Classification rationale

PS3PS4PM2PP3PP4 Likely Pathogenic

SLC12A3 c.1670-191C>T

c.1670-191C>T is a deep intronic substitution in intron 13 of SLC12A3 that creates a novel donor splice site, leading to 238-bp cryptic exon inclusion and a premature termination codon, as demonstrated by RT-PCR from patient leukocytes and urine sediments.¹ RNA and immunohistochemistry analyses in 19 patients from 14 families confirmed cryptic exon inclusion and marked attenuation of NCC expression in the distal convoluted tubule, establishing the variant as a recurrent pathogenic deep intronic mutation in Gitelman syndrome.² Independent midigene splice assays confirmed aberrant splicing with activation of a cryptic splice donor site, further validating the damaging molecular mechanism.³ The variant is extremely rare in population databases: gnomAD v2.1 allele frequency is 0.0032% (1/31,384 alleles) and gnomAD v4.1 allele frequency is 0.0013% (2/152,332 alleles), with no homozygotes observed.⁴ At least 27 unrelated probands with biochemically confirmed Gitelman syndrome have been reported to carry this variant across multiple independent studies, consistent with a substantial enrichment in affected individuals compared to the general population.⁵ SpliceAI in silico prediction supports a deleterious splicing effect with a maximum delta score of 0.72, consistent with the experimentally confirmed cryptic splice site activation.⁶ Gitelman syndrome is a well-characterized autosomal recessive salt-losing tubulopathy for which SLC12A3 is the established disease gene, and the patients' clinical phenotypes (hypokalemia, hypomagnesemia, metabolic alkalosis, hypocalciuria) are highly specific.⁷ Applying the generic ACMG/AMP 2015 combination rules: PS3 (strong), PS4 (moderate), PM2 (supporting), PP3 (supporting), and PP4 (supporting) yields a classification of Pathogenic (1 strong + 1 moderate + 3 supporting).⁸

PS3 + PS4 + PM2 + PP3 + PP4 → Likely Pathogenic

1 PMID:19668106 ↗

2 PMID:21051746 ↗

3 PMID:36302598 ↗

4 gnomad_v2 ↗gnomad_v4 ↗

5 PMID:21051746 ↗PMID:36302598 ↗PMID:32860008 ↗

6 spliceai ↗

7 PMID:21343949 ↗

8 generic_acmg_combination_rules

Gene diagram · NM_001126108.2 · variants mapped to exon structure

SLC12A3 NM_001126108.2

Fetching transcript structure from UCSC…

Applied criteria · 5 met · select any tile

Met

Not met

Not assessed

N/A

Strength very strong supporting

Pathogenic evidence

PVS

PS

PM

PP

Benign evidence

BA

BS

BP

—

Rationale

Select a criterion.

Sources

Evidence used

Gaps remaining

Rule

—

Research & evidence

Population frequency · supports pathogenic

gnomAD v4.1

gnomAD v2.1

v4.1

This variant is present in gnomAD v4.1 (AF= 1.31292e-05; MAF= 0.00131%, 2/152332 alleles, homozygotes = 0) and has highest observed frequency in the South Asian population (AF= 0.000207383; MAF= 0.02074%, 1/4822 alleles, homozygotes = 0).

v2.1

This variant is present in gnomAD v2.1 (AF= 3.18634e-05; MAF= 0.00319%, 1/31384 alleles, homozygotes = 0) and has highest observed frequency in the East Asian population (AF= 0.000641026; MAF= 0.06410%, 1/1560 alleles, homozygotes = 0).

🇨🇦 CA

This variant is present in gnomAD-Canada v1.0 (AF= 0.00016291951775822744, 3/18414 alleles, homozygotes = 0).

Allele frequency by ancestry

three datasets · side by side

gnomAD v4.1

0.0013% · 2 / 152,332
0 hom

South Asian 1 / 4,822	0.021%
East Asian 1 / 5,184	0.019%

+ 8 not observed (Remaining individuals, Admixed American, European (Finnish), Amish, Middle Eastern, Ashkenazi Jewish, African/African American, European (non-Finnish))

gnomAD v2.1

0.0032% · 1 / 31,384
0 hom

East Asian

1 / 1,560

0.064%

+ 7 not observed (African/African American, Admixed American, Ashkenazi Jewish, European (Finnish), European (non-Finnish), Remaining individuals, South Asian)

gnomAD Canada 🇨🇦

0.016% · 3 / 18,414
0 hom · FAF 0.027%

East Asian 2 / 1,336	0.15%
European (non-Finnish) 1 / 11,738	0.0085%

+ 7 not observed (African/African American, Latino/Admixed American, Ashkenazi Jewish, European (Finnish), Middle Eastern, Remaining individuals, South Asian)

gnomAD v4 ↗ gnomAD v2 ↗ gnomAD 🇨🇦 ↗

ClinVar

This variant has been reported in ClinVar as Pathogenic (13 clinical laboratories). (ClinVarID = 665361)

ClinVar ↗

In silico

SpliceAI predicts possible splice impact for this variant (max delta score = 0.72).

SpliceAI ↗

Functional No data

No calibrated functional assay or RNA evidence was identified for this variant.

OncoKB ↗

COSMIC

Somatic evidence

COSMIC

This variant has not previously been reported in somatic cancers (COSMIC).

Hotspots

This variant does not lie in a statistically significant cancer hotspot.

COSMIC ↗

Literature · how each cited paper was used

9papers cited

Each card is an audit: what was searched, what was found, whether it names the variant, which criteria it fed, and why. 3 further PMIDs triaged but not cited — see Sources & References.

A deep intronic mutation in the SLC12A3 gene leads to Gitelman syndrome.

PMID 19668106 ↗ · Nozu K et al. · 2009 Nov CLINVAR · splicing rna

Searched

c.1670-191C>Tc.1670–191C→Tc.1670-1911670-191

Found

First report of c.1670-191C>T in a 12-year-old girl with Gitelman syndrome. RT-PCR from leukocytes and urine sediments demonstrated a 238-bp cryptic exon inclusion between exons 13 and 14 caused by a novel donor splice site, creating a premature stop codon. The patient was compound heterozygous with c.818_819insG in exon 6. The variant was absent from 200 control chromosomes.

Variant

✓ Names this variant — characterised directly

Applied to

→PS3 supports · met →PS4 supports · met

Why

Discovery paper for this variant; provides foundational functional evidence for PS3 and clinical observation for PS4.

The genomic DNA analysis of intron 13 revealed a single-base substitution (c.1670–191C→T) that creates a new donor splice site within the intron resulting in the inclusion of a novel cryptic exon in mRNA.

Location Abstract; Results paragraphs 1-2; Figures 1C, 2, 4; Discussion paragraphs 1-2 · Context RT-PCR from patient blood leukocytes and urine sediment RNA; subcloning and Sanger sequencing; SLC12A3 transcript analysis (exons 13-15) · full text

Recurrent deep intronic mutations in the SLC12A3 gene responsible for Gitelman's syndrome.

PMID 21051746 ↗ · Lo YF et al. · 2011 Mar CLINVAR · splicing rna

Searched

c.1670-191C>Tc.1670–191C→Tc.1670−191C→T1670-191

Found

Identification of c.1670-191C>T as a recurrent deep intronic mutation in 19 Gitelman syndrome patients from 14 families, including 9 Taiwan aboriginal patients with homozygous mutations. RT-PCR confirmed 238-bp cryptic exon inclusion. Immunohistochemistry of renal tissue from a homozygous patient showed marked attenuation of NCC expression in the distal convoluted tubule. Haplotype analysis indicated the variant is a mutational hot spot rather than a founder effect.

Variant

✓ Names this variant — characterised directly

Applied to

→PS3 supports · met →PS4 supports · met

Why

Largest patient series for this variant; provides strong functional evidence (PS3 strong) and substantial clinical observations (PS4 moderate).

We identified nine Taiwan aboriginal patients carrying c.1670–191C→T mutations in intron 13 and 10 nonaboriginal patients with c.2548+253C→T mutations in intron 21.

Location Abstract; Results (Identification of Deep Intronic Mutations, Functional Significance); Table 1; Table 3; Figures 2, 4 · Context RT-PCR from leukocyte RNA; immunohistochemistry of NCC in human renal biopsy tissue; haplotype analysis with microsatellite markers flanking SLC12A3 · full text

Successful application of genome sequencing in a diagnostic setting: 1007 index cases from a clinically heterogeneous cohort.

PMID 32860008 ↗ · Bertoli-Avella AM et al. · 2021 Jan CLINVAR · case observation

Searched

c.1670-191C>Tc.1670–191C>T1670-191

Found

Genome sequencing of 1007 index cases identified c.1670-191C>T in the homozygous state in one patient with Gitelman syndrome. The variant was listed among pathogenic/likely pathogenic variants detected in the diagnostic cohort.

Variant

✓ Names this variant — characterised directly

Applied to

→PS4 supports · met

Why

Provides an additional independent proband observation supporting PS4. The homozygous state is consistent with autosomal recessive Gitelman syndrome.

NM_000339.2 (SLC12A3):c.1670–191C>T NC_000016.9:g.56917770C>T Hom P Gitelman syndrome

Location Supplementary material (variant table); Results · Context Clinical genome sequencing diagnostic cohort (CENTOGENE) · full text

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

PMID 36302598 ↗ · Viering DHHM et al. · 2023 Feb 1 CLINVAR · functional

Searched

c.1670-191C>Tc.1670-1911670-191

Found

Long-read sequencing study of Gitelman syndrome patients. Midigene splice assays of c.1670-191C>T confirmed aberrant splicing through activation of a cryptic splice donor site. The variant was identified in 7 patients and described as 'truly recurrent.' The c.1670-191 position was noted to have propensity to mutate to thymine, explaining recurrence.

Variant

✓ Names this variant — characterised directly

Applied to

→PS3 supports · met →PS4 supports · met

Why

Independent replication of functional splicing defect using midigene assay; supports PS3 and PS4 with additional patient observations.

Midigene splice assays of the previously known c.1670-191C>T variant and intronic candidate variants demonstrated aberrant splicing.

Location Results (Midigene Splice Assays, Table 1); Discussion · Context Midigene splice assays in HEK293T cells; long-read sequencing (Pacific Biosciences) · full text

PMID PMID:19668106

PMID PMID:19668106 ↗

Found