Variant classification summary

NM_000500.9:c.1439G>T

CYP21A2 · NP_000491.4:p.(Arg480Leu) · NM_000500.9

GRCh37: chr6:32008862 G>T · GRCh38: chr6:32041085 G>T

Gene: CYP21A2 Transcript: NM_000500.9

Final call

Likely Benign

PS3 supporting BS1 supporting benign BP4 supporting benign BP6 supporting benign

Variant details

Gene

CYP21A2

Transcript

NM_000500.9

Protein

NP_000491.4:p.(Arg480Leu)

gnomAD AF

0.0032639219510208033 (v4.1)

ClinVar

Benign

OncoKB

Classification rationale

Interpretation summary

Generated evidence synthesis

1

NM_000500.9:c.1439G>T (p.Arg480Leu) is a missense variant in exon 10 of CYP21A2, a gene associated with autosomal recessive congenital adrenal hyperplasia due to 21-hydroxylase deficiency.

2

The variant is present in gnomAD at an appreciable global allele frequency of 0.62% (v2.1, 1521/244182 alleles) and 0.33% (v4.1, 5128/1571116 alleles), exceeding the BS1 threshold (>0.3%) for a recessive disorder.

3

In silico predictions are uniformly benign: REVEL score 0.334, BayesDel score -0.316 (predicting benign), and SpliceAI detects no splice alteration (max delta 0.00), satisfying BP4.

4

ClinVar reports this variant as Benign by three clinical laboratories and Likely benign by three additional laboratories, with two VUS submissions, supporting BP6.

5

Functional studies demonstrate the variant retains ~75-80% of wild-type 21-hydroxylase activity in transfected COS-1 cells, consistent with a mild functional impact, providing PS3 at the supporting level.

6

Applying the generic ACMG/AMP 2015 combination rules (PMID:25741868), the criteria met are three supporting benign (BS1, BP4, BP6) and one supporting pathogenic (PS3). Likely benign requires at least two supporting benign criteria, which is satisfied.

Final determination: Generic ACMG/AMP 2015 fallback rules support a Likely Benign classification because either one strong benign criterion plus one supporting benign criterion, or at least two supporting benign criteria, are present.

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	NM_000500.9:c.1439G>T is a missense variant (p.Arg480Leu), not a null variant (nonsense, frameshift, canonical ±1/2 splice site, or initiation codon). Generic PVS1 framework (PMC6185798) does not apply to missense variants.	pvs1_generic_framework pvs1_variant_assessment
PS1	Not assessed	PS1 requires a different amino acid change at the same residue that has been previously established as pathogenic. No same-residue pathogenic comparator variant with a different amino acid change was identified in ClinVar or the literature for Arg480.
PS2	Not met	No confirmed de novo occurrence with both parents confirmed unaffected was identified for this variant. PS2 requires a de novo variant with maternity and paternity confirmed.
PS3	Met	Functional studies in transiently transfected COS-1 cells demonstrated that the p.Arg480Leu (R479L in older numbering) mutant retains approximately 75-80% of wild-type 21-hydroxylase activity toward both 17-hydroxyprogesterone and progesterone, consistent with a mild non-classical CAH phenotype. This partial reduction in enzyme activity supports a damaging effect at the supporting level.	PMID:17119906
PS4	Not met	PS4 requires the prevalence of the variant in affected individuals to be significantly increased compared to controls. No formal case-control study is available. The variant is present at appreciable frequency in the general population (gnomAD global AF 0.33-0.62%), which precludes PS4 applicability.	gnomad_v2 gnomad_v4
PS5	Not assessed	No different pathogenic variant at the same residue reported from a reputable source was identified.
PM1	Not met	PM1 requires location in a mutational hot spot or critical functional domain without benign variation. While Arg480 lies in the C-terminal region (exon 10) near the heme-binding domain, the variant is present in gnomAD at appreciable frequency (AF 0.33-0.62% globally, up to 6% in African population, with 1 homozygote in v4.1), indicating that benign variation exists at this position. Cancer Hotspots analysis confirms the residue is not in a statistically significant hotspot.	gnomad_v2 gnomad_v4
PM2	Not met	PM2 requires the variant to be absent or at extremely low frequency in population databases (<0.1%). The variant is present in gnomAD v2.1 at global AF 0.62% and in v4.1 at global AF 0.33%, both well above the 0.1% threshold.	gnomad_v2 gnomad_v4
PM5	N/A	No same-residue comparator variant with a different pathogenic missense change was identified; automated PM5 candidate harvesting was unable to confirm classic same-residue PM5 semantics.	pm5_candidates
PM6	Not met	PM6 requires an assumed de novo occurrence without confirmation of both parents. No verified de novo report with at least one parent tested negative was confirmed for this variant.
PP1	Not met	PP1 requires cosegregation with disease in multiple affected family members. No verified segregation data across multiple families was identified for this variant.
PP2	Not met	PP2 requires a missense variant in a gene with a low rate of benign missense variation where missense variants are a common disease mechanism. CYP21A2 has multiple known benign missense variants (e.g., R102K, N493S), and the variant itself is observed at appreciable population frequency, inconsistent with a low rate of benign missense variation.	gnomad_v2 gnomad_v4
PP3	Not met	PP3 requires multiple lines of computational evidence supporting a deleterious effect. All in silico predictors suggest a benign effect: REVEL score 0.334 (below 0.5 pathogenic threshold), BayesDel score -0.316 (negative, predicts benign), and SpliceAI max delta 0.0 (no predicted splice impact).	revel bayesdel spliceai
PP4	Not assessed	PP4 requires the patient's phenotype or family history to be highly specific for a disease with a single genetic etiology. No individual patient phenotype data was available for assessment.
PP5	Not met	PP5 requires a reputable source to have recently reported the variant as pathogenic. ClinVar reports this variant predominantly as Benign (3 laboratories) and Likely benign (3 laboratories), with 2 VUS submissions. No reputable source reports it as pathogenic.	clinvar
BA1	Not met	BA1 requires allele frequency >1% in population databases. The global allele frequency in gnomAD v2.1 is 0.62% and in v4.1 is 0.33%, both below the 1% threshold. Note: the highest subpopulation frequency (African/African American) is 6.08% (v2.1) and 5.87% (v4.1), which would satisfy BA1; however, using global AF as the primary metric, BA1 is not met.	gnomad_v2 gnomad_v4
BS1	Met	BS1 requires allele frequency >0.3% in population databases for a recessive disorder. The variant is present in gnomAD v2.1 at global AF 0.62% (1521/244182 alleles) and in v4.1 at global AF 0.33% (5128/1571116 alleles). Both exceed the 0.3% threshold. Additionally, the variant was observed in heterozygous state in 1 of 92 healthy individuals in a Hungarian population study (PMID:19505723).	gnomad_v2 gnomad_v4 PMID:19505723
BS2	Not assessed	BS2 requires observation in a healthy adult in a state (homozygous for recessive) where full penetrance is expected at an early age. One homozygote is present in gnomAD v4.1, but the clinical status of this individual is unknown, precluding definitive assessment.	gnomad_v4
BS3	Not met	BS3 requires well-established functional studies showing no damaging effect. Functional studies demonstrate that p.Arg480Leu reduces 21-hydroxylase activity to approximately 75-80% of wild-type (PMID:17119906), which is a damaging effect. BS3 does not apply because functional evidence supports pathogenicity, not benignity.	PMID:17119906
BS4	Not met	BS4 requires lack of segregation in affected family members. No evidence of non-segregation was identified.
BP1	Not met	BP1 applies when a missense variant occurs in a gene where primarily truncating variants cause disease. CYP21A2-related congenital adrenal hyperplasia is caused by both missense and truncating variants; missense variants are a well-established disease mechanism. BP1 is not applicable.
BP2	N/A	CYP21A2 is an autosomal recessive gene. BP2 applies to observations in trans with a pathogenic variant in dominant disorders, or in cis with a pathogenic variant in any inheritance pattern. For recessive disorders, observation in trans with a pathogenic variant is expected and does not support benignity.
BP4	Met	BP4 requires multiple lines of computational evidence suggesting no impact. REVEL score is 0.334 (below 0.5 pathogenic threshold), BayesDel score is -0.316 (negative, predicts benign), and SpliceAI predicts no splice impact (max delta 0.00). All three in silico tools consistently predict a benign effect.	revel bayesdel spliceai
BP5	Not assessed	BP5 requires the variant to be found in a case with an alternate molecular basis for disease. No such evidence was identified.
BP6	Met	BP6 applies when a reputable source reports the variant as benign without access to the underlying evidence. ClinVar reports this variant as Benign by 3 clinical laboratories, Likely benign by 3 additional laboratories, and VUS by 2 laboratories. The majority clinical consensus across multiple submitters supports a benign interpretation.	clinvar
BP7	N/A	BP7 applies to synonymous variants with no predicted splice impact. NM_000500.9:c.1439G>T is a missense variant (p.Arg480Leu), not a synonymous variant.

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