PRF1 And Expression Of Porforin And Granzyme B Of Hemphagocytic Lymphohistiocytosis

Objective: To analyze the correlation between genetic variants of PRF1 and no PRF1 mutations of HLH and expression level of perforin and granzymeB protein, and further determine the relationship between PRF1 gene variants no PRF1 mutations of HLH and cytotoxic T lymphocyte/natural killer(CTL/NK) cell function in famililal hemophagocytic lymphohistiocytosis(FHL2).Methods: (1)Eight children of FHL2 (P1-P8) after treatment, as well as parents and siblings of P1-P5 were collected from the first affiliated hospital of guangxi medical university pediatrics, and thirty healthy children came for physical examination were designated as controls. (2)PRF1?UNC13D (unc-13 homolog D)?STX11(syntaxin11)?STXBP2 (syntaxin binding protein 2)exons were amplified by PCR and followed by direct sequencing.Then they were compared with the sequences in gene bank to determine relatively nucleotide variations and amino acid variations. (3)Bioinformatics analysis of mutant PRF1 was performed by ExPASy online system. (4)Perforin and granzyme B expression on cytotoxic lymphocyte was detected by flow cytometry, and natural killer cell cytotoxic activity also be detected by flow cytometry.Results: (1)Three out of eight FHL2 children harbored heterozygous missense of PRF1 exons: P1 had compound heterozygous missense mutations(R4C and R33H) and P2 had heterozygous mutations (V50L) ,P3 had heterozygous mutations (R489W) ,which confirmed the diagnosis of FHL2. The father (F1) and younger brother (B1) of P1 also had compound heterozygous missense mutation (R4C/R33H), the mother (M2) and younger brother (B2) of P2 had V50L mutation,the father (F3) of P3 had no R489W mutation and the mother of P3 did not participate in this research ,so mutation of R4C/R33H of P1 inherited from paternal line, and V50L mutation of P2 came from maternal line, R489W mutation of P3 came from maternal line; the other 3 genes had no mutations, so can be clearly diagnosed as FHL2. (2)Comparing to control group,perforin expression of CD8+ T cells and natural killer (NK) cells of P1?1?B1?P2?M2 and B2 decreased significantly, but there was no significant difference between two groups in terms of granzyme B expression.(3)P1-P8 natural killer cell cytotoxic activity no different from control group.Conclusions: (1)R4C/R33H compound heterozygous mutation and V50L heterozygous mutation all cause lower expression of perforin on CTL/NK cells,and may be predisposing factors for familial hemophagocytic lymphohistiocytosis ; other genes had no mutation. (2) The expression level descend of perforin was detected by flow cytometry, which could help to rapid diagnosis of FHL.