- 學位論文
利用毛細管電泳快速鑑定α-球蛋白基因套數及其在甲型海洋性貧血症之臨床應用
Rapid identi␣cation of the copy number of a␣-globin genes by capillary electrophoresis analysis
摘要
甲型海洋性貧血症是人類常見的單一基因遺傳疾病之一，東南亞地區包括台灣更是高盛行率地區，國人甲型海洋性貧血症帶兩個α-球蛋白鏈基因缺失率約有5%左右。病人因紅血球血色素中α-球蛋白鏈的合成發生問題，使得紅血球的體積變小且每個紅血球內的血色素含量降低，造成小球性及低血色素性貧血。正常情況下，每個紅血球母細胞內應有四個α-球蛋白鏈基因，以αα/αα表示，包含製造α-球蛋白鏈的兩個HBA2和兩個HBA1基因。如果紅血球內α-球蛋白鏈製造不足，則會因嚴重程度不等而產生不同型的甲型海洋性貧血症。依照α-球蛋白鏈基因缺失，臨床上可分為以下幾大類：(1)單一α-球蛋白鏈基因缺失，以-α/αα表示，這些帶基因缺陷患者臨床上沒有任何症狀，一般的血液學檢查也無異樣。(2)兩個α-球蛋白鏈基因缺失，以--/αα或-α/-α表示，這些帶基因缺陷患者臨床上也通常不會出現明顯症狀，只有血液學檢查可出現輕微貧血。(3)三個α-球蛋白鏈基因缺失，以--/-α表示，這些帶基因缺陷患者有中度的貧血，又稱為血色素H症。(4)四個α-球蛋白鏈基因完全缺失，以--/--表示。這些患兒在胎兒時期因紅血球內完全無法製造α球蛋白鏈，所以在子宮內即產生很嚴重的溶血、缺氧及全身水腫的現象，形成所謂的胎兒水腫而死亡，這同時也會對母體健康造成極大危害。因此，甲型海洋性貧血症的產前診斷至關重要。傳統上，我們利用全血球計數、家族史、含鐵狀態及血色素電泳來幫助診斷甲型海洋性貧血症，晚近更發展利用聚合酶鏈鎖反應方法做基因型的測定。在本研究中，我們利用甲型海洋性貧血症患者α-球蛋白鏈的基因缺損大多以較大段基因缺失為主這個特性發展出一個新的診斷方法。以聚合酶鏈鎖反應方法合併毛細管電泳方式偵測α-球蛋白鏈基因的基因套數，來快速診斷甲型海洋性貧血症之α-球蛋白鏈基因缺失及其分型。 本研究利用已知的體染色體上的KRIT基因及性染色體上的CYBB基因之基因套數作為α-球蛋白鏈基因套數分析之內部參考基因 。我們收集高雄醫學大學院附設醫院66位甲型海洋性貧血症患者檢體，包括10位患者有單一α-球蛋白鏈基因缺失，其中6位為-α3.7及4位-α4.2之基因缺失型態； 48位患者有兩個α-球蛋白鏈基因缺失，其中27位為東南亞型、16位為菲律賓型、5位為泰國型之基因缺失型態；7位患者有三個α-球蛋白鏈基因缺失之血色素H症；以及一位患兒有四個α-球蛋白鏈基因缺失之嚴重胎兒水腫；另外有46位正常人為正常對照組。所有甲型海洋性貧血症患者均以聚合酶鏈鎖反應方法測定α-球蛋白鏈基因型作為確診。 在正常的男性血球細胞中，HBA1/KRIT1，HBA2/KRIT1，HBA1/CYBB及HBA2/CYBB 基因套數之比例分別為2/2，2/2，2/1及2/1，在正常的女性血球細胞中基因套數之比例則為2/2，2/2，2/2及2/2；在帶有單一α-球蛋白鏈基因缺失的男性血球細胞中，HBA1/KRIT1，HBA2/KRIT1，HBA1/CYBB及HBA2/CYBB 基因套數之比例分別為2/2，1/2，2/1及1/1，女性血球細胞中基因套數之比例則為2/2，1/2，2/2及1/2；在帶有兩個α-球蛋白鏈基因缺失的男性血球細胞中，HBA1/KRIT1，HBA2/KRIT1，HBA1/CYBB及HBA2/CYBB 基因套數之比例分別為1/2 ，0/2，1/2 及0/2;女性血球細胞中基因套數之比例則為1/2，1/2，1/2及1/2；在帶有三個α-球蛋白鏈基因缺失血色素H症的男性血球細胞中，HBA1/KRIT1，HBA2/KRIT1，HBA1/CYBB及HBA2/CYBB基因套數之比例分別為1/2，0/2，1/1及0/1，女性血球細胞中基因套數之比例則為1/2，0/2，1/2及0/2；在帶有四個α-球蛋白鏈基因缺失之嚴重胎兒水腫患兒則無法偵測HBA1及HBA2基因套數。 本研究結果顯示，我們所建立的方法可以有效且快速正確地分析α-球蛋白鏈基因的基因套數。利用我們的方式所得到的結果與聚合酶鏈鎖反應做基因型的測定相當吻合。另外我們的新方法還能夠偵測產前診斷時胎兒組織所受之少量母體核苷酸汙染的情形，並且能夠分析出罕見複雜型多α-球蛋白鏈基因套數的患者。
並列摘要
論文英文摘要 Alpha-thalassemia is one of the most common autosomal recessive disorders in the world and characterized by a microcytic hypochromic anemia. Alpha-thalassemia has a high prevalence rate in Southeast Asia area including Taiwan with a rate of 5%. A majority of the genetic defects of α-thalassemia are deletions of the α-globin genes. Normal individuals have four functioning α-globin genes and are defined as αα/αα. According to the defects of the α-globin, the clinical phenotypes of α-thalassemia could be divided into four groups. Individuals with loss of one α-globin gene (-α/αα) have 3 functional α-globin genes. Loss of one gene constitutes a silent carrier state without any clinical symptoms. Alpha thalassemia trait occurs with loss of two α-globin genes, namely -α/-α, --/αα. Such individuals are characterized with mild to moderate microcytic anemia. Individuals with hemoglobin (Hb) H disease show deletions of three α-globin genes. Such individuals are characterized with more severe anemia and splenomegaly. Lastly, the loss of four α-globin genes causes Hb Barts hydrops in individuals, which result in intrauterine death. The initial laboratory examinations for the diagnosis of α-thalassemia include a complete blood cell count, red cell indices, iron status and hemoglobin electrophoresis. To identify the copy number of α-globin genes in α-thalassemia, we developed a novel method using multiplex polymerase chain reaction (PCR) in combination with the CE analysis. Total 66 α-thalassemia patients with α-globin gene deletions including 6 with -α3.7 and 4 -α4.2 types of one-gene deletion, 27 with Southeast Asia type (- -SEA), 16 Filipino (- -FIL) type, 5 Thai (- -THAI) types of two-gene deletions, 7 Hb H disease with three-gene deletions, and 1 Hb Barts hydrops fetalis with four-gene deletions were included in this study. 46 normal controls were also obtained at Kaohsiung Medical University Hospital. The copy number ratios of HBA1/KRIT1, HBA2/KRIT1, HBA1/CYBB and HBA2/CYBB were around 2/2, 2/2, 2/1 and 2/1 in normal male individuals, and around 2/2, 2/2, 2/2 and 2/2 in normal female individuals. The copy number ratios of HBA1/KRIT1, HBA2/KRIT1, HBA1/CYBB and HBA2/CYBB were around 2/2, 1/2, 2/1 and 1/1 in male patients, and around 2/2, 1/2, 2/2 and 1/2 in female patients with a genotype of one α-globin-gene deletion, -α/αα, α-thalassemia. The copy number ratios of HBA1/KRIT1, HBA2/KRIT1, HBA1/CYBB and HBA2/CYBB were around 1/2, 0/2, 1/2 and 0/2 in male patients, and around 1/2, 1/2, 1/2 and 1/2 in female patients with a genotype of two α-globin-gene deletions, --/αα, α-thalassemia. The copy number ratios of HBA1/KRIT1, HBA2/KRIT1, HBA1/CYBB and HBA2/CYBB were around 1/2, 0/2, 1/1 and 0/1 in patients and around 1/2, 0/2, 1/2 and 0/2 in female patients with a genotype of three α-globin-gene deletions, --/-α, α-thalassemia. There is no HBA1 and HBA2 copy number detection in a fetus with a four α-globin-gene deletions, --/--, hemoglobin Barts hydrops fetalis α-thalassemia. In the present study, we established a rapid and efficient method to measure the copy number of two α-globin genes, HBA1 and HBA2, in α-thalassemia patients, determine low level of maternal DNA contamination in the fetus specimen for prenatal diagnosis and defect of rare multiplicated α-globin genes in the individuals. The proposed method provides a rapid detection of the common α-globin gene deletions. Sixty-six α-thalassemia patients and 46 normal controls were included in the present study. The obtained results showed good correlation with those obtained by gap PCR. Moreover, a low amount of maternal cell contamination in the fetus specimen for the prenatal diagnosis of hemoglobin Barts hydrops fetalis as well as the rare multiplicated α-globin genes can be identified using this method.