In silico analysis of rare pathogenic missense variants in the SLC6A2 gene

Background. The sodium-dependent noradrenaline transporter (NET), encoded by the SLC6A2 gene, plays a key role in the regulation of noradrenergic transmission and is associated with orthostatic intolerance, attention-deficit/hyperactivity disorder (ADHD), depression, and autonomic dysfunctions. The emergence of cryo-EM structures of human NET in complex with noradrenaline and antidepressants, along with new machine-learning algorithms, has enabled systematic in silico screening of known missense variants of the gene and the identification of previously uncharacterized pathogenic substitutions that may explain cases of idiopathic orthostatic intolerance, treatment-resistant depression, paradoxical responses to therapy, or hereditary autonomic disorders.

Aim of the study was to analyze missense variants of the SLC6A2 gene registered in the NCBI database (assembly GRCh38.p14), to identify variants with the highest predicted pathogenicity scores, and to perform their structural-functional characterization using modern bioinformatics tools.

Materials and methods. From 725 missense single-nucleotide variants (SNVs) of unknown clinical significance in the SLC6A2 gene (GRCh38), biallelic variants with the highest pathogenicity scores (AlphaMissense ≥0.99 and LIST-S2 ≥0.99) were selected. Two rare variants (rs1397308523 (Pro108Thr) and rs759975667 (p.Gln314Arg) underwent comprehensive in silico analysis.

Results and discussion. Both substituted residues are highly conserved (ConSurf 9/9). Predictions of topology (DeepTMHMM) and domain organization (PROSITE) revealed no differences from the wild type. MutPred2 assigned overall pathogenicity scores of 0.87 and 0.91, respectively. I-Mutant 2.0 and MUpro predicted decreased protein stability (ΔΔG –1.11 and –0.94 kcal/mol).

Conclusions. The variants rs1397308523 and rs759975667, which have the highest predicted pathogenicity among missense substitutions in SLC6A2, represent priority targets for functional studies and screening in clinical cohorts.

1. Alexandrov A.I., Mileni M., Chien E.Y., Hanson M.A., Stevens R.C. Microscale fluorescent thermal stability assay for membrane proteins. Structure. 2008; 16 (3): 351-359. https://doi.org/10.1016/j.str.2008.02.004

2. Ashkenazy H., Abadi S., Martz E., Chay O., Mayrose I., Pupko T., Ben-Tal N.. ConSurf 2016: an improved methodology to estimate and visualize evolutionary conservation in macromolecules. Nucleic Acids Res. 2016; 44 (W1): 344-350. https://doi.org/10.1093/nar/gkw408

3. Bayles R., Harikrishnan K.N., Lambert E., Baker E.K., Agrotis A., Guo L., Jowett J.B., Esler M., Lambert G., El-Osta A. Epigenetic modification of the norepinephrine transporter gene in postural tachycardia syndrome. Arterioscler. Thromb. Vasc. Biol. 2012; 32 (8): 1910-1916. https://doi.org/10.1161/ATVBAHA.111.244343

4. Brown C.R., Shetty M., Foster J.D. Palmitoylation regulates norepinephrine transporter uptake, surface localization, and total expression with pathogenic implications in postural orthostatic tachycardia syndrome. J. Neurochem. 2025; 169 (2): e16241. https://doi.org/10.1111/jnc.16241

5. Bymaster F.P., Katner J.S., Nelson D.L., Hemrick-Luecke S.K., Threlkeld P.G., Heiligenstein J.H., Morin S.M., Gehlert D.R., Perry K.W. Atomoxetine increases extracellular levels of norepinephrine and dopamine in prefrontal cortex of rat: a potential mechanism for efficacy in attention deficit/ hyperactivity disorder. Neuropsychopharmacology. 2002; 27 (5): 699-711. https://doi.org/10.1016/S0893-133X(02)00346-9

6. Capriotti E., Fariselli P., Casadio R. I-Mutant2.0: predicting stability changes upon mutation from the protein sequence or structure. Nucleic. Acids Res. 2005; 33: 306-310. https://doi.org/10.1093/nar/gki375

7. Cheng J., Novati G., Pan J., Bycroft C., Žemgulytė A., Applebaum T., Pritzel A., Wong L.H., Zielinski M., Sargeant T., Schneider R.G., Senior A.W., Jumper J., Hassabis D., Kohli P., Avsec Ž. Accurate proteome-wide missense variant effect prediction with AlphaMissense. Science. 2023; 381 (6664): eadg7492. https://doi.org/10.1126/science.adg7492

8. Cheng J., Randall A., Baldi P. Prediction of protein stability changes for single-site mutations using support vector machines. Proteins. 2006; 62 (4): 1125-1132. https://doi.org/10.1002/prot.20810

9. Fichna J.P., Humińska-Lisowska K., Safranow K., Adamczyk J.G., Cięszczyk P., Żekanowski C., Berdyński M. Rare Variant in the SLC6A2 Encoding a Norepinephrine Transporter Is Associated with Elite Athletic Performance in the Polish Population. Genes (Basel). 2021; 12 (6): 919. https://doi.org/10.3390/genes12060919

10. Hahn M.K., Robertson D., Blakely R.D. A mutation in the human norepinephrine transporter gene (SLC6A2) associated with orthostatic intolerance disrupts surface expression of mutant and wild-type transporters. J. Neurosci. 2003; 23 (11): 4470-4478. https://doi.org/10.1523/JNEUROSCI.23-11-04470.2003

11. Hallgren J., Tsirigos K.D., Pedersen M.D. DeepTMHMM predicts alpha and beta transmembrane proteins using deep neural networks. bioRxiv; 2022. https://doi.org/10.1101/2022.04.08.487609

12. Li L.B., Chen N., Ramamoorthy S., Chi L., Cui X.N., Wang L.C., Reith M.E. The role of N-glycosylation in function and surface trafficking of the human dopamine transporter. J. Biol. Chem. 2004; 279 (20): 21012-21020. https://doi.org/10.1074/jbc. M311972200

13. Malhis N., Jacobson M., Jones S.J.M., Gsponer J. LIST-S2: taxonomy based sorting of deleterious missense mutations across species. Nucleic. Acids Res. 2020; 48: 154-161. https://doi.org/10.1093/nar/gkaa288

14. Michelson D., Adler L., Spencer T., Reimherr F.W., West S.A., Allen A.J., Kelsey D., Wernicke J., Dietrich A., Milton D. Atomoxetine in adults with ADHD: two randomized, placebocontrolled studies. Biol. Psychiatry. 2003; 53 (2): 112- 120. https://doi.org/10.1016/s0006-3223(02)01671-2

15. Miranda M., Dionne K.R., Sorkina T., Sorkin A. Three ubiquitin conjugation sites in the amino terminus of the dopamine transporter mediate protein kinase C-dependent endocytosis of the transporter. Mol. Biol. Cell. 2007; 18 (1): 313-23. https://doi.org/10.1091/mbc.e06-08-0704

16. Paczkowski F.A., Bönisch H., Bryan- Lluka L.J. Pharmacological properties of the naturally occurring Ala(457)Pro variant of the human norepinephrine transporter. Pharmacogenetics. 2002; 12 (2): 165-73. https://doi.org/10.1097/00008571-200203000-00010

17. Pejaver V., Urresti J., Lugo-Martinez J., Pagel K.A., Lin G.N., Nam H.J., Mort M., Cooper D.N., Sebat J., Iakoucheva L.M., Mooney S.D., Radivojac P. Inferring the molecular and phenotypic impact of amino acid variants with MutPred2. Nat. Commun. 2020; 11 (1): 5918. https://doi.org/10.1038/s41467-020-19669-x

18. Pörzgen P., Bönisch H., Brüss M. Molecular cloning and organization of the coding region of the human norepinephrine transporter gene. Biochem. Biophys. Res. Commun. 1995; 215 (3): 1145-1150. https://doi.org/10.1006/bbrc.1995.2582

19. Proft F., Kopf J., Olmes D., Hempel S., Schmidt B., Riederer P., Deckert J., Pfuhlmann B., Reif A., Unterecker S. SLC6A2 and SLC6A4 variants interact with venlafaxine serum concentrations to influence therapy outcome. Pharmacopsychiatry. 2014; 47 (7): 245-50. https://doi.org/10.1055/s-0034-1390412

20. Ramoz N., Boni C., Downing A.M., Close S.L., Peters S.L., Prokop A.M., Allen A.J., Hamon M., Purper-Ouakil D., Gorwood P. A haplotype of the norepinephrine transporter (Net) gene Slc6a2 is associated with clinical response to atomoxetine in attention-deficit hyperactivity disorder (ADHD). Neuropsychopharmacology. 2009; 34 (9): 2135- 2142. https://doi.org/10.1038/npp.2009.39

21. Richards S., Aziz N., Bale S., Bick D., Das S., Gastier-Foster J., Grody W.W., Hegde M., Lyon E., Spector E., Voelkerding K., Rehm H.L. ACMG Laboratory Quality Assurance Committee. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet. Med. 2015; 17 (5): 405-424. https://doi.org/10.1038/gim.2015.30

22. Rott R., Szargel R., Shani V., Hamza H., Savyon M., Abd Elghani F., Bandopadhyay R., Engelender S. SUMOylation and ubiquitination reciprocally regulate α-synuclein degradation and pathological aggregation. Proc. Natl. Acad. Sci. U S A. 2017; 114 (50): 13176-13181. https://doi.org/10.1073/pnas.1704351114

23. Shannon J.R., Flattem N.L., Jordan J., Jacob G., Black B.K., Biaggioni I., Blakely R.D., Robertson D. Orthostatic intolerance and tachycardia associated with norepinephrine-transporter deficiency. N. Engl. J. Med. 2000; 342 (8): 541-549. https://doi.org/10.1056/NEJM200002243420803

24. Sigrist C.J., de Castro E., Cerutti L., Cuche B.A., Hulo N., Bridge A., Bougueleret L., Xenarios I. New and continuing developments at PROSITE. Nucleic. Acids. Res. 2013; 41: D344-7. https://doi.org/10.1093/nar/gks1067

25. Singh N., Hazari P.P., Mittal P., Yadav S.K., Kumar N., Mishra G., Dahiya S., Mishra A.K. Role of selective serotonin reuptake inhibitors, serotoninnorepinephrine reuptake inhibitors and psychedelics in the treatment of major depressive disorder: A perspective on mechanistic insight and current status. Eur. J. Pharmacol. 2025; 1001: 177737. https://doi.org/10.1016/j.ejphar.2025.177737

26. Tan J., Xiao Y., Kong F., Zhang X., Xu H., Zhu A., Liu Y., Lei J., Tian B., Yuan Y., Yan C. Molecular basis of human noradrenaline transporter reuptake and inhibition. Nature. 2024; 632 (8026): 921-929. https://doi.org/10.1038/s41586-024-07719-z

27. Torres G.E., Yao W.D., Mohn A.R., Quan H., Kim K.M., Levey A.I., Staudinger J., Caron M.G. Functional interaction between monoamine plasma membrane transporters and the synaptic PDZ domaincontaining protein PICK1. Neuron. 2001; 30 (1): 121- 134. https://doi.org/10.1016/s0896-6273(01)00267-7

28. von Heijne G. Proline kinks in transmembrane alpha-helices. J. Mol. Biol. 1991; 218 (3): 499-503. https://doi.org/10.1016/0022-2836(91)90695-3

29. Wu H.F., Hamilton C., Porritt H., Winbo A., Zeltner N. Modelling neurocardiac physiology and diseases using human pluripotent stem cells: current progress and future prospects. J. Physiol. 2025; 603 (7): 1865-1885. https://doi.org/10.1113/JP286416

30. Wu H.F., Saito-Diaz K., Huang C.W., McAlpine J.L., Seo D.E., Magruder D.S., Ishan M., Bergeron H.C., Delaney W.H., Santori F.R., Krishnaswamy S., Hart G.W., Chen Y.W., Hogan R.J., Liu H.X., Ivanova N.B., Zeltner N. Parasympathetic neurons derived from human pluripotent stem cells model human diseases and development. Cell Stem. Cell. 2024; 31 (5): 734-753. https://doi.org/10.1016/j.stem.2024.03.011

31. Yeh Y.W., Chen C.J., Jang F.L., Kuo S.C., Chen C.Y., Liang C.S., Ho P.S., Yen C.H., Shyu J.F., Wan F.J., Lu R.B., Huang S.Y. SLC6A2 variants may predict remission from major depression after venlafaxine treatment in Han Chinese population. J. Psychiatr. Res. 2015; 61: 33-39. https://doi.org/10.1016/j.jpsychires.2014.11.017

32. Zhang H., Yin Y.L., Dai A., Zhang T., Zhang C., Wu C., Hu W., He X., Pan B., Jin S., Yuan Q., Wang M.W., Yang D., Xu H.E., Jiang Y. Dimerization and antidepressant recognition at noradrenaline transporter. Nature. 2024; 630 (8015): 247-254. https://doi.org/10.1038/s41586-024-07437-6

33. Zhao S., Tran V.H. Postural Orthostatic Tachycardia Syndrome. StatPearls. Treasure Island (FL): StatPearls Publishing, 2025.