ClinVar Genomic variation as it relates to human health

PAH-specific ACMG/AMP criteria applied: PP3: Deleterious in SIFT, Polyphen2, MutationTaster; PP4_Moderate: A300S found on 56/588 hyperphenylalaninemic patients, BH4 deficiency excluded. Upgraded per ClinGen Metabolic WG. (PMID:21147011); PM3_VeryStrong: Detected with c.1066-11G>A (P), R261Q(P/LP), L48S (P), R176X (P) in 20 patients. (PMID:21147011); PS3: A300S in vitro PAH activity of 31% (PMID:17935162). In summary this variant meets criteria to be classified as pathogenic for phenylketonuria in an autosomal recessive manner based on the ACMG/AMP criteria applied as specified by the PAH Expert Panel: (PP3, PP4_Moderate, PM3_VeryStrong, PS3).

The c.898G>A (p.Ala300Ser) variant is well-documented in the literature and is associated with mild phenylketonuria phenotype that is responsive to BH4 treatment. Across a selection of available literature, the p.Ala300Ser variant has been identified in a homozygous state in at least two patients, in a compound heterozygous state in at least 20 patients, and in 40 patients whose zygosity was not explicitly stated but are presumed to be compound heterozygous (Eisensmith et al. 1992; Spaapen et al. 2001; Blau and Erlandsen 2004; Zurflüh et al. 2008; Bercovich et al. 2008; Couce et al. 2013). Control data are unavailable for this variant, which is reported at a frequency of 0.00072 in the European (Non-Finnish) population of the Exome Aggregation Consortium. Functional studies showed that the p.Ala300Ser variant did not affect enzyme activity, but resulted in severe misfolding and destabilization of the PAH protein (Gersting et al. 2008). Based on the collective evidence, the p.Ala300Ser variant is classified as pathogenic for phenylalanine hydroxylase deficiency.

Variant summary: The PAH c.898G>T (p.Ala300Ser) variant involves the alteration of a conserved nucleotide within the catalytic domain. 4/5 in silico tools predict a damaging outcome for this variant. This variant was found in 54/121010 control chromosomes at a frequency of 0.0004462, which does not exceed the estimated maximal expected allele frequency of a pathogenic PAH variant (0.0079057). This variant has been reported as one of the most common pathogenic variants that associate with a milder phenotype, which is consistant with the finding that p.Ala300Ser retains 31% of enzyme activity (Zurfluh_2008). In addition, multiple clinical diagnostic laboratories/reputable databases classified this variant as pathogenic. Taken together, this variant is classified as pathogenic.

The c.898G>T (p.A300S) alteration is located in coding exon 8 of the PAH gene. This alteration results from a G to T substitution at nucleotide position 898, causing the alanine (A) at amino acid position 300 to be replaced by a serine (S). Based on data from gnomAD, the T allele has an overall frequency of 0.05% (152/282410) total alleles studied. The highest observed frequency was 0.69% (71/10350) of Ashkenazi Jewish alleles. This alteration has been reported in individuals both homozygous and compound heterozygous with a second disease-causing allele and is typically associated with mild PKU or hyperphenylalaninemia (Bercovich, 2008; Dobrowolski, 2011; Couce, 2013; Réblová, 2013; Trunzo, 2013; Danecka, 2015; Jeannesson-Thivisol, 2015). Multiple studies have found that patients with this alteration are BH4-responsive (Zurflüh, 2008; Jeannesson-Thivisol, 2015; Shen, 2016). This amino acid position is highly conserved in available vertebrate species. The p.A300 amino acid is located in the catalytic domain of the protein. The serine substitution is too large for the side chain and destabilization is due to the change of polarity of the catalytic domain's core (reviewed in Blau, 2004). Functional analysis demonstrated the p.A300S alteration reduces protein activity to approximately 30% of wild type activity (Zurflüh, 2008; Shen, 2016). When co-expressed with other disease-causing alleles, residual activity has been reported from 5.2-18% of normal (Danecka, 2015; Shen, 2016). It has been determined that this alteration actually leads to conformational destabilization of the protein and increased degradation while the enzyme function remains intact (Gersting, 2008). This alteration is predicted to be deleterious by in silico analysis. Based on the available evidence, this alteration is classified as pathogenic.

NM_000277.1(PAH):c.898G>T(A300S) is classified as pathogenic in the context of phenylalanine hydroxylase deficiency and may be associated with classic, variant, or non-PKU hyperphenylalaninemia. Sources cited for classification include the following: PMID 9298832, 18538294, 23500595, 18299955, 16198137, 21147011, 15557004, 23792259, 24350308, and 17935162. Classification of NM_000277.1(PAH):c.898G>T(A300S) is based on the following criteria: This is a well-established pathogenic variant in the literature that has been observed more frequently in patients with clinical diagnoses than in healthy populations. Please note: this variant was assessed in the context of healthy population screening.

This variant was classified as: Pathogenic. The following ACMG criteria were applied in classifying this variant: PS1,PM1,PM5,PP2,PP3,PP5.

This sequence change replaces alanine, which is neutral and non-polar, with serine, which is neutral and polar, at codon 300 of the PAH protein (p.Ala300Ser). This variant is present in population databases (rs5030853, gnomAD 0.7%), and has an allele count higher than expected for a pathogenic variant. This missense change has been observed in individuals with PAH deficiency-related diseases (PMID: 22330942, 23764561, 25155776, 25596310, 27682710). ClinVar contains an entry for this variant (Variation ID: 92751). Advanced modeling of protein sequence and biophysical properties (such as structural, functional, and spatial information, amino acid conservation, physicochemical variation, residue mobility, and thermodynamic stability) performed at Invitae indicates that this missense variant is not expected to disrupt PAH protein function with a negative predictive value of 80%. Experimental studies have shown that this missense change affects PAH function (PMID: 15557004, 17935162, 18538294, 25596310, 26803807). For these reasons, this variant has been classified as Pathogenic.

The p.Ala300Ser variant in PAH is a well-established pathogenic variant in patients with phenylketonuria (Trefz 2009 PMID: 18956252, Heintz 2013 PMID: 23559577, Danecka 2015 PMID: 25596310), and is frequently associated with milder course and responsiveness to BH4 (Trefz 2009 PMID: 18956252, Heintz 2013 PMID: 23559577). In vitro functional studies provide some evidence that the p.Ala300Ser variant impacts protein function (Shen 2016 PMID: 26803807). This variant has been identified in 0.6% (67/10130) of Ashkenazi Jewish chromosomes by the Genome Aggregation Database (gnomAD, http://gnomad.broadinstitute.org; dbSNP rs5030853). Although this variant has been seen in the general population, its frequency is low enough to be consistent with a recessive carrier frequency. Computational prediction tools and conservation analysis suggest that the p.Ala300Ser variant may impact the protein, though this information is not predictive enough to determine pathogenicity. In summary, this variant meets criteria to be classified as pathogenic for phenylalanine hydroxylase deficiency in an autosomal recessive manner. ACMG/AMP Criteria applied PS3; PS4; PP3.

Typically associated with either mild hyperphenylalaninemia or mild PKU (PMID: 25596310, 26666653, 8831077); 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: 23500595, 26803807, 27682710, 30963030, 18538294, 30667134, 30487145, 34828281, 25087612, 11486900, 24082139, 1301187, 23559577, 25750018, 27469133, 27121329, 28676969, 29431110, 29102225, 30037505, 31355225, 34426522, 31589614, 33101986, 32778825, 33465300, 25596310, 17935162, 26666653, 8831077, 35405047, 36646061, 37189584, 36537053, 36845377, 25155776)

PAH: PM3:Very Strong, PS3, PM1, PM2, PM5, PP4:Moderate, PP3

ACMG Criteria: PS3, PM3, PM5, PP2, PP5; Variant was found in compound heterozygous state with NM_000277.3:c.688G>A.

Based on the classification scheme VCGS_Germline_v1.3.4, this variant is classified as Pathogenic. Following criteria are met: 0102 - Loss of function is a known mechanism of disease in this gene and is associated with phenylketonuria and hyperphenylalaninaemia, non-PKU mild (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 alanine to serine. (I) 0251 - This variant is heterozygous. (I) 0304 - Variant is present in gnomAD <0.01 for a recessive condition (v2: 152 heterozygotes, 0 homozygotes). (SP) 0501 - Missense variant consistently predicted to be damaging by multiple in silico tools or highly conserved with a major amino acid change. (SP) 0601 - Variant is located in the well-established functional biopterin_H domain (DECIPHER; PMID: 18538294). (SP) 0703 - Other missense variants comparable to the one identified in this case have moderate previous evidence for pathogenicity. p.(Ala300Val) has been classified as likely pathogenic by an expert panel (ClinVar) and has been reported as compound heterozygous in association with moderate-severe PKU (PMID: 8533759). Another missense variant, p.(Ala300Gly), has been reported once by a clinical laboratory as likely pathogenic (ClinVar). (SP) 0801 - This variant has strong previous evidence of pathogenicity in unrelated individuals. It has frequently been reported as pathogenic and reviewed by an expert panel (ClinVar). (SP) 1201 - Heterozygous variant detected in trans with a second pathogenic heterozygous variant (NM_000277.2(PAH):c.808A>G; p.(Arg270Gly)) in a recessive disease. (I) 1205 - This variant has been shown to be maternally inherited (by trio analysis). (I) Legend: (SP) - Supporting pathogenic, (I) - Information, (SB) - Supporting benign

GenomeConnect assertions are reported exactly as they appear on the patient-provided report from the testing laboratory. GenomeConnect staff make no attempt to reinterpret the clinical significance of the variant.