The GLA NM_000169.3:c.644A>G (NP_000160.1:p.(Asn215Ser)) missense variant has not previously been reported in somatic cancers (COSMIC). Experimental evidence: Three independent controlled studies show reduced or intermediate alpha-galactosidase A activity and abnormal N215S intracellular processing. Based on available guidelines, this variant is considered to be a Variant of Uncertain Significance (VUS).
Five-step scale from benign to pathogenic. This variant is uncertain. Select a step to read what it means.
Uncertain.The recorded classification is uncertain significance.
Gene-level background from the recorded interpretation.
Words with a dotted underline have a short definition. Hover or tap them.GLA encodes alpha-galactosidase A, a lysosomal enzyme that breaks down certain fatty sugar molecules and related glycolipids. Changes that impair the enzyme can cause Fabry disease, a rare inherited lysosomal storage disorder in which these substances build up in cells. GLA has no established role in cancer.
The recorded framework is Generic ACMG/AMP 2015 with ClinGen SVI recommendations. The criteria and strengths shown below come from the saved assessment; this page does not recalculate the classification.
The applied criteria and their strengths are shown as assessed under the stated framework.
Met (strong): three independent controlled studies show reduced or intermediate alpha-galactosidase A activity and abnormal N215S intracellular processing.
Met at supporting: gnomAD v4.1 grpmax FAF is 3.071e-05, below the PM2 threshold of 0.0001, with zero homozygotes.
No benign criterion marked as applied in the recorded assessment.
VUS: PS3 (strong) plus PM2 (supporting) do not satisfy a generic ACMG/AMP 2015 pathogenic, benign, likely pathogenic, or likely benign combination rule.
Evidence summaries and captured source records, where available. Missing entries should not be interpreted as negative findings.
gnomAD v4.1: 38 / 1,203,813 alleles. Recorded AF 3.15664e-05. Recorded homozygote count 0.
This variant is present in gnomAD v2.1 (AF= 5.45191e-06; MAF= 0.00055%, 1/183422 alleles, homozygotes = 0) and has highest observed frequency in the European (non-Finnish) population (AF= 1.22128e-05; MAF= 0.00122%, 1/81881 alleles, homozygotes = 0).
Not available in gnomAD-Canada v1.0.
Recorded SpliceAI maximum delta score: 0.002. Refer to the recorded PP3/BP4 rationale for interpretation.
23 Pathogenic. ClinVar API review status: criteria provided, multiple submitters, no conflicts.
No standalone functional evidence summary is recorded in evidence_summary.functional.
This variant has not previously been reported in somatic cancers (COSMIC).
This variant does not lie in a statistically significant hotspot.
ACMG/AMP criteria under Generic ACMG/AMP 2015 with ClinGen SVI recommendations, with the data and sources behind each one.
28 criteria with recorded statuses: 2 met · 9 not met · 13 not assessed · 4 not applicable. Open any row for the full reasoning, the rule and the evidence behind it.
PS3 strong: controlled functional studies show reduced enzyme activity and abnormal N215S processing.
PM2 supporting: the variant is rare in gnomAD, with grpmax FAF 3.071e-05 and zero homozygotes.
No criteria match.
PS3 is met at strong strength. Three independent variant-specific functional studies provide concordant damaging evidence for GLA p.Asn215Ser (N215S; c.644A>G). In HEK-293 cells, N215S showed 15.7±2.4% of wild-type alpha-galactosidase A activity without AT1001, with a statistically significant increase after chaperone treatment and corresponding increased protein abundance (PMID:21598360). In HEK293T cells, site-directed N215S expression produced intermediate enzyme activity relative to wild-type and mock-transfected controls across three separate transfections, with the authors attributing the effect to defective post-translational N-glycosylation and abnormal trafficking (PMID:21972175). In purified enzyme expressed in COS-7 cells, catalytic Km and Vmax were similar to wild type, but the variant showed abnormal intracellular abundance and degradation behavior, including increased protein/activity after ERAD inhibition and pharmacological chaperone treatment, demonstrating a biologically relevant processing defect (PMID:17555407). Together, the quantitative controls, reproducibility across distinct cellular and biochemical systems, and concordant reduction or abnormal handling of the gene product support a damaging functional effect. The studies are not documented as validated clinical diagnostic-laboratory assays, so no stronger-than-strong interpretation is assigned.
PS3: Well-established in vitro or in vivo functional studies supportive of a damaging effect on the gene or gene product. ... Functional studies that have been validated and shown to be reproducible and robust in a clinical diagnostic laboratory setting are considered the most well-established.
PM2 is met at supporting strength. The generic SVI threshold supplied for PM2 is <=0.0001. In the default all-comers gnomAD v4.1 dataset, the variant has total AF 3.156636454333024e-05 (38/1,203,813 alleles), grpmax FAF=3.071e-05, and zero homozygotes; the filtering frequency is below 0.0001. The gnomAD v2.1 all-comers exome dataset is concordant at 5.451908713240506e-06 (1/183,422 alleles), with zero homozygotes. No VCEP specifies use of a non-cancer or exome-only subset, so the all-comers totals are the governing default; the available v2.1 non-cancer exome result is also rare at 5.806593968110186e-06.
PM2 (supporting): <= 0.0001 — SVI recommendation to downgrade PM2 to Supporting (ClinGen SVI 2020) (PMID:25741868)
The governing rule requires a significantly increased prevalence in affected individuals and, under Note 1, an RR or OR >5.0 with a confidence interval that does not include 1.0. The PS4-classified paper PMID:10666480 was checked against its abstract: it describes molecular analysis in 30 unrelated Fabry families and the exact N215S variant in two unrelated affected families, but it does not report unaffected or population controls, a control count, or a case-control frequency comparison. Therefore no OR or 95% CI can be calculated from the reported evidence. gnomAD provides a population frequency (38/1,203,813 alleles overall; 37/888,895 in NFE) but no affected-case frequency, so it cannot establish the required case-control comparison.
The prevalence of the variant in affected individuals is significantly increased compared to the prevalence in controls. ... Note 1: Relative risk (RR) or odds ratio (OR), as obtained from case-control studies, is >5.0 and the confidence interval around the estimate of RR or OR does not include 1.0.
NM_000169.3:c.644A>G is a missense variant, so the governing generic rule selects the REVEL path only; SpliceAI is not evidence for missense PP3. REVEL was not available for this variant, so the applicable computational evidence is indeterminate and PP3 is not met. The BayesDel score is not used because no generic ACMG-strength calibration is established for BayesDel.
Multiple lines of computational evidence support a deleterious effect on the gene or gene product (conservation, evolutionary, splicing impact, etc.).
The governing rule concerns a reputable source reporting the variant as pathogenic when the underlying evidence is unavailable for independent evaluation. Under the specified generic application rule, PP5 is restricted to an exact-variant ClinVar expert-panel classification of Pathogenic or Likely pathogenic. ClinVar variation 10730 is an exact match for NM_000169.3:c.644A>G (p.Asn215Ser), but the audit reports zero expert-panel submissions; its Pathogenic assertions are ordinary laboratory, aggregate, or non-expert submissions. The literature reports are not a qualifying ClinVar expert-panel classification, so PP5 is not met.
Reputable source recently reports variant as pathogenic, but the evidence is not available to the laboratory to perform an independent evaluation.
BA1 is not met. Under the generic ACMG/AMP stand-alone threshold, the relevant frequency is the gnomAD v4.1 grpmax FAF of 3.071e-05 (0.003071%), based on 38/1,203,813 total alleles; this is far below the supplied BA1 threshold of >=0.05 (5%). The gnomAD v4.1 highest point estimate, 4.16247e-05 in non-Finnish Europeans, is not used for BA1 because the governing guidance requires the filtering allele frequency rather than popmax point estimate. No VCEP or exome-only/non-cancer source requirement overrides the generic default, and the gnomAD v2.1 all-comers exome frequency is likewise low at 5.45191e-06.
BA1 (stand-alone): >= 0.05 — ACMG/AMP 2015 stand-alone benign allele frequency (PMID:25741868)
BS1 is not met. The relevant gnomAD v4.1 filtering frequency is grpmax FAF=3.071e-05 (0.003071%), which is below the generic BS1 default threshold of >=0.01 (1%). The non-Finnish European popmax point estimate of 4.16247e-05 is not substituted for grpmax FAF. Because the observed FAF is already far below the generic threshold, no maximum-credible-frequency calculation is needed to reject BS1 on this path; there is no elevated ancestry-specific frequency requiring interpretation through prevalence, penetrance, or allelic heterogeneity.
BS1 (strong): >= 0.01 — Generic SVI default; per-gene derivation preferred (Whiffin et al. 2017) (PMID:28518168)
BS3 is not met. The available variant-specific functional studies do not show no damaging effect on GLA protein function or splicing. Instead, controlled cellular assays show substantially reduced or intermediate alpha-galactosidase A activity for N215S, and biochemical studies identify abnormal intracellular processing, protein abundance, or trafficking. The presence of residual activity and partial pharmacological rescue does not establish normal function and is not sufficient for BS3.
BS3: Well-established in vitro or in vivo functional studies show no damaging effect on protein function or splicing.
BP1 is not supported because the available GLA disease literature documents numerous disease-associated missense variants, including N215S and experimentally studied missense substitutions, while the materials do not show that disease is caused primarily by truncating variants. The documented missense disease mechanism conflicts with applying BP1 to this missense variant.
Missense variant in a gene for which primarily truncating variants are known to cause disease.
NM_000169.3:c.644A>G is a missense variant, so the governing generic rule selects the REVEL path only; SpliceAI is not evidence for missense BP4. REVEL was not available for this variant, so the applicable computational evidence is indeterminate and BP4 is not met. The SpliceAI max delta of 0.002 and the BayesDel score are not used for this missense BP4 assessment.
Multiple lines of computational evidence suggest no impact on gene or gene product (conservation, evolutionary, splicing impact, etc)
The governing rule concerns a reputable source reporting the variant as benign when the underlying evidence is unavailable for independent evaluation. Under the specified generic application rule, BP6 is restricted to an exact-variant ClinVar expert-panel classification of Benign or Likely benign. ClinVar variation 10730 is an exact match but has zero expert-panel submissions and no qualifying benign expert-panel classification; the available record is non-expert Pathogenic submissions. BP6 is not met.
Reputable source recently reports variant as benign, but the evidence is not available to the laboratory to perform an independent evaluation.
The variant is NP_000160.1:p.(Asn215Ser), but the available variant-specific reports describe the same c.644A>G allele and do not establish a pathogenic p.Asn215Ser allele caused by a different nucleotide change. The exact alternate-nucleotide comparison required for PS1 is therefore unavailable.
Same amino acid change as a previously established pathogenic variant regardless of nucleotide change.
No case-specific documentation establishes that the proband has the disease, lacks a family history, and carries a de novo NM_000169.3:c.644A>G variant with both maternity and paternity confirmed. The available literature reports unrelated affected families but does not provide the required parental-origin testing for this proband.
De novo (both maternity and paternity confirmed) in a patient with the disease and no family history.
No VCEP covers GLA, vcep_materials.json has no authoritative domain table, and the available evidence does not establish that residue 215 lies in a well-established critical functional domain or mutational hotspot without benign variation. Cancer Hotspots and COSMIC provide no hotspot result for GLA N215 or this variant.
Located in a mutational hot spot and/or critical and well-established functional domain (e.g. active site of an enzyme) without benign variation.
Fabry disease due to GLA is described as X-linked recessive. The variant-specific study reports six N215S hemizygous males and four N215S heterozygotes, but provides no second pathogenic GLA variant, parent or offspring testing, or demonstrated phase. The required detection of NM_000169.3:c.644A>G in trans with a pathogenic variant therefore cannot be established from the available evidence, so PM3 is not assessed.
For recessive disorders, detected in trans with a pathogenic variant...Note: This requires testing of parents (or offspring) to determine phase.
The case evidence identifies p.Asn215Ser, but no different missense substitution at residue 215 with an independently established pathogenic classification is documented. The PM5 candidate file contains zero same-residue candidates and explicitly does not establish PM5 semantics, so PM5 cannot be applied from it.
Novel missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before.
No case-specific documentation establishes an assumed de novo NM_000169.3:c.644A>G variant in the proband. The available evidence does not identify parental testing, parental origin, or a proband-specific disease presentation, so PM6 cannot be applied.
Assumed de novo, but without confirmation of paternity and maternity.
The evidence includes reports of NM_000169.3:c.644A>G in unrelated Fabry disease families, but it does not provide variant-positive and variant-negative results for affected relatives, the number of informative meioses, or case-specific phenotype-genotype segregation. Therefore, co-segregation cannot be assessed.
Co-segregation with disease in multiple affected family members in a gene definitively known to cause the disease. ... May be used as stronger evidence with increasing segregation data.
The evidence supports GLA loss of function as a disease mechanism, and multiple disease-associated missense variants are reported, but the available materials do not establish the gene-level combination of a low benign missense rate and missense as a common disease mechanism under the generic PP2 definition. No validated benign-missense rate or gene-level calibration is supplied.
Missense variant in a gene that has a low rate of benign missense variation and where missense variants are a common mechanism of disease.
The governing rule requires the patient's phenotype or family history to be highly specific for a disease with a single genetic etiology. The case evidence supplied here does not provide a patient-specific phenotype, family history, or a sufficiently specific clinical description for this variant case. Variant-specific Fabry disease reports in the literature cannot substitute for the patient's phenotype, so PP4 cannot be assessed.
Patient’s phenotype or family history is highly specific for a disease with a single genetic etiology.
BS2 is not assessed. The available gnomAD records show zero homozygotes, but they do not identify a documented healthy adult hemizygote or establish that any observed carrier was clinically unaffected at an age when full penetrance would be expected. For this X-linked GLA disorder, population allele counts alone cannot establish the healthy-adult observation required by BS2, particularly without a documented age and phenotype assessment.
Observed in a healthy adult individual for a recessive (homozygous), dominant (heterozygous), or X-linked (hemizygous) disorder, with full penetrance expected at an early age.
No family pedigree or genotype-phenotype dataset documents affected relatives who lack NM_000169.3:c.644A>G. Consequently, lack of segregation cannot be distinguished from unavailable family testing, phenocopy, variable expression, or an uninformative pedigree.
Lack of segregation in affected members of a family.
The available evidence describes an X-linked recessive Fabry disease context rather than a fully penetrant dominant GLA disorder. No affected-proband observation identifies NM_000169.3:c.644A>G with a pathogenic variant in trans, and no cis pathogenic variant is reported. The BP2 allelic configuration is therefore not assessable from the available evidence.
Observed in trans with a pathogenic variant for a fully penetrant dominant gene/disorder or observed in cis with a pathogenic variant in any inheritance pattern.
The governing rule requires the variant to be found in a case with an alternate molecular basis for disease. The supplied evidence documents Fabry disease reports and population observations but does not identify an alternate molecular diagnosis or a case in which another molecular cause explains the disease. BP5 therefore cannot be assessed.
Variant found in a case with an alternate molecular basis for disease.
PVS1 (ClinGen SVI PVS1 recommendations, PMC6185798, pvs1_generic_framework) applies only to null variant classes — nonsense, frameshift, canonical ±1/2 splice-consensus, translation-initiation-codon loss, and stop-loss variants — each expected to trigger nonsense-mediated decay or otherwise abolish normal protein function. NM_000169.3:c.644A>G in GLA is a missense substitution predicted to produce NP_000160.1:p.(Asn215Ser). As a result, no null-variant mechanism (nonsense-mediated decay, truncation, or canonical splice disruption) is triggered by this variant class, so PVS1's structural prerequisite is not met by this variant class. No VCEP criteria specification covers PVS1 for GLA, so the generic ACMG/AMP 2015 definition applies. This determination was made deterministically from the variant's consequence class, prior to LLM adjudication.
deterministic_prefilter: requires_null_variant_class
PM4 (Richards et al. 2015, PMID:25741868) applies to protein length changes: in-frame insertions/deletions in a non-repetitive region, or stop-loss variants that extend the protein past the reference stop codon. NM_000169.3:c.644A>G in GLA is a missense substitution predicted to produce NP_000160.1:p.(Asn215Ser). As a result, no change in protein length occurs, so there is nothing for this criterion to evaluate, so PM4's structural prerequisite is not met by this variant class. No VCEP criteria specification covers PM4 for GLA, so the generic ACMG/AMP 2015 definition applies. This determination was made deterministically from the variant's consequence class, prior to LLM adjudication.
deterministic_prefilter: requires_stop_loss_or_nonrepeat_indel
BP3 (Richards et al. 2015, PMID:25741868) applies to in-frame insertions/deletions located in a repetitive region without a known function. NM_000169.3:c.644A>G in GLA is a missense substitution predicted to produce NP_000160.1:p.(Asn215Ser). As a result, the variant does not alter protein length within a repeat region, so there is nothing for this criterion to evaluate, so BP3's structural prerequisite is not met by this variant class. No VCEP criteria specification covers BP3 for GLA, so the generic ACMG/AMP 2015 definition applies. This determination was made deterministically from the variant's consequence class, prior to LLM adjudication.
deterministic_prefilter: requires_repeat_region_indel
BP7 (Richards et al. 2015, PMID:25741868) is defined for a synonymous (silent) variant for which splicing prediction algorithms predict no impact to the splice consensus sequence and the nucleotide is not highly conserved. NM_000169.3:c.644A>G in GLA is a missense substitution predicted to produce NP_000160.1:p.(Asn215Ser). As a result, the encoded protein sequence is altered, so the criterion's silent-variant premise does not hold regardless of any splicing prediction, so BP7's structural prerequisite is not met by this variant class. No VCEP criteria specification covers BP7 for GLA, so the generic ACMG/AMP 2015 definition applies. This determination was made deterministically from the variant's consequence class, prior to LLM adjudication.
deterministic_prefilter: requires_synonymous
Every interpreted variant in GLA, mapped onto the exon structure of NM_000169.3. Select a row to find it on the plot.
| Protein | Location | Classification | Link |
|---|
| European (non-Finnish)37 / 888,895 | 4.2e-05 |
| Remaining individuals1 / 47,343 | 2.1e-05 |
| European (non-Finnish)1 / 81,881 | 1.2e-05 |
This variant is present in gnomAD v4.1 (AF= 3.15664e-05; MAF= 0.00316%, 38/1203813 alleles, homozygotes = 0) and has highest observed frequency in the European (non-Finnish) population (AF= 4.16247e-05; MAF= 0.00416%, 37/888895 alleles, homozygotes = 0); grpmax FAF= 3.071e-05.
This variant is present in gnomAD v2.1 (AF= 5.45191e-06; MAF= 0.00055%, 1/183422 alleles, homozygotes = 0) and has highest observed frequency in the European (non-Finnish) population (AF= 1.22128e-05; MAF= 0.00122%, 1/81881 alleles, homozygotes = 0).
Not available in gnomAD-Canada v1.0.
NM_000169.3:c.644A>G is a missense variant, so the governing generic rule selects the REVEL path only; SpliceAI is not evidence for missense PP3. REVEL was not available for this variant, so the applicable computational evidence is indeterminate and PP3 is not met. The BayesDel score is not used because no generic ACMG-strength calibration is established for BayesDel.
Multiple lines of computational evidence support a deleterious effect on the gene or gene product (conservation, evolutionary, splicing impact, etc.).
NM_000169.3:c.644A>G is a missense variant, so the governing generic rule selects the REVEL path only; SpliceAI is not evidence for missense BP4. REVEL was not available for this variant, so the applicable computational evidence is indeterminate and BP4 is not met. The SpliceAI max delta of 0.002 and the BayesDel score are not used for this missense BP4 assessment.
Multiple lines of computational evidence suggest no impact on gene or gene product (conservation, evolutionary, splicing impact, etc)
BP7 (Richards et al. 2015, PMID:25741868) is defined for a synonymous (silent) variant for which splicing prediction algorithms predict no impact to the splice consensus sequence and the nucleotide is not highly conserved. NM_000169.3:c.644A>G in GLA is a missense substitution predicted to produce NP_000160.1:p.(Asn215Ser). As a result, the encoded protein sequence is altered, so the criterion's silent-variant premise does not hold regardless of any splicing prediction, so BP7's structural prerequisite is not met by this variant class. No VCEP criteria specification covers BP7 for GLA, so the generic ACMG/AMP 2015 definition applies. This determination was made deterministically from the variant's consequence class, prior to LLM adjudication.
deterministic_prefilter: requires_synonymous
SpliceAI predicts no significant splice impact for this variant (max delta score = 0.00). BayesDel score = 0.105764.
9 paper records listed. 3 cited in the classification.
The mutant enzymes detected in variant patients (A20P, E66Q, M72V, I91T, R112H, F113L, N215S, Q279E, M296I, M296V and R301Q), and those found mostly in mild classic patients (A97V, A156V, L166V and R356W) appeared to have normal Km and Vmax values.
Location Abstract; Results, Table 1; Results, Table 2
Context Biochemical characterization of purified mutant human alpha-galactosidase A expressed in transiently transfected COS-7 cells, including kinetic assays, pH-stability testing, ERAD-inhibitor treatment, and DGJ treatment.
As shown in Figure 3, mutant forms that were increased by AT1001 in the enzyme assay consistently showed robust increases in a-Gal A protein levels (e.g., R301Q [c.902G4A] and N215S [c.644A4G]).
Location Table 1, p.N215S row; Results section, ‘AT1001 Increases Mutant a-Gal A Protein Levels’; Figure 3
Context Transient expression of site-directed GLA mutants in HEK-293 cells followed by 4–5 days of AT1001 incubation, alpha-Gal A enzyme activity measurement, concentration-response analysis, and Western blotting; three independent experiments were reported for N215S.
The p.N215S alpha-Gal A variant used for control expression showed an intermediate value of enzyme activity.
Location Table 3; Results, paragraph 4; Discussion
Context Site-directed mutant GLA plasmid transiently over-expressed in HEK293T cells for 72 hours; soluble lysates were assayed with wild-type and mock-transfected controls; three separate transfections.
The nature of the molecular lesions in the alpha-Gal A gene in 30 unrelated families was determined to provide precise heterozygote detection, prenatal diagnosis, and define genotype-phenotype correlations.
Abstract
including four with the N215S mutation, from classic hemizygotes (28), from cardiac variant hemizygotes with the N215S mutation (6)
Summary; Results, 'Urinary CTH' and 'Urinary CDH' paragraphs; Table 2
Fabry disease is a rare lysosome storage disease featuring X-linked recessive inheritance.
Abstract
Screenshots represent source records captured during evidence collection. Select one to view the recorded source content.
The evidence statements recorded by the interpretation pipeline. They have not been reinterpreted by the page.
This variant is present in gnomAD v4.1 (AF= 3.15664e-05; MAF= 0.00316%, 38/1203813 alleles, homozygotes = 0) and has highest observed frequency in the European (non-Finnish) population (AF= 4.16247e-05; MAF= 0.00416%, 37/888895 alleles, homozygotes = 0); grpmax FAF= 3.071e-05.
This variant is present in gnomAD v2.1 (AF= 5.45191e-06; MAF= 0.00055%, 1/183422 alleles, homozygotes = 0) and has highest observed frequency in the European (non-Finnish) population (AF= 1.22128e-05; MAF= 0.00122%, 1/81881 alleles, homozygotes = 0).
Not available in gnomAD-Canada v1.0.
This variant has been reported in ClinVar as Pathogenic (23 clinical laboratories). (ClinVarID = 10730)
SpliceAI predicts no significant splice impact for this variant (max delta score = 0.00). BayesDel score = 0.105764.
This variant does not lie in a statistically significant hotspot. This variant has not previously been reported in somatic cancers (COSMIC).
This variant does not lie in a statistically significant hotspot.