Quantitative determination of SLC2A1 variant functional effects in GLUT1 deficiency syndrome

Abstract

Objective: The goal of this study is to demonstrate the utility of a growth assay to quantify the functional impact of single nucleotide variants (SNVs) in SLC2A1, the gene responsible for Glut1DS.

Methods: The functional impact of 40 SNVs in SLC2A1 was quantitatively determined in HAP1 cells in which SLC2A1 is required for growth. Donor libraries were introduced into the endogenous SLC2A1 gene in HAP1-Lig4KO cells using CRISPR/Cas9. Cell populations were harvested and sequenced to quantify the effect of variants on growth and generate a functional score. Quantitative functional scores were compared to 3-OMG uptake, SLC2A1 cell surface expression, CADD score, and clinical data, including CSF/blood glucose ratio.

Results: Nonsense variants (N = 3) were reduced in cell culture over time resulting in negative scores (mean score: -1.15 ± 0.17), whereas synonymous variants (N = 10) were not depleted (mean score: 0.25 ± 0.12) (P < 2e-16). Missense variants (N = 27) yielded a range of functional scores including slightly negative scores, supporting a partial function and intermediate phenotype. Several variants with normal results on either cell surface expression (p.N34S and p.W65R) or 3-OMG uptake (p.W65R) had negative functional scores. There is a moderate but significant correlation between our functional scores and CADD scores.

Interpretation: Cell growth is useful to quantitatively determine the functional effects of SLC2A1 variants. Nonsense variants were reliably distinguished from benign variants in this in vitro functional assay. For facilitating early diagnosis and therapeutic intervention, future work is needed to determine the functional effect of every possible variant in SLC2A1.

Figure 1

GLUT1 protein diagram showing the locations of variants from exon 2 and 3 using DOG plotter. Variants identified in patients with clinical data are highlighted in red (Table 1), all others are blue (Table S1).

Figure 2

Functional scores of 40 SLC2A1 single nucleotide variants derived from HAP1 growth assay. (A) Scores for individual variants (with 95th percentile error bars derived from Miseq data) are shown sorted from lowest score (most negative) to highest. (B) Functional scores of groups of nonsense (N = 3), missense (N = 27), and synonymous (N = 10) variants. The black dot is the mean of the variants. (C) Density plot showing the distribution of individual score for each replicate of variant data within each group.

Figure 3

Functional scores for variants altering three amino acid residues (Y28, G31, and N34) in exon 2. Data presented is the % CI as calculated from Miseq sequencing data.

Figure 4

The functional scores of 40 SLC2A1 variants are inversely correlated with CADD scores (Spearman's correlation: −0.62). Scores are derived from Miseq data.

Figure 5

Receiver operating characteristic curve for GLUT1 growth assay in HAP1 cells yields an AUC of 1.