Publication: LRBA Eksikliği Tanısında Akan Hücre Ölçerde Protein İfadesi
Abstract
Giriş: Lipopolysaccharide-responsive beige-like anchor (LRBA) inhibitör bir protein olan sitotoksik T lenfosit antijen-4 (CTLA4)'ün hücre yüzeyinde ifade edilmesinde rol oynar. Yakın zamanda tanımlanmış olan LRBA eksikliği immün yetmezlik ve otoimmüniteye yol açar. LRBA eksikliğinin tanısı mutasyon analizi ve protein ifadesinin belirlenmesi ile konur. Bu çalışmada, LRBA eksikliği olan hastalarımızda uyaranlı ve uyaransız ortamda akan hücre ölçer ile hücre içi LRBA protein ifadesi değerlendirilmiştir.Gereç ve Yöntem: Çalışmaya LRBA eksikliği olan beş hasta ve yedi sağlıklı kontrol alındı. Periferik kan mononükleer hücrelerde forbol miristat asetat ve ionomisin ile uyaranlı ve/veya uyaransız olarak LRBA ifadesi ölçüldü. Ortalama florasan yoğunluğu farkı (?MFI) hesaplandı. Bulgular: Uyaransız durumda ortalama florasan yoğunluğu farkı sağlıklı kontrollerde ortalama 24±9 iken LRBA hastalarında 4.8±2.8 olarak ölçüldü. İki hasta ve bir sağlıklı kontrolde eşzamanlı uyaransız ve uyaranlı ölçümlerin karşılaştırılması sonucu elde edilen değişime bakıldığında; uyaransız durumda ?MFI değeri Hasta-3 (H3)'de 4.8, H4'de 4.6 ve sağlıklı kontrolde 20,1 saptanırken, uyaranlı durumda bu değer H3'de 20.8, H4'de 19 ve sağlıklı kontrolde 49.6 bulundu. Sonuç: Akan hücre ölçer ile LRBA ifadesinin değerlendirilmesi hızlı ve kolay bir yöntem olup mutasyon tanılı hastalarımızda etkin ve güvenilir sonuç vermiştir.
Objective: Lipopolysaccharide-responsive beige-like anchor (LRBA) plays a role in cell surface expression of inhibitory cytotoxic T lymphocyte-associated protein-4 (CTLA-4) protein. Recently identified LRBA deficiency leads to immune deficiency and autoimmunity and is diagnosed by mutation analyses and protein expression. Herein, we quantified stimulated and unstimulated intracellular LRBA protein expression by flow cytometry in LRBA deficiency patients.Materials and Methods: Five LRBA deficient patients and seven healthy controls were evaluated. The LRBA expressions were assessed in peripheral-blood-mononuclear-cells in the presence or absence of phorbol-miristat-acetate and ionomycin stimulation. The difference in mean-fluorescence-intensity (?MFI) was calculated. Results: The differences in mean-fluorescence-intensity values of LRBA by flow cytometry were 24±9 for the healthy controls and 4.8±2.8 for the patients. ?MFIs were 20.8 for P3, 19 for P4 and 49.6 for healthy controls with stimulants and 4.8, 4.6 and 20.1 respectively without stimulants. Conclusion: As a rapid and widely available assay, flow cytometric assessment of intracellular LRBA expression has been found to be an effective and reliable method in the identification of LRBA deficiency.
Objective: Lipopolysaccharide-responsive beige-like anchor (LRBA) plays a role in cell surface expression of inhibitory cytotoxic T lymphocyte-associated protein-4 (CTLA-4) protein. Recently identified LRBA deficiency leads to immune deficiency and autoimmunity and is diagnosed by mutation analyses and protein expression. Herein, we quantified stimulated and unstimulated intracellular LRBA protein expression by flow cytometry in LRBA deficiency patients.Materials and Methods: Five LRBA deficient patients and seven healthy controls were evaluated. The LRBA expressions were assessed in peripheral-blood-mononuclear-cells in the presence or absence of phorbol-miristat-acetate and ionomycin stimulation. The difference in mean-fluorescence-intensity (?MFI) was calculated. Results: The differences in mean-fluorescence-intensity values of LRBA by flow cytometry were 24±9 for the healthy controls and 4.8±2.8 for the patients. ?MFIs were 20.8 for P3, 19 for P4 and 49.6 for healthy controls with stimulants and 4.8, 4.6 and 20.1 respectively without stimulants. Conclusion: As a rapid and widely available assay, flow cytometric assessment of intracellular LRBA expression has been found to be an effective and reliable method in the identification of LRBA deficiency.