Molecular genetics of GLUT1DS Italian pediatric cohort: 10 novel 2 related-disease variants and structural analysis

15 GLUT1 deficiency syndrome (GLUT1DS1; OMIM #606777) is a rare genetic metabolic 16 disease, characterized by infantile-onset epileptic encephalopathy, global developmental delay, 17 progressive microcephaly and movement disorders (e.g. spasticity and dystonia). It is caused 18 by heterozygous mutations in the SLC2A1 gene, which encodes the GLUT1 protein, a glucose 19 transporter across the blood-brain barrier (BBB). Most commonly these variants arise de novo 20 resulting in sporadic cases, although several familial cases with AD inheritance pattern have 21 been described. 22 Twenty-seven Italian pediatric patients clinically suspect of GLUT1DS from both sporadic and 23 familial cases have been enrolled. 24 We detected by sequencing analysis 25 different variants causing GLUT1DS. 40% of the identified variants out of reported genotypic exon mutational hotspot in the SLC2A1 gene. possible correlations between mutation type and clinical and biochemical data observed in our GLUT1DS cohort, revealing that splice site and frameshift variants are related to a more severe phenotype and low CSF parameters.

missense, frameshift and splice site variants. Their X-ray structure analyses strongly suggested 27 the potential pathogenic effects of these novel disease-related mutations, broadening the 28 genotypic spectrum heterogeneity found in the SLC2A1 gene. Moreover, 24% is located in a 29 vulnerable region of the GLUT1 protein that involves transmembrane 4 and 5 helices encoded 30 by exon 4, confirming a mutational hotspot in the SLC2A1 gene. Lastly, we investigated 31 possible correlations between mutation type and clinical and biochemical data observed in our 32 GLUT1DS cohort, revealing that splice site and frameshift variants are related to a more severe 33 phenotype and low CSF parameters.

35
Author summary 36 We investigated the molecular data of 27 pediatric patients clinically suspect of GLUT1 37 deficiency syndrome. By performing trios sequencing analysis, we highlighted ten novel 38 disease-related variants, and their X-ray structure analyses, suggesting the pathogenic effects 39 of these identified mutations. Moreover, the wide clinical and genetic heterogeneity observed 40 in our cohort allowed possible correlations between mutation type and clinical and biochemical 41 data. This analysis enabled to delineate that splice site and frameshift variants are related to a

Results
. CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in Clinical and biochemical features 102 The main clinical and biochemical characteristics of 27 patients are shown in Table 1. . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in  159 . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in  . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted September 27, 2022. ; https://doi.org/10.1101/2022.09.26.509448 doi: bioRxiv preprint inherited from the unaffected father (I-1). To determine the impact of this amino acid 178 substitution in position 9, the pathogenic score was evaluated by nineteen different in silico 179 predictors, and it was predicted to be damaging by seventeen of them (Table S1). This score is 180 consistent with the amino acid change from a polar threonine to a non-polar methionine, in a  (Table S1).

217
The Gly130Ser mutation occurs at the narrow interface between TMH4 and TMH2, replacing 218 a glycine, which lacks a side chain, with a cysteine whose side chain could sterically clash with 219 TMH2, thereby destabilizing the helical packing of the transporter (Fig. S1b). 223 Family 4. The proband (III-2, patient 11) of this family was a 17-year-old girl with no seizure 224 history, but recurrent paroxysmal exercise induced dyskinesia since the age of 6. This 225 movement disorder was characterized by worsening due to fatigue and fasting. The mother (II-. CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in Family 5. The proband (II-1, patient 16) was a 7-year-old girl presenting recurrent absence 251 seizures since the age of 6 months. Moreover, since the age of 2, she is affected by exercise-252 induced dyskinesia. The mother (I-2), a 40-year woman, affected by seizure since childhood.

274
During adolescence those episodes worsened in terms of frequency and intensity, occurring . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted September 27, 2022. ; https://doi.org/10.1101/2022.09.26.509448 doi: bioRxiv preprint daily and in his twenties, these episodes became less frequent. The paternal grandfather (III-2)   276 showed recurrent PED, right lower limb was mainly affected (rarely arms were involved). It  (Table S1). This score is consistent with the 290 amino acid change in position 455 from a polar threonine to a non-polar alanine, in a highly 291 conserved residue of IC5 involved in the GLUT1 cavity.  the consequent skipping of exon 2, thus leading to exon 1-3 junction (Fig. 3). In particular, the 315 in-frame loss of exon 2 causes the completely loss of the first transmembrane helix (TMH1) of 316 the GLUT1 (predicted by protein topology THMM in silico tool), which is highly involved in 317 glucose uptake, probably due to its important role for the protein state transition [23].

318
The novel frameshift mutations identified in our patients cause the introduction of a PTC, 319 predicted to lead to the synthesis of non-functional truncated protein, or more likely to NMD.

320
It is likely that these mutations result in 50% loss of GLUT1 protein and thus, lead to severe 321 impairment of glucose transport into brain. These molecular findings were also supported by 322 both biochemical and clinical data of patients 7,14, 18 and 27: the CSF/blood glucose ratios . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in  323 were 0,36, 0,37, 031 and 0,37 mg/dL, respectively; all patients had the early-onset severe 324 phenotype in combination with intellectual disability and/or movement disorders.

325
The amino acid changes in the novel missense variants identified in this study were evaluated 326 by nineteen different in silico predictors, and they were predicted to be damaging by most of 327 them (Table S1-S2). Additionally, in order to rationalize how these novel disease-related 328 missense mutations may impact the structure and function of human GLUT1 (hGLUT1), we 329 mapped them on its X-ray crystallographic structure in complex with Nonyl-β-D-Glucoside

330
[33, 34] (PDB ID: 6THA) (Fig. 4a). The latter is a detergent bearing a glucoside group which elements of hGLUT1 (Fig. 4 b-e). The Leu67Pro mutation, located on TMH2, may cause a kink 338 of the α-helix due to the rigidity of the cyclic pyrrolidine side chain of proline (Fig. 4b). Despite 339 not being directly involved in ligand binding, this mutation could affect the overall protein helix 340 bundle flexibility and therefore the glucose transport efficiency. The Gly75Glu variant, also 341 found in TMH2, corresponds to the replacement of a small hydrophobic glycine residue with a 342 negatively charged glutamic acid on the membrane-facing side of TMH2 (Fig. 4b). Here, the 343 mutation could in principle affect not only the folding, but also the localization of hGLUT1 in 344 the phospholipidic membrane. Gln283Lys is found right in the internal cavity of the hGLUT1 345 transporter where glucose transits (Fig. 4c). Here, the WT residue Gln283 forms a hydrogen 346 bond network with Asn288, likely involved in stabilizing the glucose head group of the Nonyl-347 β-D-Glucoside, mimicking the natural ligand. The replacement with a longer and charged . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in  . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in  (Fig. 6a).
. CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in    with several potential detrimental consequences on hGLUT1 structure stability. The Gly130Cys mutation occurs 686 at the narrow interface between TMH4 and TMH2, replacing a glycine, which lacks a side chain, with a cysteine 687 whose side chain could sterically clash with TMH2, thereby destabilizing the helical packing of the transporter.

688
The Val165Ile mutation changes a smaller valine to a larger isoleucine at the interface between two helices and 689 relatively close to the substrate binding cavity. This mutation could potentially affect the helical packing and 690 maybe to a lesser extent the interaction with glucose. Although the mutation Val166del is a single aminoacidic 691 deletion (in frame), it shifts the register of TMH5 residues, misplacing the outward-facing hydrophobic residues 692 and the inward-facing polar residues with potentially devastating consequences on protein folding and function.
. CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in Nonyl-β-D-Glucoside . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted September 27, 2022. ; https://doi.org/10.1101/2022.09.26.509448 doi: bioRxiv preprint