIF4E3, NUP37, NUP50, NUP88, TPR, USP15, IRF8, and JAK1 are involved in different mechanisms of viral control, for example, the regulation of viral entry into the host cell or recognition of viral nucleic acids and proteins. Only two of nine variants were also associated in an independent German cohort of sporadic PF (75 patients, 150 controls), aligning with our hypothesis that antiviral host genes play a major role in EPF due to a specific virus–human interaction in the endemic region. Moreover, CCL5, P4HB, and APOBEC3G mRNA levels were increased (p < 0.001) in CD4+ T lymphocytes of EPF patients. Because there is limited or no evidence that these genes are involved in autoimmunity, their crucial role in antiviral responses and the associations that we observed support the hypothesis of a viral trigger for EPF, presumably a still unnoticed flavivirus. This work opens new frontiers in searching for the trigger of EPF, with the potential to advance translational research that aims for disease prevention and treatment

Investigação integrativa das vias de morte celular no pênfigo

Orientadora: Profa. Dra. Angelica Beate Winter BoldtCoorientadores: Profa. Dra. Jennifer Elisabeth Hundt e Prof. Dr. Gabriel Adelman CippolaTese (doutorado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Genética. Defesa : Curitiba, 31/03/2022Inclui referênciasResumo: Pênfigo é um grupo de doenças autoimunes bolhosas que causam dolorosas lesões cutâneas. As doenças pertencentes a este grupo são caracterizadas pela acantólise e pela produção de autoanticorpos contra proteínas de adesão celular. Apesar da relevância patogênica dos autoanticorpos IgG no processo acantolítico, os mecanismos exatos que levam os queratinócitos à morte permanecem desconhecidos. A apoptose tem sido sugerida como uma via que apresenta um papel importante em algumas dermatoses como no pênfigo foliáceo (PF) e no pênfigo vulgar (PV), antes ou após a perda de adesão celular. Dados os mecanismos subjacentes pouco compreendidos que levam à morte de queratinócitos no pênfigo, revisamos a literatura para moléculas e/ou características envolvidas nas 12 vias de morte celular descritas pelo Comitê de Nomenclatura em Morte Celular, ocorrendo em pacientes com pênfigo, linhagens celulares ou em miniórgãos de pele tratados com soros ou IgG de pacientes com pênfigo ou em modelo animal de pênfigo. Encontramos 61 estudos mencionando 97 moléculas envolvidas em vias de morte celular no pênfigo. A maioria dessas investigações se concentrou apenas na apoptose, mas quatro estudos recentes, utilizando ensaios de TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) e/ou microscopia eletrônica, desqualificaram essa via como um evento prévio de acantólise. Em seguida, investigamos em PF endêmico, variantes genéticas de genes cujos produtos estão envolvidos nas diferentes vias de morte celular. Neste estudo, encontramos 10 variantes associadas (p<0.005) pertencentes a seis vias de morte celular: apoptose; morte celular imunogênica; necroptose; necrose; partanatos e piroptose. Também investigamos em PV e nas formas endêmica e esporádica de PF, variantes genéticas em genes nucleares cujos produtos codificam subunidades da cadeia respiratória mitocondrial ou seus fatores de montagem. Nesta análise, encontramos associação de uma variante do gene do componente essencial do complexo da cadeia respiratória I (subunidade A1 da ubiquinona oxidorredutase) e outra do complexo IV (fator de montagem do citocromo c oxidase heme A) exclusivamente com PF endêmico (p<0.005). Além disso, realizamos uma caracterização ultraestrutural do miniórgão de pele humano utilizado para avaliar as proteínas candidatas, seguida de sua identificação nas biópsias de PF endêmico e esporádico. No miniórgão de pele humana, encontramos o receptor 1 do componente C5a (C5a) do complemento (C5aR1) aumentado (p<0.0001), assim como em biópsias de PF endêmico e esporádico (p<0.0001). Ainda, encontramos C5a aumentado no soro de pacientes de PF e PV (p<0.0001). Para avaliar uma das vias de morte celular, investigamos a presença de armadilhas extracelulares de neutrófilos (NETs) no soro de pacientes de PF e PV, mas não observamos diferenças significativas. Uma validação funcional dos resultados encontrados nesta tese pode contribuir para o entendimento da etiologia do pênfigo e o desenvolvimento de novas drogas e alvos terapêuticos para a doença. Novas investigações relevantes sobre o pênfigo e as vias de morte celular, além da apoptose, devem ser feitas para superar os desafios de compreensão da etiopatogênese do pênfigo.Abstract: Pemphigus is a group of bullous autoimmune diseases that cause painful skin lesions. The diseases belonging to this group are characterized by acantholysis and the production of autoantibodies against cell adhesion proteins. Despite the pathogenic relevance of IgG autoantibodies in the acantholytic process, the exact mechanisms that lead keratinocytes to death remain unknown. Apoptosis has been suggested as a pathway playing an important role in some dermatoses such as pemphigus foliaceus (PF) and pemphigus vulgaris (PV), before or after loss of cell adhesion. Given the poorly understood underlying mechanisms that lead to keratinocyte death in pemphigus, we reviewed the literature for molecules and/or features involved in the 12 cell death pathways described by the Nomenclature Committee on Cell Death, occurring in patients with pemphigus, in cell lines, or in skin mini-organs treated with sera or IgG from patients with pemphigus or in an animal model of pemphigus. We found 61 studies mentioning 97 molecules involved in cell death pathways in pemphigus. Most of these investigations focused only on apoptosis, but four recent studies using TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assays and/or electron microscopy disqualified this pathway as an event prior to acantholysis. Next, we investigated in endemic PF, variants of genes whose products are involved in different cell death pathways. In this study, we found 10 associated variants (p<0.005) belonging to six cell death pathways: apoptosis; immunogenic cell death; necroptosis; necrosis; partanates and pyroptosis. We also investigated, in PV and in the endemic and sporadic forms of PF, variants in nuclear genes whose products encode subunits of the mitochondrial respiratory chain or their assembly factors. In this analysis, we found a variant in the gene of an essential component of respiratory chain complex I (ubiquinone oxidoreductase A1 subunit) and another of complex IV (cytochrome c oxidase heme assembly factor A) exclusively associated with endemic PF (p<0.005). In addition, we performed an ultrastructural characterization of the human skin mini-organ used to evaluate candidate proteins, followed by their identification in biopsies of endemic and sporadic PF. We found the complement C5a receptor 1 (C5aR1) increased (p<0.0001) in human skin mini-organ, as well as in biopsies of endemic and sporadic PF (p<0.0001). Furthermore, the C5a component of the complement (C5a) was increased in serum of PF and PV patients (p<0.0001). To assess one of the cell death pathways, we investigated the presence of neutrophil extracellular traps (NETs) in serum of PF and PV patients, but no significant differences were observed. A functional validation of the results found in this thesis may contribute to the understanding of the etiology of pemphigus and the development of new drugs and therapeutic targets for the disease. Further relevant investigations regarding pemphigus and cell death pathways beyond apoptosis must be conducted to overcome the challenges of understanding the etiopathogenesis of pemphigus

Marked to Die-Cell Death Mechanisms for Keratinocyte Acantholysis in Pemphigus Diseases

Pemphigus is a group of blistering autoimmune diseases causing painful skin lesions, characterized by acantholysis and by the production of autoantibodies against, mainly, adhesion proteins. We reviewed the literature for molecules and/ or features involved in the 12 cell death pathways described by Nomenclature Committee on Cell Death, taking place in pemphigus patients, cell lines, or human skin organ cultures treated with sera or IgG from pemphigus patients or in pemphigus mouse models, and found 61 studies mentioning 97 molecules involved in cell death pathways. Among the molecules, most investigated were pleiotropic molecules such as TNF and CASP3, followed by FASL and CASP8, and then by FAS, BAX, BCL2, and TP53, all involved in more than one pathway but interpreted to function only within apoptosis. Most of these previous investigations focused only on apoptosis, but four recent studies, using TUNEL assays and/or electron microscopy, disqualified this pathway as a previous event of acantholysis. For PV, apoptolysis was suggested as a cell death mechanism based on pathogenic autoantibodies diversity, mitochondrial dysfunction, and p38 MAPK signaling. To answer those many questions that remain on cell death and pemphigus, we propose well-controlled, statistically relevant investigations on pemphigus and cell death pathways besides apoptosis, to overcome the challenges of understanding the etiopathology of pemphigus diseases

Association of a new <i>FCN3</i> haplotype with high ficolin-3 levels in leprosy

<div><p>Leprosy is a chronic inflammatory disease caused by <i>Mycobacterium leprae</i> that mainly affects the skin and peripheral nervous system, leading to a high disability rate and social stigma. Previous studies have shown a contribution of genes encoding products of the lectin pathway of complement in the modulation of the susceptibility to leprosy; however, the ficolin-3/<i>FCN3</i> gene impact on leprosy is currently unknown. The aim of the present study was to investigate if <i>FCN3</i> polymorphisms (rs532781899: <i>g</i>.<i>1637delC</i>, rs28362807: <i>g</i>.<i>3524_3532insTATTTGGCC</i> and rs4494157: <i>g</i>.<i>4473C>A</i>) and ficolin-3 serum levels play a role in the susceptibility to leprosy. We genotyped up to 190 leprosy patients (being 114 (60%) lepromatous), and up to 245 controls with sequence-specific PCR. We also measured protein levels using ELISA in 61 leprosy and 73 controls. <i>FCN3</i> polymorphisms were not associated with disease, but ficolin-3 levels were higher in patients with <i>FCN3 *2B1</i> (C<i>ins</i>A) haplotype (p = 0.032). Median concentration of ficolin-3 was higher in leprosy <i>per se</i> (26034 ng/mL, p = 0.005) and lepromatous patients (28295 ng/mL, p = 0.016) than controls (18231 ng/mL). In addition, high ficolin-3 levels (>33362 ng/mL) were more common in leprosy <i>per se</i> (34.4%) and in lepromatous patients (35.5%) than controls (19.2%; p = 0.045 and p = 0.047, respectively). Our results lead us to suggest that polymorphisms in the <i>FCN3</i> gene cooperate to increase ficolin-3 concentration and that it might contribute to leprosy susceptibility by favoring <i>M</i>. <i>leprae</i> infection.</p></div

Sparking Fire Under the Skin? Answers From the Association of Complement Genes With Pemphigus Foliaceus

Skin blisters of pemphigus foliaceus (PF) present concomitant deposition of autoantibodies and components of the complement system (CS), whose gene polymorphisms are associated with susceptibility to different autoimmune diseases. To investigate these in PF, we evaluated 992 single-nucleotide polymorphisms (SNPs) of 44 CS genes, genotyped through microarray hybridization in 229 PF patients and 194 controls. After excluding SNPs with minor allele frequency &lt;1%, out of Hardy–Weinberg equilibrium in controls or in strong linkage disequilibrium (r2 ≥ 0.8), 201 SNPs remained for logistic regression. Polymorphisms of 11 genes were associated with PF. MASP1 encodes a crucial serine protease of the lectin pathway (rs13094773: OR = 0.5, p = 0.0316; rs850309: OR = 0.23, p = 0.03; rs3864098: OR = 1.53, p = 0.0383; rs698104: OR = 1.52, p = 0.0424; rs72549154: OR = 0.55, p = 0.0453). C9 (rs187875: OR = 1.46, p = 0.0189; rs700218: OR = 0.12, p = 0.0471) and C8A (rs11206934: OR = 4.02, p = 0.0323) encode proteins of the membrane attack complex (MAC) and C5AR1 (rs10404456: OR = 1.43, p = 0.0155), a potent anaphylatoxin-receptor. Two encode complement regulators: MAC-blocking CD59 (rs1047581: OR = 0.62, p = 0.0152) and alternative pathway-blocking CFH (rs34388368: OR = 2.57, p = 0.0195). One encodes opsonin: C3 (rs4807895: OR = 2.52, p = 0.0239), whereas four encode receptors for C3 fragments: CR1 (haplotype with rs6656401: OR = 1.37, p = 0.0382), CR2 (rs2182911: OR = 0.23, p = 0.0263), ITGAM (CR3, rs12928810: OR = 0.66, p = 0.0435), and ITGAX (CR4, rs11574637: OR = 0.63, p = 0.0056). Associations reinforced former findings, regarding differential gene expression, serum levels, C3, and MAC deposition on lesions. Deregulation of previously barely noticed processes, e.g., the lectin and alternative pathways and opsonization-mediated phagocytosis, also modulate PF susceptibility. The results open new crucial avenues for understanding disease etiology and may improve PF treatment through additional therapeutic targets

Linkage disequilibrium between <i>FCN3</i> single nucleotide polymorphisms.

<p>LD was calculated based on the data for controls and leprosy patients. Black squares represent high LD and white low LD as measured by the correlation coefficient (r²) between sites, which values are shown inside of the squares. SNP identifiers are indicated on the abscissas.</p