Molecular and clinical characterization of a founder mutation causing G6PC3 deficiency

Abstract

G6PC3 deficiency is a monogenic immunometabolic disorder that causes syndromic congenital neutropenia. Patients display heterogeneous extra-hematological manifestations, contributing to delayed diagnosis. Here, we investigated the origin and functional consequence of the G6PC3 c.210delC variant found in patients of Mexican origin. Based on the shared haplotypes amongst carriers of the c.210delC mutation, we estimated that this variant originated from a founder effect in a common ancestor. Furthermore, by ancestry analysis, we concluded that it originated in the indigenous Mexican population. At the protein level, we showed that this frameshift mutation leads to an aberrant protein expression in overexpression and patient-derived cells. G6PC3 pathology is driven by the intracellular accumulation of the metabolite 1,5-anhydroglucitol-6-phosphate (1,5-AG6P) that inhibits glycolysis. We characterized how the variant c.210delC impacts glycolysis by performing extracellular flux assays on patient-derived cells. When treated with 1,5-anhydroglucitol (1,5-AG), the precursor to 1,5-AG6P, patient-derived cells exhibited markedly reduced engagement of glycolysis. Finally, we compared the clinical presentation of patients with the mutation c.210delC and all other G6PC3 deficient patients reported in the literature to date, and we found that c.210delC carriers display all prominent clinical features observed in prior G6PC3 deficient patients. In conclusion, G6PC3 c.210delC is a loss-of-function mutation that arose from a founder effect in the indigenous Mexican population. These findings may facilitate the diagnosis of additional patients in this geographical area. Moreover, the in vitro 1,5-AG-dependent functional assay used in our study could be employed to assess the pathogenicity of additional G6PC3 variants.

Keywords: G6PC3 deficiency; Inborn errors of immunity; founder effect; metabolic dysfunction; primary immunodeficiency; severe congenital neutropenia.

Figure 1. A common ancestor in patients with the G6PC3c.210delC mutation.

A) Familial segregation of the mutation in four unrelated, non-consanguineous families. WT: wild type; M: mutant. B) Sanger sequencing results of healthy control and patient 1 (P1) in the region spanning the G6PC3 c.210delC mutation. Amino acid changes resulting from the mutation are annotated above the graphs. C) Age estimation for the G6PC3 c.210delC variant, based on the lengths of shared ancestral haplotype blocks upstream and downstream of the G6PC3 stop-gain allele (marked in red on chr17). The computationally inferred haplotypes from Father 3 (P3-F) and Father 4 (P4-F) are denoted with an asterisk.

Figure 2. The G6PC3 c.210delC mutation is estimated to be of indigenous American ancestry.

Principal component analysis (PCA) of ancestry on A) the whole genome for c.210delC mutation carriers, using reference genomes from the combined 1000 Genomes Project (1kGP) and Human Genome Diversity Project (HGDP) dataset and B) for chromosome 17. C) Local ancestry estimates across carriers of the c.210delC variant. The mutation locus (chr17:44,071,175) is marked in red on chromosome 17. Mutated alleles are labeled with red asterisks. Abbreviations: AFR: African ancestry; AMR: Admixed American ancestry; EUR: European ancestry.

Figure 3. The G6PC3 c.210delC mutation leads to a complete loss of protein expression.

Schematic representation of A) G6PC3 gene; each box represents and exon and the mutation is indicated in red and B) G6PC3 protein structure with nine transmembrane domains in the endoplasmic reticulum. Red stars indicate the active site. The lower panel shows the predicted consequence of the c.210delC (p.F71Sfs*46) mutation. The out-of-frame sequence resulting from the premature stop codon is indicated in red. CP: cytoplasm; L: lumen. C) RT-qPCR of G6PC3 mRNA expression in HEK293T cells either non-transfected (NT), transfected with the empty vector (EV), or transfected with plasmids encoding wildtype (WT) or F71Sfs*46 (M) G6PC3 with a 6xHis tag at either the N- or C-terminal. GUS was used as a control for gene expression. n=3. D) Western blot of transfected HEK293T cell lysates using an anti-His Tag antibody. GAPDH was used as a loading control. E) RT-qPCR analysis for G6PC3 mRNA expression in EBV-B cells from patients and healthy controls. Data are presented as mean ± SD. ***p≤ 0.001 in a Student’s t-test.F) G6PC3 protein expression by western blot in membrane protein fraction of EBV-B cell. ATP1A1 was used as a membrane protein loading control.

Figure 4. Impaired glycolysis in cells from G6PC3 deficient patients.

A) Measurement of extracellular acidification rate (ECAR) in response to glucose (gluc), ATP synthase inhibitor oligomycin (oligo), and glycolytic inhibitor 2-deoxy-glucose (2-DG) in EBV-B cells from patients and healthy controls pretreated with 2-DG or 1,5-AG. Representative time courses of a patient (P1) and healthy control (HC1) are shown. B) Quantification of glycolysis rate and glycolytic capacity in EBV-B cells from four healthy controls and two patients. Data are presented as mean ± SD and represent three independent experiments. Statistical analysis was performed using two-way ANOVA with Sidak correction. ***p≤ 0.001, ****p≤ 0.0001.

Figure 5. Frequencies of the most prominent clinical features observed in G6PC3 deficient patients with the c.210delC mutation (n=14) and those with other mutations (n=112).