# Long-Read Sequencing Identifies Novel Pathogenic Intronic Variants in Gitelman Syndrome

Daan H.H.M. Viering, Marguerite Hureaux, Kornelia Neveling, Femke Latta, Michael Kwint, Anne Blanchard, Martin Konrad, René J.M. Bindels, Karl-Peter Schlingmann, Rosa Vargas-Poussou, et al.

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## To cite this version:

Daan H.H.M. Viering, Marguerite Hureaux, Kornelia Neveling, Femke Latta, Michael Kwint, et al. Long-Read Sequencing Identifies Novel Pathogenic Intronic Variants in Gitelman Syndrome. Journal of the American Society of Nephrology, 2023, 34 (2), pp.333-345. 10.1681/ASN.2022050627. hal-04053345

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# Long-Read Sequencing Identifies Novel Pathogenic Intronic Variants in Gitelman Syndrome

Daan H.H.M. Viering<sup>1</sup>,<sup>†</sup> Marguerite Hureaux,<sup>2,3,4</sup> Kornelia Neveling,<sup>5</sup> Femke Latta,<sup>1</sup> Michael Kwint,<sup>5</sup> Anne Blanchard,<sup>2,6,7</sup> Martin Konrad,<sup>8</sup> René J.M. Bindels,<sup>1</sup> Karl-Peter Schlingmann<sup>9</sup>,<sup>8</sup> Rosa Vargas-Poussou<sup>10</sup>,<sup>2,3,6</sup> and Jeroen H.F. de Baaij<sup>1</sup>

Due to the number of contributing authors, the affiliations are listed at the end of this article.

## ABSTRACT

**Background** Gitelman syndrome is a salt-losing tubulopathy characterized by hypokalemic alkalosis and hypomagnesemia. It is caused by homozygous recessive or compound heterozygous pathogenic variants in *SLC12A3*, which encodes the Na<sup>+</sup>-Cl<sup>-</sup> cotransporter (NCC). In up to 10% of patients with Gitelman syndrome, current genetic techniques detect only one specific pathogenic variant. This study aimed to identify a second pathogenic variant in introns, splice sites, or promoters to increase the diagnostic yield.

**Methods** Long-read sequencing of *SLC12A3* was performed in 67 DNA samples from individuals with suspected Gitelman syndrome in whom a single likely pathogenic or pathogenic variant was previously detected. In addition, we sequenced DNA samples from 28 individuals with one variant of uncertain significance or no candidate variant. Midigene splice assays assessed the pathogenicity of novel intronic variants.

**Results** A second likely pathogenic/pathogenic variant was identified in 45 (67%) patients. Those with two likely pathogenic/pathogenic variants had a more severe electrolyte phenotype than other patients. Of the 45 patients, 16 had intronic variants outside of canonic splice sites (nine variants, mostly deep intronic, six novel), whereas 29 patients had an exonic variant or canonic splice site variant. Midigene splice assays of the previously known c.1670-191C>T variant and intronic candidate variants demonstrated aberrant splicing patterns.

**Conclusion** Intronic pathogenic variants explain an important part of the missing heritability in Gitelman syndrome. Long-read sequencing should be considered in diagnostic workflows for Gitelman syndrome.

JASN 34: ●●●–●●●, 2022. doi: https://doi.org/10.1681/ASN.2022050627

Pathogenic variants in *SLC12A3* cause Gitelman syndrome, which is a recessively inherited renal tubulopathy. The disease is characterized by hypochloremic metabolic alkalosis, hypokalemia, hypomagnesemia, and hypocalciuria. Muscle cramps, paresthesias, nocuria, muscle weakness, and fatigue are common clinical manifestations of Gitelman syndrome.¹ The molecular mechanism of Gitelman syndrome is defined by loss of function of the thiazide-sensitive sodium chloride cotransporter (NCC), encoded by *SLC12A3*.² A defect in this transporter interferes with normal sodium and chloride reabsorption in the distal convoluted tubule. A Gitelman syndrome–like phenotype may also be found in other genetic diseases that reduce sodium reabsorption in the distal

convoluted tubule. Examples include diseases that affect the genes *CLCNKB*, *HNF1B*, *MT-TI*, *MT-TF*, *KCNJ10*, or *KCNJ16*.3–8

Received May 30, 2022. Accepted October 17, 2022.

D.H.H.M.V., M.H., R.V.-P., and J.H.F.d.B. contributed equally to this work.

Published online ahead of print. Publication date available at www.jasn.org.

**Correspondence:** Dr. Jeroen H.F. de Baaij, Department of Physiology, Radboud Institute for Molecular Life Sciences, Radboudumc, P.O. Box 9101, 6500HB, Nijmegen, The Netherlands. Email: jeroen.debaaij@radboudumc.nl

Copyright © 2022 by the American Society of Nephrology
In some patients with apparent Gitelman syndrome, a genetic diagnosis cannot be established. Previous studies demonstrate that, in 8%–31% of all patients, only one pathogenic variant in *SLC12A3* can be found.⁹⁻¹⁴ Recent studies showed heterozygous carriers of one pathogenic variant in *SLC12A3* may display a weak intermediate phenotype. Blanchard *et al*.¹⁵ compared 81 heterozygous carriers of a pathogenic/likely pathogenic *SLC12A3* variant with 82 healthy noncarriers and 79 patients with Gitelman syndrome of similar age, body mass index, and sex ratio. They found heterozygous and noncarriers were clinically and biochemically very similar. No differences were observed in BP or in plasma electrolytes, although a subtle intermediate phenotype was observed in heterozygous carriers, characterized by slightly higher serum calcium concentrations (0.04 mmol/L higher in carriers than in controls; *P*=0.01).¹⁵ Additionally, they found a trend of increased plasma aldosterone (*P*=0.06) and lower plasma PTH (*P*=0.08). Later, Wan *et al*.¹⁶ studied 5812 Old Order Amish subjects and demonstrated potassium levels were lower in heterozygous carriers of the c.1924C>G (p.R642G) variant in *SLC12A3* (4.05 versus 4.20 mmol/L; *P*=2.39×10⁻⁹). The lowest serum potassium documented in this group of 147 heterozygous carriers was 3.5 mmol/L.¹⁶ Taken together, it is unlikely that a single pathogenic heterozygous *SLC12A3* variant can explain all symptoms of Gitelman syndrome. Alternatively, four studies on Taiwanese and Japanese patients showed that three intronic variants can explain missing heredity.¹⁰,¹⁴,¹⁷,¹⁸ On the other hand, a very recent, but small, study by Rossanti *et al*.¹⁹ investigated *SLC12A3* introns in 13 patients with genetically unresolved Gitelman syndrome and found that the additional yield might be moderate: only one additional likely pathogenic variant was identified. Thus, it is currently unclear whether the findings of the first four studies translate to other populations and whether other intronic variants are still missed.

Recent advances in genetic techniques allow detection of such intronic variants and structural and promoter variants, thus increasing diagnostic yield for monogenic disorders. For example, single-molecule molecular inversion probes (smMIPs)²⁰ increased diagnostic yield in ABCA4-related disease,²¹,²² whole-genome sequencing in Alagille syndrome,²³ and long-read sequencing²⁴,²⁵ in neurodevelopmental disorders.²⁶ Notably, a previous study by Vargas-Poussou *et al*.⁹ in 448 unrelated patients with Gitelman syndrome detected a large number of structural variants in *SLC12A3*. However, the technique used in this study, multiplex ligation-dependent probe amplification (MLPA) screening of *SLC12A3* exons, is primarily suited to detect exonic deletions and not suited to detect inversions, insertions, or intronic deletions. Of the aforementioned three techniques, long-read sequencing has the highest sensitivity for detecting such structural variations. Furthermore, long-read sequencing has the potential to assess variant phasing.²⁴

Some genetic variants detected by long-read sequencing, smMIPs, or genome sequencing require further investigation

## Significance Statement

Gitelman syndrome is caused by biallelic pathogenic variants in *SLC12A3*, which encodes the thiazide-sensitive sodium-chloride cotransporter (NCC). A subset of patients with Gitelman syndrome has only one specific pathogenic variant identified. In this study, long-read sequencing identified 46 previously undetected variants in 95 patients with suspected Gitelman syndrome. A midigene splice assay confirmed the pathogenicity of intronic variants. The data show that both intronic and exonic variants were missed previously and that former detection of one *SLC12A3* variant predicts identification of an additional variant. The findings advocate long-read sequencing, complemented with a midigene splice assay, for intronic variants, as a second-tier diagnostic test in patients with one pathogenic *SLC12A3* variant.

to determine pathogenicity, especially for intronic variants. Pathogenicity of newly identified intronic variants can be assessed using a midigene approach.²²,²⁷⁻³² This technique incorporates the genomic sequence containing the variant into an expression vector to examine aberrant splicing defects.

In this study, we systematically screened the intronic regions of *SLC12A3* in a large cohort of patients with Gitelman syndrome. We hypothesized that, in patients in whom only one pathogenic variant had been found in *SLC12A3*, a second pathogenic intronic variant, promoter variant, or structural variant could explain the disease. We used long-read sequencing to examine nonexon regions of *SLC12A3* and performed variant phasing to demonstrate whether two variants are on opposite alleles.

## METHODS

### Inclusion and Ethical Approval

Two cohorts, together comprising 120 patients with a clinical suspicion of Gitelman syndrome, were included in this study. Both cohorts were enriched for patients with one pathogenic or likely pathogenic variant, and contained some patients without a candidate variant or only one variant of uncertain significance (VUS). The first cohort, containing 95 individuals without genetic diagnosis, is from a tertiary referral center in Paris, France: Hôpital Européen Georges-Pompidou. Samples were collected between 1997 and 2021, and patients had previously been investigated by Sanger sequencing, a tubulopathy gene panel,³³ and/or MLPA (the latter to screen for deletions in *SLC12A3*). The second cohort, containing 25 patients without a genetic diagnosis, is from University Hospital Münster (Münster, Germany). Samples were collected between 1997 and 2007, and all of these patients had previously been screened by single-strand conformation polymorphism (SSCP).³⁴ Further cohort details can be found in the Supplemental Appendix. The study was performed in accordance with the declaration of Helsinki, and informed consent was obtained from all subjects before inclusion.
## Clinical Data

Clinical data were obtained as part of routine clinical care at the two medical centers. Electrolytes were measured in serum (sodium, potassium, calcium, magnesium, chloride, and bicarbonate) and urine (sodium, potassium, potassium-creatinine ratio, calcium, magnesium, calcium-creatinine ratio). Reference values in the two centers were similar. Provided potassium-creatinine ratios are before supplementation. We also collected the following information: birth year, sex, family history, consanguinity, age at presentation, age at exploration, main symptoms, creatinine, renin levels, and aldosterone levels.

## Generation of Long-Range PCR Amplicons

Producing long-range PCR amplicons of a gene of interest allows long-read sequencing for many patients simultaneously. To achieve this, primers were designed using Primer-BLAST for amplicons of approximately 10–11 kb. Primers were chosen to produce seven overlapping PCR amplicons that would span all introns of *SLC12A3* and approximately 3.5 kb directly upstream of *SLC12A3* (primer sequences in Supplemental Table 1). Genomic DNA was isolated from whole blood. The LongAmp Taq polymerase (catalog number M0287L; NEB) was used for PCR reactions, and the protocol was optimized individually for every patient and every amplicon (Supplemental Appendix). Of the 120 patient DNA samples, 95 patients were selected for long-read sequencing on the basis of DNA quality. In these patients, amplicon generation was successful for at least six of the seven different amplicons.

## Long-Read DNA Sequencing and Bioinformatic Analyses

All seven amplicons of each sample were pooled equimolarly. Library preparation was done per sample, according to the protocol "Procedure and Checklist—Preparing SMRTbell Libraries using PacBio Barcoded Adapters for Multiplex SMRT Sequencing" (catalog number 100-538-700-02; Pacific Biosciences), assigning individual barcodes to each sample (Supplemental Appendix). All samples were sequenced together on a single 8M SMRTcell (catalog number 101-389-001; Pacific Biosciences) using a Sequel IIe system (Pacific Biosciences). After the run, generation of circular consensus reads, demultiplexing, and mapping of these reads to GRCh37/hg19 was performed using SMRTLink 10.0. Bam files were loaded into the Sequence Pilot software (JSIMedical Systems) to perform variant calling (Supplemental Appendix). Additionally, bam files were visually inspected in the Integrated Genome Viewer.

Variants with a minor allele frequency >0.5% were excluded from further analysis. We evaluated pathogenicity manually for all coding and canonic splice site variants in *SLC12A3* by using the American College for Medical Genetics criteria³⁵ and Association for Clinical Genomic Science specifications.³⁶ This allows classification of variants into five classes: (1) benign; (2) likely benign; (3) VUS; (4) likely pathogenic, i.e., >90% certainty of pathogenicity; and (5) pathogenic, i.e., >99% certainty of pathogenicity.

Due to the large number of intronic variants with a minor allele frequency <0.5%, only a subset of variants was selected for the midigene splice assays (details in Supplemental Appendix). Selection was primarily made on the basis of a predicted effect on splicing by spliceAI and/or SQUIRLS.

Canonic splice site variants were defined as variants affecting -1, -2, +1, and +2 positions of the exon-intron boundary. Noncanonic splice site variants were defined as intronic variants within 8 bp from the splice donor site, or within 100 bp from the splice acceptor site, except for the -1, -2, +1, and +2 positions. All other intronic variants were considered deep intronic variants. Assessing whether variants are on the same or opposite alleles (variant phasing) was performed manually in Integrated Genome Viewer in all samples with any combination of two VUS, likely pathogenic, or pathogenic variants.

## Midigenes

In short, midigenes were created by cloning a PCR amplicon obtained with patient DNA into a splice assay expression vector that was validated in previous studies (pCIneo-RHOexon3-RHOexon5; kind gift from Prof. Frans Cremers).³² Cloning was done using Gateway Cloning technology (MAN0000282). Primers used are listed in Supplemental Table 1. Midigenes were made for all selected intronic variants (Supplemental Table 2). For deep intronic variants known from literature only, we performed mutagenesis on the wild-type expression vectors to obtain mutant vectors. Mutagenesis primers are listed in Supplemental Table 1; genomic locations of midigenes are depicted in Supplemental Figure 1.

## Splice Assays

HEK293 cells were grown in poly-l-lysin-coated plates and transfected with a wild-type or mutant midigene by using lipofectamine (catalog number 11668019; Invitrogen), which was repeated for all investigated intronic variants. RNA was isolated from transfected cells and reverse transcribed with an M-MLV reverse transcription (catalog number 28025; Invitrogen). cDNA was PCR amplified (using primers listed in Supplemental Table 2) and run on a 1.5%-2.5% (wt/vol) agarose gel. Lastly, bands were excised and their sequences were assessed using Sanger sequencing (Supplemental Appendix).

## Statistical Analyses

Statistical significance of any difference in the studied parameters between patients with two likely pathogenic/pathogenic variants, with one likely pathogenic/pathogenic variant, and without any likely pathogenic/pathogenic variant was determined using an ANOVA with Tukey-corrected multiple comparisons. Urinary calcium-creatinine ratios were compared for adults only, given the variation in reference values for children at different ages.³⁷ Statistical significance was defined as $P<0.05$.
RESULTS

Long-Read Sequencing of SLC12A3 Increased Diagnostic Yield

To examine intronic variants in patients with unresolved Gitelman syndrome, seven amplicons of 10–11 kb in size were designed to contain the exons and introns of *SLC12A3*, spanning from chromosome 16: 56,892,479 to chromosome 16: 56,947,674 (GRCh37/hg19). Long-range PCR successfully generated six or seven amplicons in 96 patients with unresolved Gitelman syndrome. Of these 96 patients, 95 were selected for long-read sequencing, together with one patient who had been diagnosed (positive control; Figure 1A). Analysis of the long-read sequencing data showed a high number of high-fidelity reads that were approximately 10–11 kb in size (Supplemental Figure 2). Coverage of all amplicons ranged between 100× and 10,000×.

Long-read sequencing identified a new likely pathogenic or new pathogenic variant in 46 patients (47%; Figure 1, B and C, Supplemental Table 3). In most of these patients (45), one likely pathogenic or pathogenic variant had been

detected previously. In patients in whom no likely pathogenic or pathogenic variant had previously been detected, long-read sequencing detected a new likely pathogenic or pathogenic variant in only one patient.

In total, long-read sequencing identified 31 additional pathogenic variants, 15 additional likely pathogenic variants, and three additional VUS (Figure 1D). Of the 46 newly identified likely pathogenic or pathogenic variants, 16 variants were located at a noncanonic splice site or deep intronically (38%). Additionally, six canonic splice site variants and 22 previously undetected exonic variants were identified (Figure 1D, Supplemental Table 4). Various reasons were identified for the fact that some exonic and splice site variants were previously missed (Supplemental Table 4). Most of the patients in whom a variant had been missed were previously sequenced by Sanger sequencing or investigated with SSCP; only two patients had previously been investigated by next-generation sequencing. Lastly, two structural variants were found: a 1190-bp deletion deleting exon 9, and a 28-bp deletion removing the canonic splice site between exon 2 and intron 2.

<figure><img src="image_3.png" /><figcaption>**Figure 1.** Long-read sequencing identifies a second variant in a large number of patients. (A) Flowchart of study before sequencing. (B) Number of patients in whom an additional variant was identified by long-read sequencing, when one likely pathogenic or pathogenic variant was known before study start. (C) Number of patients in whom an additional variant was identified by long-read sequencing, when no likely pathogenic or pathogenic variant was known before study start. (D) The variants additionally identified by long-read sequencing. Only pathogenic variants, likely pathogenic variants, and VUS are shown. Likely pathogenic variant, >90% certainty of pathogenicity; pathogenic variant, >99% certainty of pathogenicity.</figcaption></figure>
Moreover, long-read sequencing was able to detect all variants that had been found by previous investigations, except for three patients. Variants were missed in these three patients because the sequencing data contained variant information of one allele only (in two patients), or because a copy number variant extended beyond the genomic region covered in this study (i.e., a deletion of exon 26 in one patient).

Variant phasing in 23 patients demonstrated that the two identified variants (VUS, likely pathogenic or pathogenic) were located on opposite alleles in 22 patients (Supplemental Figure 3A, Supplemental Table 3). In the remaining patient, the two variants were in *cis*. In 27 other patients with two identified variants (VUS, likely pathogenic or pathogenic), variant phasing was not possible due to a missing amplicon or due to the absence of genetic variants in regions of ampli-con overlap.

Variant phasing also showed a possible founder effect for the novel c.852 + 243C>T variant, while showing different allelic backgrounds (i.e., no shared haploblock) for patients with the c.1670-191C>T and c.602-16G>A variants (Supplemental Figure 3).

## Known and Novel Intronic Variants Cause Aberrant mRNA Splicing

Sixteen intronic variants were evaluated with a midigene splice assay to assess their effect on mRNA splicing (Figure 2A). We found six novel variants that are likely pathogenic, and provide supporting data for the pathogenicity of five known variants (Figure 2B, Table 1). The two algorithms SQUIRLS and SpliceAI had similar accuracy in predicting pathogenicity (67% predictions correct versus 73% predictions correct, when including possibly pathogenic scores as pathogenic for SpliceAI; Supplemental Table 5).³⁸,³⁹

Different mechanisms of aberrant splicing were observed (Figure 3, Table 1, Supplemental Figure 4). Seven variants led to inclusion of a pseudoexon by creating a splice site or activating a cryptic splice site, two variants led to exon elongation, one variant to exon skipping, and three variants created multiple aberrant transcripts with different effects. An interesting example is the c.2548 + 255G>A variant that leads to inclusion of the same 90-bp pseudoexon that is created by the known c.2548 + 253C>T variant. Inclusion of the 90-bp pseudoexon would induce a p.Gly849_Gly850insValPheTrpLeu* truncating protein change. Another example is the c.2447-38_2447-20del variant, which is predicted by Mercer et al.⁴⁰ to delete two high-confidence branch points. This leads to usage of a more proximal, cryptic splice acceptor site in almost 100% of transcripts, leading to an 86-bp elongation of exon 21 on the 5' side, leading to a direct insertion of a stop codon in isoforms NP_000330.3 and NP_001119579.2 (p.Ala815_Leu816ins* and p.Ala814_Leu815ins*, respectively), and causing a truncating frameshift in isoform NP_001119580.2 (p.Val807Glufs*35). The c.2951 + 825C>T variant, located in the last intron, showed faint accessory bands on agarose gel, but these accounted for

only 12%–30% of the total amount of transcript in mutant (Supplemental Figure 4A) and was therefore considered a VUS.

All variant classifications were submitted to ClinVar (submission number SUB11476558; Supplemental Table 6).

## Patients with a Diagnosis Differ Biochemically from Patients Who Are Undiagnosed

Patients in whom two likely pathogenic/pathogenic variants were found after sequencing differed in several biochemical parameters from patients in whom only one or zero likely pathogenic/pathogenic variants were identified (Figure 4, Supplemental Table 7). ANOVA testing showed significant differences for age at diagnosis (*P*=0.02), potassium (*P*=0.0006), magnesium (*P*=0.004), and chloride (*P*=0.0007). More specifically, patients in whom two likely pathogenic/pathogenic variants were found were, on average, 12 (95% CI, -23 to -2) years younger than patients in whom only one likely pathogenic/pathogenic variant was found. Serum potassium was, on average, 0.56 mmol/L lower in patients with two variants versus patients with one variant (95% CI, -0.19 to -0.93 mmol/L), serum magnesium was an average 0.12 mmol/L lower (95% CI, -0.02 to -0.23 mmol/L), and mean serum chloride was 3.1 mmol/L lower (95% CI, -0.27 to -5.9 mmol/L). No significant differences were observed for serum sodium, bicarbonate, or calcium levels; for urinary calcium-creatinine ratio in adults; or potassium-creatinine ratio. No significant difference was found between patients with one likely pathogenic/pathogenic variant and those without likely pathogenic/pathogenic variants for any of the parameters.

## DISCUSSION

In approximately 8%–31% of patients with symptoms of Gitelman syndrome, only one pathogenic variant is currently found, depending on the techniques used.⁹⁻¹⁴ Here, we show that resequencing of exons and introns can help to identify a second pathogenic variant in a significant number of patients. Long-read sequencing identified a second variant in 67% of patients for whom one likely pathogenic or pathogenic SLC12A3 variant was previously found (45 patients). Of these patients, 16 (36%) had a likely pathogenic or pathogenic intronic variant; in most patients, the variant was located >20 bp into the intron. We identified novel intronic candidate variants and confirmed pathogenicity for six of these variants by an SLC12A3 midigene splice assay. Moreover, patients in whom two likely pathogenic or pathogenic variants are found have a more severe phenotype than other patients.

This study provides evidence for the pathogenicity of six novel intronic variants. Most of these variants are located deep in introns, and were previously missed by commonly used sequencing techniques that cover only exons and exon-intron boundaries. These findings demonstrate the importance of
<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /><figcaption>**Figure 2.** Identification of 11 unique intronic likely pathogenic/pathogenic variants in SLC12A3. (A) Overview of identified candidate diagnostic variants and summary of which variants were tested by the midigene splice assay. The number of unique variants can be different from the total number of candidate diagnostic variants because the same unique variant was sometimes identified in multiple individuals. (B) Intron-exon structure of the SLC12A3 gene, which contains 26 exons and spans approximately 50 kb. The text describes the location of known and novel likely pathogenic/pathogenic intronic variants on the SLC12A3 transcript NM_000339.3. Previously known variants are depicted in gray; novel variants identified in this study are depicted in black. The top part shows deep intronic variants; the lower part above the gene shows noncanonic splice site variants. The location of the seven PCR amplicons obtained from patient DNA before sequencing is shown in gray at the bottom. Variant notation follows Human Genome Variation Society nomenclature. Chr16, chromosome 16.</figcaption></figure>

genetic screening of introns for rare renal diseases. Except for a recent small study,¹⁹ no renal disease genes have been studied comprehensively for pathogenic (deep) intronic variants. Excellent examples of the potential of intron sequencing can be drawn from studies on retinal disease genes.²²,⁴¹⁻⁴³ A study by Khan *et al*.²² showed that, in 22% of patients with Stargardt disease in whom two variants were found, one variant was a deep intronic variant, which is in a similar range as the 31% that we find in this study.

To tackle the diagnostic gap, an earlier study in a Taiwanese population suggested screening for known intronic variants in patients with Gitelman syndrome in whom one pathogenic variant is identified.¹⁴ Importantly, most intronic variants that we identified in our population were novel; six of 16 patients had a variant that had never been reported before in Gitelman syndrome. This suggests that screening for known or recurrent variants is not sufficient in those patients, at least not in a French/German population. Only the
<table><caption>Table 1. Intronic variants tested by midigene splice assay</caption><thead><tr><th>Variant (HGVS)</th><th>Intron</th><th>Classification</th><th>References</th><th>Putative Mechanism on the Basis of Midigene Splice Assay</th></tr></thead><tbody><tr><td>c.283-438_283-387dup</td><td>1</td><td>Likely pathogenic</td><td>This study</td><td>Variable length pseudoexon inclusion, by creating a novel splice acceptor site and activating a cryptic acceptor site at c.283-456; cryptic splice donor sites are present at c.283-73 and c.283-94</td></tr><tr><td>c.430-472_430-461del</td><td>2</td><td>Likely benign</td><td>This study</td><td>No aberrant splicing</td></tr><tr><td>c.602-16G&gt;A</td><td>4</td><td>Likely pathogenic</td><td>This study (two patients)<sup>40,69</sup></td><td>5' exon 5 elongation by 14 bp, by creating a novel splice acceptor site</td></tr><tr><td>c.602-11T&gt;A</td><td>4</td><td>Likely pathogenic</td><td><sup>a</sup></td><td>5' exon 5 elongation by 9 bp, by creating a novel splice acceptor site</td></tr><tr><td>c.852 + 243C&gt;T</td><td>6</td><td>Likely pathogenic</td><td>This study (two patients)</td><td>Variable length pseudoexon inclusion, by creating a novel splice donor site</td></tr><tr><td>c.1095 + 4A&gt;G</td><td>8</td><td>Likely pathogenic</td><td>This study<sup>9</sup></td><td>Skipping of exon 8 (131 bp) by silencing the splice donor site of exon 8</td></tr><tr><td>c.1567 + 297T&gt;G</td><td>12</td><td>Likely pathogenic</td><td><sup>18,19</sup></td><td>108-bp pseudoexon inclusion, by creating a new splice acceptor site; usage of cryptic splice donor site at c.1567 + 405</td></tr><tr><td>c.1670-191C&gt;T</td><td>13</td><td>Pathogenic</td><td>This study (7 patients)<sup>14,17-19</sup></td><td>238-bp pseudoexon inclusion, by activating a cryptic splice donor site at c.1670-192; usage of a cryptic splice acceptor site at c.1670-430</td></tr><tr><td>c.2037 + 87A&gt;G</td><td>16</td><td>Likely pathogenic</td><td>This study</td><td>3' exon 16 elongation by 82 bp, by activating a cryptic splice donor site</td></tr><tr><td>c.2368 + 185C&gt;T</td><td>19</td><td>Likely benign</td><td>This study</td><td>No aberrant splicing</td></tr><tr><td>c.2369-191C&gt;T</td><td>19</td><td>Likely benign</td><td>This study</td><td>No aberrant splicing</td></tr><tr><td>c.2447-38_2447-20del</td><td>20</td><td>Likely pathogenic</td><td>This study</td><td>5' exon 21 elongation by 86 bp (not counting the 19 deleted base pairs), by deleting two high-confidence branch points</td></tr><tr><td>c.2548 + 253C&gt;T</td><td>21</td><td>Likely pathogenic</td><td><sup>14,19</sup></td><td>90-bp pseudoexon inclusion, by activating a cryptic splice donor site at c.2548 + 251; usage of a cryptic splice acceptor site at c.2548 + 161 in some transcripts and 3' elongating exon 21 until c.2548 + 251 in others</td></tr><tr><td>c.2548 + 255G&gt;A</td><td>21</td><td>Likely pathogenic</td><td>This study</td><td>90-bp pseudoexon inclusion, by silencing a cryptic splice donor site at c.2548 + 255 and activating a cryptic splice donor site at c.2548 + 251; usage of a cryptic splice acceptor site at c.2548 + 161</td></tr><tr><td>c.2549-218_2549-215del</td><td>21</td><td>Likely benign</td><td>This study</td><td>No aberrant splicing</td></tr><tr><td>c.2951 + 825C&gt;T</td><td>25</td><td>VUS</td><td>This study</td><td>Possible inclusion of pseudoexon in some transcripts, results inconclusive</td></tr><tr><td>c.2952-33A&gt;G</td><td>25</td><td>VUS</td><td>This study</td><td>Not assessed</td></tr></tbody></table>

Conclusions from the midigene splice assays. Variant notation follows the HGVS recommendations, the positions indicate the cDNA position on SLC12A3, NM_000339.3. Classification follows the American College for Medical Genetics recommendations and Association for Clinical Genomic Science specifications.³⁵,³⁶ HGVS, Human Genome Variation Society.

c.1670-191C>T variant was truly recurrent in our study. We detected this variant in seven patients, and it has been reported previously in eight families.¹⁴,¹⁷,¹⁸

To determine the pathogenicity of the novel intronic mutations, an *in vitro* splice assay was developed for SLC12A3 using midigenes.²²,²⁷⁻³² We showed that six of the identified rare intronic variants are indeed likely pathogenic. Assessment of one variant located in the last intron, an intron that is more difficult to assess with midigenes,³² gave inconclusive results. The splicing algorithms SQUIRLS and SpliceAI each predicted 67%–73% of the splicing effects correctly.³⁸,³⁹ The limited prediction accuracy is in line with results from benchmarking studies.⁴⁴,⁴⁵ Thus, experimental evidence is still needed to classify variants as likely pathogenic or pathogenic.³⁵,³⁶

The midigene splice assay gives insight in the mis-splicing mechanism of known and novel pathogenic intronic variants. We show that six variants lead to inclusion of a pseudoexon due to creation of, or activation of, cryptic splice sites. As noted previously by Nozu *et al.*,¹⁸ the presence of repeat elements in the introns of SLC12A3 might provide cryptic splice sites that can be activated with a single intronic mutation.⁴⁶,⁴⁷ For the c.1670-191C>T variant, this predisposition is probably aggravated by the propensity of the c.1670-191 position to mutate into a thymine. This nucleotide is followed by a guanine (a CpG dinucleotide) and located in a region that is rich in cytosines followed by guanines (a CpG island), which greatly increases the mutation rate at this base.⁴⁸ Furthermore, we demonstrate that five variants direct the splicing

<footnote><sup>a</sup>This variant had been found in a patient with a Gitelman syndrome phenotype before conception of this study. However, because the clinical significance was not known, we included it for midigene testing.</footnote>
<figure><img src="image_6.png" /></figure>

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<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /><figcaption>**Figure 3.** Midigene splice assays show incorrect splicing of mutant DNA. Results of the midigene splice assays, obtained by transfection of HEK293 cells with the mutant (mut) and wild-type (WT) SLC12A3 midigenes, followed by RNA isolation and RT-PCR. (A, C, and E) Gel electrophoresis images of mutant versus wild-type midigene mRNA of the three recurrent pathogenic intronic variants. (B, D, and F) Exon-intron structure of mutant and wild-type mRNA of the same three intronic variants. Black depicts the structure of wild-type RNA; red depicts the structure of mutant mRNA. (G) Exon-intron structure of mutant mRNA of all remaining variants. Full gel images and additional Sanger sequencing results are available in Supplemental Figure 4. Dotted black lines indicate normal alternative splicing events.</figcaption></figure>
A

<figure><img src="image_13.png" /></figure>

B

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C

<figure><img src="image_15.png" /></figure>

D

<figure><img src="image_16.png" /></figure>

E

<figure><img src="image_17.png" /></figure>

F

<figure><img src="image_18.png" /></figure>

G

<figure><img src="image_19.png" /></figure>

H

<figure><img src="image_20.png" /></figure>

I

<figure><img src="image_21.png" /><figcaption>Figure 4. Patients with two likely pathogenic/pathogenic variants differ biochemically from patients with one or zero variants.</figcaption></figure>

(A–I) Serum and urinary electrolyte values in patients in whom two likely pathogenic/pathogenic variants were identified after long-read sequencing versus patients in whom only one or zero likely pathogenic/pathogenic variants were identified. Dotted lines represent upper and lower limit of normal. For urinary calcium-creatinine ratio, a lower limit of normal was not available; therefore, only the upper limit of normal for adults is depicted in (H) (children are not included in the graph). For urinary potassium-creatinine ratio, a reference value is only available to differentiate renal potassium wasting (>2 mmol/mmol) from nonrenal wasting (<2 mmol/mmol, panel I). LP, likely pathogenic; P, pathogenic. \*P<0.05, \*\*P<0.05, \*\*\*P<0.0005.
machinery away from canonic splice sites, resulting in exon elongation, intron retention, or exon skipping. Although not investigated here, exon skipping has also been shown to occur with specific *SLC12A3* mis-sense variants.⁴⁹

In contrast to an earlier study by Vargas-Poussou *et al*.,⁹ this study identified significant differences in several serum electrolytes between patients with two likely pathogenic/pathogenic versus patients with one or zero likely pathogenic/pathogenic variants. The milder biochemical phenotype in patients with one identified variant could, in theory, mean some patients are symptomatic despite having a monoallelic defect. However, when heterozygous carriers were recently characterized biochemically, their phenotype was much milder than for patients included in this study.¹⁵,¹⁶ Together, this suggests at least a substantial proportion of patients without biallelic variants have a different disorder.

Alternatively, some patients might have a genetic defect in a different gene. After all, the electrolyte disturbances found in Gitelman syndrome can be encountered in several other genetic diseases; examples include pathogenic variants in *CLCNKB*, *KCNJ10*, *KCNJ16*, *MT-TI*, *MT-TF*, *HNF1B*, *FXYD2*, and *ATP1A1*.³⁻⁶,⁵⁰⁻⁵³ A significant proportion of our cohort was not evaluated for pathogenic variants in all of these genes. Lastly, our approach might have missed variants in *SLC12A3* or variants in close proximity to it due to amplification. Moreover, it cannot be excluded that large structural variants have gone unnoticed, or that more distantly located variants were missed that would influence transcription of *SLC12A3*.

Targeted long-read sequencing is one of multiple techniques that can be used to investigate intronic regions. We chose targeted single-molecule real-time long-read sequencing because of its potential to uncover “small” structural variants and its sequencing accuracy.²⁴,⁵⁴,⁵⁵ Unexpectedly, we only found one structural variant in one patient (a 1190-bp deletion that includes exon 9). This can probably be attributed to the fact that 65 of 95 patients had already been screened with MLPA before start of the study, which would have picked up most copy number variations and some structural variation disrupting exons.⁹ We did detect three intronic “medium-small” deletions/insertions (19–52 bp in length) that could have been missed with short-read technology.⁵⁶ This might be one of the reasons that we had a higher detection rate than a 13-patient study that was published while preparing this manuscript.¹⁹

Identifying additional exonic variants in resequencing studies is not unusual²²,⁵⁷⁻⁵⁹ and can be explained by several factors. Of the total 95 patients, 19 had previously been investigated with SSCP only, which has a sensitivity of approximately 80%–90%.⁶⁰,⁶¹ Other variants had been missed because of technical challenges associated with Sanger sequencing. Lastly, updated annotations led to reclassification of previously dismissed variants.

An important challenge for all PCR amplification-based sequencing technologies is the occurrence of amplification bias.⁶²,⁶³ Amplification bias will inherently also occur in the targeted tubulopathy gene panel that is used by many

clinicians and researchers.³³ By looking at the apparent zygosity of variants in regions where amplicons overlap, we observed that amplification bias is also present in our data. However, the fact that long-read sequencing reidentified 96% of the previously known likely pathogenic or pathogenic variants, and additionally identified 22 exonic variants that had been missed before, suggests amplification bias is only a minor limitation in targeted long-read sequencing of *SLC12A3*.

A last advantage of long-read sequencing is the possibility to evaluate variant phasing.²⁴ In 22 patients, the two identified variants located in *trans* (44%) and, in one other patient, the two variants were actually positioned in *cis*. Secondly, the c.1670-191C>T occurred on a different allelic background in approximately 70% of patients, excluding a founder effect for those patients,⁶⁴ in line with a study in a Taiwanese population.¹⁴ Lastly, variant phasing allowed us to reclassify one VUS as likely pathogenic.

A limitation of our midigene splice assay is that the degree of mis-splicing might be difficult to assess. For example, our study shows residual normally spliced mRNA for the c.1670-191C>T variant *in vitro*, whereas urinary mRNA analysis by Lo *et al*.¹⁴ suggests that 100% of mis-splicing occurs *in vivo*. Confirming our results, a very recent study by Rossanti *et al*.¹⁹ tested four known intronic variants, including the c.1670-191C>T variant, using a minigene splice assay and found the same aberrant splicing patterns. An explanation for the quantitative splicing differences can be found in the source of mRNA, i.e., a urinary versus a kidney cell model; similar challenges have been observed for some variants in the well-studied *ABCA4* gene.⁶⁵,⁶⁶ Our results suggest the *SLC12A3* midigene splice assay should be used as a qualitative assay rather than as a quantitative assay. Another limitation is the amount of missing data in the phenotypic analyses (>50% for most parameters) due to the retrospective nature of the study, which presents a possible source of bias for the phenotypic analyses.

Our study illustrates two important indications for *SLC12A3* long-read sequencing. First, long-read sequencing detects both intronic and additional exonic variants in patients in whom only one likely pathogenic or pathogenic variant was identified previously. Other techniques that detect intronic variants, such as smMIPs and short-read whole-genome sequencing, may provide an alternative to long-read sequencing.²²,⁴¹,⁶⁷ Secondly, we suggest considering long-read sequencing in patients for which parental DNA is not available, because variant phasing can often provide insight on whether two candidate variants are indeed located on opposite alleles.³⁶,⁶⁸ In contrast, the diagnostic yield in patients in whom at most one VUS was previously detected in *SLC12A3* was low in this study (second variant found in only one patient). This low yield does not support routine sequencing of *SLC12A3* introns in all patients with suspected Gitelman syndrome. Additionally, we suggest the inclusion of the intronic c.1670-191C>T variant in Sanger and gene panel sequencing approaches because this variant is recurrent in different populations.¹⁴,¹⁷,¹⁸
In conclusion, long-read sequencing of SLC12A3 improves diagnostic yield in Gitelman syndrome. Long-read sequencing, and midigene testing of candidate variants, should be considered in patients with a Gitelman phenotype, especially when conventional sequencing has identified one pathogenic variant.

## DISCLOSURES

R. Bindels reports serving on the scientific board of the Dutch Kidney Foundation, and serving in an advisory or leadership role for RegMed XB (Regenerative Medicine Crossing Borders). J. De Baaij reports receiving research funding from the Dutch Diabetes Research Foundation and Dutch Kidney Foundation. M. Konrad reports having consultancy agreements with Otsuka. M. Kwint reports having ownership interest in Pacific Biosciences. R. Vargas-Poussou reports serving in an advisory or leadership role for Advicenne. All remaining authors have nothing to disclose.

## FUNDING

This work was financially supported by the IMAGEN project, which is cofunded by the PPP Allowance made available by Health~Holland (Top Sector Life Sciences & Health) grant LSHM20009 (IMplementation of Advancements in GENetic Kidney Disease), to stimulate public-private partnerships, and Nierstichting (Dutch Kidney Foundation) grant 20OP+018. Additionally, we received support from ZonMW under the framework of the European Joint Programme on Rare Diseases (EJP RD) grant EJPRD2019-40. This project has received funding from the European Union's Horizon 2020 research and innovation program under the EJP RD COFUND-EJP N° 825575 and the H2020 European Research Council grant 101040682 IN-THE-KIDNEY.

## ACKNOWLEDGMENTS

The authors are grateful to the patients for their participation in this study.

We would like to express strong appreciation for Lars Vermeltfoort and Mostafa Shaarawy, who became very skillful in performing and troubleshooting the midigene splice assays during their internships. We thank Simone van den Heuvel, for her great help with practical aspects of sequencing preparations, and Ronny Derks, for his help with the long-read data analysis. We thank the Radboudumc Genome Technology Center for their facilities and support. Furthermore, we express our gratitude to Prof. Frans Cremers and Janine Reurink whose advice was of tremendous help during the midigene splice assays, and for providing the DNA vectors.

Several authors of this publication are members of the European Reference Network for Rare Kidney Diseases (ERKNet), project identification number 739532.

## AUTHOR CONTRIBUTIONS

R.J.M. Bindels, J.H.F. de Baaij, M. Konrad, K. Neveling, K.-P. Schlingmann, and R. Vargas-Poussou provided supervision; A. Blanchard, M. Hureaux, M. Kwint, F. Latta, K. Neveling, K.-P. Schlingmann, R. Vargas-Poussou, and D.H.H.M. Viering were responsible for investigation; J.H.F. de Baaij, M. Hureaux, and D.H.H.M. Viering wrote the original draft; J.H.F. de Baaij,

K. Neveling, R. Vargas-Poussou, and D.H.H.M. Viering conceptualized the study; and all authors reviewed and edited the manuscript.

## DATA SHARING STATEMENT

Genetic data are deposited to the ClinVar repository under accession number SUB11476558. Patient data cannot be shared.

## SUPPLEMENTAL MATERIAL

This article contains the following supplemental material online at http://jasn.asnjournals.org/lookup/suppl/doi:10.1681/ASN.2022050627/-/DCSupplemental.

Supplemental Appendix. Supplementary methods.

Supplemental Table 1. Primers.

Supplemental Table 2. Midigene and primers used per variant.

Supplemental Table 3. Per patient description of identified pathogenic variants, likely pathogenic variants and variants of uncertain significance.

Supplemental Table 4. Additional variants found in exons and proximity.

Supplemental Table 5. Bioinformatic predictions of splicing defects.

Supplemental Table 6. Per variant pathogenicity evaluation.

Supplemental Table 7. Per patient description of clinical phenotype.

Supplemental Figure 1. Sequencing quality checks confirm correct amplicon length.

Supplemental Figure 2. Genomic location of amplicons for midigene synthesis.

Supplemental Figure 3. Variant phasing provides information on allelic background of recurrent variants.

Supplemental Figure 4. Full gel image and Sanger sequencing results of intronic pathogenic variants.

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## AFFILIATIONS

¹Department of Physiology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, the Netherlands

²Reference Center for Hereditary Kidney and Childhood Diseases (Maladies Rénales Héréditaires de l'Enfant et de l'Adulte, MARHEA), Paris, France

³Department of Genetics, Hôpital Européen Georges-Pompidou, Assistance Publique Hôpitaux de Paris, Paris, France

⁴Paris CardioVascular Research Center, Institut National de la Santé et de Recherche Médicale (INSERM) U970, Paris City University, Paris, France

⁵Department of Human Genetics, Radboud University Medical Center, Nijmegen, the Netherlands

⁶Clinical Investigations Center, Hôpital Européen Georges-Pompidou, Assistance Publique Hôpitaux de Paris, Paris, France

⁷Centre de Recherche des Cordeliers, Sorbonne Université, INSERM, University of Paris, Centre National de la Recherche Scientifique (CNRS), Paris, France

⁸Department of General Pediatrics, University Children's Hospital, Münster, Germany