Variant classification summary

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

Likely Pathogenic

PS3 strong PS4 moderate PM2 supporting PP3 supporting PP4 supporting

Variant details

Gene

SLC12A3

Transcript

NM_001126108.2

Protein

NP_001119580.2:p.?

gnomAD AF

1.3129217761205787e-05 (v4.1)

ClinVar

Pathogenic

OncoKB

Classification rationale

Interpretation summary

Generated evidence synthesis

1

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.

2

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.

3

Independent midigene splice assays confirmed aberrant splicing with activation of a cryptic splice donor site, further validating the damaging molecular mechanism.

4

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.

5

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.

6

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.

7

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.

8

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).

Final determination: Generic ACMG/AMP 2015 fallback rules support a Likely Pathogenic classification based on the observed combination of pathogenic criteria.

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 substitution (c.1670-191C>T, intron 13) outside canonical ±1,2 splice consensus. Does not fall into standard ClinGen SVI PVS1 null-variant buckets (nonsense, frameshift, canonical splice). Proven splicing effect captured by PS3.	pvs1_generic_framework
PS1	N/A	PS1 applies to same amino acid change from different nucleotide substitutions; not applicable to deep intronic variants.
PS2	Not assessed	No de novo data available for this variant in the reviewed literature.
PS3	Met	Well-established functional studies across three independent publications demonstrate that c.1670-191C>T creates a novel donor splice site in intron 13, resulting in 238-bp cryptic exon inclusion, a premature termination codon, and loss of functional NCC protein. RT-PCR from patient leukocytes and urinary sediments (PMID:19668106), RNA and immunohistochemistry analyses in 19 patients showing marked attenuation of NCC expression in the distal convoluted tubule (PMID:21051746), and midigene splice assays (PMID:36302598) all consistently confirm the damaging splicing effect.	PMID:19668106 PMID:21051746 PMID:36302598
PS4	Met	The variant has been observed in at least 27 unrelated probands with Gitelman syndrome across multiple independent studies (19 patients in PMID:21051746, 7 patients in PMID:36302598, 1 homozygous patient in PMID:32860008), while being extremely rare in population databases (gnomAD v4.1 AF = 0.0013%). The prevalence in affected individuals is substantially enriched compared to general population controls.	PMID:21051746 PMID:36302598 PMID:32860008 gnomad_v2 gnomad_v4
PS5	N/A	No different pathogenic variant has been reported at the same nucleotide position (c.1670-191). PS5 requires a different pathogenic change at the same residue/nucleotide.
PM1	N/A	Deep intronic variant outside established functional protein domains. PM1 is designed for coding-region mutational hot spots and critical functional domains (e.g., active sites); not applicable to deep intronic positions even when recurrently mutated.
PM2	Met	Variant is absent or at extremely low frequency in large population cohorts. gnomAD v2.1: 1/31,384 alleles (AF = 0.00319%); gnomAD v4.1: 2/152,332 alleles (AF = 0.00131%); gnomAD-Canada: absent. All allele frequencies are well below the 0.1% PM2 threshold. No homozygotes observed in population databases.	gnomad_v2 gnomad_v4 gnomad_canada
PM5	N/A	Intronic variant; PM5 requires a different pathogenic missense change at the same amino acid residue. Protein consequence could not be determined for this deep intronic variant.
PM6	Not assessed	No de novo data available for this variant in the reviewed literature.
PP1	Not assessed	No segregation data available. Cosegregation with disease in families was not specifically analyzed in the reviewed publications.
PP2	N/A	PP2 applies only to missense variants in genes with low benign missense variation. This is a deep intronic substitution, not a missense variant.
PP3	Met	SpliceAI predicts a deleterious splicing impact with a maximum delta score of 0.72, consistent with creation of a novel splice site. The in silico prediction is corroborated by functional evidence (PS3).	spliceai
PP4	Met	The variant has been identified in multiple unrelated patients with Gitelman syndrome, a well-characterized disorder with SLC12A3 as the primary genetic etiology. The clinical phenotype (hypokalemia, hypomagnesemia, metabolic alkalosis, hypocalciuria) is highly specific for this gene.	PMID:19668106 PMID:21051746 PMID:21343949
PP5	Not met	ClinVar reports this variant as Pathogenic (Variation ID: 665361) by 13 clinical laboratories. However, primary evidence from publications is available and has been independently evaluated for PS3, PS4, and PM2. Applying PP5 would constitute double-counting of evidence; PP5 is intended for situations where the primary evidence is not accessible to the evaluating laboratory.	clinvar
BA1	Not met	Allele frequency in gnomAD (0.00319% v2.1, 0.00131% v4.1) is well below the 1% BA1 threshold.	gnomad_v2 gnomad_v4
BS1	Not met	Allele frequency in gnomAD (0.00319% v2.1, 0.00131% v4.1) is well below the 0.3% BS1 threshold.	gnomad_v2 gnomad_v4
BS2	Not met	No homozygous observations in gnomAD (v2.1: 0 homozygotes; v4.1: 0 homozygotes). Gitelman syndrome is autosomal recessive; BS2 would require homozygous observation in a healthy adult. Only heterozygous carriers identified in population databases.	gnomad_v2 gnomad_v4
BS3	Not met	Functional studies consistently demonstrate a damaging effect on splicing (cryptic exon inclusion → premature termination codon → loss of NCC function). BS3 requires functional studies showing no damaging effect, which is contradicted by the available evidence.	PMID:19668106 PMID:21051746 PMID:36302598
BS4	Not assessed	No segregation data available. Lack of cosegregation in affected family members cannot be evaluated from the reviewed publications.
BP1	N/A	BP1 applies only to missense variants in genes where primarily truncating variants cause disease. This is a deep intronic substitution, not a missense variant.
BP2	Not assessed	No phasing data available to determine whether the variant has been observed in cis or trans with other pathogenic SLC12A3 variants. Compound heterozygosity with different SLC12A3 mutations has been reported (PMID:19668106, PMID:21051746) but this is consistent with autosomal recessive inheritance rather than indicating benign effect.
BP4	Not met	SpliceAI predicts a deleterious splicing impact (max delta = 0.72), contradicting BP4 which requires multiple lines of computational evidence suggesting no impact.	spliceai
BP5	Not assessed	No data available on alternate molecular basis for disease in patients carrying this variant.
BP6	Not met	ClinVar reports this variant as Pathogenic (Variation ID: 665361) by 13 clinical laboratories. BP6 requires a reputable source reporting the variant as benign, which is contradicted.	clinvar
BP7	N/A	BP7 applies only to synonymous (silent) variants with no predicted splicing impact. This is a deep intronic substitution, not a synonymous variant.

Disclaimer: The content and results provided by LYFE Sciences are for research and educational purposes only and must not be used as a substitute for professional medical judgment, diagnosis, or treatment. Always consult a qualified healthcare professional before making any clinical decisions.