Hereditary nonspherocytic hemolytic anemia caused by red cell glucose-6-phosphate isomerase (GPI) deficiency in two Portuguese patients: Clinical features and molecular study

Abstract

Glucose-6-phosphate isomerase (GPI) deficiency cause hereditary nonspherocytic hemolytic anemia (HNSHA) of variable severity in individuals homozygous or compound heterozygous for mutations in GPI gene. This work presents clinical features and genotypic results of two patients of Portuguese origin with GPI deficiency. The patients suffer from a mild hemolytic anemia (Hb levels ranging from 10 to 12.7 g/mL) associated with macrocytosis, reticulocytosis, hyperbilirubinemia, hyperferritinemia and slight splenomegaly. Genomic DNA sequencing revealed in one patient homozygosity for a new missense mutation in exon 3, c.260G > C (p.Gly87Ala), and in the second patient compound heterozygosity for the same missense mutation (p.Gly87Ala), along with a frameshift mutation resulting from a single nucleotide deletion in exon 14, c.1238delA (p.Gln413Arg fs*24). Mutation p.Gln413Arg fs*24 is the first frameshift null mutation to be described in GPI deficiency. Molecular modeling suggests that the structural change induced by the p.Gly87Ala pathogenic variant has direct impact in the structural arrangement of the region close to the active site of the enzyme.

Introduction

Glucose-6-phosphate isomerase (GPI, EC 5.3.1.9) is a homodimeric enzyme that catalyses the interconversion of glucose-6-phosphate (G6P) and fructose-6-phosphate (F6P), in the second reaction step of the Embden–Meyerhof glycolytic pathway [1].

The enzyme is present ubiquitously in most organisms and expressed in all tissues, which has several physiological consequences in addition to its essential role in carbohydrate metabolism. GPI belongs to the moonlighting family of proteins having multiple functions/activities [2]. Molecular cloning and sequencing revealed that GPI share the same sequence to the protein known as neuroleukin (NLK), a neurotrophic factor that mediates differentiation and survival of embryonic spinal and sensory neurons [3], [4]. GPI has been shown to work as autocrine motility factor (AMF), a tumor-secreted cytokine which stimulates cell migration and metastasis in an autocrine manner in various tumor cells [5], [6], [7]. GPI also shares sequence homology with the differentiation and maturation inducer for human myeloid leukemia HL-60 cells to terminal monocytic cells which indicates that GPI and maturation factor (MF) also share a common biological function, regulating differentiation and proliferation of human myeloid leukemic cells [2], [8]. It was also found that chronic arthritis spontaneously developed by the K/BxN T cell receptor-transgenic mouse, with many features of human rheumatoid arthritis disease, is initiated by T cell recognition of GPI enzyme [9]. Moreover, immunization with human recombinant GPI protein induced arthritis in several mice models [10], [11], [12]. Recent literature also suggests a positive correlation between anti-GPI autoantibody and the arthritis disease in humans [13].

GPI deficiency (OMIM: 172400) is the second most frequent erythroenzymopathy in glycolysis and since the first report of the disease [14] about fifty cases have been reported throughout the world [1], [15]. Deficiency of the enzymatic activity occurs in individuals homozygous or compound heterozygous for GPI gene mutations and affects mostly erythrocytes causing hereditary nonspherocytic hemolytic anemia (HNSHA). Diagnosis is based on determination of the GPI activity in the red blood cells by enzyme quantitative assay. The major clinical features include variable degrees of jaundice, slight-to-moderate splenomegaly, an increased incidence of gallstones, and mild to severe anemia that is normochromic in most of the cases [1]. In severe cases GPI deficiency was associated with hydrops fetalis and neonatal death [16]. Few patients present with neuromuscular dysfunctions defined by muscle weakness and mental retardation [17].

The gene encoding GPI is located on chromosome 19q13.1 [18], contains 18 exons [19], and the cDNA of 1.9 kb translates a protein of 558 amino acids. The molecular characterization of GPI deficient variants shows that the gene defects are mostly missense mutations leading to protein instability or negatively influence the enzyme catalytic activity [1], [15]. Until now 34 GPI pathogenic variants have been documented [1], [20], [21] (http://www.biobase-international.com), including 28 missense, three nonsense, two splicing and one recently described frameshift mutation (submitted).

A Portuguese GPI deficient patient was previously reported with hemolytic anemia associated with hyperbilirubinemia and splenomegaly, showing severe neurological impairment [22], however, at that time, molecular analysis could not be performed. In this work we present the clinical features and genotypic analysis of two additional unrelated Portuguese patients with GPI deficiency.