ClinVar Genomic variation as it relates to human health

PAH-specific ACMG/AMP criteria applied: PP4_Moderate: Seen on 3 PKU alleles, BH4 deficiency was ruled out. Upgraded per ClinGen Metabolic WG. (PMID:8268925); PM3_VeryStrong: A403V found with 4 pathogenic variants . Upgraded per ClinGen SVI workgroup. (PMID:9429153); PS3: In vitro A403V mutant protein activity was ~43% wt. (PMID:21820508). 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: (PP4_Moderate, PM3_VeryStrong, PS3).

The c.1208C>T (p.Ala403Val) missense variant in the PAH gene is a well-established pathogenic variant associated with mild hyperphenylalaninemia and BH4-responsive phenylketonuria (Aulehla-Scholz et al., 2003; Fiori et al., 2005; Daniele et al., 2007; Zurflüh et al., 2008). Multiple in vitro functional studies have demonstrated that this variant results in reduced enzymatic activity to approximately 32% (Bénit et al., 1999; Blau et al., 2002; Cerreto et al., 2011). This variant is reported at low frequency in the population databases (Exome Sequencing Project = 0.058%; 1000 Genomes = 0.2%; and ExAC = 0.0.093%). Multiple in silico algorithms predict this variant to have a deleterious effect (GERP = 5.63; CADD = 26.8; PolyPhen = 0.996). Multiple reputable diagnostic laboratories have reported this variant as a well-established pathogenic variant (Emory Genetics Laboratory, Partners HealthCare Personalized Medicine, and GeneDx). Therefore, this collective evidence supports the classification of the c.1208C>T (p.Ala403Val) as a Pathogenic variant for Phenylketonuria. We have confirmed this finding in our laboratory using Sanger sequencing.

The PAH c.1208C>T (p.Ala403Val) missense variant is well-described as a pathogenic mild, BH4-responsive variant. Across a selection of the available literature, the p.Ala403Val variant has been identified in affected individuals of various ethnic origins including in six in a homozygous state, in 91 in a compound heterozygous state and in one in a heterozygous state (Spaapen et al. 2001; Desviat et al. 2001; Aulehla-Scholz et al. 2003; Zurflüh et al. 2008; Kasnauskiene et al. 2008; Sterl et al. 2013; Djordjevic et al. 2013; Couce et al. 2013; Trunzo et al. 2013; Bik-Multanowski et al. 2013). The p.Ala403Val was reported in two of 320 controls and is reported at a frequency of 0.005112 in the Ashkenazi Jewish population of the Genome Aggregation Database. The Ala403 residue is highly conserved. Functional studies demonstrated that the p.Ala403Val variant resulted in residual PAH enzyme activity between 12% and 32% of wild type (Zurflühet al. 2008; Danecka et al. 2015). Based on the collective evidence the p.Ala403Val variant is classified as pathogenic for phenylalanine hydroxylase deficiency. This variant was observed by ICSL as part of a predisposition screen in an ostensibly healthy population.

NM_000277.1(PAH):c.1208C>T(A403V) is classified as pathogenic in the context of phenylalanine hydroxylase deficiency and can be associated with variant or non-PKU hyperphenylalaninemia. Sources cited for classification include the following: PMID 23430547, 18299955, 17096675, 22513348, 23792259, 16198137, 17935162, 19062537, 24350308 and 22526846. Classification of NM_000277.1(PAH):c.1208C>T(A403V) 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.

A heterozygous missense variant was identified, NM_000277.2(PAH):c.1208C>T in exon 12 of 13 of the PAH gene. This substitution is predicted to create a minor amino acid change from alanine to valine at position 403 of the protein, NP_000268.1(PAH):p.(Ala403Val). The alanine at this position has high conservation (100 vertebrates, UCSC), and is located within the biopterin hydroxylase domain. In silico software predictions of the pathogenicity of this variant are conflicting (Polyphen, SIFT, CADD, Mutation Taster). The variant is present in the gnomAD population database at a frequency of 0.06%% (164 heterozygotes, 0 homozygotes). The variant has previously been reported as pathogenic in patients with mild hyperphenylalaninemia and mild phenylketonuria (ClinVar, Jeannesson-Thivol E. et al. 2015). In addition, functional studies show that this variant results in approximately 43% residual activity compared to wild-type (Cerreto M. et al. 2011). Based on information available at the time of curation, this variant has been classified as PATHOGENIC.

This sequence change replaces alanine, which is neutral and non-polar, with valine, which is neutral and non-polar, at codon 403 of the PAH protein (p.Ala403Val). This variant is present in population databases (rs5030857, gnomAD 0.5%), and has an allele count higher than expected for a pathogenic variant. This missense change has been observed in individual(s) with BH4-responsive mild hyperphenylalaninemia (HPA), mild form of phenylketonuria, and/or non-PKU HPA (PMID: 2575001, 8268925, 8739972, 8830172, 9429153, 17096675, 25596310). In at least one individual the data is consistent with being in trans (on the opposite chromosome) from a pathogenic variant. It is commonly reported in individuals of Spanish, Italian, or Israeli ancestry (PMID: 8268925, 8739972, 8830172, 9429153, 25596310). ClinVar contains an entry for this variant (Variation ID: 92731). 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: 21820508, 23500595, 23559577). For these reasons, this variant has been classified as Pathogenic.

The p.Ala403Val variant in PAH is a well-established pathogenic variant for phenylalanine hydroxylase deficiency/phenylketonuria (PKU) and is associated with a milder phenotype (Zekanowski 1997, Benit 1999, Bardelli 2002, Kasnauskiene 2002, Daniele 2007, Groselj 2012, Georgiou 2012, Bik-Multanowski 2013, Polak 2013). This variant was identified in 0.5% (51/10150) of Ashkenazi Jewish chromosomes by the Genome Aggregation Database (gnomAD, http://gnomad.broadinstitute.org). It has also been reported in ClinVar (Variation ID 92731) and is classified as pathogenic by the ClinGen PAH Expert Panel. In vitro functional studies refunctional studies support an impact on protein function (Cerreto 2011 PMID:21820508). In addition, computation prediction tools support an impact to the protein. In summary, the p.Ala403Val variant in PAH meets criteria to be classified as pathogenic for PKU in an autosomal recessive manner. ACMG/AMP criteria applied: PM3_VeryStrong, PS3, PP3.

The c.1208C>T (p.A403V) alteration is located in coding exon 12 of the PAH gene. This alteration results from a C to T substitution at nucleotide position 1208, causing the alanine (A) at amino acid position 403 to be replaced by a valine (V). Based on data from gnomAD, the T allele has an overall frequency of 0.06% (164/282804) total alleles studied. The highest observed frequency was 0.51% (53/10368) of Ashkenazi Jewish alleles. The c.1208C>T (p.A403V) alteration has been detected in the homozygous state, and in conjunction with another alteration in PAH , in multiple individuals with phenylalanine hydroxylase deficiency and is primarily associated with mild hyperphenylalaninemia phenotypes (Guldberg, 1993; Zekanowski, 1997; Benit, 1999; Aulehla-Scholz, 2003; Daniele, 2007; Berkovich, 2008; Couce, 2013; Aldámiz-Echevarría, 2016). In addition, clinical studies have indicated that this is a BH4-responsive allele (Zurfluh, 2008; Aldámiz-Echevarría, 2016). This amino acid position is highly conserved in available vertebrate species. Functional studies have demonstrated a significant reduction in enzymatic activity for the A403V variant (Benit, 1999; Blau, 2004; Cerreto, 2011; Himmelreich, 2018). This alteration is predicted to be deleterious by in silico analysis. Based on the available evidence, this alteration is classified as pathogenic.

Variant summary: The c.1208C>T variant affects a conserved nucleotide, resulting in amino acid change from Ala to Val. 4/5 in-silico tools predict this variant to be damaging. This variant is found in 65/121306 control chromosomes at a frequency of 0.0005358, which does not exceed maximal expected frequency of a pathogenic allele (0.0079057). This variant was one of the most common pathogenic variant found in European PKU or MHP patients. The phenotype of these patients is likely to be mild and they respond to BH4 treatment. Two independent groups showed PAH p.A403V activity is 30-40%, which is consistant with the mild phenotype seen in the patients carrying this variant. In addition, multiple clinical laboratories/reputable databases/literatures classified this variant as pathogenic. Taken together, this variant was classified as pathogenic.

Associated with mild hyperphenylalaninemia and 30-40% residual phenylalanine hydroxylase enzyme activity (Bnit et al., 1999); 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: 31980526, 30747360, 27121329, 25596310, 30487145, 30667134, 30037505, 29499199, 8268925, 28956315, 25750018, 17935162, 12409276, 27469133, 27830119, 27175728, 23942198, 12501224, 8889590, 15557004, 23764561, 24033266, 17096675, 12640344, 22330942, 22513348, 25087612, 23500595, 23430547, 23559577, 21953985, 21820508, 12644360, 11486900, 10479481, 8739972, 18346471, 22526846, 23792259, 16051511, 18482855, 9686365, 9298832, 18299955, 11096279, 9429153, 10598814)

In the published literature, it has been reported along with a second pathogenic variant in multiple individuals affected with BH4-responsive mild hyperphenylalaninemia (PMID: 8739972 (1996), 8889590 (1996), 21820508 (2011), 23430547 (2013), 25596310 (2015)). Functional studies have shown this variant has reduced enzyme activity (PMID: 10479481 (1999), 21820508 (2011), 30037505 (2018)). Furthermore, the variant is associated with BH4-responsiveness (PMID: 16290003 (2005), 21820508 (2011)). Therefore, the variant is classified as pathogenic.

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

The PAH c.1208C>T variant is predicted to result in the amino acid substitution p.Ala403Val. This variant has been commonly reported to be causative for hyperphenylalaninemia (e.g., Guldberg et al. 1993. PubMed ID: 8268925; Bénit et al. 1999. PubMed ID: 10479481; Spaapen et al. 2001. PubMed ID: 11486900; Table S3, Hillert et al. 2020. PubMed ID: 32668217) and is considered a mild hyperphenylalaninemia variant (Figure S4, Hillert et al. 2020. PubMed ID: 32668217). The p.Ala403Val substitution has been reported to reduce PAH enzyme activity to ~30% of wild-type and lead to a PAH protein that is responsive to tetrahydrobiopterin (BH4) (Zurflüh et al. 2008. PubMed ID: 17935162). In the ClinVar database, this variant is classified as pathogenic by the ClinGen PAH Variant Curation Expert Panel as well as multiple outside laboratories (https://www.ncbi.nlm.nih.gov/clinvar/variation/92731/). In summary, we interpret this variant as pathogenic.

The PAH p.Ala403Val variant has been reported in multiple individuals with Phenylketonuria (PKU) from Italian, Argentine, Slovak, French, Russian and Israeli backgrounds; this variant is generally associated with a mild phenotype, as well as non-PKU mild hyperphenylalaninemia in one homozygote (Gundorova_2019_PMID:30668579, Jeannesson-Thivisol_2015_PMID:26666653, Bercovich_2008_PMID:18299955, Bardelli_2002_PMID:12409276 , Polak_2013_PMID:23764561, Enacan_2019, Daniele_2009_PMID_19292873). The variant was identified in dbSNP (ID: rs5030857) and ClinVar (classified as pathogenic for Phenylketonuria by ClinGen PAH Variant Curation Expert Panel, Laboratory for Molecular Medicine, Invitae and 12 other laboratories). The variant was identified in control databases in 164 of 282804 chromosomes at a frequency of 0.0005799 (Genome Aggregation Database March 6, 2019, v2.1.1). The variant was observed in the following populations: Ashkenazi Jewish in 53 of 10368 chromosomes (freq: 0.005112), European (non-Finnish) in 96 of 129168 chromosomes (freq: 0.000743), Other in 3 of 7228 chromosomes (freq: 0.000415), Latino in 8 of 35394 chromosomes (freq: 0.000226), African in 3 of 24958 chromosomes (freq: 0.00012) and South Asian in 1 of 30614 chromosomes (freq: 0.000033), but was not observed in the East Asian or European (Finnish) populations. The p.Ala403 residue is conserved across mammals and other organisms, and computational analyses (PolyPhen-2, SIFT, AlignGVGD, BLOSUM, MutationTaster) suggest that the variant may impact the protein; however, this information is not predictive enough to assume pathogenicity. The variant occurs outside of the splicing consensus sequence and 2 of 4 in silico or computational prediction software programs (SpliceSiteFinder, MaxEntScan, NNSPLICE, GeneSplicer) predict a greater than 10% difference in splicing; this is not very predictive of pathogenicity. Functional studies of the p.A403V variant have demonstrated reduced enzyme activity (only 43±4% of wild-type enzyme activity), altered oligomerization and lower inferred melting temperatures compared to the wild type protein indicating a loss in stability (Cerreto_2011_PMID:21820508). There is also evidence that patients with this variant would likely respond to BH4, a compound that helps convert phenylalanine to other essential molecules in the body (Cerreto_2011_PMID:21820508; Daniele_2008_PMID:18346471; Gundorova_2019_PMID:30668579; Bardelli_2002_PMID:12409276; Polak_2013_PMID:23764561). In summary, based on the above information this variant meets our laboratory’s criteria to be classified as pathogenic.