The PAH NM_000277.3:c.1222C>T (NP_000268.1:p.(Arg408Trp)) missense variant has previously been reported in somatic cancers (COSMIC; COSV61018595, n = 3 times). Experimental evidence: P.Arg408Trp PAH activity was 1.85% of wild type, below the VCEP PS3 threshold of 50%. Based on available guidelines, this variant is considered to be Likely Benign. This variant is considered to be Pathogenic by the VCEP (ClinGen PAH Variant Curation Expert Panel; ClinVar ID 577).
Five-step scale from benign to pathogenic. This variant is likely benign. Select a step to read what it means.
Likely benign.A likely benign classification under the recorded framework.
Gene-level background from the recorded interpretation.
Words with a dotted underline have a short definition. Hover or tap them.The PAH gene provides instructions for phenylalanine hydroxylase, an enzyme that converts phenylalanine into tyrosine and helps the body break down phenylalanine. Reduced activity of this enzyme causes phenylketonuria (PKU), an inherited condition in which phenylalanine can build up to harmful levels. PAH has no established role in cancer.
The recorded framework is ClinGen PAH VCEP v2.0. 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 at supporting: p.Arg408Trp PAH activity was 1.85% of wild type, below the VCEP PS3 threshold of 50%.
Met at Moderate: REVEL score 0.887 falls within the VCEP PP3 missense interval of 0.773–0.932.
Not applicable: the PAH VCEP prohibits PP5 use regardless of pathogenic assertions from reputable sources.
Met at strong: gnomAD v2.1 grpmax FAF is 0.0027554, exceeding the VCEP BS1 threshold of 0.002 (0.2%).
Met at Supporting: SpliceAI maximum delta score 0.092 is below the VCEP BP4 no-splice-impact threshold of <0.1.
Met at Supporting: BP4 is met, and the exon-12 variant is outside the first and last 3 exon bases required by the VCEP.
Likely Benign: BS1 (strong) plus BP4 (supporting) satisfy the PAH VCEP's Rule18, with BP7 (supporting) providing additional benign evidence.
Evidence summaries and captured source records, where available. Missing entries should not be interpreted as negative findings.
gnomAD v4.1: 2,136 / 1,613,656 alleles. Recorded AF 0.0013237. Recorded homozygote count 0.
This variant is present in gnomAD v2.1 (AF= 0.000890988; MAF= 0.08910%, 252/282832 alleles, homozygotes = 0) and has highest observed frequency in the European (non-Finnish) population (AF= 0.00171864; MAF= 0.17186%, 222/129172 alleles, homozygotes = 0); grpmax FAF= 0.0027554.
Not available in gnomAD-Canada v1.0.
Recorded REVEL score: 0.887. Refer to the recorded PP3/BP4 rationale for interpretation.
33 Pathogenic · 2 pathogenic. ClinVar API review status: reviewed by expert panel.
No standalone functional evidence summary is recorded in evidence_summary.functional.
This variant has previously been reported in somatic cancers (COSMIC; COSV61018595, n = 3 times).
This variant does not lie in a statistically significant hotspot.
ACMG/AMP criteria under ClinGen PAH VCEP v2.0, with the data and sources behind each one.
28 criteria with recorded statuses: 6 met · 5 not met · 8 not assessed · 9 not applicable. Open any row for the full reasoning, the rule and the evidence behind it.
Within the requested codon, residue, and domain criteria, PM1 is not met because residue 408 is outside every PAH VCEP-listed PM1 region; PS1 and PM5 remain not assessed because their required comparator evidence is unavailable; PP2 and BP1 are VCEP-defined not applicable.
The variant is p.Arg408Trp, and the VCEP's explicit PM1 location list governs despite literature describing the residue more broadly as part of the catalytic domain.
No segregation or de novo criterion is met from the documented evidence; PM6 is not applicable under the PAH VCEP specification.
The available ClinVar segregation statement lacks the quantitative family data required by the VCEP PP1 thresholds.
No criteria match.
The PAH VCEP functional-assay reference identifies NM_000277.3:c.1222C>T, p.(Arg408Trp), as PS3 with a pathogenic result and assigns PS3_supporting. Its Table S8 row reports p.Arg408Trp residual activity of 1.85% of wild type, based on two assay sources/measurements. This is below the VCEP PS3_supporting threshold of 50% of wild-type activity. The VCEP assay table also records approved PAH functional assays as PS3_supporting, while the available evidence does not establish the VCEP requirements for PS3_moderate or stronger; therefore PS3 is applied at supporting strength only.
In vitro enzyme activity ≤50% compared to wild type controls * with ≤10 benign/pathogenic variant controls used in assay
The PAH VCEP PP3 Moderate rule applies to missense variants with REVEL scores from 0.773 to 0.932. NM_000277.3:c.1222C>T is a missense variant encoding p.(Arg408Trp), and its REVEL score is 0.887, so PP3 is met at Moderate strength. The VCEP PP3 splice path requires SpliceAI >0.5; the maximum score here is 0.092, so that path is not met and is not counted alongside REVEL.
Applicable as described in Pejaver et al (PMID: 36413997): REVEL score 0.773 - 0.932 for missense variants
Expert panel ClinGen PAH Variant Curation Expert Panel classified as Pathogenic.
This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee.
The PAH VCEP defines BS1 Strong as allele frequency >=0.002 (0.2%). In gnomAD v2.1 all-comers, the maximum reported population frequency is grpmax FAF 0.0027554 (0.27554%), which exceeds the threshold. The gnomAD v4.1 grpmax FAF is 0.00160006, but the v2.1 all-comers population value independently meets the VCEP rule.
Allele frequency >=0.002 (0.2%)
The PAH VCEP BP4 Supporting rule includes a splice-prediction path for no predicted impact on splicing by SpliceAI with a score <0.1. This missense variant has SpliceAI scores DS_AG 0.004, DS_AL 0.092, DS_DG 0.006, and DS_DL 0.071; the maximum delta score is 0.092, which is <0.1. The REVEL score of 0.887 does not meet the VCEP BP4 missense interval of 0.183–0.290, so BP4 is applied only through the qualifying SpliceAI path.
Applicable as described in Pejaver et al. * REVEL score of 0.183 - 0.290 for missense variants * In frame deletion or insertion predicted benign by PROVEAN, MutationTaster, and MutPred-InDel * No predicted impact on splicing by SpliceAI (score \<0.1)
The PAH VCEP BP7 Supporting rule requires BP4 to be met and specifies that exonic variants must be outside the first and last 3 bases of the exon. BP4 is met through SpliceAI, and NM_000277.3:c.1222C>T is an exonic missense variant in exon 12, positioned outside those exon-edge bases; therefore BP7 is met at Supporting strength. The rule's exonic-location path does not limit BP7 to synonymous variants.
Per SVI recommendations (PMID: 36865205), use BP7 only if BP4 is met; for variants with experimental evidence supporting that they do not alter splicing, use BP7\_strong (RNA) * intronic variants must be outside +7/-21 nt * exonic variants must be outside first and last 3 bases of exon
The PAH VCEP's applicable PM1 rule limits qualifying locations to its listed active-site residues, substrate-binding residues 46-48 and 63-69, and cofactor-binding residues His285, His290, Glu330, 246-266, 280-283, 322-326, and 377-379; p.Arg408Trp is at residue 408 and is in none of those listed regions. The VCEP rule also says not to apply PM1 if PP3_Strong applies; PP3_Strong is not established here because the available REVEL score is 0.887, below the VCEP's 0.932 threshold. Although papers describe Arg408Trp as in the catalytic domain, that location is not one of the PAH VCEP's specified PM1 regions.
* Active site residues in PAH include: Tyr138, Arg158, Val245, Tyr268, Thr278, Pro279, Glu289, Ala300, Asp315, Phe331, Ala345, Gly346, Ser349, Tyr377 * Substrate binding residues in PAH are: 46-48, 63-69 * Cofactor binding residues in PAH are: His285, His290, Glu330, 246-266, 280-283, 322-326, 377-379 * Do not apply if PP3\_Strong applies
The PAH VCEP defines PM2 Supporting as allele frequency <0.0002 (0.02%). The observed all-comers frequencies are above this cutoff: gnomAD v2.1 total AF 0.000890988 and gnomAD v4.1 total AF 0.0013237; their maximum population frequencies are 0.0027554 and 0.00160006, respectively. Therefore PM2 is not met.
* Threshold <0.0002 (0.02%) The 0.0002 cutoff is based on disease frequency of 1:12,000 and the most common PAH pathogenic variant, R408W, the ExAC frequency is 0.0006594 (ExAC MAF: 0.001109 74/66718 European Non-Finnish) and gnomAD overall: 0.0009056 (gnomAD MAF: 0.001728 219/126,700 European Non-Finnish).
The PAH VCEP defines BA1 as an allele frequency >=0.015 (1.5%). The highest reported all-comers population frequency is the gnomAD v2.1 grpmax FAF of 0.0027554 (0.27554%), while gnomAD v4.1 grpmax FAF is 0.00160006 (0.160006%); both are below 0.015. No BA1 evidence is met.
An allele frequency >=0.015 (1.5%), which is calculated with genetic heterogeneity of 90% to account for defects of BH4 metabolism, and penetrance of 80% to account for individuals who come to attention after becoming clinically symptomatic.
The PAH VCEP permits BS2 Strong only when the variant is observed in the homozygous state in a healthy adult. gnomAD v2.1 reports 0 homozygotes and gnomAD v4.1 reports 0 homozygotes; population allele counts do not establish a qualifying healthy-adult homozygote.
Only to be used when variant is observed in the homozygous state in a healthy adult.
The VCEP BS3 rule requires in vitro enzyme activity greater than 85% of wild type in the specified expression-system context. The VCEP functional-assay reference instead records p.Arg408Trp activity of 1.85% of wild type, and the directly relevant biochemical studies report markedly reduced or absent activity. The available functional evidence therefore contradicts the BS3 threshold rather than supporting benign function.
In vitro enzyme activity >85% compared to wild type * Expression systems: placing the mutant (and wildtype) cDNA into plasmid vectors and introducing these into host cells. Transiently transfected human or other mammalian host cells are the closest available approximation to the in vivo situation (e.g., COS cells) (Trunzo, et al. Gene. 2016. 594:138-143).
The PAH VCEP restricts PS1 to a previously classified (likely) pathogenic variant with the same predicted splicing impact. The available evidence gives SpliceAI scores for c.1222C>T (maximum delta 0.092) but does not identify a different previously classified (likely) pathogenic comparator with a documented matching splicing impact, so the VCEP rule cannot be evaluated.
Same predicted splicing impact as a previously classified (likely) pathogenic variant Applicable as described in Walker et al. (PMID: 36865205)
The case evidence does not document a de novo event in a proband, confirmation of both maternity and paternity, absence of family history, or a known pathogenic variant in trans. Therefore, the VCEP PS2 requirement cannot be evaluated.
The PAH VCEP specifies PM3 for recessive disease when the variant is detected in trans with a pathogenic variant and notes that parental or offspring testing is required to determine phase. The available case evidence does not document a co-occurring pathogenic PAH variant in an affected proband, phase, or inheritance information for NM_000277.3:c.1222C>T (p.Arg408Trp). Therefore PM3 cannot be assigned.
For recessive disorders, detected in trans with a pathogenic variant ... Note: This requires testing of parents (or offspring) to determine phase. ... Applicable as described in SVI recommendations for in trans criterion
The PAH VCEP permits PM5 for a novel missense change at a residue where a different missense change is pathogenic or, at supporting strength, likely pathogenic. The available PM5 comparator search reports zero same-residue candidates and no expert-panel or non-expert-majority pathogenic/likely pathogenic comparator candidates at Arg408; therefore the required different missense comparator is not established.
Applicable as described. ... Applicable when the different missense change is likely pathogenic.
A ClinVar submission contains a general statement that the variant was observed to segregate with disease in related individuals, but it provides no count of affected segregations, unaffected segregations, families, meioses, or phenotype details. The VCEP thresholds therefore cannot be applied to assign PP1 strength.
The PAH VCEP permits PP4 at supporting strength for persistently elevated plasma phenylalanine above 120umol/L (2mg/dL) without BH4-pathway exclusion testing, or at moderate strength when that elevation is accompanied by normal urine pterins and DHPR activity or exclusion by BH4-pathway sequencing. The available case evidence does not document a patient's persistent plasma phenylalanine concentration, urine pterins, DHPR activity, or BH4-pathway sequencing, so neither VCEP path can be evaluated.
Plasma phenylalanine concentration persistently above 120 µmol/L (2mg/dL), and either normal urine pterins and normal DHPR activity, or sequencing of genes in the BH4 cofactor metabolism pathway to exclude a defect of BH4 cofactor metabolism. ... A plasma phenylalanine concentration persistently above 120umol/L (2mg/dL) without analysis of urine pterins, DHPR activity, or sequencing to exclude defects of BH4 cofactor metabolism.
The available evidence does not document an affected family member who lacks the variant or provide a pedigree with non-segregation data. Therefore, the VCEP BS4 criterion cannot be evaluated.
The PAH VCEP lists BP5 as applicable as described, corresponding to the criterion for a variant found in a case with an alternate molecular basis for disease. The available evidence does not document an affected case carrying this variant together with a separate molecular diagnosis that explains the phenotype, so BP5 cannot be assigned.
Applicable as described
The PAH VCEP PVS1 rule is limited to nonsense or frameshift variants upstream or downstream of c.1285, canonical splice-site variants, initiator-codon variants, or qualifying RNA evidence. NM_000277.3:c.1222C>T is a missense variant in exon 12, encoding p.(Arg408Trp), so it is outside the variant types named by the VCEP PVS1 rule; NMD, exon criticality, and protein-length-change considerations do not create a PVS1 path for this missense change.
Applicable as described in Tayoun et al. 2018. * Any nonsense or frameshift variant occurring upstream of c.1285 * Any canonical splice site predicted to disrupt reading frame and undergo nonsense mediated decay PVS1 (RNA): splicing assay data - assays demonstrating a variant leads to aberrant splicing profile that can be categorized against a PVS1 decision tree * Use the PVS1 decision tree to determine code strength * Applicable as described in Walker et al. (PMID: 36865205)
The PAH VCEP explicitly states that PS4 is not applicable for PAH and directs proband counting to PM3. Literature reviewed for the PS4 lead included variant-level PAH patient observations, but those observations cannot override the VCEP applicability restriction.
This criterion is not applicable for PAH. For proband counting, use PM3 criterion.
The PAH VCEP PM4 entry states 'Applicable as described.' NM_000277.3:c.1222C>T is a missense substitution producing p.(Arg408Trp), not a protein-length-changing variant; it has no in-frame insertion/deletion or stop-loss consequence. Therefore the VCEP PM4 criterion is outside its named consequence scope for this variant.
Applicable as described
The PAH VCEP marks PM6 as not applicable. No PM6 evidence is assigned.
Assumed de novo, but without confirmation of paternity and maternity.
The PAH VCEP marks PP2 not applicable for this framework, so no gene-level missense-mechanism evidence can activate PP2 under the governing specification.
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 PAH VCEP marks BP1 not applicable for this framework, so the benign criterion for a missense variant in a primarily truncating-disease gene cannot be used.
Missense variant in a gene for which primarily truncating variants are known to cause disease.
The PAH VCEP explicitly marks BP2 as not applicable. Accordingly, BP2 is not assessed regardless of whether phase, cis/trans observations, or co-occurring pathogenic variants are available.
Not Applicable
The PAH VCEP CSPEC explicitly designates BP3 as Not Applicable. Accordingly, BP3 cannot be assigned to this variant, regardless of the absence or presence of repeat-region evidence.
Not Applicable
The PAH VCEP explicitly marks BP6 as not applicable and states that the criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. No benign assertion can therefore be assigned as BP6 under this framework.
This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee.
Every interpreted variant in PAH, mapped onto the exon structure of NM_000277.3. Select a row to find it on the plot.
| Protein | Location | Classification | Link |
|---|
| Remaining individuals114 / 62,498 | 0.0018 |
| European (non-Finnish)1960 / 1,179,634 | 0.0017 |
| European (Finnish)35 / 64,024 | 0.00055 |
| Ashkenazi Jewish8 / 29,600 | 0.00027 |
| African/African American14 / 74,992 | 0.00019 |
| Middle Eastern1 / 6,058 | 0.00017 |
| Admixed American3 / 60,002 | 5e-05 |
| South Asian1 / 91,054 | 1.1e-05 |
| European (non-Finnish)222 / 129,172 | 0.0017 |
| Remaining individuals6 / 7,226 | 0.00083 |
| European (Finnish)13 / 25,124 | 0.00052 |
| Ashkenazi Jewish4 / 10,370 | 0.00039 |
| African/African American7 / 24,962 | 0.00028 |
This variant is present in gnomAD v4.1 (AF= 0.0013237; MAF= 0.13237%, 2136/1613656 alleles, homozygotes = 0) and has highest observed frequency in the Remaining individuals population (AF= 0.00182406; MAF= 0.18241%, 114/62498 alleles, homozygotes = 0); grpmax FAF= 0.00160006.
This variant is present in gnomAD v2.1 (AF= 0.000890988; MAF= 0.08910%, 252/282832 alleles, homozygotes = 0) and has highest observed frequency in the European (non-Finnish) population (AF= 0.00171864; MAF= 0.17186%, 222/129172 alleles, homozygotes = 0); grpmax FAF= 0.0027554.
Not available in gnomAD-Canada v1.0.
The PAH VCEP PP3 Moderate rule applies to missense variants with REVEL scores from 0.773 to 0.932. NM_000277.3:c.1222C>T is a missense variant encoding p.(Arg408Trp), and its REVEL score is 0.887, so PP3 is met at Moderate strength. The VCEP PP3 splice path requires SpliceAI >0.5; the maximum score here is 0.092, so that path is not met and is not counted alongside REVEL.
Applicable as described in Pejaver et al (PMID: 36413997): REVEL score 0.773 - 0.932 for missense variants
The PAH VCEP BP4 Supporting rule includes a splice-prediction path for no predicted impact on splicing by SpliceAI with a score <0.1. This missense variant has SpliceAI scores DS_AG 0.004, DS_AL 0.092, DS_DG 0.006, and DS_DL 0.071; the maximum delta score is 0.092, which is <0.1. The REVEL score of 0.887 does not meet the VCEP BP4 missense interval of 0.183–0.290, so BP4 is applied only through the qualifying SpliceAI path.
Applicable as described in Pejaver et al. * REVEL score of 0.183 - 0.290 for missense variants * In frame deletion or insertion predicted benign by PROVEAN, MutationTaster, and MutPred-InDel * No predicted impact on splicing by SpliceAI (score \<0.1)
The PAH VCEP BP7 Supporting rule requires BP4 to be met and specifies that exonic variants must be outside the first and last 3 bases of the exon. BP4 is met through SpliceAI, and NM_000277.3:c.1222C>T is an exonic missense variant in exon 12, positioned outside those exon-edge bases; therefore BP7 is met at Supporting strength. The rule's exonic-location path does not limit BP7 to synonymous variants.
Per SVI recommendations (PMID: 36865205), use BP7 only if BP4 is met; for variants with experimental evidence supporting that they do not alter splicing, use BP7\_strong (RNA) * intronic variants must be outside +7/-21 nt * exonic variants must be outside first and last 3 bases of exon
SpliceAI predicts no significant splice impact for this variant (max delta score = 0.09). REVEL score = 0.887. BayesDel score = 0.530452.
8 paper records listed. 3 cited in the classification.
p.R408W c.1222C4T 35 (5.6) 1.85 Catalytic
Location Table 1, continued, ‘BH4-nonresponsive mutations’; Results and Discussion
Context BIOPKUdb analysis of 315 patients who responded to BH4 loading tests, compared with PAHdb and population genotype datasets; residual activity was an average from different cell systems.
S310Y and R408W eluted as high-molecular-weight aggregates without any detectable tetramers.
Location Results, ‘Disturbed Oligomerization Manifests as Aggregation or Impaired Tetramer Assembly,’ paragraph 2; Table 1 and Figure 2B
Context Recombinant human PAH variants expressed in E. coli as N-terminal MBP-PAH fusion proteins and assessed by chromatography for oligomerization, with enzyme activity and structural effects evaluated.
The p.R408W enzyme is dysfunctional in nearly all biochemical parameters, as evidenced by disease severity in homozygous and hemizygous patients.
Location Results, Table 1 and Tables 2–4; Results section ‘Correlation of PAH enzyme biochemistry with disease presentation’; Discussion
Context Recombinant full-length human tetrameric PAH expressed in E. coli and characterized by steady-state enzyme kinetics, BH4-binding fluorescence, and kinetic unfolding at 37°C; clinical data came from 14 patients carrying p.R408W.
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= 0.0013237; MAF= 0.13237%, 2136/1613656 alleles, homozygotes = 0) and has highest observed frequency in the Remaining individuals population (AF= 0.00182406; MAF= 0.18241%, 114/62498 alleles, homozygotes = 0); grpmax FAF= 0.00160006.
This variant is present in gnomAD v2.1 (AF= 0.000890988; MAF= 0.08910%, 252/282832 alleles, homozygotes = 0) and has highest observed frequency in the European (non-Finnish) population (AF= 0.00171864; MAF= 0.17186%, 222/129172 alleles, homozygotes = 0); grpmax FAF= 0.0027554.
Not available in gnomAD-Canada v1.0.
This variant has been reported in ClinVar as Pathogenic (33 clinical laboratories) and as pathogenic (2 clinical laboratories) and as Pathogenic by ClinGen PAH Variant Curation Expert Panel (expert panel). (ClinVarID = 577)
SpliceAI predicts no significant splice impact for this variant (max delta score = 0.09). REVEL score = 0.887. BayesDel score = 0.530452.
This variant does not lie in a statistically significant hotspot. This variant has previously been reported in somatic cancers (COSMIC; COSV61018595, n = 3 times).
This variant does not lie in a statistically significant hotspot.