G6PD Potenza: A Novel Pathogenic Variant Broadening the Mutational Landscape in the Italian Population

Abstract

Background: Glucose 6 phosphate dehydrogenase (G6PD) is a rate-limiting enzyme of the pentose phosphate pathway. The loss of G6PD activity in red blood cells increases the risk of acute haemolytic anaemia under oxidative stress induced by infections, some medications, or fava beans. More than 200 single missense mutations are known in the G6PD gene. A 41-year-old woman with a family history of favism coming from the Basilicata region (Italy) was evaluated at our hospital for G6PD abnormalities. Methods: DNA was extracted from a peripheral blood sample and genotyped for the most common G6PD pathogenic variants (PVs). Positive results obtained by Restriction Fragment Length Polymorphism (RFLP), as per practice in our laboratory, were then reconfirmed in Sanger sequencing. Results:RFLP analysis highlighted a variant compatible with the G6PD Cassano variant. Confirmatory testing by Sanger unexpectedly identified a novel variant: c.1357G>A, p.(Val453Met) (NM_001360016.2); the same variant was found in the patient's mother. In silico models predicted a deleterious effect of this variant at the protein level. The novel G6PD variant was named "G6PD Potenza" on the basis of the patient's regional origin. Conclusions: This case describes a novel G6PD variant. It also highlights how the Sanger sequencing technique still represents an indispensable confirmatory standard method for variants that could be misinterpreted by only using a "first-level" approach, such as the RFLP. We stress that the evaluation of clinical manifestations in G6PD-deficient patients is of primary importance for the classification of each new G6PD mutation, in agreement with the new WHO guidelines.

Keywords: G6PD deficiency; Potenza; Sanger sequencing; acute haemolytic anaemia.

Figure 1

The sequencing results of the G6PD gene analysis. The blue arrows indicate the position of the G6PD Cassano, instead the red arrows indicate the position of the novel nucleotide change identified in this study. The patient resulted as a heterozygote for the c.1357 G>A variant in the G6PD gene; her mother was a heterozygote for the same variant. The figure demonstrates the proximity of the new variant to G6PD Cassano in a heterozygous patient shown as an example.

Figure 2

Capillary electrophoresis analysis of the enzymatic digestions of Potenza and Cassano PCR products. The figure shows the results obtained from the tape station capillary electrophoresis of the enzymatic digestions, performed with samples of the wild-type control (panel A), the patient (B), heterozygous G6PD Cassano (panel C), and the patient’s mother (panel D). Clear different profiles emerged from the wild-type and Potenza or Cassano analyses. However, the presence of two independent fragment patterns belonging to the presence of Potenza and Cassano variants is not evident. The altered pattern was also identified in the patient’s mother’s sample (panel D).

Figure 3

Schematic representation of restriction sites of NlaIII enzyme, obtained with NEBcutter V2.0.

Figure 4

A schematic representation of the alteration in the G6PD protein functional domains. The variants Cassano (c.1347G>C p.Q449H) and Potenza (c.1357G>A p.Val453Met) are highlighted in black in the protein domains. The crystallographic structure of the human G6PD enzyme, assembled from https://swissmodel.expasy.org, (accessed on 10 August 2024).