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Genome mining yields putative disease-associated ROMK variants with distinct defects [1]

['Nga H. Nguyen', 'Department Of Biological Sciences', 'University Of Pittsburgh', 'Pittsburgh', 'Pennsylvania', 'United States Of America', 'Srikant Sarangi', 'Inc.', 'Waltham', 'Massachusetts']

Date: 2023-12

Bartter syndrome is a group of rare genetic disorders that compromise kidney function by impairing electrolyte reabsorption. Left untreated, the resulting hyponatremia, hypokalemia, and dehydration can be fatal, and there is currently no cure. Bartter syndrome type II specifically arises from mutations in KCNJ1, which encodes the renal outer medullary potassium channel, ROMK. Over 40 Bartter syndrome-associated mutations in KCNJ1 have been identified, yet their molecular defects are mostly uncharacterized. Nevertheless, a subset of disease-linked mutations compromise ROMK folding in the endoplasmic reticulum (ER), which in turn results in premature degradation via the ER associated degradation (ERAD) pathway. To identify uncharacterized human variants that might similarly lead to premature degradation and thus disease, we mined three genomic databases. First, phenotypic data in the UK Biobank were analyzed using a recently developed computational platform to identify individuals carrying KCNJ1 variants with clinical features consistent with Bartter syndrome type II. In parallel, we examined genomic data in both the NIH TOPMed and ClinVar databases with the aid of Rhapsody, a verified computational algorithm that predicts mutation pathogenicity and disease severity. Subsequent phenotypic studies using a yeast screen to assess ROMK function—and analyses of ROMK biogenesis in yeast and human cells—identified four previously uncharacterized mutations. Among these, one mutation uncovered from the two parallel approaches (G228E) destabilized ROMK and targeted it for ERAD, resulting in reduced cell surface expression. Another mutation (T300R) was ERAD-resistant, but defects in channel activity were apparent based on two-electrode voltage clamp measurements in X. laevis oocytes. Together, our results outline a new computational and experimental pipeline that can be applied to identify disease-associated alleles linked to a range of other potassium channels, and further our understanding of the ROMK structure-function relationship that may aid future therapeutic strategies to advance precision medicine.

Bartter syndrome is a rare genetic disorder characterized by defective renal electrolyte handing, leading to debilitating symptoms and, in some patients, death in infancy. Currently, there is no cure for this disease. Bartter syndrome is divided into five types based on the causative gene. Among these subtypes, Bartter syndrome type II results from genetic variants in the gene encoding the ROMK protein, which is expressed in the kidney and assists in regulating sodium, potassium, and water homeostasis. Prior work established that some disease-associated ROMK mutants misfold and are destroyed soon after their synthesis in the endoplasmic reticulum (ER). Because a growing number of drugs have been identified that correct defective protein folding and/or potentiate ion transport, we wished to identify an expanded cohort of putative disease-associated ROMK mutants. To this end, we developed a pipeline that employs computational analyses of human genome databases along with genetic and biochemical assays. Next, we confirmed the identity of known variants and uncovered previously uncharacterized ROMK variants that are potentially associated with Bartter syndrome type II. Further analyses indicated that select mutants are targeted for ER-associated degradation, while another mutant compromises ROMK function. This work sets-the-stage for continued mining of loss-of-function alleles in ROMK as well as other potassium channels, and may position select Bartter syndrome mutations for correction using emerging pharmaceuticals.

Funding: This work was supported by grant GM131732 from the National Institutes of Health (NIH) to JLB, by grant DK079307 and DK137329 (Pittsburgh Center for Kidney Research) from the NIH to TRK, by grant DK129285 from the NIH to TRK and S. Sheng, and by award ID 826608 from the American Heart Association to NHN. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

The rapid growth of human genome sequence data and improved curation of existing databases have facilitated the identification of disease-linked genes as well as uncharacterized disease-causing mutations. To date, ROMK mutations associated with Bartter syndrome type II were primarily identified via clinical studies [ 4 , 12 , 26 , 27 ], but numerous uncharacterized disease-linked ROMK mutations likely remain unearthed in human databases. We now report on the use of two computational approaches to uncover additional ROMK variants that are potentially associated with Bartter syndrome type II. First, we examined ROMK missense mutations in two NIH-supported databases, the Trans-Omics for Precision Medicine (TOPMed) study [ 28 ] and the ClinVar database [ 29 ], using an algorithm that predicts mutation severity and pathogenecity. This algorithm, known as Rhapsody [ 30 ], utilizes evolutionary conservation along with structural and dynamic features. We previously validated Rhapsody’s predictive power to probe the potential impact of both known disease-associated and randomly selected ROMK variants [ 31 ]. Second, we performed in silico association analyses to identify links between ROMK variants in the UK Biobank and disease-associated phenotypes [ 32 – 34 ] using the REVEAL: Biobank computational platform [ 35 – 38 ]. As a result of these complementary approaches, we report here on the identification and characterization of a cohort of ROMK variants using yeast, X. laevis oocytes, and tissue culture cells. Ultimately, we discovered new variants that 1) are unstable and targeted for ERAD, 2) are poorly expressed at the cell surface, and 3) exhibit defective channel function. The identification of a common allele from the two computational approaches validates the complementary nature of these methods and outlines a new pipeline to assess other identified disease-associated mutations in ROMK, an effort that may aid in the development of precision medicines to treat those with Bartter syndrome type II.

The ERAD pathway represents a first-line defense in the secretory pathway to recognize and deliver misfolded proteins to the ubiquitin-proteasome system (UPS) in the cytosol. During ERAD, molecular chaperones, such as heat shock protein 70 (Hsp70), recognize and target misfolded proteins for extraction (or “retrotranslocation”) from the ER lumen and ER membrane into the cytosol and then for ubiquitination, which serves as a prelude to proteasome-dependent degradation [ 16 – 21 ]. Retrotranslocation requires a AAA + -ATPase, known as Cdc48 in yeast, or p97 (also known as Valosin Containing Protein; VCP) in higher cells [ 22 , 23 ]. In a study utilizing a yeast expression system and human cell lines, we showed that Hsp70 and Cdc48 were required for the degradation of Bartter syndrome-linked mutant ROMK species, whereas wild-type ROMK was relatively stable (15). In addition, the expression of ROMK in a yeast strain lacking two endogenous potassium channels (trk1Δtrk2Δ) restored yeast growth on low potassium media [ 24 , 25 ]. As a result, ROMK folding, trafficking to the plasma membrane (where it functions), and potassium transport can be assayed in yeast. Together, these data indicate that the yeast system effectively monitors the efficacy of ROMK biogenesis and provides a facile growth assay, allowing one to screen for defective ROMK mutants in a quantitative and high-throughput manner.

In theory, defects in ROMK might arise from a lack of expression, altered protein folding and/or tetramerization, accelerated degradation of poorly folded/assembled subunits, inefficient transport to the cell surface, and/or poor channel (i.e., potassium transport) activity. Indeed, early studies in X. laevis oocytes and COS-7 cells demonstrated that some Bartter syndrome type II-associated mutants were absent from the cell surface and others were defective for potassium transport [ 12 – 14 ]. Later work by our group showed that four disease-causing ROMK mutations that cluster in a cytosolic, β sheet-rich immunoglobulin-like domain cause the protein to misfold in the endoplasmic reticulum (ER) [ 15 ], an outcome that targets ROMK for ER associated degradation (ERAD).

One among several causes of Bartter syndrome arises from defects in a potassium channel residing on the apical surface of two segments of the nephron: the thick ascending limb and the cortical collecting duct [ 4 ]. The channel, ROMK (also known as Kir1.1), is encoded by KCNJ1 and was the first inwardly rectifying potassium (Kir) channel identified [ 5 – 7 ]. Like other Kir channels, ROMK functions as a tetramer [ 8 ] and exhibits a larger inward current than outward current; all family members also share a common structure that contains two transmembrane domains (TMD) and cytoplasmic N- and C-terminal domains [ 9 ]. In the kidney, ROMK plays a central role in mediating potassium efflux, which in turn provides a crucial source of potassium to facilitate sodium reabsorption through the NKCC2 transporter in the thick ascending limb. Furthermore, ROMK-dependent potassium secretion generates a lumen positive transepithelial potential that drives paracellular sodium absorption [ 10 ]. Mutations in ROMK give rise to Bartter syndrome type II, also called antenatal Bartter syndrome, since patients often present prenatally (e.g., with excessive amniotic fluid). Among these individuals, observed features include a failure to thrive, renal salt wasting and volume depletion, early post-natal hyperkalemia, hypercalcuria, nephrocalcinosis, and arrhythmias, which together contribute to a high infant mortality rate [ 11 ].

First identified in 1962, Bartter syndrome is group of rare, life-threatening disorders caused by defects in or impaired function of electrolyte channels within the kidney, compromising renal sodium and potassium handling and resulting in excessive electrolyte and water excretion [ 1 ]. To date, therapies for Bartter syndrome include electrolyte supplements and non-steroidal anti-inflammatory drugs, which are limited to only mitigating the symptoms. Although disease severity, presentation, and age of onset vary, Bartter syndrome can lead to a failure to thrive, sudden cardiac arrest, and even death [ 2 , 3 ].

Results

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[1] Url: https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1011051

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