Clinicaland Molecular Analysis Of HIGM And Ipex

Part one: Analysis of Clinical and Molecular Characteristics of HIGM in 12 Patients from 12 Unrelated Chinese FamiliesObjective: To analyze the genes encoding the CD40L, CD40, AID, UNG, NEMO and CD40L, CD40 protein expression in 12 Chinese patients from 12 unrelated families with the hyper-IgM phenotype. To detect the relatitonship among lymphocyte sybsets of Treg, Th1 and Th17 freqency and autoimmunity in HIGM. Methods: The CD40L, CD40, AID, UNG, NEMO gene mutations were screened through direct sequencing of exon and cDNA specific polymerase chain reaction (PCR) products. Flow cytometry was used to determine the CD40L and CD40 expression on activated T lymphocytes. Treg, Th1 and Th17 freqency were detected by FCM in patient with HIGM including the petients after bone marrow transplantation. Results: In the present study, molecular defects involved in the HIGM in 12 patients were investigated. We identified 8 distinct CD40L mutations, 6 of which had not been previously described. All 8 patients with CD40L mutation showed no production of CD40L protein on activated T cells. The frequency of regulatory T cells decresed when compared with healthy control (1.265±0.4801 N=6 VS 2.718±0.3963 N=12 P=0.04), so the proportion of Th1/Treg also decreased (21.39±14.64 N=6 VS 10.50±2.596 N=12). Besides, the frequency of Th17 also trend to dcreased. Two out of the eight genetically defined patients received umbilical cord blood stem cell transplantation from unrelated donor and achieved clinical remission. The expression of CD40L on the PBMC restored. The Treg and Th17 cells increased. Conclusion: We demonstrated 8 XHIM patients with CD40L mutations, six of wich were novel mutations. All of the mutations led to null protein expression of CD40L. The decreased Treg frequency and proportion of Th1/Treg may associate with antoimmunity in XHIM patients. Part two: Analysis of Clinical and Molecular Characteristics of IPEX in 3 Patients from 3 Unrelated Chinese FamiliesObjective To investigate variations in FOXP3 gene and its expression in male children presented with IPEX phenotype. Methods Five male children presented with early-onset severe enteropathy, rash, with or without insulin-dependent diabetes mellitus (IDDM) were subjected to the detection of FOXP3 expression on CD4+CD25+ T cells and frequency of regulatory T cells (Tregs) by flow cytometry. The cDNA and relative coding exons including promoter region of FOXP3 gene were amplified by PCR and sequenced. The candidate mutation sites were compared with those of 100 healthy controls to exclude polymorphism. Results Genetic analysis revealed 1 insertion and 2 missense mutations out of the total 5 children. P1 presented classical IPEX clinical phenotype. A novel frameshift insertion occurred in exon 11 (p.N361KfsX1) which led to complete abrogation of Tregs. P2 showed incomplete IPEX form and carryied a missense mutation in exon 11 (p.M370I) with slightly increased frequency of Tregs, whereas P3 presented relatively mild classical manifestations of IPEX and was demonstrated a previously reported missense mutation in exon 10 (p.F324L) with decreased frequency of Tregs. Conclusions: Three cases of IPEX presented as different clinical form with 2 novel mutations were identified from three unrelated families in China. Our limited data indicates somewhat correlation between genotype and phenotype of this disease as well. Part three: the relationships between suppression function of natural mutated FOXP3 and IPEX clinical characterizationsObjective To analyze the relationship between the suppression function of natural mutated FOXP3 and IPEX clinical characterizations.Methods FOXP3 gene CDS region was amplified by PCR with the template of normal cDNA and then cloned into pReceiver-12 vector. The mutated FOXP3 expression vectors were constructed by site-directed mutagenesis. The pReceiver-12 vector and IL-2 promoter luciferase reporter vector were cotransfected to Jurkat T cells and stimulated with 50 ng/ml of phorbol myristate acetate and 500 ng/ml ionomycin for 6–7 hrs before lysing cells and analyzed by means of dual luciferase assay normalized with Renilla luciferase activity according to the manufacturer's protocol. Results Eleven mutated foxp3 protein were constructed and expressed (N326Kfs1X; V408M; A384T; R337Q; F324L; 251delE; L242P; R146W; P187L; T108M; M370I). The mutated FOXP3 of 251delE, N326Kfs1, L242P, M370I and R146W absolutely lost suppression function and the others partly lost. The analysis of clinical characterization shows no relationship with the FOXP3 suppression function except that the severe mutation 251delE, N326Kfs1 always showed triad of IPEX and severely suppression function defect. Conclusions: Severe FOXP3 mutations are associated with severe suppression function defect and triad of IPEX. The clinical phenotype was determined by gene bachground and environment and FOXP3 gene analysis was the final diagnosis.