ClinVar Genomic variation as it relates to human health

The p.Pro392Leu variant in SQSTM1 has been reported in at least 100 individuals with Paget disease of the bone (PDB) and was found to segregate with PDB in >40 individuals across 11 families (Laurin 2002, Morissette 2006, Seton 2016). In vi tro and in vivo functional studies support an impact on protein function (Layfie ld 2004, Hiruma 2008, Chamoux 2009, Kurihara 2011; Daroszewska 2018); however, s ome of these studies suggest that the variant is not sufficient to cause disease on its own, and that environmental stimuli may play a role in its phenotypic ex pression (Hiruma 2008, Kurihara 2011). This variant has also been identified in 0.14% (179/126230) of European chromosomes by gnomAD, including 2 homozygous ind ividuals (http://gnomad.broadinstitute.org). This appreciable frequency is consi stent with the reduced penetrance of PDB as well as the high prevalence of PDB i n European populations (estimates range from 0.2-10% across different patient po pulations; Morrisette 2006, Valenzuela 2017). It should be noted that this varia nt has also been reported in patients with frontotemporal dementia (FTD; van der Zee 2014, Le Ber 2013), amyotrophic lateral sclerosis (ALS; Teyssou 2013, Kwok 2014, van der Zee 2014, Morgan 2017), and myopathy (Lee 2018, Niu 2018). However , the role of the SQSTM1 gene in these disorders is not well established. In sum mary, the p.Pro392Leu variant meets criteria to be classified as pathogenic for PDB in an autosomal dominant manner with reduced penetrance. Additional data is required to determine if this variant contributes to other disorders.

SQSTM1: PS3, PS4:Moderate, PM5:Supporting, PP3

The SQSTM1 c.1175C>T (p.Pro392Leu) missense variant is the most commonly observed SQSTM1 variant in individuals with Paget disease of bone (PDB) to date (Seton et al. 2016). Across a selection of the literature, the p.Pro392Leu variant has been identified in a homozygous state in four PDB patients and in a heterozygous state in 101 individuals with familial PDB and in 211 individuals with sporadic PDB (Laurin et al. 2002; Michou et al. 2003; Hocking et al. 2004; Lucas et al. 2005; Morissette et al. 2006; Chung et al. 2008; Rea et al. 2009; Visconti et al. 2010; Seton et al. 2016). Morissette et al. (2006) calculated the penetrance of this variant to be 50% among all age groups, and approximately 80% for individuals over the age of 60. The p.Pro392Leu variant was absent from 1186 controls but is reported at a frequency of 0.02404 in the Puerto Ricans from Puerto Rico population of the 1000 Genomes Project. This frequency is high but is consistent with the disease prevalence and reduced penetrance seen among younger individuals. Several studies identified a shared ancestral haplotype among most individuals carrying the p.Pro392Leu variant allele, indicating that the variant is likely a founder mutation in populations of Western European descent (Laurin et al. 2002; Lucas et al. 2005; Morissette et al. 2006; Chung et al. 2008; Rea et al. 2009). Functional studies demonstrated that the p.Pro392Leu variant disrupted protein interaction and reduced binding affinity for ubiquitin (Cavey et al. 2005; Rea et al. 2006; Chamoux et al. 2009; Kurihara et al. 2011; Sundaram et al. 2011). Based on the collective evidence, the p.Pro392Leu variant is classified as pathogenic for Paget disease of bone. This variant was observed by ICSL as part of a predisposition screen in an ostensibly healthy population.

ACMG criteria used to clasify this variant: PS4_MOD, PM1, PP3_MOD, PM2_SUP, BS2

Observed in multiple unrelated patients with Paget disease of bone (PDB), frontotemporal dementia (FTD), and/or amyotrophic lateral sclerosis (ALS) in published literature (Almeida et al., 2016; Le Ber et al., 2013; McCann et al., 2020; Teyssou et al., 2013, Tripolszki et al., 2019; Acosta-Uribe et al. 2022); however, variant has been observed in controls; Published mouse models expressing the P392L equivalent variant (P394L in the murine sqstm1 gene) developed a skeletal disorder similar to PDB (Daroszewska et al., 2011); however studies using affected patient cells and a different mouse model suggest that the P392L variant may be insufficient to cause PDB on its own, and additional genetic and/or environmental factors may be required (Kurihara et al., 2011; Seton et al., 2016; Hiruma et al., 2008); 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: 24899140, 15493999, 18765443, 19589897, 12857745, 26713335, 28430856, 23942205, 25241215, 33612544, 32329415, 32893041, 33325387, 34426522, 11992264, 21195346, 26839080, 24042580, 32409511, 23417734, 33015249, 30889971, 31000212, 30729484, 30671590, 30726512, 31462833, 28889094, 32978683, 29457785, 27594680, 27275741, 29948344, 29599744, 30886882, 25708934, 30594595, 15125799, 26627873, 31634910, 31616248, 32176830, 31530427, 35241069, 35260199, 21515589, 31475037, 35229101, 36327984, 35769265, 35708843, 36918542, 35355568, 36035996, 35330074, 36133075)

PP3, PM1

This sequence change replaces proline, which is neutral and non-polar, with leucine, which is neutral and non-polar, at codon 392 of the SQSTM1 protein (p.Pro392Leu). This variant is present in population databases (rs104893941, gnomAD 0.1%), including at least one homozygous and/or hemizygous individual. This missense change has been observed in individual(s) with Paget disease of the bone (PMID: 11473345, 11992264, 15125799, 17229007). It has also been observed to segregate with disease in related individuals. The penetrance of this variant appears to be reduced and increases with age. In one study, the penetrance was 50% across all age groups but 17% below the age of 50 (PMID: 17229007). Of note, this variant has also been observed in individuals with frontotemporal dementia (PMID: 24899140, 24042580), amyotrophic lateral sclerosis (PMID:23417734, 23942205, 24899140, 28430856), and myopathy (PMID:29457785, 29599744), but the association with these diseases is unclear. ClinVar contains an entry for this variant (Variation ID: 8108). 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 SQSTM1 protein function. Experimental studies have shown that this missense change affects SQSTM1 function (PMID: 15765181, 16813535, 19589897, 21195346, 21878516). For these reasons, this variant has been classified as Pathogenic.

The SQSTM1 p.P392L variant is a well-known variant and was identified in 59 of 356 proband chromosomes (frequency: 0.1657) from individuals or families with Paget’s disease of bone (PDB) and was not identified in 582 control chromosomes from healthy individuals (Laurin_2002_PMID:11992264, Hocking_2002_PMID:12374763, Seton_2016_PMID:26713335). The p.P392L variant was also identified in patients with PDB in the Dutch, Italian, French-Canadian, and Australian populations and was also shown to segregate with disease in 20 families from Quebec with PDB (Laurin_2002_PMID:11992264; Morissette_2006_PMID:17229007; Hocking_2002_PMID:12374763; Eekhoff_2004_PMID:15146436; Falchetti_2004_PMID:15125799; Good_2004_PMID:15207768). In addition to Paget’s disease of bone, the p.P392L variant has been identified in patients with Amyotrophic Lateral Sclerosis (ALS), Frontotemporal Dementia (FTD), and Distal Myopathy (Fecto_2011_PMID:22084127, Teyssou_2013_PMID:23417734, Le Ber_2013_PMID:24042580, Kwok_2014_PMID:23942205, van der Zee_2014_PMID:24899140, Niu_2018_PMID:29599744). Some of these patients with ALS did not have a personal/family history of PDB or they did; one family had three affected individuals with FTD and PDB and other unaffected family members did not carry the variant (Fecto_2011_PMID:22084127, Le Ber_2013_PMID:24042580, Kwok_2014_PMID:23942205, Niu_2018_PMID:29599744). The p.P392L variant is located in the highly conserved ubiquitin-associated (UBA) domain in the p62 protein which is involved in multiubiquitin chain binding. An NMR structure of the UBA domain with the p.P392L mutant showed no effect on the interaction of the UBA domain with multiubiquitin chain binding (Ciani_2003_PMID:12857745). An in vitro assay showed the p.P392L mutation does not affect the binding properties of UBA, however it had subtle local effects on the UBA domain structure (Layfield_2004_PMID:15493999). Other in vitro functional expression studies in E. coli showed the p.P392L mutation caused loss of monoubiquitin binding and impaired K48-linked polyubiquitin binding (Cavey_2005_PMID:15765181). There are conflicting in vivo studies on mice with the p.P392L mutation and association with PDB (Hiruma_2008_PMID:18765443, Daroszewska_2011_PMID:21515589). Chamoux et al. conducted in vivo studies on mature osteoclasts from PDB patients and healthy donors that carried or do not carry the p.P392L variant. Results showed the p62 protein was significantly more expressed in PDB patient than in healthy donors, regardless of whether the p.P392L variant was present or not. However, overexpression of p.P392L mutated p62 led to the formation of more osteoclasts which contained more nuclei than non-transfected cells or cells overexpressing wild-type p62. The p62 p.P392L mutation contributed to increased activation of other proteins (Chamoux_2009_PMID:19589897). Other studies speculate environmental and genetics factors involved in the formation of disease in those with PDB and distal myopathy (Niu_2018_PMID:29599744, Kurihara_2011_PMID:21195346). The variant was identified in dbSNP (ID: rs104893941), LOVD 3.0 and ClinVar (conflicting interpretations of pathogenicity: uncertain significance by GeneDx, pathogenic by Laboratory for Molecular Medicine and Illumina, likely pathogenic by Division of Human Genetics, Children's Hospital of Philadelphia , and benign by Invitae, associated with the conditions Paget disease, Amyotrophic lateral sclerosis and/or frontotemporal dementia 1, and Frontotemporal dementia and/or amyotrophic lateral sclerosis 3). The variant was identified in control databases in 259 of 282398 chromosomes (2 homozygous) at a frequency of 0.000917 increasing the likelihood this could be a low frequency benign variant (Genome Aggregation Database March 6, 2019, v2.1.1). The variant was observed in the following populations: Other in 15 of 7228 chromosomes (freq: 0.002075), European (non-Finnish) in 173 of 128718 chromosomes (freq: 0.001344), Latino in 45 of 35440 chromosomes (freq: 0.00127), European (Finnish) in 9 of 25118 chromosomes (freq: 0.000358), South Asian in 9 of 30616 chromosomes (freq: 0.000294), African in 6 of 24960 chromosomes (freq: 0.00024) and Ashkenazi Jewish in 2 of 10366 chromosomes (freq: 0.000193), but was not observed in the East Asian population. The p.Pro392 residue is not conserved in mammals and computational analyses (PolyPhen-2, SIFT, AlignGVGD, BLOSUM, MutationTaster) provide inconsistent predictions regarding the impact to the protein; this information is not very predictive of pathogenicity. The variant occurs outside of the splicing consensus sequence and three of four in silico or computational prediction software programs (SpliceSiteFinder, MaxEntScan, NNSPLICE, GeneSplicer) do not predict a greater than 10% difference in splicing; this is not very predictive of pathogenicity. In summary, based on the above information the clinical significance of this variant cannot be determined with certainty at this time although we would lean towards a more pathogenic role for this variant. This variant is classified as likely pathogenic.