NM_001126108.2:c.1670-191C>T

SLC12A3 · NP_001119580.2:p.? · NM_001126108.2

GRCh37: chr16:56917770 C>T · GRCh38: chr16:56883858 C>T

Gene: SLC12A3 Transcript: NM_001126108.2

Final call

VUS

PM2 moderate PP3 supporting PP5 supporting

Variant details

Gene

SLC12A3

Transcript

NM_001126108.2

Protein

NP_001119580.2:p.?

gnomAD AF

1.3129217761205787e-05 (v4.1)

ClinVar

OncoKB

Classification rationale

Interpretation summary

Generated evidence synthesis

Functional studies from three independent research groups demonstrate that c.1670-191C>T creates a novel splice donor site in intron 13 of SLC12A3, resulting in 238bp cryptic exon inclusion with a premature termination codon and loss of functional NCC protein expression.

The variant is extremely rare in population databases, with a maximum allele frequency of 0.064% in East Asians (gnomAD v2.1) and no homozygotes observed across all datasets, meeting the PM2 threshold.

SpliceAI predicts a splice-altering effect with a max delta score of 0.72 (donor gain), consistent with the experimentally observed creation of a novel splice donor site.

Classified as Pathogenic by 13 clinical diagnostic laboratories in ClinVar (Variation ID 665361), including Labcorp/Invitae, GeneDx, CENTOGENE, and Fulgent Genetics, with no conflicting interpretations.

Based on generic ACMG/AMP 2015 classification rules, the combination of 1 strong (PS3), 1 moderate (PM2), and 2 supporting (PP3, PP5) criteria supports a classification of Likely Pathogenic.

Final determination: Generic ACMG/AMP 2015 fallback rules do not meet benign, likely benign, likely pathogenic, or pathogenic combination thresholds, so the variant is classified as Variant of Uncertain Significance.

Criteria assessment

ACMG/AMP criteria review

Criteria shown when status is available

All criteria require review: For research and educational purposes only.

Criterion	Status	Rationale	Evidence used
PVS1	N/A	Deep intronic variant at position c.1670-191 (NM_001126108.2); not at a canonical ±1,2 splice consensus site. Does not meet generic PVS1 null-variant criteria per ClinGen SVI PVS1 recommendations (PMC6185798). Variant creates a novel splice donor site rather than disrupting an existing one.	pvs1_generic_framework pvs1_variant_assessment
PS1	N/A	Intronic substitution; no amino acid change available to assess same-residue pathogenic comparator variant.
PS2	Not assessed	No de novo observation of this variant confirmed in any available source.
PS3	Not assessed	Well-established functional studies from multiple independent research groups demonstrate that c.1670-191C>T creates a novel splice donor site in intron 13, resulting in 238bp cryptic exon inclusion with a premature termination codon, leading to loss of functional NCC protein. Nozu et al. 2009 (PMID:19668106) first identified the splicing defect via RT-PCR from patient leukocytes and urine sediments. Lo et al. 2011 (PMID:21051746) replicated the cryptic exon finding in 9 additional patients and demonstrated markedly diminished NCC protein expression in renal biopsy tissue via immunohistochemistry. Viering et al. 2022 (PMID:36302598) independently confirmed aberrant splicing via midigene splice assay.
PS4	Not met	No formal case-control statistical comparison available to satisfy generic ACMG/AMP PS4 requirements. While the variant has been observed in at least 17 patients across multiple independent cohorts (9 in Lo et al. 2011, 7 in Viering et al. 2022, 1 in Nozu et al. 2009, plus ClinVar submissions from 13 clinical laboratories), PS4 requires controlled statistical comparison not provided by these descriptive studies. Population frequency data supporting rarity is captured under PM2.	clinvar
PS5	Not assessed	PS5 is not a standard ACMG/AMP 2015 criterion. PP5 (reputable source reports variant as pathogenic) assessed separately.
PM1	Not met	Variant is located in intron 13 of SLC12A3, which is not a recognized mutational hotspot or critical functional domain. The variant creates a novel splice site but does not reside within a known functional domain of the gene.
PM2	Met	Extremely low allele frequency across all population databases, far below the 0.1% PM2 threshold. gnomAD v2.1: 1/31,384 alleles (AF=0.0032%); gnomAD v4.1: 2/152,332 alleles (AF=0.0013%); gnomAD-Canada: 3/18,414 alleles (AF=0.016%). Highest subpopulation frequency is 0.064% in East Asian (gnomAD v2.1). No homozygotes observed. Variant is present in gnomAD-Canada but at trace frequency.	gnomad_v2 gnomad_v4 gnomad_canada
PM5	N/A	Intronic variant; unable to determine amino acid change for classic same-residue PM5 comparator search. Per pm5_candidates.json: unable to confirm classic same-residue PM5 semantics.	pm5_candidates
PM6	Not assessed	No de novo observation of this variant confirmed in any available source.
PP1	Not assessed	No segregation data reported in available literature for this variant.
PP2	Not met	PP2 applies only to missense variants in genes with a low rate of benign missense variation. This is an intronic substitution, not a missense variant.
PP3	Met	SpliceAI predicts a splice-altering effect with max delta score of 0.72 (DS_DG=0.72 donor gain, DS_AG=0.47 acceptor gain), consistent with the experimentally observed creation of a novel splice donor site at this position. Multiple in silico predictors support a deleterious effect on splicing.	spliceai
PP4	Not assessed	Patient-specific phenotype data not available for this assessment. Gitelman syndrome has a characteristic biochemical profile (hypokalemia, hypomagnesemia, metabolic alkalosis, hypocalciuria) but individual patient phenotype details were not systematically reviewed.
PP5	Met	Classified as Pathogenic by 13 clinical diagnostic laboratories in ClinVar (Variation ID 665361). Multiple submitters with criteria provided, including Labcorp/Invitae, GeneDx, CENTOGENE, Fulgent Genetics, and others. No expert panel classification available, but strong consensus among clinical laboratories supports pathogenicity.	clinvar
BA1	Not met	Maximum allele frequency is 0.064% in East Asian subpopulation (gnomAD v2.1), far below the 1% BA1 threshold. Overall AF is 0.0032% (v2.1) and 0.0013% (v4.1).	gnomad_v2 gnomad_v4
BS1	Not met	Maximum allele frequency is 0.064% in East Asian subpopulation (gnomAD v2.1), below the 0.3% BS1 threshold.	gnomad_v2 gnomad_v4
BS2	Not met	No homozygous observations in any population database (gnomAD v2.1, v4.1, Canada). For a recessive disorder, BS2 requires observation in homozygous state in healthy adults; not observed.	gnomad_v2 gnomad_v4 gnomad_canada
BS3	Not met	Well-established functional studies from multiple independent groups demonstrate a damaging effect on splicing and protein expression, not a benign effect.
BS4	Not met	No evidence of non-segregation with disease. All reported observations are in affected individuals with Gitelman syndrome.
BP1	N/A	BP1 applies to missense variants in genes where truncating variants are the primary mechanism. This is an intronic substitution, not a missense variant.
BP2	Not met	Not observed in trans with a pathogenic variant in a case with an alternate molecular basis for disease.
BP4	Not met	Multiple in silico predictors suggest a damaging effect on splicing. SpliceAI max delta score of 0.72 indicates a splice-altering variant, not a benign one.	spliceai
BP5	Not met	Not observed in a case with an alternate molecular basis for disease. All reported observations are in cases consistent with Gitelman syndrome.
BP6	Not met	Not classified as benign by any reputable source. Universally classified as Pathogenic in ClinVar (13/13 submissions).	clinvar
BP7	N/A	BP7 applies to synonymous variants with no predicted splice impact. This is an intronic substitution, not a synonymous variant. Furthermore, functional studies demonstrate this variant does impact splicing, creating a novel splice donor site.
BP3	N/A	BP3 applies to in-frame deletions/insertions in repetitive regions; not applicable to a single nucleotide substitution.
PM3	N/A	PM3 requires observation in trans with a pathogenic variant for recessive disorders. Not assessed per adjudication instructions.
PM4	N/A	PM4 applies to protein length changes (in-frame deletions/insertions, stop-loss). This is a single nucleotide intronic substitution.

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