Screening And Clinical Characteristics Analysis Of PRF1 And UNC13D Gene Mutations In Children With Hemophagaoytic Lymphohistiocytosis

ObjectiveTo understand the mutations of PRF1 and UNC13 D genes in children with hemophagocytic lymphohistiocytosis.Analyze the characteristics of clinical data,and improve the understanding of the diagnosis and treatment of hemophagocytic lymphohistiocytosis.Method(1)39 children with HLH who were diagnosed in the pediatric department of the First Affiliated Hospital of Guangxi Medical University from August 1,2016 to January 31,2019 were selected as the research objects.The peripheral blood of children was collected using EDTA anticoagulation tubes,and the clinical data of the children were collected.(2)The exons of PRF1 and UNC13 D genes were amplified,using the polymerase chain reaction(PCR)to amplify the exons of PRF1 and UNC13 D genes.Then the PCR products were subjected to agarose gel electrophoresis to detect the accuracy of PCR results.Finally,it was sequenced and compared with the human PRF1 and UNC13 D gene sequences of the gene bank.(3)The mutations in 39 children wereanalyzed,and software predicted the effect of mutation site on its protein function,initially explores the gene variants in PRF1 and UNC13 D.(4)Retrospective analysis of clinical data of the child,including demographic characteristics,pathogenic factors,clinical symptoms,signs,Laboratory indicators and treatment effects.Result(1)The proportion of males and females in the 39 children was 0.95:1;the median age of males was 1.83 years,and the median age of female children was1.46 years old.There was no significant difference between males and females,P=0.512.The main age of onset of HLH in children is concentrated within three years of age,which accounts for 64% of the children;children under ten years of age are fewer,accounting for only 8%.(2)A total of five point mutations and one deletion mutation were detected in the PRF1 gene,including two missense mutations,three synonymous mutations.The two missense mutations were heterozygous mutation on exon 2c.10C>T(p.Arg4Cys)and heterozygous mutation on exon 2 c.178C>T(p.Pro60Ser).The three synonymous mutations were heterozygous mutation on exon 2 c.216C>T(1 case),heterozygous mutation on exon 3(13 cases)c.822C>T,homozygous mutation(24 cases)and heterozygous mutation(13cases)on exon 3 c.900C>T.One deletion mutation was a c.35-38del(TCCT)nucleotide deletion.(3)UNC13D gene detected ten point mutations,of which six point mutations were missense mutations and four point mutations were synonymous mutations.The six missense mutations were heterozygous mutation on exon 13 c.1228A>C(p.Ile410 Leu,1 case),heterozygous mutation on exon 22 c.2044C>G(p.Arg682 Gly,1 case),heterozygous mutation c.2131G>A on exon23(p.Val711 Met,1 case),heterozygous mutation(5 cases)and homozygous mutation(4 cases)on exon 27 c.2599A>G(p.Lys867Glu),homozygous mutation on exon 30 c.2897G>A(p.Arg966 Gln,1 case),and heterozygous mutation one exon 31 c.3079G>A(p.Gly1027 Arg,1case).Four synonymous mutations were heterozygous mutation on exon 11(16 cases)and homozygous mutation(6 cases)c.888G>C,heterozygous mutation on exon 19 c.1744C>T(1case),heterozygous mutation(12 cases)and homozygous mutation(1 case)on exon 21 c.1977C>T,heterozygous mutation(17 cases)and homozygous mutation(6 cases)on exon 32 c.3198A>G.(4)PolyPhen-2 predicted that PRF1 p.Pro60Ser(P60S)and UNC13 D p.Arg682Gly(R682G)might be pathogenic;PROVEAN predicted that UNC13 D p.Gly1027Arg(G1027R)might be pathogenic.(5)39 patients(100%)had fever before admission,30 cases(77%)had respiratory symptoms,8 cases(21%)had digestive symptoms,32 cases(82%)had hepatomegaly,28 Case(72%)with splenomegaly,and 19 cases(49%)with superficial lymphadenopathy.There were 30 cases(77%)with two or three lines of hematopenia,33 cases(85%)with decreased NK cell activity,and 38 cases(97%)with serum ferritin ?500 g/L.Twenty-six patients(67%)were diagnosed with infection-related HLH,20 patients(51%)were infected with virus,and 17 patients(44%)were infected with Epstein-Barr virus.38 patients of the 39 children with HLH underwent EBV nucleic acid quantification,and 29 patients were positive for EBV,with a positive rate of 76%(29/38).(6)There was no statistically significant difference in efficacy between the mutant group and the non-mutant group(P=0.196),and it could not be considered that gene mutations(including missense mutations and deletion mutations)have an impact on the prognosis of the children.(7)After 2-3 weeks of treatment,Hb(t=1.764,P=0.044),PLT(Z=2.143,P=0.016)and SF(Z=2.184,P=0.015)in the remission group were all higher than those in the non-remission group,and the difference was statistically significant.Suggesting that Hb,PLT and SF levels after 2-3 weeks of treatment could predict the prognosis.(8)Among the 37 patients,25 patients had fever before VP-16,and were higher than 38 °C.The 25 patients were changed according to the body temperature after 24 hours of VP-16.Divided into two groups,the treatment effect of the body with temperature dropped below 37.6 °C(including 37.6 °C)was better than the child with temperature still higher than 37.6 °C,the difference was statistically significant(P = 0.003).Conclusion(1)About 41% of 39 children are find to have missense or deletion mutations of PRF1 and UNC13 D genes.The incidence of UNC13 D mutations is higher than that of PRF1;c.2599A>G is more common in UNC13 D mutations,and c.10C>T is more common in PRF1 mutations.(2)This study find a novel deletion mutation of c.35-38del(TCCT)in the PRF1 gene of one patient.(3)Among the missense mutations of PRF1 and UNC13 D genes in the children of FHL,single allele heterozygous mutations are the most common.(4)It has not been found that carrying PRF1 and UNC13 D gene mutations(including missense mutations and deletion mutations)have an impact on the prognosis of children.(5)The main age of onset of HLH in children is within 3 years old,and EBV infection is the most common trigger.(6)After 2-3 weeks of treatment,the levels of hemoglobin,platelet andserum ferrit have a certain evaluation effect on the treatment of children.(7)The change of body temperature after 24 hours of VP-16 has a certain predictive effect on the prognosis of children.