驗認創新的p110δ 和Btk 基因的突變/多型性及分析其衍生蛋白質特性來闡明他們在原發性B細胞免疫缺陷的致病

Abstract

此計劃的目的為尋找在未知原因的原發性B 細胞免疫缺陷病童的致病機轉是否為p110δ 基因突變所造成。但因根據外國資料, 原發 性B 細胞免疫缺陷的病童大部(約85%)為 Btk 基因突變所引起, 所以應須先行檢查Btk 基因； 不過, 因為目前台灣尚未有方便且可 靠的分子生物學方法來在原發B 細胞免疫缺 陷病童中確定診斷為XLA 疾病﹐也無XLA 病童的Btk 基因突變型本土資料。在國科會 補助下(NSC91-2314-13-002-189/NSC92- 2314-B-002-211) ﹐ 我們已建立用genomic DNA-PCR and direct sequencing 的方法來從 患有原發B 細胞免疫缺陷疾病的病童中篩檢 及確定其Btk 基因突變位置及多型式從而來 證實診斷為XLA 疾病。目前, 從所篩檢病童 中, 已成功確立4 個病童其Btk 基因突變位 置及型式以及其家族的基因篩檢。 同時, 亦 已建立用RT-PCR and direct sequencing 的方 法來篩檢在Btk 基因檢查確定無突變但仍不 知原因的原發B 細胞免疫缺陷病童的PI3 kinase p110δ基因是否有多型性及突變以證 實p110δ基因的突變可能是某些未知原因的 原發性B 細胞免疫缺陷的致病機轉。目前, 在3 位病人發現其p110δ基因有一整段 Exon14 刪除。這段被刪除的基因位於PI3 kinase 的accessory domain, 它在PI3 和 PI4- kinase 是conserved 的,其功能為受脢質的呈 現。

This study is to explore the possibility that some patients with defects in B-cell immunodeficiency of unknown etiology might have mutations in PI3 kinase p110δ. Although, according to the foreign data, the majority of primary B-cell immunodeficiency (about 85%) is due to Btk mutation. We need to exclude the possibility of Btk gene mutation before we check the p110δgene. However, at present, there is still no convenient and reliable molecular method for definite diagnosis of XLA in patients with primary B-cell immunodeficiency in Taiwan. There is no Btk gene mutation and polymorphism data for Taiwanese XLA patients, either. Under this grant support of NSC (NSC91-2314-13-002- 189/NSC92-2314-B-002-211), we have established the methods to screen the patients with primary B-cell defect by genomic DNAPCR and direct sequencing analysis to identify the Btk gene mutations and then establish the diagnosis of the XLA patients. Until now, we have already successfully identified 4 patients of primary B-cell immunodeficiency with Btk gene mutation and screened their family members. We also have established the RTPCR and direct sequencing methods to screen the potential p110 δ gene polymorphism and mutation in the remaining primary B-cell immunodeficiency patients without Btk mutations. Until now, we have identified three patient with 2 different sizes of his 3rd PCR amplified fragment. After cloning and comparison with human p110δsequence, the nucleotides 2,007-2,150 were completely missed in the shorter band. These missed nucleotides coded for the entire Exon 14(inreading frame deletion). The missed Exon 14 is located in PI3K accessory domain which is conserved in all PI3 and PI4-kinase with the function for substrate presentation.