A somatic mosaicism in the <i>G6PD</i> gene inducing a late onset chronic non‐spherocytic hemolytic anemia
Authors: Lucile Couronné, Gérard Tertian, Audrey Boutron, Véronique Picard, Hakim Ouled‐Haddou, Patricia Hughes, Olivier Hermine, Claude Préhu, Gil Tchernia, Loïc Garçon
Journal: American Journal of Hematology (2017)

With more than 400 million people affected, G6PD deficiency is the most common human red cells enzyme defect. More than 140 mutations have been identified. Localized on chromosome Xq28, the G6PD gene encodes the first enzyme of the pentose phosphate pathway. The active form is a homodimer or a tetramer and is widely expressed in all cells. Because of the absence of mitochondria in red cells, this enzymatic pathway represents the sole way to produce NADPH in erythrocytes and to preserve a constant stock of reduced glutathione, the major source of reducing power against oxidative stress.1 G6PD deficiency typically leads to an acute intravascular hemolysis occurring when red cells are exposed to an increased oxidative stress such as infection, drugs, or the classical fava bean (Vicia faba) intake. However, in rare patients, very low enzymatic level induces the class I G6PD deficiency according to the WHO classification, ie, a chronic non-spherocytic hemolytic anemia. These cases are sporadic and correspond to de novo germinal mutations occurring recurrently in “hot spots,” mostly in the exon 10 of the G6PD gene, implicated in the dimer formation and the stability of the active enzyme.2 These sporadic mutations correspond to single amino-acid substitutions or in frame deletions, but no null mutation has been reported yet, probably because such mutation would be lethal; indeed, a minimal residual G6PD activity is essential during embryogenesis.3 We report here the case of 65 years old Caucasian man referred in 1993 for hemolytic anemia. No personal or familial history of anemia was reported. Nine and five years before, the hemoglobin (Hb) level and the mean corpuscular volume (MCV) were normal. At diagnosis, the anemia was moderate (Hb 119 g/L), macrocytic (MCV 104 fL), and regenerative (reticulocyte count: 550 G/L) with hemolytic features. Platelets and leucocytes counts were normal. No spleen enlargement was noted. With a follow-up of 17 years, we noticed a progressive worsening of the anemia, requiring 8-16 packed red cell transfusions per year. Concomitantly, macrocytosis increased up to 144 fL despite acid folic intake. Several bone marrow examinations ruled out any dysplastic changes and repeated cytogenetics analyses showed no emerging clone. He died at the age of 82 from heart failure. At diagnosis, the first screening tests were negative for various acquired or constitutional causes of hemolysis, including immune hemolytic anemia, paroxystic nocturnal hemoglobinuria (PNH), membrane red cells disorders, or hemoglobinopathies. Finally, dosage of G6PD activity in red cells displayed repeatedly very low levels from 0.6 to 1.5 UI/g of Hb (normal range 5.3-7.9). Sequencing the G6PD gene from peripheral blood revealed the presence of a single nucleotide substitution at the junction between exon and intron 10 (Figure 1A, upper). This substitution (G to A) led to the abolition of a splicing donor site, resulting in a shorter protein with aberrant C-terminus. A, Presence of a mutation (G to A) at the junction between exon 10 and intron 10 in the G6PD gene in hematopoietic cells obtained from peripheral blood (upper). This mutation was not present in skin fibroblasts (lower). B, RFLP showing that the BAN I restriction enzymes was able to cleave the PCR products from control DNA or fibroblasts, but failed to cleave the DNA of hematopoietic cells obtained from peripheral blood, because of the abolition of the restriction site induced by the mutation. C, Measurement of the G6PD activity in skin fibroblasts, granulocytes, lymphocytes, and red cells from the patient in comparison to normal control showing a drastic decrease of the activity restricted to the patient's hematopoietic cells, but a normal level in his skin fibroblasts. While occurring at the germline level, it is known that severe mutations in the G6PD gene can lead to two distinct situations: in the worst case, the residual level is not sufficient for a normal embryogenesis and such mutation would be lethal since G6PD activity, as a source of reducing power against oxidative stress, is essential during embryogenesis.3 In the best case (ie, a sufficient level of activity in non-hematopoietic cells), such mutation would induce a chronic anemia from birth that should remain steady. Given the surprisingly late onset of anemia and the further dramatic worsening of hematological parameters over follow-up in our patient, we therefore hypothesized that the mutation occurred somatically in hematopoietic cells. We sequenced the G6PD gene in skin fibroblasts and could not detect the mutation (Figure 1A, lower). Since the mutation abolished a BAN I restriction site, we confirmed this observation using RLFP after PCR amplification of the exon 10–intron 10 junction (Figure 1B). Enzyme activities were then measured in both cell types and were found normal in skin fibroblasts, but nearly undetectable in all the blood cells we tested, including red cells and leukocytes (Figure 1C). Our report is the first description of a somatic mosaicism involving the G6PD gene in the hematopoietic cells. One explanation could be that the mutation was a mark of a clonal hematopoiesis in a context of an underlying myelodysplastic syndrome (MDS). Indeed, acquired mutations in genes implicated in red cell physiology, including red cell membrane skeleton,4 hemoglobin synthesis, or structure,5 have been long recognized in MDS. However, little is known about red cell enzymes activities in MDS and no mutation in the corresponding genes, restricted to the hematopoietic lineages and leading to acquired alterations in their function or expression has been reported yet. In this view, we checked whether any mark of MDS was detectable in our patient. Sequential bone marrow aspirations and cytogenetics studies failed to detect any dysplastic changes or clonal evolution during the follow-up. High-throughput genomic studies have recently revealed existence of clonal hematopoiesis of indeterminate potential (CHIP) in apparently healthy persons. CHIP is defined by the presence of detectable somatic clonal mutations in genes recurrently mutated in hematologic malignancies but without known hematologic malignancy, cytopenias, or dysplastic hematopoiesis.6 We therefore investigated whether the acquired G6PD mutation could be associated with CHIP in our patient by performing a comparative WES analysis between in vitro-cultured progenitor hematopoietic cells and skin fibroblasts (cf. Supporting Information data). We confirmed the presence of the G6PD mutation only in the hematopoietic cells but could not detect any additional somatic variants, including mutations classically associated with CHIP. Of note, variant allele frequency of G6PD mutation was close to 100% (178 over 179 reads), which was in accordance with the very low enzyme level observed in red cells. Altogether, these data do not support the presence of a clonal “myelodysplastic-like” hematopoiesis in our patient. One can therefore suggest an alternative mechanism by which progressive extrinsic selection of a non-malignant mutated clone would result in the late appearance of the phenotype and progressive worsening of the hemolytic anemia, as described in PNH. Nothing to report. Additional Supporting Information may be found in the online version of this article. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.