分子病検査診断「症例呈示と病態解析」(5)

異常血色素およびサラセミアの遺伝子診断
−特に広範囲遺伝子欠失型の欠失断端の決定−

服　部　幸　夫*　山　城　安　啓*
　　　　　　　　　　　　　　　　　　　　　　
Gene Diagnosis of Hemoglobinopathies
−The Determination of Breakpoints for a Large Deletion−

Yukio HATTORI, MD* and Yasuhiro YAMASHIRO, PhD*

A large number of mutations leading to hemoglobinopathies(abnormal hemoglobins, thalassemias) have been discovered. Gene diagnosis for a point mutation or a deletion/insertion of a few nucleotides is now readily performed. For a large deletion, once the precise breakpoints are unraveled, the same type of deletion is promptly diagnosed by gap PCR. However, a number of new types of large deletions remain unexamined. They need meticulous Southern blot analysis and/or cloning. Here we present a new technique to determine their precise breakpoints. One of the two breakpoints needs to be first assigned within the 5-kb portion estimated by gene dosage by quantitative PCR. The other breakpoint is left unexamined. The genome DNA is digested with one of eight kinds of endonucleases and subjected to recombination with pUC18 cloning vector digested with the same endonuclease. The gap PCR is subsequently performed between the common primers of pUC18 and five arbitrary primers within the aforementioned 5-kb portion. An abnormal gap PCR product, if detected by electrophoresis, discloses precise 5’ and 3’ breakpoints after direct sequencing. This method successfully disclosed the breakpoints for two εγδβ-thalassemias, one δβ-thalassemia and one β-thalassmeia in a relatively short period. All are new mutations. This method uses neither cloning procedures nor Southern blot, but employs gene dosage estimation and PCR. Thus, it is relatively simple. The progress of the genome project facilitated analysis of any large deletions.
[Rinsho Byori 51 : 550〜555, 2003]

*Faculty of Health Sciences, Yamaguchi University School of Medicine, Ube 755-8505

【Key Words】abnormal hemoglobin(異常Hb)，α-thalassemia(αサラセミア)，β-thalassemia(βサラセミア)，gene diagnosis(遺伝子診断)，PCR

*山口大学医学部保健学科病態検査学講座(〒755-8554 宇部市南小串1-1-1)

E-mail :hattori-ygc@umin.ac.jp