Vives Corrons J L, Zarza R, Aymerich J M, Boixadera J, Carrera A, Colomer D, Corbella M, Castro M, Crespo J M, Del Arco A, Erkiaga S, Font L, González I, Juncá J, Lausin A, Manrubia E, Martín Núñez G, Murga M J, Oliva E, Pérez de Mendiguren B, Pujades M A, Remacha A, Rovira A, Villegas A
Hospital Clinic i Provincial, Laboratorio Central de Hematología, Barcelona.
Sangre (Barc). 1997 Oct;42(5):391-8.
G6PD deficiency is the most frequent enzymopathy-producing genetic polymorphism in humans. Up to now, over 400 putative variants of G6PD have been distinguished on the basis of biochemical characterization of the deficient enzyme. Analysis of the G6PD gene has made possible a precise classification of the G6PD molecular variants by identification of about 80 different point mutations causing much of the phenotypic heterogeneity. In the Spanish population, the analysis of G6PD has led to the identification of 15 different point mutations that underlay the phenotypic heterogeneity of G6PD previously reported by biochemical analysis. The purpose of the study has been to identify the genetic mutation responsible of the G6PD deficiency and to improve the knowledge of its genetic homogeneity.
From 50 Spanish males with G6PD deficiency 34 came from out consultation and 16 from the Spanish Study Group on Red Cell Pathology (GEHBTA-Eritropatología) The methods employed included screening of prevalent mutations by ER-PCR, SSCP-PCR, genetic segmentation and biochemical characterization of the deficient enzyme.
In 31 cases the mutations were characteristic of the four most frequent polymorphic variants found in Spain (G6PD A-376G/202A, G6PD Mediterranean 563T G6PD Union 1360T and G6PD Seattle 344C). Since these mutations either create or abolish a specific site recognized by a restriction endonuclease (RE), they can be rapidly detected by RE digestion of a PCR-amplified product (PCR-RE). In patients where none of these mutations were present (17 cases), the G6PD gene was subjected to PCR single-strand conformation polymorphism (PCR-SSCP) analysis combined with direct PCR-sequencing. By using this procedure, 9 new mutations have been identified, five of them have been also found in other geographical areas and were associated with favism (G6PD A-376G/968C, G6PD Santamaria 376G/542T, G6PD Aures 143C and G6PD Chatham 1003A) or chronic haemolytic anaemia (G6PD Tomah 1153C). The other four mutations are unique and not reported so far: Three of them are associated with favism (G6PD Málaga 542T, G6PD Murcia 209G and G6PD Valladolid 406T) and one with chronic haemolytic anaemia (G6PD Madrid 1155G). The remaining eight cases are under study.
The present study confirms the marked genetic heterogeneity of G6PD deficiency in Spain and demonstrate that the PCR-RE analysis is an easy tool for rapid diagnosis of the molecular defect in subjects with the common forms of G6PD deficiency. Furthermore the fact that G6PD A-376G/202A is the most common variant within Spanish population and the finding of G6PD Aures 43C and G6PD Santamaría 76G/542T, who are polymorphic in Algeria is consistent with a significant gene flow from Africa to Europe through Spain.
葡萄糖-6-磷酸脱氢酶（G6PD）缺乏症是人类中最常见的导致酶病的基因多态性。到目前为止，基于缺陷酶的生化特征，已鉴别出400多种G6PD的假定变体。对G6PD基因的分析通过鉴定约80种导致大部分表型异质性的不同点突变，使得对G6PD分子变体进行精确分类成为可能。在西班牙人群中，对G6PD的分析已导致鉴定出15种不同的点突变，这些点突变是先前通过生化分析报道的G6PD表型异质性的基础。本研究的目的是鉴定导致G6PD缺乏症的基因突变，并增进对其基因同质性的了解。
50名患有G6PD缺乏症的西班牙男性，其中34名来自外部会诊，16名来自西班牙红细胞病理学研究组（GEHBTA-红细胞病理学）。所采用的方法包括通过酶切反应-聚合酶链反应（ER-PCR）、单链构象多态性聚合酶链反应（SSCP-PCR）、基因分型以及缺陷酶的生化特征分析来筛查常见突变。
在31例病例中，突变是西班牙发现的四种最常见多态性变体的特征（G6PD A-376G/202A、G6PD地中海型563T、G6PD联合型1360T和G6PD西雅图型344C）。由于这些突变要么产生要么消除一种被限制性内切酶（RE）识别的特定位点，因此可以通过对聚合酶链反应扩增产物（PCR-RE）进行RE消化来快速检测。在不存在这些突变的患者（17例）中，对G6PD基因进行聚合酶链反应单链构象多态性（PCR-SSCP）分析并结合直接聚合酶链反应测序。通过使用该程序，已鉴定出9种新突变，其中5种在其他地理区域也有发现，并且与蚕豆病（G6PD A-376G/968C、G6PD圣玛丽亚型376G/542T、G6PD奥雷斯型143C和G6PD查塔姆型1003A）或慢性溶血性贫血（G6PD托马型1153C）相关。其他4种突变是独特的，迄今为止尚未见报道：其中3种与蚕豆病相关（G6PD马拉加型542T、G6PD穆尔西亚型209G和G6PD巴利亚多利德型406T），1种与慢性溶血性贫血相关（G6PD马德里型1155G）。其余8例正在研究中。
本研究证实了西班牙G6PD缺乏症存在明显的基因异质性，并表明PCR-RE分析是快速诊断常见形式G6PD缺乏症患者分子缺陷的简便工具。此外，G6PD A-376G/202A是西班牙人群中最常见的变体，以及在阿尔及利亚具有多态性的G6PD奥雷斯型43C和G6PD圣玛丽亚型76G/542T的发现，与通过西班牙从非洲到欧洲的显著基因流动一致。
