Molecular and Clinical Characterization of a Founder Mutation Causing G6PC3 Deficiency

Abstract

G6PC3 deficiency is a monogenic immunometabolic disorder that causes severe congenital neutropenia type 4. 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 descent. Based on the shared haplotypes amongst mutation carriers, we estimated that this variant originated from a founder effect in a common ancestor. Furthermore, by ancestry analysis, we concluded that it appeared 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 Epstein-Barr Virus-immortalized B (EBV-B) cells. The neutropenia observed in G6PC3-deficient patients is driven by the intracellular accumulation of the metabolite 1,5-anhydroglucitol-6-phosphate (1,5-AG6P) that inhibits glycolysis. We characterized how the c.210delC variant impacts glycolysis by performing extracellular flux assays on patient-derived EBV-B cells. When treated with 1,5-anhydroglucitol (1,5-AG), the precursor to 1,5-AG6P, patient 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, and we found that the c.210delC carriers display all prominent clinical features observed in prior 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: Founder effect; G6PC3 deficiency; Inborn errors of immunity; Metabolic dysfunction; Primary immunodeficiency; Severe congenital neutropenia.

Fig. 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 a 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 frameshift 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

Fig. 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) on chromosome 17. (C) Local ancestry estimation 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. AFR: African ancestry; AMR: Admixed American ancestry; EUR: European ancestry

Fig. 3

The G6PC3 c.210delC mutation leads to a complete loss of protein expression. Schematic representation of (A)the G6PC3 gene with each box representing an exon and the mutation indicated in red, and (B) the 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 blotting of transfected HEK293T cell lysates using an anti-His Tag antibody. GAPDH was used as a loading control. (E) RT-qPCR of G6PC3 mRNA expression in EBV-B cells from patients and healthy controls. Data are presented as mean ± SD and represent three independent experiments. ***p ≤ 0.001 in a Student’s t-test. (F) G6PC3 protein expression by western blotting in membrane protein fraction of EBV-B cells. ATP1A1 was used as a membrane protein loading control

Fig. 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 two patients (P) and four healthy controls (HC) pretreated with 2-DG or 1,5-AG. (B) Quantification of glycolysis rate and glycolytic capacity in EBV-B cells from four healthy controls and two patients. (C) Western blotting analysis of LAMP2 glycosylation pattern in EBV-B cells. Cells were left untreated (UT), or treated with 2-DG or 1,5-AG for five days before the preparation of whole cell lysates. GAPDH was used as a loading control. Data are presented as mean ± SD and represent three independent experiments. Statistical analysis was performed using two-way ANOVA with Šidák correction. ***p ≤ 0.001, ****p ≤ 0.0001

Fig. 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)