Analysis of ankyrin gene mutations in hereditary spherocytosis

我们分析了36例日本遗传性球形红细胞增多症（HS）患者锚蛋白基因的所有外显子（外显子1 -外显子42）和启动子区域。从外周血白细胞中提取基因组DNA，用PCR（聚合酶链反应）扩增DNA片段，采用非ri单链构象多态性（SSCP）方法筛选锚蛋白突变。对sscp异常的DNA片段进行直接测序分析。结果发现19个锚蛋白基因突变：2个无义突变，6个错义突变，1个缺失突变（内含子中8个碱基缺失），1个内含子碱基替换，9个沉默突变。两个无义突变（E886X: GAG - TAG; K1140X: AAG - TAG）是尚未报道的新突变，可能直接参与锚蛋白缺乏，导致潜在细胞骨架蛋白组装减少。虽然6个错义突变是HS的主要缺陷还是仅仅是多态性还需要进一步的研究，但值得注意的是，有3例患者是错义突变（I1075T: ATC - ACC）的纯合子。我们发现锚蛋白基因突变是日本HS患者的病因之一，发生率可能为10 - 20%。

We analyzed all exons (exon 1 - exon 42) and promotor region of ankyrin gene in 36 Japanese patients with hereditary spherocytosis (HS). Genomic DNAs were extracted from peripheral blood leukocytes and PCR (polymerase chain reaction)-amplified DNA fragments were subjected to screen ankyrin mutations by non-RI SSCP (single strand conformation polymorphism) method. The DNA fragments showing abnormal SSCPs were analyzed by direct DNA sequencing. As the results, we found 19 ankyrin gene mutations : 2 nonsense mutations, 6 missense mutations, 1 deletional mutation (deletion of 8 bases in intron), 1 intronic base substitution, and 9 silent mutations. Two nonsense mutations (E886X : GAG - TAG ; K1140X : AAG - TAG) were novel mutations that have not been reported, and may be directly involved in ankyrin deficiency which result in reduced assembly of underlying cytoskeletal proteins. Although further investigation should be needed to clarify whether 6 missense mutations are the primary defects of HS or mere polymorphisms, it was a notable evidence that 3 patients were a homozygote of the missense mutation (I1075T : ATC - ACC). We showed ankyrin gene mutations are one of the cause of Japanese patients with HS and these incidence may be 10 - 20%.