Research On The Intron Mutations Of Gene PRF 1 And UNC13D In Children With Hemophagocytic Lymphohistiocytosis

Objective:To investigate the intronic mutations of the genes PRF1 and UNC13D in Chinese pediatric patients with hemophagocytic lymphohistiocytosis (HLH), and to explore the relationship between the possible mutations and HLH.Methods:48 pediatric patients with HLH from Guangxi, Guangzhou, Wuhan, Suzhou in China, as well as 50 healthy children as a control group were recruited in this study. The introns of PRF1 and UNC13D gene were genotyped by polymerase chain reaction (PCR), which was followed by direct sequencing.Results:(1)Four single nucleotide polymorphisms (SNPs) were revealed in the introns of PRF1 among 48 cases, including g.5179de1G (rs55974750) in 25 cases (52.08%), g.5452C>T (rs3758562) in 46 cases (95.83%), g.5936T>C (rs6480460) in 47 cases (97.91%), g.7745T>C (rs80019657) in 10 cases (20.83%). All the SNPs above had no statistical difference in allelic frequencies between the cases and the controls (P>0.05). Simultaneously, other two SNPs g.7494C>T(rs10999426) and g.7473G>A (rs10999427) were detected only in two healthy children (4%).Their allelic frequencies between the case group and the control group had no statistical difference (P>0.05). Haplotype analysis showed there was a pairwise linkage disequilibrium between rs10999426 and rs10999427 (D-1, r2=1), but the distribution of the haplotypes had no statistical difference between the case group and the control group.(2) Twenty SNPs were detected in the introns of UNC13D in this study, including g.5527G>A (rs3744011) in 29 cases(60.4%), g.5556C>T (rs3744010) in 8 cases (16.7%), g.630C>T (rs9903200) in 10 cases (20.8%), g.6433G>A (rs3744009) in 10 cases (20.83%),g.6585C>G (rs3744008) in 7 cases (14.6%),g.6628G>A (rs142996967) in 2 cases (4.2%),g.6942G>A (rsl 1870883) in 8 cases (16.7%),g.6983C>T (rs3744006) in 35 cases (72.9%),g.8657T>G (rs8067076) in 29 cases(60.4%),g.882C>A (rs6501838) in 2 cases (4.2%),g.9072G>A (rs7208603) in 2 cases (4.2%),g.9358C>T (rs7226310) in 1 case (2.1%),g.9381C>T (rs201406035) in 2 cases (4.2%),g.13899A>G (rs3744026) in 26 cases (54.2%),g.14141T>C (rs3744024) in 27 cases (56.3%),g.14803G>A (rs17581728) in 7 cases (14.6%),g.15529C>T (rs7212635) in 28 cases (58.3%),g.16806C>T (rs113796451) in 5 cases (10.4%),g.19188C>T (rs2290768) in 28 cases (58.3%),g.1939C>G (rs2290769) in 9 cases (18.8%).The allelic frequency of rs8067076 in case group was statistically different from the control group (P=0.046<0.05).Haplotype analysis showed there was a pairwise linkage disequilibrium between rs6501838 and rs7208603 (D-1, r2=1), but their allelic frequencies and the distribution of the haplotypes had no statistical difference between the case group and the control group (P>0.05).The rest 17 SNPs had no statistical differences in allelic frequencies between the case group and the control group (P>0.05). Simultaneously,4 mutations in deep intron 1were detected in 4 cases relatively, which were IVS1+>T238C、IVS1+394C>T、 IVS1+466G>A,IVS1+498C>A. In addition, one mutation IVS29+62A>T was detected in 6 cases. They were all heterozygosis ones which were not detected in the control group. The allelic frequency of IVS29+62A>T in the case group was statistically different from the control group (P=0.034<0.05)Conclusion:(1) Four SNPs in the introns of PRF1 gene and nineteen SNPs in the introns of UNC13D gene were not associated with HLH possibly.(2) There was a pairwise linkage disequilibrium between rs 10999426 and rs 10999427, but the A-T haplotype might not play a protective role in the development of HLH. However, one SNP in gene UNC13D (rs8067076) was likely to be protective factor related to HLH.(3) One pathological intron mutation reported previously (IVS1+394C>T) and 4 novel intronic mutations (TVS1+238C>T,IVS1+466G>A, IVS1+498C>A,IVS29+62A>T) in the deep introns of gene UNC13D were only discovered in the HLH group, which were low in incidence. However, it is necessary to make deep research on the relationship of the latter ones with HLH.