Search For The Potential "Second-hit" Mechanism Underlying The Onset Of Familial Hemophagocytic Lymphohistiocytosis Type 2 By Whole-exome Sequencing Analysis

Objective:Searching for the reason why siblings with the same perforin mutation have different disease status, one was ill while the other was completely healthy, and exploring the other possible mechanism of the disease.Methods:Investigate the patient of adult familial hemophagocytic lymphohistocytosis type 2 (FHL2) and their family members, collect clinical data and complete detection of molecular biology, immunology and whole-exome sequencing, then the data of whole-exome sequencing were analyzed by bioinformatics for underlying additional mutations associated with FHL2.Results:After collecting clinical data, testing NK cell activity, EBV-DNA, perforin, HLH next generation sequencing, the patient was diagnosed with adult FHL2, and the patient's parents, brother and son were analyzed PRF1 by Sanger sequencing, then the pedigree was depicted. The FHL2 patient and her asymptomatic brother carried the same heterozygous missense (c.916G>A) and frameshift mutation (c.65de1C) in PRF1. Through whole-exome sequencing, heterozygous spontaneous somatic mutations within FASTKD3, HOXA10 and SVEP1 were found exclusively in the proband. Germline gene mutations:PCDH18, CKD11B, MAGEC1, NOS1, and PPP1R14BP3 were detected in an autosomal recessive model; MLL3, PCDH18, ANKRD36, HNRNPC, PCMTD1, and MAGEC1 were detected in an autosomal dominant model. Notably, PCDH18 was the common gene screened by the two model, and the proband was exclusively detected with a homozygous missense mutation (S1006L) in the PCDH18 gene, whereas all her family members were found to have a heterozygous mutation (S1006L) of the PCDH18 gene. Moreover, the mutation was predicted to be probably damaging.Conclusions:This study is the first to perform whole-exome sequencing analysis to search for the potential "second-hit" mechanism underlying the onset of FHL2. We found a novel heterozygous mutation combination (c.916G>A and c.65delC) in PRF1. The exclusive detection of a homozygous germline mutation in the PCDH18 gene (c.3017C>T) in the proband, strongly supports the presence of a "second-hit" germline mutation besides PRF1 that might be potentially an important mechanism for triggering the onset of FHL2.Objective:To preliminary explore the mechanism ofPCDH18 mutation screened by the whole exome sequencing of FHL2 pedigrees in the onset of FHL2Methods:PyMOL software was used to predict the effects of PCDH18-S1006L mutation on the three-dimensional structure of the protocadherin-18, and the total Gibb's free energy change that resulted from the mutation was calculated. The Wild-type and mutated (S1006L) PCDH18 overexpression plasmids were constructed, and transfected into 293 and HepG2 cells to investigate the effects of mutations on the transcriptional level and translational levels, and then transfected into CTLL-2 cells to observe the effects of mutations on the activation and apoptosis of the cells.Immunohistochemistry was used to evaluate the PCDH18 protein expression in proband's tissues samples and her asymptomatic brother and normal controls.Results:We built the three-dimensional structure of the protocadherin-18, both wild-type and mutated PCDH18, and found that the mutation caused replacement of Serine with Leucine, which changed the amino acid from hydrophilic into hydrophobic and hydrogen bonds may be reduced to two.. The mutant domain is likely to be strongly altered, causing the protein structural unstable. The total Gibb's free energy change (A AG) that resulted from the mutation was 8.08kcal/mol (>2kcal/mol), which also showed that the protein structure was unstable. Thewild-type, mutated (S1006L) PCDH18or negative control plasmids were transfected into 293 and HepG2 cellsto observe the effects of mutations on the transcriptional level and translational levels, and results demonstrated that there was no significant difference in the mRNA expression in the 293 cells or in the HepG2 cells, while the S1006L mutant protein expression reduced significantly compared with WT controls. Thewild-type, mutated or negative control plasmids were transfected intoCTLL-2 cells to investigate the effects of mutations on functions, and the data indicated that the wild-type PCDH18exerted an inhibitory effect on activation and increased the apoptosis rates of CTLL-2 cells with or without rIL-2 compared with empty vector. The mutated type, however, could counteract these two effects. Immunohistochemistry data demonstrated that the expression level of PCDH18 protein was much lower in the proband than in her asymptomatic brother and normal controls.Conclusions:Through functional research, we found that S1006L mutation could cause the protein structural unstable, the protein expression reduced, and might enhance the activation and apoptosis arrest of cytotoxic T lymphocytes, led to the onset of FHL2 cooperating with PRF1 mutation.Objective:To study the relationship among onset age of late-onset FHL, genetic defects and EBV-DNA.Methods:Clinical data of clinical suspicion HLHpatients were collected, combined with clinical course, HLH next generation sequencing results to identify late-onset FHL; then their familymembers were investigated and pedigrees were drawn, and EBV-DNA were tested in the four late-onset FHL family. R language was used to draw the picture depicted the relationship among onset age of late-onset FHL, genetic defects and EBV-DNA.Results:Four late-onset FHL patients were identified by combining analysis of clinical data, HLH-2004 regimen effect, and HLH next generation sequencing results. Then family investigations were carried out and pedigrees were depicted. R language was used to drawn a figure to observe the relationship of onset age of late-onset FHL, genetic defects and EBV-DNA; the figure indicated that patients with similar genetic defects, the higher EBV-DNA is,the younger onsetageis, and vice versa; those who have similar EBV-DNA, the stronger the degree of genetic defect is, the smaller onsetageis, and vice versa. Thus, the onset age of late-onset FHL patients is determined by both the degree of genetic defects and EBV-DNA.Conclusions:The onset age of late-onsetFHL depends on the degree of their genetic defects and EBV infection, the stronger degree of genetic defect and higher EBV-DNA, the smaller the age of onset; while moderate genetic defects or relatively lower EBV-DNA or simultaneously with the both, late-onset of the disease will occur. Part of asymptomatic persons who have genetic defect, without external triggers, they may not develop FHL for the whole life.