ClinVar Genomic variation as it relates to human health

PAH-specific ACMG/AMP criteria applied: PP3: in silico analysis supportive of damaging effect; PM3_Strong: In trans with R261Q (PMID 21147011), and in trans with IVS2+1G>A (PMID 12655554) (PMID:21147011; PMID:12655554); PP4_Moderate: BH4 deficiency excluded (PMID:21147011). In summary this variant meets criteria to be classified as likely pathogenic for phenylketonuria in an autosomal recessive manner based on the ACMG/AMP criteria applied as specified by the PAH Expert Panel: (PP3, PM3_Strong, PP4_Moderate).

In silico analysis supports that this missense variant has a deleterious effect on protein structure/function; This variant is associated with the following publications: (PMID: 15171997, 12655554, 34426522, 30389586, 32778825, 29771303, 21147011, 31355225)

Variant summary: PAH c.355C>T (p.Pro119Ser) results in a non-conservative amino acid change located in the C-terminal domain (IPR019774) of the encoded protein sequence. Five of five in-silico tools predict a damaging effect of the variant on protein function. 4/4 computational tools predict no significant impact on normal splicing. However, these predictions have yet to be confirmed by functional studies. The variant allele was found at a frequency of 0.00029 in 251358 control chromosomes, predominantly at a frequency of 0.0023 within the South Asian subpopulation in the gnomAD database. This frequency is not significantly higher than expected for a pathogenic variant in PAH causing Phenylalanine Hydroxylase Deficiency (Phenylketonuria) (0.00029 vs 0.0079), allowing no conclusion about variant significance. c.355C>T has been reported in the literature in multiple individuals affected with Phenylalanine Hydroxylase Deficiency (Phenylketonuria) or Hyperphenylalaninemia (e.g. Lindner_2003, Dobrowolski_2011, Esfahani_2018, Kuznetcova_2019, Su_2019). These data indicate that the variant is very likely to be associated with disease. Eight clinical diagnostic laboratories have submitted clinical-significance assessments for this variant to ClinVar after 2014 without evidence for independent evaluation (pathogenic n=2, likely pathogenic n=5, VUS n=1). Based on the evidence outlined above, the variant was classified as pathogenic.

This sequence change replaces proline, which is neutral and non-polar, with serine, which is neutral and polar, at codon 119 of the PAH protein (p.Pro119Ser). This variant is present in population databases (rs398123292, gnomAD 0.2%). This missense change has been observed in individual(s) with hyperphenylalaninemia (PMID: 12655554, 21147011, 30389586, 31355225). ClinVar contains an entry for this variant (Variation ID: 92741). An algorithm developed to predict the effect of missense changes on protein structure and function (PolyPhen-2) suggests that this variant is likely to be disruptive. For these reasons, this variant has been classified as Pathogenic.

The c.355C>T (p.P119S) alteration is located in exon 4 (coding exon 4) of the PAH gene. This alteration results from a C to T substitution at nucleotide position 355, causing the proline (P) at amino acid position 119 to be replaced by a serine (S). Based on data from gnomAD, the T allele has an overall frequency of 0.029% (72/251358) total alleles studied. The highest observed frequency was 0.229% (70/30616) of South Asian alleles. This alteration has been reported compound heterozygous with a second alteration in PAH in multiple patients with biochemically confirmed PKU or hyperphenylalaninemia (Lindner, 2003; Esfahani, 2019; Su, 2019). This amino acid position is highly conserved in available vertebrate species. This alteration is predicted to be deleterious by in silico analysis. Based on the available evidence, this alteration is classified as pathogenic.

This variant is associated with phenylketonuria and hyperphenylalaninemia (PMIDs: 17924342 (2007), 21147011 (20111), and 35405047 (2022)). The variant has been reported as homozygous (PMID: 36537053 (2022)) or compound heterozygous with other pathogenic PAH variants in multiple individuals with PAH-related conditions (PMIDs: 31355225 (2019), 30389586 (2019), and 32668217 (2020)). Analysis of this variant using bioinformatics tools for the prediction of the effect of amino acid changes on protein structure and function yielded predictions that this variant is damaging. Based on the available information, this variant is classified as likely pathogenic.

Based on the classification scheme VCGS_Germline_v1.3.4, this variant is classified as Likely pathogenic. Following criteria are met: 0102 - Loss of function is a known mechanism of disease in this gene and is associated with phenylketonuria (MIM#261600). (I) 0106 - This gene is associated with autosomal recessive disease. (I) 0200 - Variant is predicted to result in a missense amino acid change from proline to serine. (I) 0252 - This variant is homozygous. (I) 0304 - Variant is present in gnomAD (v2) <0.01 for a recessive condition (72 heterozygotes, 0 homozygotes). (SP) 0309 - An alternative amino acid change at the same position has been observed in gnomAD (v3) (1 heterozygotes, 0 homozygotes). (I) 0501 - Missense variant consistently predicted to be damaging by multiple in silico tools and highly conserved with a moderate amino acid change. (SP) 0600 - Variant is located in the annotated Biopterin_H domain (DECIPHER). (I) 0708 - Another missense variants comparable to the one identified in this case has conflicting previous evidence for pathogenicity. p.(Pro199Leu) has been reported in ClinVar once as a variant of unknown significance and once as likely pathogenic. (I) 0801 - This variant has strong previous evidence of pathogenicity in unrelated individuals. This variant has been reported eight times in ClinVar as pathogenic or likely pathogenic and reviewed by the ClinGen expert panel as likely pathogenic. This variant likely represents a hypomorphic allele as it has only been observed in affected individuals when it is in trans with another variant, homozygotes appear to be unaffected (PMID: 29771303). (SP) 1209 - This variant has been shown to be both maternally and paternally inherited (biallelic) (by trio analysis). (I) Legend: (SP) - Supporting pathogenic, (I) - Information, (SB) - Supporting benign

The observed missense c.355C>T(p.Pro119Ser) variant in PAH gene has been reported previously in homozygous and compound heterozygous states in individuals affected with Phenylketonuria/Hyperphenylalaninemia (Kuznetcova I, et al., 2019; Su Y, et al., 2019; Esfahani MS & Vallian S., 2019). The p.Pro119Ser variant has been reported with allele frequency of 0.03% in gnomAD Exomes. This variant has been reported to the ClinVar database as Uncertain Significance / Pathogenic / Likely Pathogenic (multiple submissions). Multiple lines of computational evidences (Polyphen - Probably damaging, SIFT - Damaging and MutationTaster - Disease causing) predict a damaging effect on protein structure and function for this variant. The reference amino acid change at this position on PAH gene is predicted as conserved by GERP++ and PhyloP across 100 vertebrates. The amino acid Pro at position 119 is changed to a Ser changing protein sequence and it might alter its composition and physico-chemical properties. Additional functional studies will be required to prove the pathogenicity of this variant. For these reasons, this variant has been classified as Likely Pathogenic. In absence of another reportable variant in PAH gene, the molecular diagnosis is not confirmed