Objective: To analyze the correlation between genetic variants of PRF1 and expression level of perforin and granzyme B protein, and further determine the relationship between PRF1 gene variants and cytotoxic T lymphocyte/natural killer (CTL/NK) cell function in famililal hemophagocytic lymphohistiocytosis (FHL2).
Methods: Eight children of FHL2 (P1-P8) after treatment, as well as parents and siblings of P1-P5 were included, and thirty healthy children came for physical examination were designated as controls. PRF1, Unc13D, STX11, STXBP2, RAB27A, LYST, SH2D1A, BIRC4 exons were amplified by PCR and followed by direct sequencing. Bioinformatics analysis of mutant PRF1 was performed by ExPASy online system. Perforin and granzyme B expression on cytotoxic lymphocyte was detected by flow cytometry.
Results: ① Three 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. ② Comparing to control group, perforin expression of CD8(+) T cells and natural killer (NK) cells of P1, F1, B1, P2, M2 and B2 decreased significantly, but there was no significant difference between two groups in terms of granzyme B expression.
Conclusions: R4C/R33H compound heterozygous mutation and V50L heterozygous mutation all cause lower expression of perforin on CTL/NK cells, and may be causative mutations for familial hemophagocytic lymphohistiocytosis.
目的: 了解穿孔素1(PRF1）基因突变型的噬血细胞性淋巴组织细胞增生症（HLH）患儿穿孔素和颗粒酶B的表达水平，初步探讨PRF1基因突变与患儿免疫细胞功能和临床表现的关系。
方法: 将8例（例1~8）治疗后HLH患儿、5例（例1~5）患儿的父母及同胞纳入研究，以30名门诊体检正常健康儿童作为正常对照；采用PCR法分段扩增PRF1、Unc13D、STX11、STXBP2、RAB27A、LYST、SH2D1A、BIRC4基因并直接测序；通过ExPASy网站在线分析系统进行PRF1蛋白构象生物信息学分析；采用流式细胞术检测细胞毒T淋巴细胞（CD8+ T细胞）和NK细胞穿孔素和颗粒酶B的表达。
结果: ①8例患儿中有3例存在PRF1外显子编码区杂合错义突变：例1为复合杂合错义突变R4C和R33H，其父、兄也存在相同突变；例2为杂合错义突变V50L，其母、弟也存在相同突变；例3为杂合错义突变R489W，其父不存在R489W，其母未参与检测，推测其突变来自母亲。例1、2、3可明确诊断为家族性HLH第2亚型（FHL2）。②例1及其父、兄以及例2及其母、弟的外周血CD8+ T细胞穿孔素阳性率（0~1.48%）和NK细胞的穿孔素阳性率（8.69%~32.10%）较正常对照组明显减少，但两组间颗粒酶B表达未见明显差异。
结论: R4C和R33H复合杂合突变以及V50L杂合突变均可导致CD8+ T淋巴细胞和NK细胞穿孔素表达减少，为FHL的致病突变。