ClinVar Genomic variation as it relates to human health

PAH-specific ACMG/AMP criteria applied: PM3_VeryStrong: Detected with Y414C (P), R408W (P), P281L (P), IVS10nt-11 (P), R252W (P/LP), and R243Q(P). (PMID:12501224; PMID:1301201; PMID:10767174); PP3: Predicted dleterious in SIZFT, Polyphen2, MutationTaster. REVEL=0.985; PP4_Moderate: Detected in a patient with mild PKU. BH4 deficiency excluded. Upgraded per ClinGen PAH EP. (PMID:12501224); PS3: 25% mutant enzyme activity in COS cells as compared in wt (PMID:1301201). 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: (PM3_VeryStrong, PP3, PP4_Moderate, PS3).

The PAH c.194T>C (p.I65T) missense variant has been found in the homozygous and compound heterozygous state in individuals with a range of clinical phenotypes including hyperphenylalaninemia; mild phenylketonuria, and classic phenylketonuria (PMID: 9254847; 1301201; 10767174; 12501224).

NM_000277.1(PAH):c.194T>C(I65T) is classified as likely 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 11524738, 23500595, 11326337, 1301187, 17935162, 15557004, 8533759, and 9781015. Classification of NM_000277.1(PAH):c.194T>C(I65T) is based on the following criteria: This variant 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.

Common pathogenic PAH variant most often reported in association with mild-moderate phenylketonuria (PKU) (Pey et al., 2007; Zurfluh et al, 2008; Danecka et al. 2015). It has also been identified in an individual with classic PKU (Couce et al., 2013).; Functional studies demonstrate I65T is associated with reduced phenylalanine hydroxylase activity compared to wild-type (Danecka et al., 2015); 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: 1301187, 17924342, 1301201, 22975760, 30963030, 23500595, 17935162, 11326337, 23559577, 12655546, 25596310, 19036622, 11461190, 21953985, 27121329, 28645531, 25087612, 27264808, 26803807, 19194782, 29030855, 30648773, 30747360, 31980526, 30275481, 31589614, 32668217, 32853555)

This sequence change replaces isoleucine, which is neutral and non-polar, with threonine, which is neutral and polar, at codon 65 of the PAH protein (p.Ile65Thr). This variant is present in population databases (rs75193786, gnomAD 0.05%). This missense change has been observed in individual(s) with PAH-related conditions (PMID: 12655546, 23500595, 25596310). ClinVar contains an entry for this variant (Variation ID: 636). 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: 12655546, 17935162, 26803807). For these reasons, this variant has been classified as Pathogenic.

The c.194T>C (p.I65T) alteration is located in coding exon 3 of the PAH gene. This alteration results from a T to C substitution at nucleotide position 194, causing the isoleucine (I) at amino acid position 65 to be replaced by a threonine (T). Based on data from the Genome Aggregation Database (gnomAD), the PAH c.194T>C alteration was observed in 0.03% (83/282610) of total alleles studied, with a frequency of 0.06% (72/129008) in the European (non-Finnish) subpopulation. No homozygotes were observed. The c.194T>C (p.I65T) alteration has been described homozygous and compound heterozygous with a second allele in multiple unrelated families with phenotypes ranging from hyperphenylalaninemia to classic PKU (Couce, 2013; Desviat, 2004; John, 1992; Muntau, 2002; Rivera, 2000). The p.I65 amino acid is conserved in available vertebrate species. In vitro functional studies determined protein expressing the c.194T>C (p.I65T) alteration decreased PAH activity to less than 50% of wild type activity. When observed with a second deleterious alteration, residual PAH activity ranged between 5.5% to 48% (John, 1992; Shen, 2016). The p.I65T alteration is predicted to be deleterious by in silico analysis. Based on the available evidence, this alteration is classified as pathogenic.

Based on the classification scheme VCGS_Germline_v1.3.4, this variant is classified as pathogenic. The 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 isoleucine to threonine. (I) 0251 - This variant is heterozygous. (I) 0304 - Variant is present in gnomAD (v2) <0.01 for a recessive condition (83 heterozygotes, 0 homozygotes). (SP) 0309 - An alternative amino acid change at the same position has been observed in gnomAD (v2) (2 heterozygotes, 0 homozygotes). (I) 0501 - Missense variant consistently predicted to be damaging by multiple in silico tools or highly conserved with a major amino acid change. (SP) 0600 - Variant is located in the annotated ACT domain (DECIPHER). (I) 0801 - This variant has strong previous evidence of pathogenicity in unrelated individuals. This variant is a well-reported pathogenic variant (ClinVar). It is one of the most common pathogenic variants reported in individuals with phenylketonuria (PMID: 33465300) and is associated with classic PKU (BIOPKU database). (SP) Legend: (SP) - Supporting pathogenic, (I) - Information, (SB) - Supporting benign

PM3_VeryStrong+PP4_Moderate+PS3

PP3, PP4_moderate, PM2_moderate, PM3_very_strong

Variant summary: The PAH c.194T>C (p.Ile65Thr) variant located in the ACT domain (via InterPro) involves the alteration of a conserved nucleotide, which 5/5 in silico tools predict a damaging outcome. The variant of interest was observed in the large, broad control population, ExAC, with an allele frequency of 22/121240 (1/5509), which does not exceed the estimated maximal expected allele frequency for a pathogenic PAH variant of 1/126. Multiple publications cite the variant in affected individuals, predominantly as compound heterozygous and was indicated to cause a mild PKU phenotype. In addition, multiple clinical diagnostic laboratories and databases cite the variant as "pathogenic." Therefore, the variant of interest has been classified as "Pathogenic."

Functional studies provide strong evidence of the variant having a damaging effect on the gene or gene product (PMID: 1301201, PS3_S). In silico tool predictions suggest damaging effect of the variant on gene or gene product (REVEL: 0.985, 3CNET: 0.998, PP3_P). A missense variant is a common mechanism associated with Phenylketonuria; PKU (PP2_P). It is observed at an extremely low frequency in the gnomAD v2.1.1 dataset (total allele frequency: 0.000294, PM2_M). The same variant was previously reported several times in trans with another pathogenic variant in this gene (PMID: 1301201, 10767174, 12501224) (PM3_VS). A different missense change at the same codon has been reported as pathogenic/likely pathogenic with strong evidence (ClinVar ID: VCV000102622,VCV000102623, PMID:22526846,9521426,12501224, PM5_M). Therefore, this variant is classified as pathogenic according to the recommendation of ACMG/AMP guideline.

ACMG classification criteria: PS3, PS4, PM1, PM2, PM3, PP3

PS3, PM3_Very Strong, PP3

The PAH c.194T>C (p.Ile65Thr) variant is associated with a variable phenotype that ranges from non-PKU hyperphenylalaninemia to classic PKU (PMID: 1301201 (1992), 12501224 (2002), 15557004 (2004)). Assessment of experimental evidence regarding the effect of this variant suggests it causes decreased enzyme activity with a residual activity of about 30% (PMID: 26803807 (2016), 12655546 (2003), 1301201 (1992)). Therefore, the variant is classified as pathogenic.

PAH: PM3:Very Strong, PM2, PP3, PS3:Supporting

The PAH c.194T>C variant is predicted to result in the amino acid substitution p.Ile65Thr. This variant has been reported in the literature as causative for phenylalanine hydroxylase deficiency (for example, see Eisensmith and Woo et al. 1992. PubMed ID: 1301187; Zschocke et al. 1995. PubMed ID: 8533759; Jeannesson-Thivisol et al. 2015. PubMed ID: 26666653; Hillert et al. 2020. PubMed ID: 32668217; Vela-Amieva et al. 2021. PubMed ID: 34828281). Based on functional studies, the p.Ile65Thr amino acid change has been reported to result in reduced PAH protein level and reduced enzyme activity, and has been classified as a BH4-responsive amino acid substitution (Zurflüh et al. 2008. PubMed ID: 17935162; Shi et al. 2012. PubMed ID: 21953985). Multiple other laboratories, as well as the ClinGen PAH Variant Curation Expert Panel, classify this variant as pathogenic or likely pathogenic (https://www.ncbi.nlm.nih.gov/clinvar/variation/636/). Based on these observations, this variant is interpreted as pathogenic.