Correlation Of Genotype, WASp Expression And Phenotype Of Wiskott-Aldrich Syndrome And Impact Of WASp Gene Mutation On Cellular Immune Function

Part one Analysis of Genotype, WASp Expression and Phenotype Correlation of Wiskott-Aldrich SyndromeObjective:To establish the largest database of Wiskott-Aldrich syndrome (WAS) in China and to further determine the correlation among genotype, phenotype and long-term outcome in this cohort.Methods:Blood samples of 21 WAS patients were collected from February 2011 to September 2012. Expression of WAS protein (WASp) was quantitatively assessed by flow cytometry (FCM) in peripheral blood mononuclear cells (PBMCs). WASp gene was amplified and directly sequenced to analyze mutations. Clinical data of 81 WAS patients diagnosed from April 2000 to September 2012 were followed up, and the correlation among genotype, phenotype and long-term outcome were analyzed.Results:Of the 81 male patients cohort,9 were assigned a score of 2 with X-linked thrombocytopenia (XLT) phenotype,56 received a score of 3 or 4 with classic Wiskott-Aldrich syndrome (WAS) phenotype,13 progressed to a score of 5 after developing autoimmune diseases (12) or malignancies (1), and the score of the remaining 3 were unknown. WASp expression of 64 patients in PBMCs detected by FCM showed absence of WASp in 40 cases, reduced expression with single peak in 18 cases and reduced expression with dual peaks in 6 cases. A total of 60 unique mutations, including 20 novel mutations and 8 hotspots were identified in the total of 78 patients. There were 17 missense mutations,17 nonsense mutations,5 insertions,16 deletions,19 splice anomalies and 4 complex mutations. Among the patients with XLT phenotype,8 were missense mutations, and the remaining one was undetermined; 5 showed reduced expression of WASp with single peak,2 showed absence of WASp expression and 2 were not tested. Among the patients with classic WAS phenotype,31 were null (nonsense, insertion or deletion) mutations,15 were splice anomalies,6 were missense mutations,2 were complex mutations, and the remaining 2 were undetermined; 32 showed absence of WASp expression,10 showed reduced expression of WASp with single peak,2 showed reduced expression of WASp with dual peaks and 12 were not tested. Among the patients scored 5,4 were nonsense mutations (including 2 patients who had spontaneous revertant mutations),4 were splice anomalies,3 were missense mutations,1 was deletion and 1 was complex mutation; 6 showed absence of WASp expression,4 showed reduced expression of WASp with dual peaks and 3 showed reduced expression of WASp with single peak.57 patients hadn’t received hematopoietic stem cell transplantation (HSCT), long-term survival rates were lower in classic WAS patients compared to XLT patients (P=0.18), lower in non-missense mutation patients compared to missense mutation patients (P=0.26,0.55), and lower in WASp negative patients compared to WASp positive patients (P=0.08,0.20).Conclusions:The phenotype of classic WAS or milder XLT and long-term outcome are potentially influenced by the effect of these defects on gene transcription and translation. Patients with missense mutations allowing expression of mutated WASp and those with splice anomalies which result in generation of multiple products including normal WASp, present the attenuated XLT phenotype and show better prognosis; classic WAS patients with complete absence of WASp are strong candidates for hematopoietic stem cell transplantation.Part two Impact of WASp Gene Mutation on Cellular Immune FunctionObjective:To analyze the impact of WASp gene mutation on the frequency of Th1, Th2, Th17 helper/effector T cells and regulatory T (Treg) cells in Wiskott-Aldrich syndrome (WAS) patients.Methods:Blood samples of 14 WAS patients,23 age-matched healthy donors (AMHDs) and 14 adult healthy donors (AHDs) were collected from March 2011 to July 2012. The frequency of Th1, Th2, Th17 and Treg cells accounted for CD4+T cells separately in peripheral blood mononuclear cells (PBMCs) were detected by flow cytometry (FCM). The imbalance of CD4+T cells subgroups was analyzed and the impact of WASp gene mutation on CD4+T cells immune function was determined.Results:The average ages of 14 WAS patients and 23 AMHDs were 1.97 years and 1.89/2.41 years separately (P=0.912/0.677), and the 14 AHDs was 30.79 years. The frequency of each subgroup accounted for CD4+T cells separately in PBMCs detected by FCM showed WAS patients’ Th1/CD4+T cell was 18.28±11.08, equal to AMHDs’(15.89±6.34, P=0.478), but significantly lower than AHDs’(31.86±11.67, P=0.004); patients’Th2/CD4+T cell was 1.20±1.02, equal to AMHDs’(0.78±0.46, P=0.177) and AHDs’(0.68±0.58, P=0.112); patients’Th17/CD4+T cell was 1.47±0.76, equal to AMHDs’(0.99±0.51, P=0.058) and AHDs’(1.96±0.85, P=0.123); patients’ Treg/CD4+T cell was 2.88±1.19, equal to AHDs’ (3.55±0.68, P=0.081), but significantly lower than AMHDs’(4.80±1.37, P=0.003).Conclusions:Lower frequency of Th1 cells in WAS patients than AHDs maybe related to the age factor, while frequency of Treg cells significantly lower than AMHDs appears to indicate the defects of Treg cells generation and function, reflecting that WASp gene mutation possibly play a critical role in the prone to autoimmune diseases. However, more in-depth research should be performed in future to confirm it and clarify its mechanism.