The role of SLC7A13/AGT1-variation in human cystinuria

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Cystinuria (CU) is an inherited kidney stone disorder with significant morbidity due to frequent recurrence and associated chronic kidney disease. Mechanistically, disturbed tubular cystine reabsorption based on the dysfunctional heterodimeric amino acid transporter rBAT/SLC3A1-BAT1/SLC7A9 leads to urinary cystine precipitation and consecutive calculus formation. While biallelic pathogenic variants of either SLC3A1 or SLC7A9 can be found in the majority of cases, no clear genotype-phenotype correlation could be established until today. Additionally, in about 10-15% of patients, genetic diagnostics are inconclusive, as none or only monoallelic variants can be identified and dominant inheritance is discussed controversially. Recently, the existence of a second heterodimeric cystine transporter consisting out of the heavy chain rBAT/SLC3A1 with the novel light chain partner AGT1/SLC7A13 has been demonstrated in S3 segments from murine tubular membranes. In a pilot-study with 132 CU-patients, we identified three AGT1-missense variants (N45K, L270F, M452T) on top of biallelic known SLC3A1 or SLC7A9 mutations. In preliminary in vitro analyses, we demonstrated a negative impact of identified SLC7A13/AGT1 patient variants on rBAT N-glycosylation, membrane expression, and subsequent cystine uptake. We therefore hypothesize that specific SLC7A13/AGT1-variants will have an impact on disease severity in human cystinuria, either by genetic modification or full penetrance. With this application, we propose the following four specific aims: i) identification and functional validation of both rare and common SLC7A13/AGT1-variants in combination with patient-specific rBAT and BAT1-variations in a cohort of 400 CU-patients recruited from the multinational EUROCYS-registry and additional major collaborators in the field, ii) evaluation of the prevalence and impact of the common SLC7A13/AGT1 variant N45K in larger CU-cohorts, iii) assessment of epigenetic modification of SLC7A13/AGT1, SLC7A9, and SLC3A1 as determinants of intra- and inter-familial disease variability in CU, and iv) application of a genotype-first approach in order to characterize human SLC7A13/AGT1-associated phenotypes in an unbiased manner via large Exome-datasets (> 200,000 samples from the UK-biobank and Geisinger-database). By successful implementation of aforementioned aims, we believe to clarify the clinical significance of SLC7A13/AGT1-germline variation and to eventually provide long-missing phenotype-genotype correlations in human CU. Unraveling the mechanisms of disturbed luminal complex expression of the AGT1-rBAT and BAT1-rBAT transporters may further pave the way for novel pharmacological treatment options supporting complex stabilization and plasma membrane abundance.