Successful use of empagliflozin to treat neutropenia in two G6PC3-deficient children: Impact of a mutation in SGLT5

Abstract

Neutropenia and neutrophil dysfunction found in deficiencies in G6PC3 and in the glucose-6-phosphate transporter (G6PT/SLC37A4) are due to accumulation of 1,5-anhydroglucitol-6-phosphate (1,5-AG6P), an inhibitor of hexokinase made from 1,5-anhydroglucitol (1,5-AG), an abundant polyol present in blood. Lowering blood 1,5-AG with an SGLT2 inhibitor greatly improved neutrophil counts and function in G6PC3-deficient mice and in patients with G6PT-deficiency. We evaluate this treatment in two G6PC3-deficient children. While neutropenia was severe in one child (PT1), which was dependent on granulocyte cololony-stimulating factor (GCSF), it was significantly milder in the other one (PT2), which had low blood 1,5-AG levels and only required GCSF during severe infections. Treatment with the SGLT2-inhibitor empagliflozin decreased 1,5-AG in blood and 1,5-AG6P in neutrophils and improved (PT1) or normalized (PT2) neutrophil counts, allowing to stop GCSF. On empagliflozin, both children remained infection-free (>1 year - PT2; >2 years - PT1) and no side effects were reported. Remarkably, sequencing of SGLT5, the gene encoding the putative renal transporter for 1,5-AG, disclosed a rare heterozygous missense mutation in PT2, replacing the extremely conserved Arg401 by a histidine. The higher urinary clearance of 1,5-AG explains the more benign neutropenia and the outstanding response to empagliflozin treatment found in this child. Our data shows that SGLT2 inhibitors are an excellent alternative to treat the neutropenia present in G6PC3-deficiency.

Keywords: 1,5-anhydroglucitol; G6PC3-deficiency; GSD1b; SGLT2-inhibitors; SGLT5; empagliflozin; glycogen Storage Disease 1b; neutropenia.

FIGURE 1

Empagliflozin improves neutropenia by lowering plasma 1,5‐AG and intracellular 1,5‐AG6P, and corrects protein glycosylation in PMNs and PBMCs from two G6PC3‐deficient patients. (A, C, E, and G) show results for individual 1 (PT1) treated for 839 days and (B, D, F and H) results for individual 2 (PT2) treated for 420 days. (A and B) Time course of absolute neutrophil counts (ANC) and 1,5‐AG concentration in plasma before and during treatment with empagliflozin (dosage indicated by the red line). (C and D) 1,5‐AG6P in neutrophils determined during treatment by liquid‐chromatography‐mass spectrometry (LC–MS) in blood cells after plasma removal (for 1,5‐AG determination) and normalized to total metabolite content (total ion current, TIC) and ANC. (E‐H) Western blots illustrating the almost complete correction of glycosylation of the protein LAMP2 in granulocytes (PMNs—E and F) and peripheral blood mononuclear cells (PBMCs—G and H) isolated before and during treatment with empagliflozin and compared with a healthy control (CT).

FIGURE 2

The replacement of the highly conserved arginine R401 by a histidine in SGLT5 is associated with a higher 1,5‐anhydroglucitol urinary clearance. (A and B) Urinary excretion over 24 hours of glucose and 1,5‐AG before and during empagliflozin treatment. (C) Glucose and 1,5‐anhydroglucitol (1,5‐AG) urinary clearance measured in 24‐h urine collections before empagliflozin treatment; measurements were repeated in triplicate (or more) for two different collections for PT1 and one collection for PT2. (D) Glucose and 1,5‐AG urinary clearance measured in 24‐hr urine collections from PT1 and PT2 on empagliflozin treatment at the indicated dose; measurements shown are respectively for n = 11, n = 5 (PT1 taking 0.18 or 0.35 mg/kg/day EMPA) or n = 8 (PT2 taking 0.24 mg/kg/day EMPA) in different collections. Note the different scale used to represent clearance in C and D. (E) DNA sequence of exon11 in the SGLT5 gene of PT2 showing the heterozygous missense change c.1243G>A (NM_001042450) corresponding to p.R401H (shown in gnomAD as R417H). The protein alignments show strict conservation of the amino acid R401 in six members of the human sodium‐glucose transport protein family (hSLC5A10: NP_001035915; hSLC5A9: NP_001011547; hSLC5A1: NP_000334; hSLC5A2: NP_003032; SMIT2: NP_001339171; SMIT1: NP_008864) as well as among the indicated SLC5A10 orthologs (NP_001035915; NP_001369197; XP_001233919; XP_041435094; XP_017211446). (E) Schematic representation of the impact of the heterozygous replacement of the arginine 401 by a histidine in SGLT5 (D). The R401H change is suggested to decrease the tubular reabsorption and increase the urinary excretion of 1,5‐AG (blue arrows) in comparison to the wild‐type SGLT5 (red arrows). The R401H replacement is likely to result in a lower renal reabsorption of 1,5‐AG, explaining the observed increase in 1,5‐AG clearance before (C) and during (D) empagliflozin treatment. During empagliflozin treatment, SGLT2, the renal glucose transporter is inhibited and the resulting glucosuria inhibits the tubular reabsorption of 1,5‐AG. This lowers plasma 1,5‐AG and intracellular 1,5‐AG6P.