rs28933973

Positions:

• chr10-70599048-G-A

Variant summary

Our verdict is Pathogenic. Variant got 16 ACMG points: 16P and 0B. PM1 PM2 PP3_Strong PP5_Very_Strong

The NM_001083116.3(PRF1):​c.673C>T​(p.Arg225Trp) variant causes a missense change involving the alteration of a non-conserved nucleotide. The variant allele was found at a frequency of 0.00000479 in 1,461,136 control chromosomes in the GnomAD database, with no homozygous occurrence. In-silico tool predicts a pathogenic outcome for this variant. Variant has been reported in ClinVar as Pathogenic (★★). Another variant affecting the same amino acid position, but resulting in a different missense (i.e. R225P) has been classified as Uncertain significance.

• Frequency:
  • Genomes: not found (cov: 33)
  • Exomes 𝑓: 0.0000048 (0 hom.)
• Consequence: PRF1 NM_001083116.3 missense
• Clinical Significance: Pathogenic criteria provided, multiple submitters, no conflicts P:10
• Conservation: PhyloP100: 0.273

Genes affected

PRF1 (HGNC:9360): (perforin 1) This gene encodes a protein with structural similarities to complement component C9 that is important in immunity. This protein forms membrane pores that allow the release of granzymes and subsequent cytolysis of target cells. Whether pore formation occurs in the plasma membrane of target cells or in an endosomal membrane inside target cells is subject to debate. Mutations in this gene are associated with a variety of human disease including diabetes, multiple sclerosis, lymphomas, autoimmune lymphoproliferative syndrome (ALPS), aplastic anemia, and familial hemophagocytic lymphohistiocytosis type 2 (FHL2), a rare and lethal autosomal recessive disorder of early childhood. [provided by RefSeq, Aug 2017]

PALD1 (HGNC:23530): (phosphatase domain containing paladin 1) Predicted to enable protein tyrosine phosphatase activity. Predicted to be involved in peptidyl-tyrosine dephosphorylation. Located in cytosol. [provided by Alliance of Genome Resources, Apr 2022]

ACMG classification

Verdict is Pathogenic. Variant got 16 ACMG points.

• PM1: In a hotspot region, there are 3 aminoacids with missense pathogenic changes in the window of +-8 aminoacids around while only 0 benign, 2 uncertain in NM_001083116.3
• PM2: Very rare variant in population databases, with high coverage
• PP3: MetaRNN computational evidence supports a deleterious effect, 0.994
• PP5: Variant 10-70599048-G-A is Pathogenic according to our data. Variant chr10-70599048-G-A is described in ClinVar as [Pathogenic]. Clinvar id is 13711.Status of the report is criteria_provided_multiple_submitters_no_conflicts, 2 stars. Variant chr10-70599048-G-A is described in Lovd as [Likely_pathogenic]. Variant chr10-70599048-G-A is described in Lovd as [Pathogenic].

Transcripts

RefSeq

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	MANE	UniProt
PRF1	NM_001083116.3	c.673C>T	p.Arg225Trp	missense_variant	3/3	ENST00000441259.2
PRF1	NM_005041.6	c.673C>T	p.Arg225Trp	missense_variant	3/3

Ensembl

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	TSL	MANE	Appris	UniProt
PRF1	ENST00000441259.2	c.673C>T	p.Arg225Trp	missense_variant	3/3	5	NM_001083116.3	P1

Frequencies

GnomAD3 genomes Cov.: 33

GnomAD3 exomes AF: 0.0000120 AC: 3 AN: 249622 Hom.: 0 AF XY: 0.0000222 AC XY: 3 AN XY: 134962

Gnomad AFR exome AF: 0.00

Gnomad AMR exome AF: 0.00

Gnomad ASJ exome AF: 0.00

Gnomad EAS exome AF: 0.0000544

Gnomad SAS exome AF: 0.00

Gnomad FIN exome AF: 0.00

Gnomad NFE exome AF: 0.0000178

Gnomad OTH exome AF: 0.00

GnomAD4 exome AF: 0.00000479 AC: 7 AN: 1461136 Hom.: 0 Cov.: 35 AF XY: 0.00000413 AC XY: 3 AN XY: 726754

Gnomad4 AFR exome AF: 0.00

Gnomad4 AMR exome AF: 0.00

Gnomad4 ASJ exome AF: 0.00

Gnomad4 EAS exome AF: 0.00

Gnomad4 SAS exome AF: 0.0000232

Gnomad4 FIN exome AF: 0.00

Gnomad4 NFE exome AF: 0.00000450

Gnomad4 OTH exome AF: 0.00

GnomAD4 genome Cov.: 33

Alfa AF: 0.0000459 Hom.: 0

ExAC AF: 0.0000247 AC: 3

EpiCase AF: 0.0000545

EpiControl AF: 0.0000593

ClinVar

Significance: Pathogenic

Submissions summary: Pathogenic:10

Revision: criteria provided, multiple submitters, no conflicts

Submissions by phenotype

Familial hemophagocytic lymphohistiocytosis 2 Pathogenic:6

Pathogenic, criteria provided, single submitter	clinical testing	Neuberg Centre For Genomic Medicine, NCGM	-	The missense variant p.R225W in PRF1 (NM_001083116.3) has been previously reported in affected patients (Madkaikar M et al). Functional studies reveal a damaging effect (Trizzino et al, 2008). It has been submitted to ClinVar as Pathogenic. The p.R225W variant is observed in 1/18,386 (0.0054%) alleles from individuals of East Asian background in gnomAD Exomes and is novel (not in any individuals) in 1000 Genomes. For these reasons, this variant has been classified as Pathogenic. -
Pathogenic, no assertion criteria provided	literature only	OMIM	Nov 01, 2013	- -
Pathogenic, criteria provided, single submitter	clinical testing	3billion	Sep 01, 2022	The variant is observed at an extremely low frequency in the gnomAD v2.1.1 dataset (total allele frequency: 0.001%). Missense changes are a common disease-causing mechanism. Same nucleotide change resulting in same amino acid change has been previously reported as pathogenic/likely pathogenic with strong evidence (ClinVar ID: VCV000013711). The variant has been observed in multiple (>3) similarly affected unrelated individuals (PMID: 23443029 , 26184781 , 26684649 , 27271812). Different missense changes at the same codon (p.Arg225Gln, p.Arg225Pro) have been reported to be associated with PRF1-related disorder (PMID: 17674359 , 29357941). Therefore, this variant is classified as Pathogenic according to the recommendation of ACMG/AMP guideline. -
Pathogenic, criteria provided, single submitter	clinical testing	Invitae	Nov 24, 2023	This sequence change replaces arginine, which is basic and polar, with tryptophan, which is neutral and slightly polar, at codon 225 of the PRF1 protein (p.Arg225Trp). This variant is present in population databases (rs28933973, gnomAD 0.006%). This missense change has been observed in individuals with familial hemophagocytic lymphohistiocytosis (PMID: 10583959, 11565555, 14757862, 27271812). ClinVar contains an entry for this variant (Variation ID: 13711). 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) has been performed at Invitae for this missense variant, however the output from this modeling did not meet the statistical confidence thresholds required to predict the impact of this variant on PRF1 protein function. Experimental studies have shown that this missense change affects PRF1 function (PMID: 15365097). For these reasons, this variant has been classified as Pathogenic. -
Pathogenic, criteria provided, single submitter	clinical testing	Illumina Laboratory Services, Illumina	Apr 27, 2017	The PRF1 c.673C>T (p.Arg225Trp) missense variant has been described in at least eight studies in which it is found in a total of 16 patients with familial hemophagocytic lymphohistiocytosis including in 11 in a homozygous state, in three in a compound heterozygous state, and in two of unknown zygosity (Stepp et al. 1999; Clementi et al. 2001; Molleran et al. 2004; Trizzino et al. 2008; Chiapparini et al. 2011; Dias et al. 2013; Tesi et al. 2015; Madkaikar et al. 2016). The variant was also found in a heterozygous state in four unaffected related individuals. The p.Arg225Trp was absent from at least 100 control chromosomes but is reported at a frequency of 0.00035 in the European American population of the Exome Sequencing Project. Functional studies demonstrated that the variant results in decreased or absent perforin expression and reduced NK cell cytolytic activity (Stepp et al. 1999; Risma et al. 2004; Voskoboinik et al. 2004; Trizzino et al. 2008). Based on the evidence, the p.Arg225Trp variant is classified as pathogenic for familial hemophagocytic lymphohistiocytosis. This variant was observed by ICSL as part of a predisposition screen in an ostensibly healthy population. -
Pathogenic, criteria provided, single submitter	clinical testing	KCCC/NGS Laboratory, Kuwait Cancer Control Center	Jul 07, 2023	a substitution of Tryptophan for Arginine in codon 225. This variant’s frequency is reported as A=0.00002 (3/120924, ExAC) and A=0.00001 (3/244444, GnomAD). It is reported in ClinVar as pathogenic and not reported in LOVD. Homozygous or compound heterozygous pathogenic mutations in the PRF1 gene are known to cause Hemophagocytic lymphohistiocytosis (HLH, OMIM 603553). Heterozygous germline pathogenic mutations in the PRF1 gene maybe associated with increased risk of non-Hodgkin Lymphoma (OMIM 605027), Wiernik et al. (2000) and Altieri et al. (2005). -

Aplastic anemia Pathogenic:1

Pathogenic, criteria provided, single submitter

clinical testing

Baylor Genetics

Mar 11, 2024

- -

Inborn genetic diseases Pathogenic:1

Pathogenic, criteria provided, single submitter

clinical testing

Ambry Genetics

Jun 18, 2021

The c.673C>T (p.R225W) alteration is located in exon 3 (coding exon 2) of the PRF1 gene. This alteration results from a C to T substitution at nucleotide position 673, causing the arginine (R) at amino acid position 225 to be replaced by a tryptophan (W). Based on data from the Genome Aggregation Database (gnomAD) database, the PRF1 c.673C>T alteration was observed in <0.01% (3/249622) of total alleles studied, with a frequency of 0.01% (1/18386) in the East Asian subpopulation. This alteration has been reported in the homozygous and compound heterozygous states in multiple unrelated patients with features of familial hemophagocytic lymphohistiocytosis (Stepp, 1999; Molleran Lee, 2004; Clementi, 2001; Trizzino, 2008; Dias, 2013; Tesi, 2015). This amino acid position is not well conserved in available vertebrate species. Cells from an affected patient showed nearly complete absence of perforin and greatly reduced cytolytic activity (Stepp, 1999; Feldmann, 2002). In addition, in vitro functional studies of the p.R225W alteration demonstrated abnormal protein function including loss of cytolytic activity, failure to traffic to rat basophil leukemia secretory granules, diminished perforin detection, and no apparent proteolytic maturation. In one study, immunohistochemistry analysis of transfected cells indicated mislocalization whereas another demonstrated punctate staining on immunohistochemistry similar to wildtype (Voskoboinik, 2004; Risma, 2006). The in silico prediction for the p.R225W alteration is inconclusive. Based on the available evidence, this alteration is classified as pathogenic. -

Familial hemophagocytic lymphohistiocytosis Pathogenic:1

Pathogenic, criteria provided, single submitter

clinical testing

Women's Health and Genetics/Laboratory Corporation of America, LabCorp

Jul 13, 2022

Variant summary: PRF1 c.673C>T (p.Arg225Trp) results in a non-conservative amino acid change located in the membrane attack complex component/perforin (MACPF) domain (IPR020864) of the encoded protein sequence. Five of five in-silico tools predict a damaging effect of the variant on protein function. The variant allele was found at a frequency of 1.2e-05 in 249622 control chromosomes (gnomAD). c.673C>T has been reported in the literature in multiple homozygous and compound heterozygous individuals affected with Familial Hemophagocytic Lymphohistiocytosis (e.g. Stepp_1999, Clementi_2001, Molleran Lee_2004, Steinburg_2006, Trizzino_2008, Blincoe_2020). These data indicate that the variant is very likely to be associated with disease. Experimental studies have shown that the variant is associated with reduced or absent expression of the mature protein and severely impaired NK function (e.g. Stepp_1999, Voskoboinik_2004, Risma_2006). Three clinical diagnostic laboratories have submitted clinical-significance assessments for this variant to ClinVar after 2014 and all classified the variant as pathogenic. Based on the evidence outlined above, the variant was classified as pathogenic. -

Autoinflammatory syndrome Pathogenic:1

Pathogenic, criteria provided, single submitter

clinical testing

Genome Diagnostics Laboratory, The Hospital for Sick Children

Feb 01, 2018

- -

Computational scores

Source: dbNSFP v4.3

Name	Calibrated prediction	Score	Prediction
AlphaMissense	Benign	0.18
BayesDel_addAF	Uncertain	0.045	T
BayesDel_noAF	Uncertain	0.0
CADD	Uncertain	24
DANN	Uncertain	1.0
DEOGEN2	Uncertain	0.74	D;D
Eigen	Benign	0.093
Eigen_PC	Benign	-0.052
FATHMM_MKL	Benign	0.14	N
LIST_S2	Uncertain	0.87	.;D
M_CAP	Uncertain	0.094	D
MetaRNN	Pathogenic	0.99	D;D
MetaSVM	Uncertain	0.52	D
MutationAssessor	Uncertain	2.7	M;M
MutationTaster	Benign	0.00039	A;A;A
PrimateAI	Benign	0.33	T
PROVEAN	Uncertain	-3.2	D;D
REVEL	Uncertain	0.58
Sift	Uncertain	0.025	D;D
Sift4G	Uncertain	0.0070	D;D
Polyphen		1.0	D;D
Vest4		0.79
MutPred		0.91		Loss of methylation at R225 (P = 0.0393);Loss of methylation at R225 (P = 0.0393);
MVP		0.88
MPC		0.14
ClinPred		0.90	D
GERP RS		2.3
Varity_R		0.30
gMVP		0.83

Splicing

Name	Calibrated prediction	Score	Prediction
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: rs28933973; hg19: chr10-72358804; COSMIC: COSV64615781;