Study Approval

This study was conducted in accordance with the Helsinki Declaration, with written informed consent obtained from the patients and their families. Approval for this study was obtained from the Vanderbilt University Medical Center Institutional Review Board (IRB), Nashville, USA.

G6PC3 Allele Age Estimation

Genomic DNA was isolated from whole blood samples using the GeneJET Whole Blood Genomic DNA Purification Kit (ThermoFisher Scientific, Waltham, MA). Primer sequences used to amplify exon 1 of G6PC3 to confirm the c.210delC mutation through sanger sequencing were as follows: Forward: 5’- AGG AAA CGC CTT TAC AAA G -3’, Reverse: 5’- GAC AGT TGC TGA AAG AGA C -3’. Blood-derived DNA collected from 10 carriers of the G6PC3 c.210delC allele (two trios and two mother-child pairs) were sequenced using Illumina short-read sequencing. The affected chromosome (chr17) was sequenced at an average read depth of 10.92x across all ten individuals. Raw reads were aligned to human reference genome build GRCh38/hg38 using Burrows-Wheeler Aligner [15] (0.7.17). PCR duplicates were subsequently identified in the aligned reads, and base quality scores were recalibrated following the Genome Analysis Toolkit (GATK) Best Practices Workflow [16]. Pre-called genomic variant call format (gVCF) from these six individuals underwent subsequent joint variant calling using the HaplotypeCaller [17] function from GATK (4.2.0.0). Finally, variant quality scores were recalibrated for small nucleotide polymorphisms (SNPs) and indels.

The jointly called variant call format (VCF) was pruned using VCFtools [18] (0.1.15) and VcfFilter from the BIOPET suite [19] (0.2). After removing variants with any missingness across the six individuals, those with a minor allele count of less than 3, a quality score of less than 30, or a minimum read depth of less than 20 were also removed.

Using this pruned variant set, the affected chromosome (chr17) was phased using SHAPEIT4 [20] (4.2.0), with 3,202 1000 Genomes samples sequenced at 30x [21] serving as the reference panel. The phased VCF was converted to hap/sample format using bcftools [22] (1.12). Using R (4.0.3), the hap file was reformatted as a 13-column text file containing the chr21 coordinate and phased variant calls for each of the 12 phased chromosomes (2 per individual) at that site. Phased variant calls could then be manually spot-checked for concordance with the aligned reads using the IGV genome browser [23] (2.9.4). As genetic data from the fathers were unavailable in two of the four pedigrees, these carrier haplotypes were inferred from the mother and proband haplotypes using a custom Python script. The lengths in centimorgans (cM) of the chr17 chromosomal arms upstream and downstream of the mutation site were calculated, and these respective lengths were used as inputs for the Mutation_age_estimation.R script developed by Gandolfo et al. [24]. A confidence coefficient of 0.95 without chance sharing correction was used, assuming a correlated genealogy.

Ancestry Inference Using Principal Component Analysis (PCA)

To determine the genetic ancestries of the G6PC3 c.210delC carriers, we combined the variant calls from 10 samples with those from 2,343 reference samples with African, American, and European ancestries from the harmonized HGDP + 1KGP dataset [25]. Variant filtering was performed according to Hardy-Weinberg equilibrium (P < 1 × 10−6) and missingness using PLINK v.1.9 [26]. Autosomal variants with a minor allele frequency > 1% were retained and pruned for linkage disequilibrium (r2 = 0.2). PCA was conducted using 157,601 variants with smartpca implemented in EIGENSOFT version 8.0.0 [27]. Eigenvectors were calculated using 2,343 reference samples, and the 10 G6PC3 c.210delC carriers were projected onto these eigenvectors. A second PCA was performed using 8,032 variants located on chr17.

Local Ancestry Estimation

Using the phase_common_static function from SHAPEIT (v5.5.1) [28], variant calls from the 10 sequenced carriers of the G6PC3 c.210delC allele were phased together with 2,343 deeply sequenced reference samples (788 of European ancestry, 1,003 of African origin, and 552 of American ancestry) from the gnomAD v3.1.2 HGDP + 1KG callset [29] (https://gnomad.broadinstitute.org/downloads#v3-hgdp-1kg). After phasing, RFMix (v2.03-r0) [30] was used to estimate local ancestry across chr17 for the 10 carriers of the G6PC3 c.210delC allele.

Establishment of Epstein-Barr Virus-Immortalized B (EBV-B) Cell Lines

For generation of EBV-B cell lines derived from patients and healthy control individuals, B cells were immortalized with EBV as previously reported [31].

Cell Culture

EBV-B cell lines were cultured in RPMI 1640 Medium without glucose (Gibco, Carlsbad, CA), supplemented with 5mM glucose (Gibco) and 10% fetal bovine serum (FBS) (Corning, Corning, NY). HEK293T cells (ATCC; CRL-3216) were cultured in DMEM medium (Sigma-Aldrich, St. Louis, MO) supplemented with 10% FBS (Corning).

Plasmid Cloning and site-directed Mutagenesis

RNA was extracted from healthy control EBV-B cells using the RNeasy Plus Mini Kit (Qiagen, Germantown, MD). RNA was reverse transcribed using the Verso cDNA Synthesis Kit (ThermoFisher Scientific). Primer sequences used to generate the full-length cDNA of G6PC3 with a His-tag right next to the start or stop codon of cDNA that encodes the G6PC3 protein were as follows: N-ter His-tag Forward: 5’- ATG CAT CAC CAT CAC CAT CAC ATG GAG TCC ACG CTG G -3’; N-ter His-tag Reverse: 5’- TCA GGA AGA GTG GAT GGG -3’; C-ter His-tag Forward: 5’- ATG GAG TCC ACG CTG G -3’; C-ter His-tag Reverse: 5’- TCA GTG ATG GTG ATG GTG ATG GGA AGA GTG GAT GGG C -3’. The products were TA-cloned into a pcDNA3.1 plasmid vector using the pcDNA 3.1/V5-His TOPO TA Expression Kit (ThermoFisher Scientific) according to the manufacturer’s protocol. Plasmid DNAs were purified from bacterial clones with a Miniprep kit (ThermoFisher Scientific). Then, constructs carrying the 210delC mutant allele were generated by site-directed mutagenesis with primers: Forward: 5’- CTC AAC CTC ATT TCA AGT GGT T -3’; Reverse: 5’- AAC CAC TTG AAA TGA GGT TGA G -3’. In brief, after the PCR reaction using PfuUltra II Fusion High-fidelity DNA Polymerase (Agilent, Santa Clara, CA), 1uL DpnI (NEB, Ipswich, MA) was added followed with incubation at 37 C for 3 hr. Mutagenesis was validated by Sanger sequencing.

HEK293T Cell Transfection

In 6-well plates, HEK293T cells were transfected in 2 mL medium with 2.5ug of pcDNA3.1 empty vector, N or C-terminal His-tagged WT G6PC3, or N- or C-terminal His-tagged 210delC mutant constructs using Lipofectamine 3000 Transfection Reagent (ThermoFisher Scientific) according to the manufacturer’s protocol for 48 hr.

G6PC3 mRNA Detection by RT-qPCR

RNA was extracted from transfected HEK293T and EBV-B cells using the RNeasy Plus Mini Kit (Qiagen). The RT-qPCR was performed using the Luna Universal One-Step RT-qPCR Kit (NEB) on the CFX96 RT-qPCR detection system (Bio-Rad, Hercules, CA). The following primers were used to target the cDNA of G6PC3: Forward: 5’- TCA AGT GGT TTC TTT TTG GAG -3’; Reverse: 5’- ATC ATG CAG TGT CCA GAA G -3’. The G6PC3 mRNA expression level in each sample was normalized to the expression level of the GUS gene transcript. Primers used to target GUS cDNA are: Forward: 5’- CTA CTT GAA GAT GGT GAT CG -3’; Reverse: 5’- CTG TTC AAA CAG ATC ACA TC -3’.

Western Blotting Analysis

Whole-cell lysates were prepared from transfected HEK293T cells or EBV-B cells with RIPA lysis buffer (150 mM NaCl, 50 mM TRIS-HCl pH 8.0, 1 mM EDTA, 0.5% sodium deoxycholate, 1% NP-40, 0.1% SDS), supplemented with protease inhibitor cocktail (ThermoFisher Scientific). Protein concentrations were quantified with DC Protein Assay (Bio-Rad). To detect His tag, hexokinases, and ADP-GK, lysates mixed with Laemmli loading buffer were incubated at 100 °C for 5 min and subjected to electrophoresis in a 10% polyacrylamide gel. For analysis of LAMP2, lysates were mixed with XT sample buffer and reducing agent and subjected to electrophoresis in a precast 4–12% Bis-Tris Protein Gel using the MOPS running buffer (Bio-Rad). The membrane protein fraction of EBV-B cells was obtained with the Mem-PER Plus Membrane Protein Extraction Kit (ThermoFisher Scientific). The membrane protein extracts were mixed with loading buffer, incubated at 37 °C for 30 min, and subjected to electrophoresis in a precast 4–20% gradient gel (Bio-Rad). Proteins were then transferred to PVDF membranes, blocked with PBS-T containing 0.05% Tween-20 and 5% non-fat dry milk.

The following antibodies were used for western blotting: HRP-conjugated Anti-His tag (Biolegend, 652503, 1:5000), Anti-G6PC3 (Invitrogen, PA5-109749, 1:1000), Anti-ATP1A1 (Invitrogen, MA5-32184, 1:1000), HRP-conjugated Anti-GAPDH (Proteintech, HRP-60004, 1:1000), Anti-HK1 (Cell signaling, 2804, 1:1000), Anti-HK2 (Proteintech, 22029-1-AP, 1:3000), Anti-HK3 (Invitrogen, PA5-29304, 1:1000), Anti-ADPGK (Proteintech, 15639-1-AP, 1:1000), and Anti-LAMP2 (Santa Cruz Biotechnology, sc-18822, 1:300). Unconjugated antibodies were detected by incubation with HRP Goat Anti-mouse IgG secondary antibody (Sigma-Aldrich, AP127P, 1:5000) or HRP Goat Anti-Rabbit IgG secondary antibody (Sigma-Aldrich, AP156P, 1:5000). Blots were developed using the Pierce ECL Western Blotting Substrate (ThermoFisher Scientific).

Extracellular Flux Assay

For EBV-B cells pretreatment prior to the glycolysis stress assay, cells were cultured in RPMI 1640 medium with 5mM glucose and 10% FBS, supplemented with either 2mM of 2-DG (Cayman Chemical, Ann Arbor, MI) or 1,5-AG (Cayman Chemical), for five days. Fresh medium was added to cells every two days. The assay was performed according to the Agilent Seahorse XF Glycolysis Stress Test Kit user guide. To summarize, EBV-B cells were counted and resuspended in Agilent Seahorse XF RPMI 1640 medium supplemented with 2mM glutamine, with 2mM of 2-DG or 1,5-AG added to corresponding conditioned cells. 150,000 live cells/well, in 180µL medium, were plated in 4–6 technical replicates on a Cell-Tak-coated plate (Corning). Extracellular acidification rate (ECAR) was measured on a Seahorse XFe 96 Analyzer using the glycolysis stress test with sequential injections of 20µL of 100mM glucose to Port A, 22µL of 15µM oligomycin to Port B, and 25µL of 500mM 2-DG to Port C of the sensor cartridge. The final concentrations of these compounds in each well are 10mM glucose, 1.5µM oligomycin, and 50mM 2-DG. The glycolysis rate was quantified as the change in ECAR before and after adding glucose. Glycolytic capacity was calculated as the difference between the maximal ECAR reached following the oligomycin injection and the non-glycolytic acidification prior to the addition of glucose.

Viability Measurement of EBV-B cells

As described above, EBV-B cells were incubated with 2 mM of 2-DG or 1,5-AG for five days. Cells were stained with Zombie NIR dye (Biolegend) at room temperature for 15 min. Data was acquired using a BD LSRFortessa III flow cytometer (BD Biosciences). Using the FlowJo software v.10.10.0, the percentage of viable cells was quantified after gating on the singlet population using forward scatter (FSC) and side scatter (SSC).