rs5030851

chr12-102852815-G-Achr12-102852815-G-Cchr12-102852815-G-T

Variant summary

Our verdict is Pathogenic. Variant got 15 ACMG points: 15P and 0B. PM1 PM2 PM5 PP3 PP5_Very_Strong

The NM_000277.3(PAH):c.842C>T(p.Pro281Leu) variant causes a missense, splice region change involving the alteration of a conserved nucleotide. The variant allele was found at a frequency of 0.000125 in 1,613,880 control chromosomes in the GnomAD database, with no homozygous occurrence. In-silico tool predicts a pathogenic outcome for this variant. 14/23 in silico tools predict a damaging outcome for this variant. 2/3 splice prediction tools predicting alterations to normal splicing. Variant has been reported in ClinVar as Likely pathogenic (★★★). Another variant affecting the same amino acid position, but resulting in a different missense (i.e. P281S) has been classified as Likely pathogenic.

• Frequency:
  • Genomes: 𝑓 0.00012 (0 hom., cov: 32)
  • Exomes 𝑓: 0.00013 (0 hom.)
• Consequence: PAH NM_000277.3 missense, splice_region
• Scores: Splicing: ADA: 0.9929
• Clinical Significance: Likely pathogenic reviewed by expert panel P:23 O:2
• Conservation: PhyloP100: 10.0

Genes affected

PAH (HGNC:8582): (phenylalanine hydroxylase) This gene encodes a member of the biopterin-dependent aromatic amino acid hydroxylase protein family. The encoded phenylalanine hydroxylase enzyme hydroxylates phenylalanine to tyrosine and is the rate-limiting step in phenylalanine catabolism. Deficiency of this enzyme activity results in the autosomal recessive disorder phenylketonuria. [provided by RefSeq, Aug 2017]

ACMG classification

Verdict is Pathogenic. Variant got 15 ACMG points.

• PM1: In a hotspot region, there are 14 aminoacids with missense pathogenic changes in the window of +-8 aminoacids around while only 1 benign, 6 uncertain in NM_000277.3
• PM2: Very rare variant in population databases, with high coverage
• PM5: Other missense variant is known to change same aminoacid residue: Variant chr12-102852816-G-A is described in ClinVar as [Likely_pathogenic]. Clinvar id is 92749.Status of the report is reviewed_by_expert_panel, 3 stars.
• PP3: Splicing scoreres supports a deletorius effect: Scorers claiming Pathogenic: dbscSNV1_ADA, dbscSNV1_RF. No scorers claiming Uncertain. Scorers claiming Benign: max_spliceai.
• PP5: Variant 12-102852815-G-A is Pathogenic according to our data. Variant chr12-102852815-G-A is described in ClinVar as [Likely_pathogenic]. Clinvar id is 589.Status of the report is reviewed_by_expert_panel, 3 stars. Variant chr12-102852815-G-A is described in Lovd as [Pathogenic]. Variant chr12-102852815-G-A is described in Lovd as [Pathogenic].

Transcripts

RefSeq

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	MANE	UniProt
PAH	NM_000277.3	c.842C>T	p.Pro281Leu	missense_variant, splice_region_variant	7/13	ENST00000553106.6
PAH	NM_001354304.2	c.842C>T	p.Pro281Leu	missense_variant, splice_region_variant	8/14

Ensembl

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	TSL	MANE	Appris	UniProt
PAH	ENST00000553106.6	c.842C>T	p.Pro281Leu	missense_variant, splice_region_variant	7/13	1	NM_000277.3	P1
PAH	ENST00000307000.7	c.827C>T	p.Pro276Leu	missense_variant, splice_region_variant	8/14	5
PAH	ENST00000635477.1	c.5C>T	p.Pro2Leu	missense_variant, splice_region_variant	1/6	5
PAH	ENST00000549247.6	n.601C>T		splice_region_variant, non_coding_transcript_exon_variant	1/6	2

Frequencies

GnomAD3 genomes AF: 0.000118 AC: 18 AN: 152154 Hom.: 0 Cov.: 32

Gnomad AFR AF: 0.0000483

Gnomad AMI AF: 0.00

Gnomad AMR AF: 0.0000655

Gnomad ASJ AF: 0.00

Gnomad EAS AF: 0.00

Gnomad SAS AF: 0.00

Gnomad FIN AF: 0.00

Gnomad MID AF: 0.00

Gnomad NFE AF: 0.000221

Gnomad OTH AF: 0.00

GnomAD3 exomes AF: 0.000103 AC: 26 AN: 251254 Hom.: 0 AF XY: 0.000103 AC XY: 14 AN XY: 135784

Gnomad AFR exome AF: 0.00

Gnomad AMR exome AF: 0.00

Gnomad ASJ exome AF: 0.00

Gnomad EAS exome AF: 0.00

Gnomad SAS exome AF: 0.00

Gnomad FIN exome AF: 0.0000924

Gnomad NFE exome AF: 0.000211

Gnomad OTH exome AF: 0.00

GnomAD4 exome AF: 0.000125 AC: 183 AN: 1461726 Hom.: 0 Cov.: 31 AF XY: 0.000129 AC XY: 94 AN XY: 727166

Gnomad4 AFR exome AF: 0.0000299

Gnomad4 AMR exome AF: 0.0000224

Gnomad4 ASJ exome AF: 0.00

Gnomad4 EAS exome AF: 0.00

Gnomad4 SAS exome AF: 0.00

Gnomad4 FIN exome AF: 0.0000374

Gnomad4 NFE exome AF: 0.000155

Gnomad4 OTH exome AF: 0.0000497

GnomAD4 genome AF: 0.000118 AC: 18 AN: 152154 Hom.: 0 Cov.: 32 AF XY: 0.000121 AC XY: 9 AN XY: 74328

Gnomad4 AFR AF: 0.0000483

Gnomad4 AMR AF: 0.0000655

Gnomad4 ASJ AF: 0.00

Gnomad4 EAS AF: 0.00

Gnomad4 SAS AF: 0.00

Gnomad4 FIN AF: 0.00

Gnomad4 NFE AF: 0.000221

Gnomad4 OTH AF: 0.00

Alfa AF: 0.000166 Hom.: 0

Bravo AF: 0.000166

ESP6500AA AF: 0.00 AC: 0

ESP6500EA AF: 0.000116 AC: 1

ExAC AF: 0.0000988 AC: 12

EpiCase AF: 0.000382

EpiControl AF: 0.000178

ClinVar

Significance: Likely pathogenic

Submissions summary: Pathogenic:23 Other:2

Revision: reviewed by expert panel

Submissions by phenotype

Phenylketonuria Pathogenic:17 Other:1

Likely pathogenic, criteria provided, single submitter	clinical testing	3billion	Jan 03, 2022	Same nucleotide change resulting in same amino acid change has been previously reported as pathogenic/likely pathogenic with strong evidence (ClinVar ID: VCV000000589, PMID:1672294, PS1_S). A different missense change at the same codon has been reported to be associated with PAH related disorder (ClinVar ID: VCV000092749, PMID:12409276,10598814,15171997, PM5_P). In silico tool predictions suggest damaging effect of the variant on gene or gene product (REVEL: 0.983, 3CNET: 0.998, PP3_P). A missense variant is a common mechanism associated with Classic phenylketonuria (PP2_P). It is observed at an extremely low frequency in the gnomAD v2.1.1 dataset (total allele frequency: 0.000103, PM2_M). Therefore, this variant is classified as likely pathogenic according to the recommendation of ACMG/AMP guideline. -
Pathogenic, criteria provided, single submitter	clinical testing	Women's Health and Genetics/Laboratory Corporation of America, LabCorp	Jan 19, 2017	Variant summary: The PAH c.842C>T (p.Pro281Leu) variant involves the alteration of a conserved nucleotide. 5/5 in silico tools predict a damaging outcome for this variant, which was confirmed by at least one in vitro study showing the activity of PAH p.P281L was <1% of wild-type PAH activity (Kayaalp_1997). This variant was found in 12/121404 control chromosomes at a frequency of 0.0000988, which does not exceed the estimated maximal expected allele frequency of a pathogenic PAH variant (0.0079057). This variant has been reported in numerous patients with classic PKU. In addition, multiple clinical diagnostic laboratories/reputable databases classified this variant as pathogenic. Taken together, this variant is classified as pathogenic. -
Pathogenic, criteria provided, single submitter	clinical testing	Myriad Genetics, Inc.	Dec 04, 2019	NM_000277.1(PAH):c.842C>T(P281L) is classified as pathogenic in the context of phenylalanine hydroxylase deficiency. Sources cited for classification include the following: PMID 22513348, 22526846, 19394257, 15503242, 20920871, 20187763, 22763404, 18299955, 17935162 and 10471838. Classification of NM_000277.1(PAH):c.842C>T(P281L) 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. -
Pathogenic, criteria provided, single submitter	clinical testing	Laboratory for Molecular Medicine, Mass General Brigham Personalized Medicine	Dec 29, 2015	The p.Pro281Leu variant in PAH was first reported in patients with classic PKU by Okano et al., (Okano 1991, Okano 1991) and has been reported in numerous patients with PKU since then (Zurfluh 2008, Danecka 2015). In vitro functional studies provide evidence that the p.Pro281Leu variant impacts protein function (Okano 1991, Zurfluh 2008, Danecka 2015, Ellingsen 1999, Reblova 2015, Shi 2012). This variant has been identified in 0.018% (12/66740) of Euroepan chromosomes by the Exome Aggregation Consortium (ExAC, http://exac.broadinstitute.org; rs5030851). Although this variant has been seen in the general population, its frequency is low enough to be consistent with a recessive carrier frequency. In summary, this variant meets our criteria to be classified as pathogenic for phenylketonuria in an autosomal recessive manner. -
Pathogenic, criteria provided, single submitter	clinical testing	Baylor Genetics	-	- -
Pathogenic, criteria provided, single submitter	clinical testing	Invitae	Jan 25, 2024	This sequence change replaces proline, which is neutral and non-polar, with leucine, which is neutral and non-polar, at codon 281 of the PAH protein (p.Pro281Leu). This variant is present in population databases (rs5030851, gnomAD 0.02%). This missense change has been observed in individual(s) with PAH-related conditions (PMID: 1672294, 21871829, 25596310; Invitae). In at least one individual the data is consistent with being in trans (on the opposite chromosome) from a pathogenic variant. ClinVar contains an entry for this variant (Variation ID: 589). 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. Experimental studies have shown that this missense change affects PAH function (PMID: 1672294, 17935162, 21953985). For these reasons, this variant has been classified as Pathogenic. -
Pathogenic, criteria provided, single submitter	clinical testing	Revvity Omics, Revvity	Nov 15, 2021	- -
Pathogenic, criteria provided, single submitter	clinical testing	Institute of Human Genetics, University of Leipzig Medical Center	Jan 01, 2019	This variant was identified as compound heterozygous. -
Pathogenic, no assertion criteria provided	literature only	OMIM	Jan 01, 1994	- -
Pathogenic, criteria provided, single submitter	clinical testing	Centogene AG - the Rare Disease Company	Nov 03, 2017	- -
Pathogenic, no assertion criteria provided	clinical testing	Payam Genetics Center, General Welfare Department of North Khorasan Province	Mar 01, 2023	- -
Pathogenic, criteria provided, single submitter	clinical testing	Fulgent Genetics, Fulgent Genetics	Oct 31, 2018	- -
Pathogenic, no assertion criteria provided	clinical testing	Natera, Inc.	Sep 16, 2020	- -
Pathogenic, criteria provided, single submitter	research	UNC Molecular Genetics Laboratory, University of North Carolina at Chapel Hill	-	The PAH c.842C>T (p.P281L) variant is located at an intron/exon boundary, and this variant has been reported as pathogenic in individuals with phenylketonuria. This variant was also shown to abolish the function of the phenylalanine hydroxylase enzyme in vitro (PMID: 1672290; 9634518; 10234516; 17935162). -
not provided, no classification provided	literature only	GeneReviews	-	- -
Pathogenic, criteria provided, single submitter	clinical testing	Greenwood Genetic Center Diagnostic Laboratories, Greenwood Genetic Center	Nov 10, 2023	PS3, PM3_Strong, PP3 -
Pathogenic, criteria provided, single submitter	clinical testing	Genome-Nilou Lab	Jul 22, 2021	- -
Likely pathogenic, reviewed by expert panel	curation	ClinGen PAH Variant Curation Expert Panel	Aug 27, 2018	PAH-specific ACMG/AMP criteria applied: PP3: ; PS3: 2% activity in bioPKU (PAH0309) (PMID:25596310; PMID:17935162); PP4_Moderate: 2 patients with moderate or classical PKU; patients with severe PKU. BH4 deficiency ruled out. (PMID:15503242; PMID:12655553); PM3: IVS4-1G>A (P/LP) (PMID:15503242). 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, PS3, PP4_Moderate, PM3). -

not provided Pathogenic:4 Other:1

Pathogenic, criteria provided, single submitter	clinical testing	Eurofins Ntd Llc (ga)	Jan 26, 2017	- -
not provided, no classification provided	literature only	DeBelle Laboratory for Biochemical Genetics, MUHC/MCH RESEARCH INSTITUTE	-	- -
Pathogenic, criteria provided, single submitter	clinical testing	GeneDx	May 18, 2020	Associated with very low (0-1%) residual phenylalanine hydroxylase enzyme activity and is considered a severe PAH variant as individuals homozygous for this variant have been reported with classic phenylketonuria (Okano et al., 1991; Rivera et al., 2011; Shi et al., 2012; Danecka et al., 2015); In silico analysis, which includes protein predictors and evolutionary conservation, supports a deleterious effect; Reported to not be responsive to tetrahydrobiopterin (BH4) therapy (Zurflh et al., 2008); This variant is associated with the following publications: (PMID: 33101986, 31589614, 32853555, 30275481, 30747360, 31355225, 30648773, 30963030, 30159272, 12501224, 24296287, 21871829, 29288420, 1672294, 29499199, 2014036, 28676969, 19194782, 22513348, 8889590, 1672290, 25750018, 17935162, 1481864, 21953985, 25596310, 25087612, 22975760, 21228398, 10471838) -
Pathogenic, criteria provided, single submitter	clinical testing	CeGaT Center for Human Genetics Tuebingen	Jun 01, 2022	PAH: PM3:Very Strong, PM1, PM2, PM5, PP3, PS3:Supporting -
Pathogenic, criteria provided, single submitter	clinical testing	Quest Diagnostics Nichols Institute San Juan Capistrano	Nov 04, 2019	In the literature, this variant has been reported as homozygous or compound heterozygous with other pathogenic PAH variants in many individuals with PAH-related diseases (PMIDs: 30747360 (2019), 29749107 (2018), 25596310 (2015), 21871829 (2011), 20082265 (2010), 18294361 (2008), and 1672294 (1991)). An in vitro study indicated that the homozygous c.842C>T (p.Pro281Leu) variant could generate a full length splicing product and a shorter splicing product without exon 8. The full length product has minimal enzyme activity and the shorter product causes the premature termination of the PAH protein (PMID 10471838 (1999)). Multiple other experimental studies have also confirmed that this variant results in diminished enzyme activity and protein expression (PMID: 25596310 (2015), 21953985 (2012), 17935162 (2008), 11161839 (2001), and 1672294 (1991)). This pathogenic variant is located in the catalytic domain of the PAH protein and is associated with classic PKU (PMID 20082265 (2010)). -

PAH-related disorder Pathogenic:1

Pathogenic, criteria provided, single submitter

clinical testing

PreventionGenetics, part of Exact Sciences

Jan 02, 2024

The PAH c.842C>T variant is predicted to result in the amino acid substitution p.Pro281Leu. This variant is a recurrent variant that has been documented as causative for phenylalanine hydroxylase deficiency and has been associated with classical phenylketonuria (PKU) (e.g., Okano et al. 1991. PubMed ID: 2014036; Ellingsen et al. 1999, PubMed ID: 10471838; Trunzo et al. 2014, PubMed ID: 24296287; Hillert et al. 2020. PubMed ID: 32668217). The p.Pro281 amino acid resides in the cofactor binding site (CBR) region of the PAH enzyme, and the p.Pro281Leu substitution has been reported to reduce enzyme activity to ~1% of control and result in a mutant PAH protein that is non-responsive to BH4 (Zurflüh et al. 2008. PubMed ID: 17935162). This variant is classified as pathogenic or likely pathogenic by multiple outside laboratories, as well as the ClinGen PAH Variant Curation Expert Panel (https://www.ncbi.nlm.nih.gov/clinvar/variation/589/). This variant is interpreted as pathogenic. -

Inborn genetic diseases Pathogenic:1

Pathogenic, criteria provided, single submitter

clinical testing

Ambry Genetics

May 21, 2021

The c.842C>T (p.P281L) alteration is located in exon 7 (coding exon 7) of the PAH gene. This alteration results from a C to T substitution at nucleotide position 842, causing the proline (P) at amino acid position 281 to be replaced by a leucine (L). Based on data from the Genome Aggregation Database (gnomAD) database, the PAH c.842C>T alteration was observed in 0.01% (29/282652) of total alleles studied, with a frequency of 0.02% (26/128978) in the European (non-Finnish) subpopulation. This mutation has been identified in numerous individuals with phenylalanine hydroxylase (PAH) deficiency (Okano, 1991; Shi, 2012; Gundorova, 2019) Activity and protein levels were approximately 1% when this variant was expressed n COS cells (Shi, 2012) The p.P281L alteration is predicted to be deleterious by in silico analysis. Based on the available evidence, this alteration is classified as pathogenic. -

Computational scores

Source: dbNSFP v4.3

Name	Calibrated prediction	Score	Prediction
AlphaMissense	Pathogenic	0.94
BayesDel_addAF	Pathogenic	0.38	D
BayesDel_noAF	Pathogenic	0.60
Cadd	Pathogenic	35
Dann	Uncertain	1.0
DEOGEN2	Pathogenic	0.99	D;D
Eigen	Pathogenic	1.1
Eigen_PC	Pathogenic	0.98
FATHMM_MKL	Pathogenic	0.99	D
LIST_S2	Pathogenic	0.99	D;D
M_CAP	Pathogenic	0.77	D
MetaRNN	Pathogenic	0.98	D;D
MetaSVM	Pathogenic	0.91	D
MutationAssessor	Pathogenic	4.9	H;.
MutationTaster	Benign	1.0	A;A
PrimateAI	Pathogenic	0.85	D
PROVEAN	Pathogenic	-9.6	D;D
REVEL	Pathogenic	0.98
Sift	Pathogenic	0.0	D;D
Sift4G	Pathogenic	0.0010	D;D
Polyphen		1.0	D;.
Vest4		0.97
MVP		1.0
MPC		0.24
ClinPred		1.0	D
GERP RS		5.7
RBP_binding_hub_radar		0.0
RBP_regulation_power_radar		1.7
Varity_R		0.99
gMVP		0.99

Splicing

Name	Calibrated prediction	Score	Prediction
dbscSNV1_ADA	Pathogenic	0.99
dbscSNV1_RF	Pathogenic	0.86
SpliceAI score (max)		0.0		Details are displayed if max score is > 0.2

Find out detailed SpliceAI scores and Pangolin per-transcript scores at spliceailookup.broadinstitute.org

Publications

LitVar

Other links and lift over

dbSNP: rs5030851; hg19: chr12-103246593;