ClinVar Genomic variation as it relates to human health

The c.607C>T (p.R203W) alteration is located in coding exon 4 of the PACS1 gene. This alteration results from a C to T substitution at nucleotide position 607, causing the arginine (R) at amino acid position 203 to be replaced by a tryptophan (W). Based on data from gnomAD, the T allele has an overall frequency of 0.001% (1/152152) total alleles studied. The highest observed frequency was 0.002% (1/41434) of African/African American alleles. This one allele was reported with low allele balance (0.2-0.25). This recurrent de novo alteration has been reported in multiple individuals with Schuurs-Hoeijmakers syndrome, with commonly reported features including developmental delay/intellectual disability, dysmorphic facial features, seizures, and other congenital malformations (Schurrs-Hoeijmakers, 2016; Deciphering Developmental Disorders Study, 2017; Stern, 2017; Gadzicki, 2015; Tenorio-Castaño, 2021). This amino acid position is highly conserved in available vertebrate species. The p.R203W substitution is positioned in the furin (cargo)-binding region of PACS1, which lies directly adjacent to the R196RKRY CK2-binding motif. This motif regulates phosphorylation status of the autoregulatory domain and PACS1 gene activation (Shuurs-Hoeijmakers, 2012). Functional analysis in zebrafish embryos with overexpression of mutant mRNA with this alteration demonstrated a significant reduction in cranial cartilaginous structures at the ventral aspect. In addition, overexpression of this altered protein resulted in defective migration of cranial-neural-crest cells in the head. Studies in human embryonic kidney cells demonstrated that expression of mutant PACS1 remains more stable than the wild-type protein leading to observed cellular aggregates (Shuurs-Hoeijmakers, 2012). The in silico prediction for this alteration is inconclusive. Based on the available evidence, this alteration is classified as pathogenic.

Recurrent pathogenics PACS1 variant.

This variant has been previously reported as a recurrent de novo change in patients with seizures, dysmorphic features, gastroesophageal reflux, cardiac abnormalities, intellectual disability, speech delay, (PMID: 28111752, 26842493). In vivo studies of the p.Arg203Trp variant in zebrafish embryos observed that it induces craniofacial defects, most likely in a dominant-negative fashion. This phenotype is driven by aberrant specification and migration of SOX10-positive cranial, but not enteric, neural-crest cells (PMID: 23159249). It is absent from the gnomAD population database and thus is presumed to be rare. The c.607C>T (p.Arg203Trp) variant affects a highly conserved amino acid and is predicted by multiple in silico tools to have a deleterious effect on protein function. Analysis of the parental samples was negative for the variant, indicating this variant likely occurred as a de novo event. Based on the available evidence, the c.607C>T (p.Arg203Trp) variant is classified as Pathogenic.

ACMG classification criteria: PS3, PS4, PM2, PM6, PP2

Based on the classification scheme VCGS_Germline_v1.1.1, this variant is classified as Pathogenic. Following criteria are met: 0104 - Dominant Negative is a mechanism of disease for this gene. (N) 0107 - This gene is known to be associated with autosomal dominant disease. (N) 0200 - Variant is predicted to result in a missense amino acid change from an arginine to a tryptophan (exon 4). (N) 0251 - Variant is heterozygous. (N) 0302 - Variant is present in gnomAD <0.001 for a dominant condition (1 heterozygote, 0 homozygotes). (P) 0501 - Missense variant consistently predicted to be damaging by multiple in silico tools or highly conserved with a major amino acid change. (P) 0603 - Missense variant in a region that is highly intolerant to missense variation (high constraint region). (P) 0704 - Comparable variant has low previous evidence for pathogenicity. Another variant in the same location p.(Arg203Gln), was found to be de novo in a patient with Schuurs-Hoeijmakers syndrome (PMID: 28975623). (P) 0801 - Strong previous evidence of pathogenicity in unrelated individuals. This variant has been reported to be de novo in more than 20 patients with Schuurs-Hoeijmakers syndrome (PMID: 23159249, 25522177, 26842493; ClinVar). (P) 0905 - No segregation evidence has been identified for this variant. (N) 1002 - Moderate functional evidence supporting abnormal protein function. In vitro assays demonstrated formation of cytoplasmic aggregates, leading to protein-trafficking defects (PMID:23159249). (P) 1203 - Variant shown to be de novo in proband (parental status confirmed). (P) Legend: (P) - Pathogenic, (N) - Neutral, (B) - Benign

PS4, PM2_SUP, PP2

Criteria applied: PS2_VSTR,PS4,PM2_SUP,PP2

The variant c.607C>T (p.Arg203Trp) in PACS1 is reported as pathogenic for Schuurs-Hoeijmakers syndrome in ClinVar (Variation ID: 39581) and as affects function in the Global Variome shared LOVD database v.3.0. There is no information on frequency in gnomAD, 1000 Genomes or NHLI Exome Sequencing Project (ESP). The nucleotide position is conserved across 35 mammalian species (GERP RS: 4.79). In silico analysis indicates that the variant might be damaging. This mutation has been firstly reported by Schuurs-Hoeijmakers et al. (2012) in 2 unrelated boys with mental retardation and a strikingly similar facial appearance. Later, different other groups reported the same de novo recurrent pathogenic variant in patients with a similar phenotype (Gadzicki et al., 2015; Martinez-Monseny et al.,2018; Dutta et al., 2019 and many others).

This variant was classified as: Pathogenic. The following ACMG criteria were applied in classifying this variant: PS1,PS3,PM1,PM2,PP2,PP3.

Same nucleotide change resulting in same amino acid change has been previously reported at least twice as de novoo in similarly affected indivisual (PMID: 25533962, 23159249, PS2, PS4_M). The variant was observed as assumed (i.e. paternity and maternity not confirmed) de novoo (3billion dataset, PM6). It is not observed in the gnomAD v2.1.1 dataset (PM2). Therefore, this variant is classified as pathogenic according to the recommendation of ACMG/AMP guideline.

The de novo p.Arg203Trp variant identified in PACS1 is a known pathogenic recurrent de novo variant that has been reported in multiple affected individuals in the literature [PMID: 26842493; PMID: 23159249; PMID: 25522177]. The variant has been reported in the ClinVar database by multiple laboratories and is classified as pathogenic (ClinVar Variation ID: 39581). The p.Arg203Trp variant has 0.000007 allele frequency in the gnomAD(V3) database (1 out of 143,260 heterozygous alleles) indicating it is not a common benign variant in the populations represented in that database. The variant affects a highly conserved residue and is predicted deleterious by multiple in silico prediction tools. In vitro functional studies have shown that the p.Arg203Trp variant forms cytoplasmic aggregates resulting in defective protein trafficking, suggestive of a dominant-negative mechanism of disease [PMID: 23159249]. Based on the available evidence, the p.Arg203Trp variant in the PACS1 gene is assessed as Pathogenic.

The c.607C>T;p.(Arg203Trp) missense change has been observed in affected individual(s) and ClinVar contains an entry for this variant (Clinvar ID: 39581; PMID: 23159249; 25522177; 26795593; 26842493; 28111752; 30588754) - PS4.Well-established in vitro or in vivo functional studies supportive of a damaging effect on the gene or gene product (PMID: 23159249) - PS3_supporting. The variant is present at low allele frequencies population databases (rs398123009– gnomAD 0.00006572%; ABraOM no frequency - http://abraom.ib.usp.br/) -PM2_supporting. The variant was assumed de novo, but without confirmation of paternity and maternity (PMID: 23159249; 25522177; 33166031) PM6_strong. In summary, the currently available evidence indicates that the variant is Pathogenic

This missense PACS1 variant at c.607C>T (p.R203W) variant in the PACS1 was seen on exome through the Texome project (R01HG011795) as a de novo variant in the patient (PS2). This recurrent de novo variant has been previously reported in more than 20 individuals with Schuurs-Hoeijmakers syndrome (PMID: 26842493, 30588754). This variant has not been observed in gnomAD (PM2). Functional studies demonstrated that this variant forms cytoplasmic aggregates with increased protein stability, consistent with a dominant-negative mechanism (PMID: 23159249) (PS3). This variant has a deleterious prediction score (CADD: 29.4) (PP3), and the evolutionary conservation of this residue is high. We classify this variant as pathogenic.

Across a selection of available literature, the PACS1 c.607C>T (p.Arg203Trp) missense variant has been found in a heterozygous state in at least 35 individuals with PACS1-related Syndrome (Schuurs-Hoeijmakers et al. 2012; Farwell et al. 2015; Schuurs-Hoeijmakers et al. 2016, Lazaridis et al. 2016; Stern et al. 2017; Tarailo-Graovac et al. 2017; Bowling et al. 2017; Geisheker et al. 2017; Pefkianaki et al. 2018; Dutta et al. 2019). This variant was identified as de novo in the affected individuals in all instances where parental samples were available. Control data are unavailable for this variant, which is absent from the Genome Aggregation Database in an area of good sequencing coverage, so the variant is presumed to be rare. Expression of the variant PACS1 mRNA in zebrafish embryos induced craniofacial defects (Schuurs-Hoeijmakers et al. 2012). The authors also showed that the p.Arg203Trp variant results in the formation of cytoplasmic aggregates and altered protein trafficking, and suggested a dominant-negative affect on the protein. Based on the collective evidence, the p.Arg203Trp variant is classified as pathogenic for PACS1-related syndrome.

Published functional studies indicate that the R203W variant perturbs normal PACS1 function by forming cytoplasmic aggregates, resulting in altered protein-trafficking, possibly suggesting a dominant-negative effect on the protein (Schuurs-Hoeijmakers et al., 2012); In silico analysis, which includes protein predictors and evolutionary conservation, supports a deleterious effect; Not observed at significant frequency in large population cohorts (gnomAD); This variant is associated with the following publications: (PMID: 25533962, 28135719, 25356970, 27959697, 26842493, 29656858, 23159249, 26795593, 26944241, 28111752, 28554332, 28628100, 28867141, 27875746, 30690871, 30588754, 30113927, 31330568, 25522177, 28975623, 28471432, 31988453, 30577886, 32963807, 33166031, 33333793, 30755392, 33144682, 32903913, 33726816, 34068396, 31785789)

This sequence change replaces arginine, which is basic and polar, with tryptophan, which is neutral and slightly polar, at codon 203 of the PACS1 protein (p.Arg203Trp). This variant is not present in population databases (gnomAD no frequency). This missense change has been observed in individual(s) with intellectual disability and/or epilepsy (PMID: 26795593, 26842493). In at least one individual the variant was observed to be de novo. ClinVar contains an entry for this variant (Variation ID: 39581). 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 PACS1 protein function. Experimental studies have shown that this missense change affects PACS1 function (PMID: 23159249). For these reasons, this variant has been classified as Pathogenic.

The PACS1 c.607C>T variant is predicted to result in the amino acid substitution p.Arg203Trp. This variant has been reported as a recurrent de novo alteration in several individuals with Schuurs-Hoeijmakers syndrome and is considered one of the defining variants of this disorder (Schuurs-Hoeijmakers et al. 2012. PubMed ID: 23159249; Seto et al. 2020. PubMed ID: 33166031). This variant has not been reported in a large population database, indicating it is rare. This variant is interpreted as pathogenic.

Submission from Simons Searchlight facilitated by GenomeConnect. Variant interpreted by the Simons Searchlight team most recently on 2019-01-14 and interpreted as Pathogenic. The reporting laboratory might also submit to ClinVar. This variant was identified in multiple probands enrolled in Simons Searchlight.