Polimorfizam HLA-DR i HLA-DQ alela kod pacijenata sa pemfigus vulgarisom

Abstract

Uvod: Pemphigus vulgaris (PV) pripada grupi organ specifičnih autoimunskih oboljenja, kod koga se stvaraju autoantitela usmerena na specifične proteine (antigene) kože i sluzokoža. Autoantitela kod pemfigusa su usmerena prema komponentama međućelijskih spojeva (dezmozomima). To su dezmoglein 3 (Dsg 3), u manjoj meri dezmoglein 1 (Dsg 1) i drugi proteini. Kao posledica vezivanja cirkulišućih antitela za proteine dezmozoma dolazi do cepanja međućelijskih spojeva (akantolize) i pojave plikova (bula) na koži i sluzokožama. Autoimunski pemfigus, kao i druge autoimunske bolesti, predstavlja multifaktorsko oboljenje, rizik od nastanka bolesti zavisi od složenih interakcija gena i faktora spoljašnje sredine. U okviru naslednih faktora u etiopatogenezi pemfigusa najviše je ispitivan kompleks gena tkivne podudarnosti koji kodira sistem humanih leukocitnih antigena (HLA). HLA je najpolimorfniji deo humanog genoma. Složenost polimorfizma HLA je posledica postojanja više genskih lokusa, velikog broja alela za većinu gena i kombinacija produkata ovih alela. HLA molekuli II klase su posebno značajni jer prikazuju peptide CD4+ T-limfocitima pod čijom kontrolom je sekrecija anti Dsg 3 i anti Dsg 1 antitela od strane B-limfocita. Do sada je dokazana udruženost pojedinih HLA alela sa većim brojem autoimunskih oboljenja uključujući i pemfigus. Ciljevi istraživanja: Ciljevi ovog istraživanja su bili da se ustanovi učestalost alela i haplotipova u HLA-DR i DQ lokusima kod pacijenata sa PV u Srbiji, da se ustanovi stepen genetske sličnosti sa obolelima od PV u drugim populacijama, kao i da se ispita korelacija između genotipova, težine bolesti i koncentracije Dsg1 i Dsg3 antitela. Ispitanici i metode: Ispitivanje je izvršeno kod 72 pacijenta sa dokazanim PV. Kod ispitanika je određena alelska učestalost u HLA-DR i DQ lokusima (DRB1*, DQB1* i DQA1*). Izolacija DNK je rađena upotrebom QIAamp DNA Blood Mini Kit-a (QIAGEN, Germany). Određivanje grupa alela ispitivanih lokusa je rađeno testovima niske/srednje rezolucije, a potom su testovima visoke rezolucije određivani aleli upotrebom prajmera specifičnih za alelsku sekvencu PCR-SSP (engl. Polymerase chain reaction with sequence-specific primers) prema preporukama proizvođača testova (BAG lich Germany i Invitrogen)...

Introduction: Pemphigus vulgaris (PV) belongs to the group of organ specific autoimmune disorders, characterized by the production of pathogenic autoantibodies against keratinocyte adhesion molecules- desmogleins, mostly Desmoglein 3 (Dsg3) and 1 (Dsg1), or other proteins. As a result of bounding autoantibodies to desmosomal proteins, separation of keratinocytes occur with clinical manifestation of blisters on the skin and mucosal membranes. PV, as other autoimmunes diseases belongs to the family of polygenic disorders, the risk of developing the disease depends on interactions of endogenous genetic factors and environmental factors. Inheritance of certain human leukocyte antigen (HLA) alleles has been suggested to be the most probable predisposing factor. HLA is the most polymorphous part of the human genome. The complexity of HLA polymorphism is due to the existence of a big number of genetic loci, alleles for most of the genes and combination of these alleles. HLA II class genes are particularly important due to peptide presentation to CD4+ T-cells and consequent secretion by B- cells of anti-Dsg3 and anti-Dsg1 antibodies. So far, associations of some HLA alleles with a great number of autoimmune diseases have been shown, including pemphigus. Aims of the investigation: Aims of this study were to determine HLA-DR and HLA-DQ allelic and haplotypic frequencies in patients with pemphigus vulgaris in Serbia; to establish similarities with other PV patients in different populations; to establish correlation between genotypes, disease activity and anti-Dsg3 and anti-Dsg1 antibodies. Individuals and methods: Investigation of allelic frequency of HLA-DR and HLA-DQ was conducted on 72 patients with diagnosed PV. DNA was extracted from blood samples using the Qiagen QIAamp DNAMini Kit (Qiagen, Germany). Allele groups were determined with low/intermediary resolution tests. High resolution was performed using polymerase chain reaction sequence-specific primers (PCRSSP) according to the manufacturer’s instructions (BAG lich Germany and Invitrogen). ARLEQUIN software package, version 3.11 was applied for estimated allele and haplotype frequencies, as well as for testing Hardy–Weinberg equilibrium...