ClinVar Genomic variation as it relates to human health

The c.143T>C (p.Leu48Ser) variant in PAH has been reported multiple individuals with mild and classic PKU (BH4 deficiency excluded). (PP4_Moderate; PMID: 26322415, 16879198, 1679030). This variant has a frequency that is higher than the suggested cutoff for PM2 by the PAH VCEP (MAF=0.00026). This variant has 39% residual activity (PS3, PMID: 17935162). This variant was detected in trans with p.G247R (LP, 2 submitters) PM3, PMID: 26322415). Computational prediction tools and conservation analysis suggest that this variant may impact the protein (PP3). Cosegregation with PKU in 2 siblings for 2 unrelated families was reported ( PMID: 23430547). In summary, this variant meets criteria to be classified as pathogenic for PAH. PAH-specific ACMG/AMP criteria applied: PP4_Moderate, PS3, PM3, PP1, PP3.

This variant is associated with a variable phenotype that ranges from hyperphenylalaninemia to classic PKU (PMIDs: 25596310 (2015), 23430547 (2013), 21953985 (2012), 16879198 (2006), and 1679030 (1991)). The variant occurs in multiple cases with a lone recessive pathogenic/likely pathogenic variant in the same gene, and several have a phenotype known to be consistent with disease. In addition, experimental studies have shown that this variant has deleterious effects on PAH enzyme activity and protein expression (PMIDs: 25596310 (2015), 23500595 (2013), 21953985 (2012), and 17935162 (2008)).

The variant is observed at an extremely low frequency in the gnomAD v2.1.1 dataset (total allele frequency: 0.012%). Missense changes are a common disease-causing mechanism. Functional studies provide strong evidence of the variant having a damaging effect on the gene or gene product (PMID: 17935162). In silico tool predictions suggest damaging effect of the variant on gene or gene product (REVEL: 0.97; 3Cnet: 0.98). The variant has been reported to be in trans with a pathogenic variant as either compound heterozygous or homozygous in at least 4 similarly affected unrelated individuals (PMID: 26481238). A different missense change at the same codon (p.Leu48Val) has been reported to be associated with PAH related disorder (PMID: 31623983). Therefore, this variant is classified as Pathogenic according to the recommendation of ACMG/AMP guideline.

This sequence change replaces leucine, which is neutral and non-polar, with serine, which is neutral and polar, at codon 48 of the PAH protein (p.Leu48Ser). This variant is present in population databases (rs5030841, gnomAD 0.03%). This missense change has been observed in individuals with classical and variant phenylketonuria (PKU) (PMID: 1679030, 9399896, 23430547, 23500595). ClinVar contains an entry for this variant (Variation ID: 608). 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 expected to disrupt PAH protein function with a positive predictive value of 80%. Experimental studies have shown that this missense change affects PAH function (PMID: 11461190, 23500595, 25596310). For these reasons, this variant has been classified as Pathogenic.

Functional studies demonstrate p.(L48S) is associated with significantly reduced enzyme activity compared to wildtype (PMID: 25596310, 26803807); 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: 21953985, 25525159, 1679030, 30963030, 34900593, 23500595, 25087612, 11461190, 17935162, 23559577, 22975760, 23430547, 26803807, 9323556, 28676969, 9399896, 31355225, 26322415, 16879198, 34426522, 31589614, 33101986, 8592329, 32778825, 33465300, 36537053, 36646061, 36672771, 35405047, 25596310, 37189584)

PAH: PM3:Very Strong, PM2, PP4:Moderate, PS3:Supporting

Variant summary: The PAH c.143T>C (p.Leu48Ser) variant located in the ACT domain (via InterPro) causes a missense change involving a conserved nucleotide, to which 5/5 in silico tools predict a damaging outcome. The variant of interest was found in the large, broad control population, ExAC, with an allele frequency of 10/121396 (1/12135), which does not exceed the estimated maximal expected allele frequency for a pathogenic PAH variant of 1/126. The variant of interest has been reported in multiple affected individuals as homozygous and compound heterozygous and had been implicated to cause a mild PKU phenotype. In addition, multiple clinical diagnostic laboratories/databases cite the variant as "pathogenic." Therefore, the variant of interest has been classified as "pathogenic."

NM_000277.1(PAH):c.143T>C(L48S) is classified as pathogenic in the context of phenylalanine hydroxylase deficiency and is associated with classic or variant PKU. Sources cited for classification include the following: PMID 16879198, 22513348, 12501224, 9521426, 8889590, 23500595, 23430547, 21953985, 17935162 and 1679030. Classification of NM_000277.1(PAH):c.143T>C(L48S) 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.‚Äã

This variant was classified as: Pathogenic. The following ACMG criteria were applied in classifying this variant: PS1,PM1,PM2,PP2,PP3.

The c.143T>C (p.L48S) alteration is located in exon 2 (coding exon 2) of the PAH gene. This alteration results from a T to C substitution at nucleotide position 143, causing the leucine (L) at amino acid position 48 to be replaced by a serine (S). Based on data from the Genome Aggregation Database (gnomAD), the PAH c.143T>C alteration was observed in 0.012% (34/282,822) of total alleles studied, with a frequency of 0.025% (9/35,440) in the Latino subpopulation. This is a recurrent mutation, reported in multiple unrelated patients with mild and classic PKU (Konecki, 1991; Couce, 2013; Djordjevic, 2013; Gundorova, 2019; Su, 2019). This amino acid position is highly conserved in available vertebrate species. Functional analysis of the p.L48S alteration, either homozygous or compound heterozygous with another PAH mutation, found that it is associated with reduced enzyme activity compared to wildtype (Waters, 2001; Danecka, 2015; Shen, 2016). The p.L48S alteration is predicted to be deleterious by in silico analysis. Based on the available evidence, this alteration is classified as pathogenic.

The PAH c.143T>C variant is predicted to result in the amino acid substitution p.Leu48Ser. This variant has been well documented as causative for phenylalanine hydroxylase deficiency (e.g., Couce et al. 2013. PubMed ID: 23500595; Heintz et al. 2013. PubMed ID: 23559577, Table S3 in Hillert et al. 2020. PubMed ID: 32668217). The c.143T>C variant has been reported to reduce the activity of the PAH protein to 39% of wild-type, and individuals with the p.Leu48Ser substitution are often found to be responsive to tetrahydrobiopterin (BH4). However, it should be noted that there is some inconsistency in BH4-responsivness by individuals carrying this particular variant (Zurflüh et al. 2008. PubMed ID: 17935162). The ClinGen PAH Variant Curation Expert Panel, as well as several other labs, classify this variant as pathogenic (https://www.ncbi.nlm.nih.gov/clinvar/variation/608/). Based on these observations, we also interpret this variant as pathogenic.