Department of Hematogenetics, National Institute of Immunohematology (Indian Council of Medical Research), Mumbai, India
Department of Hematogenetics, National Institute of Immunohematology (Indian Council of Medical Research), Mumbai, India.
J Clin Pathol. 2019 Jan;72(1):81-85. doi: 10.1136/jclinpath-2018-205420. Epub 2018 Oct 18.
Glucose-6-phosphate isomerase (GPI) deficiency is an autosomal recessive genetic disorder causing congenital haemolytic anaemia (CHA). Diagnosis of GPI deficiency by the biochemical method is unpredicted. Molecular diagnosis by identifying genetic mutation is the gold standard method for confirmation of disease, but causative genes involved in CHA are numerous, and identifying a gene-by-gene approach using Sanger sequencing is also cumbersome, expensive and labour intensive. Recently, next-generation targeted sequencing is more useful in the diagnosis of unexplained haemolytic anaemia. We used targeted next-generation sequencing (NGS) clinical panel for diagnosis of unexplained haemolytic anaemia in two Indian patients which were pending for a long time. All possible causes of haemolytic anaemia were found within normal limit. NGS by clinical exome panel revealed homozygous novel missense mutation in exon 12, c.1009G>A (p.Ala337Thr) in both patients. We further confirm by measuring red blood cell GPI activity in the patients and showed deficiency whereas parents were having intermediate activity. c.1009G>A mutation was also confirmed by Sanger sequencing of exon 12 of GPI gene. The structural-functional analysis by bioinformatics software like Swiss PDB, PolyPhen-2 and PyMol suggested that this pathogenic variant has a direct impact on the structural rearrangement at the region near the active site of the enzyme. This rapid and high-performance targeted NGS assay can be configured to detect specific CHA mutations unique to an individual defect, making it a potentially valuable method for diagnosis of unexplained haemolytic anaemia.
葡萄糖-6-磷酸异构酶（GPI）缺乏症是一种常染色体隐性遗传疾病，可导致先天性溶血性贫血（CHA）。生化方法诊断 GPI 缺乏症的预测性较差。通过识别基因突变进行分子诊断是疾病确诊的金标准方法，但涉及 CHA 的致病基因众多，采用 Sanger 测序进行逐个基因的方法也很繁琐、昂贵且劳动强度大。最近，新一代靶向测序在不明原因溶血性贫血的诊断中更有用。我们使用靶向新一代测序（NGS）临床面板诊断了两名印度患者的不明原因溶血性贫血，这些患者的诊断一直悬而未决。所有可能导致溶血性贫血的原因都在正常范围内。临床外显子组面板的 NGS 发现两名患者在第 12 外显子中均存在纯合新错义突变，c.1009G>A（p.Ala337Thr）。我们进一步通过测量患者的红细胞 GPI 活性来证实该突变导致 GPI 缺乏，而父母则具有中间活性。通过对 GPI 基因第 12 外显子的 Sanger 测序也证实了 c.1009G>A 突变。生物信息学软件（如 Swiss PDB、PolyPhen-2 和 PyMol）的结构功能分析表明，该致病性变异直接影响酶活性部位附近区域的结构重排。这种快速高效的靶向 NGS 检测可以配置为检测个体缺陷特有的特定 CHA 突变，使其成为诊断不明原因溶血性贫血的一种潜在有价值的方法。
