Oleg A. Kirillov

Objective: Type A cystinuria is caused by mutations in the SLC3A1 gene encoding rBAT, the heavy subunit of the rBAT-b^0,+AT transporter. Exon 8 of SLC3A1 gene encodes a region of the rBAT ectodomain involved in hydrophobic-core formation and stabilization of its subdomain architecture. Missense mutations in this region can disrupt its maturation in endoplasmic reticulum (ER) and promote ER-associated degradation (ERAD). The aim of this study was to characterize the structural consequences of frequent exon 8 missense variants, evaluate their effects on rBAT-b^0,+AT maturation, and assess their potential as targets for pharmacological chaperone therapy. Methods: Four amino acid substitutions in rBAT ectodomain (p.Met467Thr, p.Met467Lys, p.Ser455Leu and p.Arg456Cys) were analysed. rBAT missense variant interpretation was based on ClinVar, gnomAD, and published functional studies. Structural modeling was performed using AlphaFold 3, with visualization in UCSF Chimera and contact analysis in YASARA. Results and Discussion: Three of the four variants (M467T, M467K, and R456C) were predicted to significantly destabilize the rBAT structure, whereas S455L showed only a modest effect on global stability. The most pronounced effects were observed for M467T and M467K, which disrupted packing of the hydrophobic core. S455L mainly affected surface packing through increased hydrophobicity and loss of potential hydrogen bonds. R456C introduced a free cysteine residue that may promote aberrant disulfide bond formation. Conclusions: The identified variants are predicted to destabilize the rBAT ectodomain while likely preserving early rBAT-b^0,+AT assembly. Impaired ectodomain maturation may trigger ER quality-control mechanisms and ERAD of the complex. These variants therefore represent potential targets for future pharmacological chaperone-based approaches in type A cystinuria.

Cystinuria is an inherited disorder caused by pathogenic variants in SLC3A1 or SLC7A9, leading to impaired tubular reabsorption of cystine and recurrent cystine stone formation. Disease severity is determined by genotype, the degree of cystinuria, and patient adherence to long-term preventive therapy. Cases with long-term follow-up and molecular genetic confirmation are of particular clinical interest. We report a 36-year-old woman with severe recurrent cystine nephrolithiasis who was followed for 12 years after disease onset at the age of 24 years, shortly after her second pregnancy. Despite standard preventive measures, including high fluid intake, sodium and animal protein restriction, urinary alkalinization, and repeated attempts at standard-dose D-penicillamine therapy, the disease remained highly refractory. During follow-up, the patient experienced 6 surgical interventions and 10 episodes of spontaneous stone passage. Stone analysis repeatedly confirmed 100% cystine composition. Urinary cystine excretion ranged from 169.8 to 979 mg/day. Sanger sequencing identified a pathogenic homozygous SLC3A1 variant, c.1400TC (p.Met467Thr), associated with severe impairment of rBAT subunit trafficking and protein folding defects. Following poor tolerance of conventional thiol therapy doses, continuous low-dose D-penicillamine (250 mg/day) was initiated in February 2024 and was well tolerated. With this regimen, combined with dietary measures and high fluid intake, no new stones were detected on CT imaging over nearly 2 years of follow-up. This case illustrates a severe genotype-associated cystinuria phenotype linked to homozygous SLC3A1 c.1400TC (p.Met467Thr) and suggests that long-term low-dose D-penicillamine may be an effective and better tolerated therapeutic option in patients who cannot tolerate standard dosing regimens.

Cystinuria is a hereditary nephropathy caused by defects in the SLC3A1 and SLC7A9 genes, which encode the subunits of the heterodimeric transporter rBAT–b0,+AT. This complex mediates cystine reabsorption in the proximal renal tubules. Consequently, dysfunction of this complex leads to cystine accumulation in urine and the formation of cystine stones. A major challenge in patient management still remains delayed diagnosis, typically after the manifestation of urolithiasis, which particularly underscores the need to develop preventive and molecularly oriented approaches. The absence of effective therapies capable of modifying the course of the disease thus highlights the relevance of establishing adequate experimental models. Animal models—primarily murine—are clearly indispensable tools for both dissecting the molecular basis of cystinuria and for preclinical evaluation of potential novel therapeutic strategies. This comprehensive review summarizes all current data on animal models of cystinuria, with emphasis on their ability to reproduce important key aspects of the disease associated with dysfunction of the rBAT–b0,+AT transport complex.

Potassium channels constitute the most diverse group of ion channels and play a key role in regulating neuronal excitability and cardiac electrical activity. Despite significant progress in understanding their structure and function, the relationship between specific genetic alterations and disease phenotypes remains insufficiently systematized, which underscores the relevance of a comprehensive analysis of available data. This review summarizes findings from studies of animal models carrying mutations in potassium channel genes, aimed at elucidating their physiological roles and the molecular mechanisms of pathogenesis. Knockout models of kcna1, kcna2, and kcnq2 reproduce key features of human epileptic syndromes, including neuronal hyperexcitability, spontaneous seizures, and early mortality. Deficiency of kcnma1 is associated with impaired motor coordination and the development of cerebellar ataxia. Mutations in kcnq1 and kcne1 result in cardiac and auditory abnormalities characteristic of Jervell and Lange-Nielsen syndrome, whereas double knockout of kcne1/kcnh2 leads to a pronounced susceptibility to ventricular arrhythmias. In addition, deletion of kcnd2 and kcnd3, encoding Kv4 family channel subunits, reveals their critical role in shaping the early phase of cardiac repolarization. Thus, the systematization of data on genetically modified animal models enables the establishment of links between molecular defects in potassium channels and clinical manifestations of disease, and highlights their importance as tools for the development and testing of novel therapeutic approaches.

BACKGROUND: The CRISPR/Cas9 technology enables the generation of genetically modified founder animals (F0 generation) already at the stage of zygote editing. However, the resulting offspring are often mosaic, meaning they carry different mutations in different cells, which complicates accurate genotyping using standard tissue samples. To establish a stable knockout line, it is necessary to identify F0 individuals carrying the target mutations, which requires subsequent crossing with wild-type mice and a large-scale analysis of the F1 offspring, involving significant costs. Therefore, the efficient prediction of inheritable mutations at the F0 generation stage is a critical task. AIM: A comparative evaluation of the efficiency of the TIDE and ICE bioinformatic algorithms for analyzing Sanger sequencing data to accurately predict inheritable mutations in the vldlr gene in F0 mosaic mice. METHODS: The study was conducted on five F0 mosaic mice with mutations in the vldlr gene, obtained by microinjection of CRISPR/Cas9 components into zygotes. Genomic DNA was isolated from ear tissue, the target region of the vldlr gene was amplified by PCR and sequenced by the Sanger method. The resulting chromatograms were analyzed using the TIDE and ICE algorithms. The predictions were validated by crossing the F0 mice with wild-type mice and analyzing the inheritance of mutations in the F1 generation. RESULTS: The comparison of the algorithms showed that both programs correctly predicted all mutations that were subsequently detected in the F1 generation. In total, 31 F1 offspring were analyzed. CONCLUSION: Both tools are suitable for primary screening. The analysis of the offspring confirmed that all actually inherited mutations were predicted by both methods.

Catechol-O-methyltransferase (COMT) is one of the main mammalian enzymes involved in the metabolism of catecholamines such as dopamine and norepinephrine. Catecholamine metabolism is important for regulating normal functioning of the nervous system, including emotions and stress responses. COMT activity in the nervous system allows animals with an inactive comt gene to be used as a model for studying mental disorders. We created a new line of mice with a knockout of the comt gene using CRISPR/Cas9 technology. The concentration of neurotransmitters and their metabolites in the striatum and prefrontal cortex of comt knockout and wild-type mice was measured by high-performance liquid chromatography using an internal standard 2,3-dihydroxybenzoic acid, which is not found in native tissue, at a concentration of 100 ng/ml. Homozygous comt knockout mice were found to have a lack of homovanillic acid, the end product of dopamine metabolism, in the prefrontal cortex and striatum, while levels of the intermediate product 3,4-dihydroxyphenylacetic acid were increased. Norepinephrine levels were increased in the prefrontal cortex of comt knockout mice, while no significant differences were found in the striatum between comt knockout and wild-type mice. Despite corresponding changes in catecholamine metabolites, serotonin and dopamine concentrations were not significantly different between comt knockout and wild-type mice.