MO035COMPUTATIONAL MODELING APPROACH FOR THE COMPREHENSIVE INTERPRETATION OF RARE TUBULOPATHIES
Authors: Suzumoto Yoko , Columbano Valeria , Capolongo Giovanna , Simeoni Mariadelina , Perna Alessandra , Zacchia Miriam , Viggiano Davide , Capasso Giovambattista , Trepiccione Francesco 
Published: May 2021

Abstract

Background and Aims
Kidney plays a central role on the maintenance of water homeostasis, acid-base and electrolytes balances through the activity of different types of ion channels/transporters expressed along the nephron segments. Mutations in genes encoding these transporters could subsequently lead to aberrant transporter activities, resulting in abnormal renal handling of electrolytes, thus represent monogenic form of rare kidney diseases.
Accumulating number of mutations identified in genes responsible for such monogenic disorders demonstrated that eventual disease phenotypes may vary according to the type and localization of the mutation within the gene. Thus, careful evaluation of gene variation would be crucial prior to designing the strategy for the therapy in each case.
Here we present various mutations from our patients, identified in genes including kcnj10, SLC12A1, SLC26A4 and clcn7 which are associated with rare tubulopathies EAST/SeSAME syndrome, Bartter’s syndrome, Pendred syndrome and Fanconi syndrome, respectively.
In order to explore molecular mechanisms underlying the observed disease conditions in our patients, we have performed computational modeling analyses of these mutations in comparison with wild-type models.

Method
Three-dimensional homology models of Kir4.1, NKCC2, Pendrin and CLC-7 proteins were generated by Swiss-Model protein structure homology-modelling server (http://swissmodel. expasy.org) and I-TASSER server (https://zhanglab.ccmb.med.umich.edu/I-TASSER/). Disease-related mutations including novel mutations identified from our patients were mapped onto the three-dimensional models and compared with wild-type models in terms of atomic interactions as well as secondary, tertiary and quaternary structures. Furthermore, we assessed possible effects of missense mutations on the function of ion channels/transporters using online bioinformatic prediction tools PolyPhen-2, Mutation taster, PROVEAN and SIFT.

Results
The three-dimensional model comparison between wild-type Kir4.1 and Ala167Val variant, which is related to EAST/SeSAME syndrome, revealed that Ala167Val located at the junction between transmembrane domain 2 (TM2) and C-terminus is predicted not to interrupt the sequence of the hydrogen bonds, thus not altering the TM2 alpha-helix structure. In addition, while PolyPhen-2 and Mutation taster evaluated Ala167Val as ‘probably damaging’, PROVEAN and SIFT predicted Ala167Val as ‘neutral’ and ‘tolerated’. These observations are in line with the clinical data demonstrating the milder phenotype in patients with Ala167Val mutation compared with the ones harboring frameshift mutations leading to truncated Kir4.1 channel (Figure. 1).
Furthermore, computational modeling of wild-type NKCC2 and frameshift mutation Arg302Glyfs*3 variant clearly demonstrated that Arg302Glyfs*3 results in a loss of large part of the protein, indicating that NKCC2-Arg302Glyfs*3 is practically nonfunctional (Figure. 2).

Conclusion
Computational modeling of disease-related mutations in various ion channels/transporters represents a novel, powerful approach for comprehensive interpretation of the disease phenotypes observed in patients with rare tubulopathies. In addition, combination of in silico modeling and clinical data could provide us with further insight into molecular mechanisms underlying the renal transporter activities. Furthermore, this in silico computational modeling approach can be applicable and suggestive for novel pharmacological intervention as well as the visual disease severity assessment.