Criteria Specification Registry

Criteria Specification (CSpec) Registry is intended to provide access to the Criteria Specifications used and applied by ClinGen Variant Curation Expert Panels and biocurators in the classification of variants.

For general information about the ClinGen Expert Panels and Variant Curation please visit: Clinical Domain Working Groups. For specific inquiries regarding content correction or adding a new criteria specification refer to the Help page.

Should you encounter any issues regarding the data displayed, lack of functionality or other problems, please let us know by contacting us via email.

Criteria Specification

ClinGen Lysosomal Diseases Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for IDUA Version 1.0.0

Version : 1.0.0

Lysosomal Diseases VCEP

Released

12/5/2024

14:23:07

Rules for IDUA

Gene: IDUA (HGNC:5391) HGNC Name: alpha-L-iduronidase Transcripts: NM_000203.4 Disease: mucopolysaccharidosis type 1 (MONDO:0001586) Mode of Inheritance: Autosomal recessive inheritance

Criteria & Strength Specifications
PVS1 PVS1 PS1 PS2 PS3 PS4 PM1 PM2 PM3 PM4 PM5 PM6 PP1 PP2 PP3 PP4 PP5 BA1 BS1 BS2 BS3 BS4 BP1 BP2 BP3 BP4 BP5 BP6 BP7 Files References
Original ACMG Summary Null variant (nonsense, frameshift, canonical +/−1 or 2 splice sites, initiation codon, single or multi-exon deletion) in a gene where loss of function (LOF) is a known mechanism of disease. Caveats: • Beware of genes where LOF is not a known disease mechanism (e.g. GFAP, MYH7). • Use caution interpreting LOF variants at the extreme 3’ end of a gene. • Use caution with splice variants that are predicted to lead to exon skipping but leave the remainder of the protein intact. • Use caution in the presence of multiple transcripts. Stand Alone Very Strong All nonsense and frameshift variants will meet PVS1 (Very Strong), unless the variant is predicted to be undetected by nonsense-mediated decay (NMD) i.e. if the resulting premature termination codon is in the last exon (exon 14) or in the last 50 nucleotides of the penultimate exon (exon 13; after c.1778, codon 592). In this case, PVS1_Moderate will be applied because <10% of the primary amino acid sequence is predicted to be lost (in this case, ~9.3%). For all variants involving either the +1 or +2 position of GT donor splice sites, the exon immediately 5’ of the variant is predicted to be skipped, and for all variants involving either the -1 or -2 position of AG acceptor splice sites, the exon immediately 3’ of the variant is predicted to be skipped, unless indicated otherwise by RT-PCR or in silico prediction. For the predicted in frame/out of frame consequences for skipping any exon in IDUA, and recommended weight for PVS1, see Appendix 1. Follow the recommendations of Walker et al (PMID: 37352859) for all variants occurring in splice motifs (the donor site motif = last 3 nucleotides of an exon and first 6 nucleotides of an intron; acceptor site motif = first nucleotide of an exon and 20 nucleotides upstream from the exon boundary). If a single or multi-exon deletion results in an out-of-frame consequence, use PVS1 (Very Strong) if NMD is predicted to occur. Initiation codon variants will meet PVS1. The next in-frame methionine is at position 133. If used as a start signal, the signal sequence (amino acids 1-27) would be lost (https://www.uniprot.org/uniprotkb/P35475/entry) There are 6 variants, upstream of Met133, that are classified as pathogenic in ClinVar (data accessed July 20, 2022) (Table 1). If a deletion results in an in-frame consequence, the deletion must encompass one or more exons for PVS1 to apply. Consult Appendix 1 and use professional judgment regarding the strength of evidence to apply. For duplications, consult the PVS1 decision tree and Appendix 1 to assess the impact of single and multi-exon duplications and apply PVS1 at the appropriate strength. Modification Type: None Strong For frameshift variants at the 3’ end of IDUA that are not predicted to undergo NMD i.e. PTC downstream of c.1778, consider the length of abnormal amino acid sequence that is added due to the frameshift. If >10% of the length of the normal sequence is altered, PVS1 can be applied at strong. For all variants involving either the +1 or +2 position of GT donor splice sites, the exon immediately 5’ of the variant is predicted to be skipped, and for all variants involving either the -1 or -2 position of AG acceptor splice sites, the exon immediately 3’ of the variant is predicted to be skipped, unless indicated otherwise by RT-PCR or in silico prediction. For the predicted in frame/out of frame consequences for skipping any exon in IDUA, and recommended weight for PVS1, see Appendix 1. Follow the recommendations of Walker et al (PMID: 37352859) for all variants occurring in splice motifs (the donor site motif = last 3 nucleotides of an exon and first 6 nucleotides of an intron; acceptor site motif = first nucleotide of an exon and 20 nucleotides upstream from the exon boundary). If a deletion results in an in-frame consequence, the deletion must encompass one or more exons for PVS1 to apply. Consult Appendix 1 and use professional judgment regarding the strength of evidence to apply. For duplications, consult the PVS1 decision tree and Appendix 1 to assess the impact of single and multi-exon duplications and apply PVS1 at the appropriate strength. Modification Type: Disease-specific,Strength Moderate Any nonsense or frameshift variant that is predicted to be undetected by nonsense-mediated decay (NMD) i.e. if the resulting premature termination codon is in the last exon (exon 14) or in the last 50 nucleotides of the penultimate exon (exon 13; after c.1778, codon 592). In this case, PVS1_Moderate will be applied because <10% of the primary amino acid sequence is predicted to be lost ( ~9.3%). For frameshift variants at the 3’ end of IDUA that are not predicted to undergo NMD i.e. PTC downstream of c.1778, consider the length of abnormal amino acid sequence that is added due to the frameshift. If <10% of the length of the normal sequence is altered, PVS1 can be applied at moderate. For all variants involving either the +1 or +2 position of GT donor splice sites, the exon immediately 5’ of the variant is predicted to be skipped, and for all variants involving either the -1 or -2 position of AG acceptor splice sites, the exon immediately 3’ of the variant is predicted to be skipped, unless indicated otherwise by RT-PCR or in silico prediction. For the predicted in frame/out of frame consequences for skipping any exon in IDUA, and recommended weight for PVS1, see Appendix 1. Follow the recommendations of Walker et al (PMID: 37352859) for all variants occurring in splice motifs (the donor site motif = last 3 nucleotides of an exon and first 6 nucleotides of an intron; acceptor site motif = first nucleotide of an exon and 20 nucleotides upstream from the exon boundary). If a deletion results in an in-frame consequence, the deletion must encompass one or more exons for PVS1 to apply. Consult Appendix 1 and use professional judgment regarding the strength of evidence to apply. For duplications, see the PVS1 decision tree and Appendix 1 to assess the impact of single and multi-exon duplications and apply PVS1 at the appropriate strength. Modification Type: Disease-specific,Strength Supporting Instructions: Please consult the “PVS1 Decision Tree” (Appendix 2) for additional information. If PVS1 is applied, PM4 will not be applied. If an in-frame deletion is smaller than one exon, PVS1 does not apply; consider using PM4. Use the PVS1 decision tree to assess the impact of single and multi-exon duplications. Use SpliceAI in analysis of all canonical splice site variants (see PP3 and BP4 for details on thresholds). If there is a nearby (within +/- 20 nucleotides) splice site sequence that may reconstitute in-frame splicing, this should be taken into consideration. Consult Walker et al (PMID: 37352859) to apply PVS1 for splice motif variants. Not Applicable
PS1 PVS1 PS1 PS2 PS3 PS4 PM1 PM2 PM3 PM4 PM5 PM6 PP1 PP2 PP3 PP4 PP5 BA1 BS1 BS2 BS3 BS4 BP1 BP2 BP3 BP4 BP5 BP6 BP7 Files References
Original ACMG Summary Same amino acid change as a previously established pathogenic variant regardless of nucleotide change. Example: Val->Leu caused by either G>C or G>T in the same codon. Caveat: Beware of changes that impact splicing rather than at the amino acid/protein level. Stand Alone Very Strong Strong Same amino acid change as a previously established pathogenic variant regardless of nucleotide change.Splice region variants following Table 3 in Walker et al (PMID: 37352859). Modification Type: None Moderate Same amino acid change as a previously established likely pathogenic variant regardless of nucleotide change. Splice region variants following Table 3 in Walker et al (PMID: 37352859). Modification Type: Strength Supporting Splice region variants following Table 3 in Walker et al (PMID: 37352859). Modification Type: Strength Instructions: The classification of the other variant must be made following the Lysosomal Diseases VCEP specifications. To avoid circularity, the classification of the other variant should not use evidence from the variant being interrogated. If there is a question as to whether PS1 should be applied to variant A or variant B, use the classification of the variant with a greater level of evidence to support the classification of the variant with less evidence. When applying PS1 for amino acid changes, there should be no splicing impact for either variant, shown by splicing assay or computational predictors (SpliceAI <0.1). Not Applicable
PS2 PVS1 PS1 PS2 PS3 PS4 PM1 PM2 PM3 PM4 PM5 PM6 PP1 PP2 PP3 PP4 PP5 BA1 BS1 BS2 BS3 BS4 BP1 BP2 BP3 BP4 BP5 BP6 BP7 Files References
Original ACMG Summary De novo (both maternity and paternity confirmed) in a patient with the disease and no family history. Note: Confirmation of paternity only is insufficient. Egg donation, surrogate motherhood, errors in embryo transfer, etc. can contribute to non-maternity. Stand Alone Very Strong Strong Variant occurs de novo in an affected individual, and the biological relationship of the parent without the variant is confirmed e.g. if the father is not heterozygous for an IDUA variant that has been detected in the patient, paternity must be confirmed. Modification Type: Disease-specific Moderate Variant occurs de novo in an affected individual, and the biological relationship of the parent without the variant is not confirmed. Modification Type: Disease-specific Supporting Instructions: Note: De novo variants are rarely reported in IDUA. Not Applicable Comments: De novo variants are rarely reported in IDUA. The occurrence of de novo variants in IDUA is not a mechanism of disease for MPS 1, and the observation that a variant in IDUA has arisen de novo does not support its causality. Any de novo variants will be assessed based on the variant type, functional evidence, and in trans data as described in these guidelines.
PS3 PVS1 PS1 PS2 PS3 PS4 PM1 PM2 PM3 PM4 PM5 PM6 PP1 PP2 PP3 PP4 PP5 BA1 BS1 BS2 BS3 BS4 BP1 BP2 BP3 BP4 BP5 BP6 BP7 Files References
Original ACMG Summary Well-established in vitro or in vivo functional studies supportive of a damaging effect on the gene or gene product. Note: Functional studies that have been validated and shown to be reproducible and robust in a clinical diagnostic laboratory setting are considered the most well-established. Stand Alone Very Strong Strong Moderate When PS3_Supporting is met for enzyme activity AND there is expression data (Western blot, pulse chase) showing a clear difference in synthesis and/or processing of alpha-iduronidase, PS3 will be applied at moderate (e.g. Matte et al, 2003, PMID: 12559846; see Appendix 3). Modification Type: Disease-specific,Strength Supporting In vitro expression studies: After assessment of the parameters listed below, results from such studies can be used at PS3_Supporting (see Appendix 3 for thresholds for specific studies; <2% activity (Beesley et al, 2001, PMID: 11735025; Yogalingam et al, 2004, PMID: 15300847; Matte et al, 2003); <1% activity / enzyme abundance (Yu et al, 2020. PMID: 33198351). Were clones sequenced to verify that the variant is present and that no artifacts have been introduced during the site-directed mutagenesis process? Were appropriate controls included? Examples Negative controls: Empty vector, antisense (at least one appropriate negative control is required); Positive control: Wild type IDUA, normal cells (at least one appropriate positive control is required) Was the experiment replicated? If cells have intrinsic IDUA activity e.g., COS cells, the level of activity should be stated so that this can be taken into account. Modification Type: Disease-specific,Strength Instructions: Any variant meeting the requirements below can meet PS3 (at the appropriate strength). Note that these assays are in vitro, research-based, and may not truly reflect in vivo function. There are several studies involving expression of IDUA sequence variants in cultured cells and subsequent measurement of enzyme activity. Some studies also include analysis of IDUA synthesis and processing by Western blot. Please see Appendix 3 for details on the methodology used in the studies that included the largest number of variants (up to 8 variants per study) (Beesley et al, 2001; Yogalingam et al, 2004, PMID: 15300847; Yu et al, 2020, PMID: 33198351). Additional studies on smaller numbers of variants have also been published (e.g. Bach et al, 1993, PMID: 8328452; Tieu et al, 1995, PMID: 7550232; Lee-Chen et al, 1999, PMID: 10466419; Prommajan et al, 2011, PMID: 21364962; Ngiwsara et al, 2017, PMID: 29282708). Historically, these assays have been broadly accepted in the analysis of IDUA variants as well as other enzymes involved in metabolic disease. Note: PS3 will not be used for RT-PCR assays proving evidence that a variant impacts splicing. Instead, this evidence will be used to assign the appropriate weight of evidence for PVS1, following the guidance of Walker et al (PMID: 37352859). Not Applicable
PS4 PVS1 PS1 PS2 PS3 PS4 PM1 PM2 PM3 PM4 PM5 PM6 PP1 PP2 PP3 PP4 PP5 BA1 BS1 BS2 BS3 BS4 BP1 BP2 BP3 BP4 BP5 BP6 BP7 Files References
Original ACMG Summary The prevalence of the variant in affected individuals is significantly increased compared to the prevalence in controls. Note 1: Relative risk (RR) or odds ratio (OR), as obtained from case-control studies, is >5.0 and the confidence interval around the estimate of RR or OR does not include 1.0. See manuscript for detailed guidance. Note 2: In instances of very rare variants where case-control studies may not reach statistical significance, the prior observation of the variant in multiple unrelated patients with the same phenotype, and its absence in controls, may be used as moderate level of evidence. Stand Alone Very Strong Strong Moderate Supporting Not Applicable Comments: There are no case-control studies for MPS1. As this is a recessive disorder, the prevalence of the variant in affected individuals may not be increased compared to controls (who could be heterozygous carriers). The number of patients with the variant will be addressed by the PM3 evidence code.
PM1 PVS1 PS1 PS2 PS3 PS4 PM1 PM2 PM3 PM4 PM5 PM6 PP1 PP2 PP3 PP4 PP5 BA1 BS1 BS2 BS3 BS4 BP1 BP2 BP3 BP4 BP5 BP6 BP7 Files References
Original ACMG Summary Located in a mutational hot spot and/or critical and well-established functional domain (e.g. active site of an enzyme) without benign variation. Stand Alone Very Strong Strong Moderate Studies on functional domains (Bie et al, 2013, PMID: 24036510; Maita et al, 2013, PMID: 23959878; Saito et al, 2014, PMID: 24480078; Figueiredo et al, 2014, PMID: 25459762) have shown that the following residues are important to the function of IDUA: Active site nucleophiles: Glu182 and Glu299 Active site pocket and substrate binding: Arg89, His91, Asn181, His262, Lys264, Asp301, Gly305, Trp306, Asp349, Arg363, Asn372. PM1 will be applied to any missense substitutions or inframe deletions of the above residues. There are no benign or likely benign missense or infame deletions of these residues in ClinVar, or common missense or inframe deletions of these residues in gnomAD v4.1.0 (ClinVar and gnomAD v4.1.0 data accessed on October 30, 2024). Modification Type: Disease-specific Supporting Not Applicable
PM2 PVS1 PS1 PS2 PS3 PS4 PM1 PM2 PM3 PM4 PM5 PM6 PP1 PP2 PP3 PP4 PP5 BA1 BS1 BS2 BS3 BS4 BP1 BP2 BP3 BP4 BP5 BP6 BP7 Files References
Original ACMG Summary Absent from controls (or at extremely low frequency if recessive) in Exome Sequencing Project, 1000 Genomes or Exome Aggregation Consortium. Caveat: Population data for indels may be poorly called by next generation sequencing. Stand Alone Very Strong Strong Moderate Supporting This criterion will be applied at the supporting level based on guidance from the ClinGen Sequence Variant Interpretation Working Group. Minor allele frequency <0.025% (0.00025) in any continental population with >2000 alleles in the most recent version of gnomAD (version # will be stated in the written summary). Variants may be observed in the homozygous state because MPS1 can present in adulthood, and some variants may be hypomorphic. However, the presence and number of homozygotes should be noted. Modification Type: General recommendation,Strength Not Applicable
PM3 PVS1 PS1 PS2 PS3 PS4 PM1 PM2 PM3 PM4 PM5 PM6 PP1 PP2 PP3 PP4 PP5 BA1 BS1 BS2 BS3 BS4 BP1 BP2 BP3 BP4 BP5 BP6 BP7 Files References
Original ACMG Summary For recessive disorders, detected in trans with a pathogenic variant Note: This requires testing of parents (or offspring) to determine phase. Stand Alone Very Strong Detected in trans with a pathogenic variant. Points system based on guidance from the ClinGen Sequence Variant Interpretation Working Group. See Appendix 4 for the Lysosomal Diseases VCEP's specification of PM3. Note that points will NOT be applied for any variants of uncertain significance confirmed in trans, due to the high number of pseudodeficiency variants in IDUA. These variant interpretation guidelines should be used to determine the classification of the “other variant” in order to determine the appropriate number of points to assign. Modification Type: General recommendation,Strength Strong Detected in trans with a pathogenic variant. Points system based on guidance from the ClinGen Sequence Variant Interpretation Working Group. See Appendix 4 for the Lysosomal Diseases VCEP's specification of PM3. Note that points will NOT be applied for any variants of uncertain significance confirmed in trans, due to the high number of pseudodeficiency variants in IDUA. These variant interpretation guidelines should be used to determine the classification of the “other variant” in order to determine the appropriate number of points to assign. Modification Type: General recommendation,Strength Moderate Detected in trans with a pathogenic variant. Points system based on guidance from the ClinGen Sequence Variant Interpretation Working Group. See Appendix 4 for the Lysosomal Diseases VCEP's specification of PM3. Note that points will NOT be applied for any variants of uncertain significance confirmed in trans, due to the high number of pseudodeficiency variants in IDUA. These variant interpretation guidelines should be used to determine the classification of the “other variant” in order to determine the appropriate number of points to assign. Modification Type: None Supporting Detected in trans with a pathogenic variant. Points system based on guidance from the ClinGen Sequence Variant Interpretation Working Group. See Appendix 4 for the Lysosomal Diseases VCEP's specification of PM3. Note that points will NOT be applied for any variants of uncertain significance confirmed in trans, due to the high number of pseudodeficiency variants in IDUA. These variant interpretation guidelines should be used to determine the classification of the “other variant” in order to determine the appropriate number of points to assign. Modification Type: General recommendation,Strength Instructions: PM2_Supporting must be met in order to apply points to any case. The patient must be described as having MPS1, at a minimum. If the case meets PP4 and PM3, both criteria are applied. However, our PP4 criterion need not necessarily be met in order to apply PM3, as long as the patient is stated to have MPS1. For compound heterozygous cases, the second variant must have been classified by the Lysosomal Diseases VCEP. For rare variants that are routinely observed to be in cis with a pseudodeficiency variant, substantial additional evidence must be available to support the pathogenicity of the variant. The variant must meet PM2_Supporting, functional data should be available to support a deleterious impact, and cases considered for PM3 must have other clinical and laboratory findings supporting a diagnosis of MPS1. We strongly recommend not applying this exception for a novel variant with a single report. If multiple unrelated compound heterozygous cases have the same genotype, and the variants are not confirmed in trans, then no more than two cases should be used for assigning points (i.e. maximum of 1 point; similar to the guidance for homozygotes). This avoids over-counting evidence if the variants are actually in cis, and hence inherited together, in multiple individuals. Care must be taken to ensure that the reports do not represent the same case. For a variant to be “confirmed in trans” in a compound heterozygous patient, parental testing in at least one parent, or another appropriate molecular method (such as cloning each allele separately followed by sequencing), must have been performed. Otherwise, the phase of the variants is unknown. Parental testing is not required for homozygous cases. As noted in the SVI guidance for PM3, “To avoid circularity, in all instances (phasing confirmed or unknown), the classification of the variant on the other allele should not use evidence from the variant being interrogated.” Not Applicable
PM4 PVS1 PS1 PS2 PS3 PS4 PM1 PM2 PM3 PM4 PM5 PM6 PP1 PP2 PP3 PP4 PP5 BA1 BS1 BS2 BS3 BS4 BP1 BP2 BP3 BP4 BP5 BP6 BP7 Files References
Original ACMG Summary Protein length changes due to in-frame deletions/insertions in a non-repeat region or stop-loss variants. Stand Alone Very Strong Strong Moderate Stop loss variants, and in frame deletion/insertions of two or more amino acids but less than one exon. Modification Type: None Supporting In frame deletion/insertion of one amino acid. Modification Type: Strength Instructions: For in-frame deletions of one or more exons, use PVS1. Not Applicable
PM5 PVS1 PS1 PS2 PS3 PS4 PM1 PM2 PM3 PM4 PM5 PM6 PP1 PP2 PP3 PP4 PP5 BA1 BS1 BS2 BS3 BS4 BP1 BP2 BP3 BP4 BP5 BP6 BP7 Files References
Original ACMG Summary Novel missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before. Example: Arg156His is pathogenic; now you observe Arg156Cys. Caveat: Beware of changes that impact splicing rather than at the amino acid/protein level. Stand Alone Very Strong Strong Moderate Missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before. Stop loss variant if another stop loss variant has been determined to be pathogenic. Modification Type: None Supporting Missense change at an amino acid residue where a different missense change determined to be likely pathogenic has been seen before. Stop loss variant if another stop loss variant has been determined to be likely pathogenic. Modification Type: Strength Instructions: The classification of the other variant must be made following the Lysosomal Diseases VCEP specifications. To avoid circularity, the classification of the other variant should not use evidence from the variant being interrogated. If there is a question as to whether PM5 should be applied to variant A or variant B, use the classification of the variant with a greater level of evidence to support the classification of the variant with less evidence. There is no splicing impact for either variant, shown by splicing assay or computational predictors (SpliceAI ≤0.1). Not Applicable
PM6 PVS1 PS1 PS2 PS3 PS4 PM1 PM2 PM3 PM4 PM5 PM6 PP1 PP2 PP3 PP4 PP5 BA1 BS1 BS2 BS3 BS4 BP1 BP2 BP3 BP4 BP5 BP6 BP7 Files References
Original ACMG Summary Assumed de novo, but without confirmation of paternity and maternity. Stand Alone Very Strong Strong Moderate Supporting Not Applicable Comments: See PS2.
PP1 PVS1 PS1 PS2 PS3 PS4 PM1 PM2 PM3 PM4 PM5 PM6 PP1 PP2 PP3 PP4 PP5 BA1 BS1 BS2 BS3 BS4 BP1 BP2 BP3 BP4 BP5 BP6 BP7 Files References
Original ACMG Summary Co-segregation with disease in multiple affected family members in a gene definitively known to cause the disease. Note: May be used as stronger evidence with increasing segregation data. Stand Alone Very Strong Strong Moderate See Appendix 5 for points system and guidance (based on PMID: 38103548). Modification Type: General recommendation Supporting See Appendix 5 for points system and guidance (based on PMID: 38103548). Modification Type: General recommendation Instructions: The combination of strengths for PP1 and PP4 MUST NOT exceed strong (2 x moderate, i.e. 4 points using Bayesian system) if cases are also being counted as probands under PM3. Counting segregations: Do not count probands as a segregation. Affected segregations = # affected individuals in the family with the variants minus 1. Affected segregations are defined as affected family members (typically siblings) who harbor the variant in question and a second variant on the remaining allele. Unaffected segregations are defined as unaffected family members, typically siblings, who are at risk to inherit the two variants (or one variant in homozygosity) identified in the proband. These individuals should be either homozygous normal or heterozygous for only one variant. Unaffected, carrier parents DO NOT count as unaffected segregations. Not Applicable Comments: Sibships large enough to meet this criterion are extremely rare. In addition, because IDUA is the only gene involved in MPS 1, all patients are expected to be bi-allelic, regardless of whether the pathogenic variants can be, or have been, detected. A variant under assessment may not be the true pathogenic variant but instead in linkage disequilibrium with an unidentified pathogenic variant. For this reason, this criterion does not facilitate assessment of pathogenicity.
PP2 PVS1 PS1 PS2 PS3 PS4 PM1 PM2 PM3 PM4 PM5 PM6 PP1 PP2 PP3 PP4 PP5 BA1 BS1 BS2 BS3 BS4 BP1 BP2 BP3 BP4 BP5 BP6 BP7 Files References
Original ACMG Summary Missense variant in a gene that has a low rate of benign missense variation and where missense variants are a common mechanism of disease. Stand Alone Very Strong Strong Moderate Supporting Not Applicable Comments: Does not apply; there are benign and pathogenic missense variants in IDUA.
PP3 PVS1 PS1 PS2 PS3 PS4 PM1 PM2 PM3 PM4 PM5 PM6 PP1 PP2 PP3 PP4 PP5 BA1 BS1 BS2 BS3 BS4 BP1 BP2 BP3 BP4 BP5 BP6 BP7 Files References
Original ACMG Summary Multiple lines of computational evidence support a deleterious effect on the gene or gene product (conservation, evolutionary, splicing impact, etc.). Caveat: As many in silico algorithms use the same or very similar input for their predictions, each algorithm should not be counted as an independent criterion. PP3 can be used only once in any evaluation of a variant. Stand Alone Very Strong Strong Moderate Any missense changes with a REVEL score >0.773 will meet PP3_Moderate (Based on Pejaver et al, PMID: 3641399; Note that the VCEP has chosen not to apply PP3 at strong). Modification Type: Disease-specific Supporting Any missense changes with a REVEL score between 0.644 - 0.773 will meet PP3 (Pejaver et al., PMID: 3641399) For in frame insertions and deletions, use Mutation Taster (http://www.mutationtaster.org/ ; count if “disease-causing”), and MutPred-Indel (http://mutpredindel.cs.indiana.edu/ ; score >0.5 for “pathogenic”). Apply PP3 if both predictors indicate that the variant is deleterious. For non-canonical splice site variants (e.g., +3, -3), use SpliceAI (https://spliceailookup.broadinstitute.org/ ). A score of >0.2 is taken to indicate disruption of the splice site allowing PP3 to be applied (based on Walker et al, PMID: 37352859). Evidence for use of a cryptic splice site and the impact on the gene product should also be assessed. PP3 for splicing variants will be applied only in the absence of functional (RNA analysis) data. Modification Type: Disease-specific Instructions: We note that a publication on the use of in silico predictors for IDUA reported specificity of 88% and sensitivity of 75% for REVEL, using a 0.75 cutoff to create a binary classification (PMID: 34746235). REVEL performed well, although some other predictors, including BaysDel, had better sensitivity, specificity, and accuracy. We have decided to continue using REVEL and to follow the SVI guidance (Pejaver et al, PMID: 3641399), due to the ease of use of this predictor and the ability to apply weight of evidence based on score. Not Applicable
PP4 PVS1 PS1 PS2 PS3 PS4 PM1 PM2 PM3 PM4 PM5 PM6 PP1 PP2 PP3 PP4 PP5 BA1 BS1 BS2 BS3 BS4 BP1 BP2 BP3 BP4 BP5 BP6 BP7 Files References
Original ACMG Summary Patient’s phenotype or family history is highly specific for a disease with a single genetic etiology. Stand Alone Very Strong Strong Moderate 3 or more of the following criteria are met: Deficient IDUA activity, within the affected range (usually non-detectable or expressed affected range by lab) in fibroblasts, leukocytes, or plasma. Do not count if one or more pseudodeficiency variants are present, or if result was obtained on newborn screen without confirmatory enzyme testing. Enzyme replacement therapy results in a significant reduction in urine GAGs (either total or heparan sulfate). Urinary and/or dried blood spot GAGs expressed as either total GAGs, specific GAG (heparan sulfate, dermatan sulfate, or endogenous biomarker elevation) above normal range. Patient reported to have clinical features specific to MPS; At minimum at least 2 of the following: dysostosis multiplex, hepatosplenomegaly, arthropathy, corneal involvement, valvular thickening. Bone marrow transplant results in a significant reduction in urine GAGs (either total or heparan sulfate). Modification Type: Disease-specific,Strength Supporting 2 of the following criteria are met: Deficient IDUA activity, within the affected range (usually non-detectable or expressed affected range by lab) in fibroblasts, leukocytes, or plasma. Do not count if one or more pseudodeficiency variants are present, or if result was obtained on newborn screen without confirmatory enzyme testing. Enzyme replacement therapy results in a significant reduction in urine GAGs (either total or heparan sulfate). Urinary and/or dried blood spot GAGs expressed as either total GAGs, specific GAG (heparan sulfate, dermatan sulfate, or endogenous biomarker elevation) above normal range. Patient reported to have clinical features specific to MPS; At minimum at least 2 of the following: dysostosis multiplex, hepatosplenomegaly, arthropathy, corneal involvement, valvular thickening. Bone marrow transplant results in a significant reduction in urine GAGs (either total or heparan sulfate). Modification Type: Disease-specific Not Applicable
PP5
Original ACMG Summary Reputable source recently reports variant as pathogenic, but the evidence is not available to the laboratory to perform an independent evaluation. Not Applicable This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. PubMed : 29543229
BA1 PVS1 PS1 PS2 PS3 PS4 PM1 PM2 PM3 PM4 PM5 PM6 PP1 PP2 PP3 PP4 PP5 BA1 BS1 BS2 BS3 BS4 BP1 BP2 BP3 BP4 BP5 BP6 BP7 Files References
Original ACMG Summary Allele frequency is above 5% in Exome Sequencing Project, 1000 Genomes or Exome Aggregation Consortium. Stand Alone Any variant with Grpmax >0.005 in the most recent version of gnomAD (95% confidence interval, lower bound) (version # will be stated in the written summary) BA1 minor allele frequency cut-off calculated using http://cardiodb.org/allelefrequencyapp with prevalence = 1 in 40,000 (PMID: 33208168), genetic heterogeneity = 1.0 (IDUA is the only gene known to cause MPS1), allelic heterogeneity = 1.0, and penetrance = 1.0. Modification Type: Disease-specific Very Strong Strong Moderate Supporting Not Applicable
BS1 PVS1 PS1 PS2 PS3 PS4 PM1 PM2 PM3 PM4 PM5 PM6 PP1 PP2 PP3 PP4 PP5 BA1 BS1 BS2 BS3 BS4 BP1 BP2 BP3 BP4 BP5 BP6 BP7 Files References
Original ACMG Summary Allele frequency is greater than expected for disorder. Stand Alone Very Strong Strong Any variant with Grpmax >0.0025 in the most recent version of gnomAD (95% confidence interval, lower bound) (version # will be stated in the written summary). BS1 minor allele frequency cut-off calculated using http://cardiodb.org/allelefrequencyapp with prevalence = 1 in 40,000 (PMID: 33208168), genetic heterogeneity = 1.0 (IDUA is the only gene known to cause MPS1), allelic heterogeneity = ~0.5 (frequency of the most common known pathogenic variants in patients with MPS1 (PMID: 28595941, 29393969), and penetrance = 1.0. Modification Type: Disease-specific Moderate Supporting Instructions: Variants meeting only BS1 will be classified as likely benign. Not Applicable
BS2 PVS1 PS1 PS2 PS3 PS4 PM1 PM2 PM3 PM4 PM5 PM6 PP1 PP2 PP3 PP4 PP5 BA1 BS1 BS2 BS3 BS4 BP1 BP2 BP3 BP4 BP5 BP6 BP7 Files References
Original ACMG Summary Observed in a healthy adult individual for a recessive (homozygous), dominant (heterozygous), or X-linked (hemizygous) disorder, with full penetrance expected at an early age. Stand Alone Very Strong Strong BS2 can be applied if there is clear documentation that an individual of any age is either homozygous for the variant, or has the variant confirmed in trans with a pathogenic or likely pathogenic variant, and has normal IDUA activity. Values for IDUA activity and the reference range for the laboratory must be provided. Note: Patients with late onset MPS1 can present late in life (5^th-6^th decade), can have mild symptoms, and may remain undiagnosed. Therefore, it is possible that individuals who are homozygous for hypomorphic IDUA variants could be present in population databases. Modification Type: None Moderate Supporting Not Applicable
BS3 PVS1 PS1 PS2 PS3 PS4 PM1 PM2 PM3 PM4 PM5 PM6 PP1 PP2 PP3 PP4 PP5 BA1 BS1 BS2 BS3 BS4 BP1 BP2 BP3 BP4 BP5 BP6 BP7 Files References
Original ACMG Summary Well-established in vitro or in vivo functional studies show no damaging effect on protein function or splicing. Stand Alone Very Strong Strong Moderate Supporting The same assays outlined for PS3 will be used for BS3. Please see PS3 guidance for additional information on these assays. BS3_Supporting can be applied for expression of IDUA sequence variants in cultured cells and subsequent measurement of enzyme activity provided that there is no other evidence to suggest that the variant could be disease-causing e.g. mislocalization (see Appendix 3 for thresholds for specific studies: >10% activity (Beesley et al, 2001, PMID: 11735025; Yogalingam et al, 2004, PMID: 15300847; Matte et al, 2003); >~10% activity / enzyme abundance (Yu et al, 2020. PMID: 33198351). Modification Type: Disease-specific,Strength Instructions: Variants meeting the combination of BS3_Supporting, BP4, and PM2_Supporting will be classified as likely benign. Not Applicable
BS4 PVS1 PS1 PS2 PS3 PS4 PM1 PM2 PM3 PM4 PM5 PM6 PP1 PP2 PP3 PP4 PP5 BA1 BS1 BS2 BS3 BS4 BP1 BP2 BP3 BP4 BP5 BP6 BP7 Files References
Original ACMG Summary Lack of segregation in affected members of a family. Caveat: The presence of phenocopies for common phenotypes (i.e. cancer, epilepsy) can mimic lack of segregation among affected individuals. Also, families may have more than one pathogenic variant contributing to an autosomal dominant disorder, further confounding an apparent lack of segregation. Stand Alone Very Strong Strong Non-segregation with disease in a family i.e. variant is absent in an affected individual. Modification Type: None Moderate Supporting Not Applicable
BP1 PVS1 PS1 PS2 PS3 PS4 PM1 PM2 PM3 PM4 PM5 PM6 PP1 PP2 PP3 PP4 PP5 BA1 BS1 BS2 BS3 BS4 BP1 BP2 BP3 BP4 BP5 BP6 BP7 Files References
Original ACMG Summary Missense variant in a gene for which primarily truncating variants are known to cause disease. Stand Alone Very Strong Strong Moderate Supporting Not Applicable Comments: Does not apply. All types of variants cause MPS1.
BP2 PVS1 PS1 PS2 PS3 PS4 PM1 PM2 PM3 PM4 PM5 PM6 PP1 PP2 PP3 PP4 PP5 BA1 BS1 BS2 BS3 BS4 BP1 BP2 BP3 BP4 BP5 BP6 BP7 Files References
Original ACMG Summary Observed in trans with a pathogenic variant for a fully penetrant dominant gene/disorder or observed in cis with a pathogenic variant in any inheritance pattern. Stand Alone Very Strong Strong Moderate Supporting Not Applicable
BP3 PVS1 PS1 PS2 PS3 PS4 PM1 PM2 PM3 PM4 PM5 PM6 PP1 PP2 PP3 PP4 PP5 BA1 BS1 BS2 BS3 BS4 BP1 BP2 BP3 BP4 BP5 BP6 BP7 Files References
Original ACMG Summary In frame-deletions/insertions in a repetitive region without a known function. Stand Alone Very Strong Strong Moderate Supporting Not Applicable Comments: There are no known repetitive regions without known function in IDUA.
BP4 PVS1 PS1 PS2 PS3 PS4 PM1 PM2 PM3 PM4 PM5 PM6 PP1 PP2 PP3 PP4 PP5 BA1 BS1 BS2 BS3 BS4 BP1 BP2 BP3 BP4 BP5 BP6 BP7 Files References
Original ACMG Summary Multiple lines of computational evidence suggest no impact on gene or gene product (conservation, evolutionary, splicing impact, etc) Caveat: As many in silico algorithms use the same or very similar input for their predictions, each algorithm cannot be counted as an independent criterion. BP4 can be used only once in any evaluation of a variant. Stand Alone Very Strong Strong Moderate Supporting Missense changes with a REVEL score < 0.290 (threshold based on Pejaver et al; however, BP4 will only be applied at the supporting level). For in frame insertions and deletions, use PROVEAN (http://provean.jcvi.org/seq_submit.php ; score >-2.5), Mutation Taster (http://www.mutationtaster.org/ ; count if “polymorphism”, and MutPred-Indel (http://mutpredindel.cs.indiana.edu/ ; score <0.5. Apply BP4 if all predictors indicate that the variant is benign. For non-canonical splice site variants, use SpliceAI (https://spliceailookup.broadinstitute.org/ ) to assess the impact of variants that are not +/-1 or 2 canonical splice site variants. This criterion can be applied as PP3 (Splicing) for SpliceAI scores ≤0.1 (based on Walker et al, PMID: 37352859). If there is any evidence for possible creation of a cryptic splice site, this criterion should not be applied. Modification Type: Disease-specific Instructions: Variants meeting the combination of BS3_Supporting, BP4, and PM2_Supporting will be classified as likely benign. Not Applicable
BP5 PVS1 PS1 PS2 PS3 PS4 PM1 PM2 PM3 PM4 PM5 PM6 PP1 PP2 PP3 PP4 PP5 BA1 BS1 BS2 BS3 BS4 BP1 BP2 BP3 BP4 BP5 BP6 BP7 Files References
Original ACMG Summary Variant found in a case with an alternate molecular basis for disease. Stand Alone Very Strong Strong Moderate Supporting Instructions: There is no known alternate molecular basis for deficiency of alpha-L-iduronidase activity, other than variants in IDUA. Not Applicable Comments: There is no known alternate molecular basis for deficiency of alpha-L-iduronidase activity, other than variants in IDUA.
BP6
Original ACMG Summary Reputable source recently reports variant as benign, but the evidence is not available to the laboratory to perform an independent evaluation. Not Applicable This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. PubMed : 29543229
BP7 PVS1 PS1 PS2 PS3 PS4 PM1 PM2 PM3 PM4 PM5 PM6 PP1 PP2 PP3 PP4 PP5 BA1 BS1 BS2 BS3 BS4 BP1 BP2 BP3 BP4 BP5 BP6 BP7 Files References
Original ACMG Summary A synonymous variant for which splicing prediction algorithms predict no impact to the splice consensus sequence nor the creation of a new splice site AND the nucleotide is not highly conserved. Stand Alone Very Strong Strong Experimental evidence (RT-PCR, RNA Seq, minigene) shows that the variant does not impact splicing. BP7_Strong (RNA) will be used only under strict circumstances in which it is clear that the allele with the variant is expressed at the normal level to avoid counting a “normal” result because the allele with the variant is absent due to nonsense-mediated decay (Walker et al, PMID: 37352859). Modification Type: Strength Moderate Supporting BP7 can be applied if the variant is synonymous, unless the variant is in the first nucleotide or last three nucleotides of an exon, AND BP4 is met i.e. SpliceAI predicts no impact on splicing (score ≤ 0.10) Modification Type: None Instructions: If a variant meets BP7, BP4 can also be applied. Not Applicable

Rules for Combining Criteria

Pathogenic

1 Very Strong (PVS1, PM3_Very Strong) AND ≥ 1 Strong (PVS1_Strong, PS1, PS2, PM3_Strong)

1 Very Strong (PVS1, PM3_Very Strong) AND ≥ 2 Moderate (PVS1_Moderate, PS1_Moderate, PS2_Moderate, PS3_Moderate, PM1, PM3, PM4, PM5, PP3_Moderate, PP4_Moderate)

1 Very Strong (PVS1, PM3_Very Strong) AND 1 Moderate (PVS1_Moderate, PS1_Moderate, PS2_Moderate, PS3_Moderate, PM1, PM3, PM4, PM5, PP3_Moderate, PP4_Moderate) AND 1 Supporting (PS1_Supporting, PS3_Supporting, PM2_Supporting, PM3_Supporting, PM4_Supporting, PM5_Supporting, PP3, PP4)

1 Very Strong (PVS1, PM3_Very Strong) AND ≥ 2 Supporting (PS1_Supporting, PS3_Supporting, PM2_Supporting, PM3_Supporting, PM4_Supporting, PM5_Supporting, PP3, PP4)

≥ 2 Strong (PVS1_Strong, PS1, PS2, PM3_Strong)

1 Strong (PVS1_Strong, PS1, PS2, PM3_Strong) AND ≥ 3 Moderate (PVS1_Moderate, PS1_Moderate, PS2_Moderate, PS3_Moderate, PM1, PM3, PM4, PM5, PP3_Moderate, PP4_Moderate)

1 Strong (PVS1_Strong, PS1, PS2, PM3_Strong) AND 2 Moderate (PVS1_Moderate, PS1_Moderate, PS2_Moderate, PS3_Moderate, PM1, PM3, PM4, PM5, PP3_Moderate, PP4_Moderate) AND ≥ 2 Supporting (PS1_Supporting, PS3_Supporting, PM2_Supporting, PM3_Supporting, PM4_Supporting, PM5_Supporting, PP3, PP4)

1 Strong (PVS1_Strong, PS1, PS2, PM3_Strong) AND 1 Moderate (PVS1_Moderate, PS1_Moderate, PS2_Moderate, PS3_Moderate, PM1, PM3, PM4, PM5, PP3_Moderate, PP4_Moderate) AND ≥ 4 Supporting (PS1_Supporting, PS3_Supporting, PM2_Supporting, PM3_Supporting, PM4_Supporting, PM5_Supporting, PP3, PP4)

Likely Pathogenic

1 Very Strong (PVS1, PM3_Very Strong) AND 1 Moderate (PVS1_Moderate, PS1_Moderate, PS2_Moderate, PS3_Moderate, PM1, PM3, PM4, PM5, PP3_Moderate, PP4_Moderate)

1 Strong (PVS1_Strong, PS1, PS2, PM3_Strong) AND 1 Moderate (PVS1_Moderate, PS1_Moderate, PS2_Moderate, PS3_Moderate, PM1, PM3, PM4, PM5, PP3_Moderate, PP4_Moderate)

1 Strong (PVS1_Strong, PS1, PS2, PM3_Strong) AND ≥ 2 Supporting (PS1_Supporting, PS3_Supporting, PM2_Supporting, PM3_Supporting, PM4_Supporting, PM5_Supporting, PP3, PP4)

≥ 3 Moderate (PVS1_Moderate, PS1_Moderate, PS2_Moderate, PS3_Moderate, PM1, PM3, PM4, PM5, PP3_Moderate, PP4_Moderate)

2 Moderate (PVS1_Moderate, PS1_Moderate, PS2_Moderate, PS3_Moderate, PM1, PM3, PM4, PM5, PP3_Moderate, PP4_Moderate) AND ≥ 2 Supporting (PS1_Supporting, PS3_Supporting, PM2_Supporting, PM3_Supporting, PM4_Supporting, PM5_Supporting, PP3, PP4)

1 Moderate (PVS1_Moderate, PS1_Moderate, PS2_Moderate, PS3_Moderate, PM1, PM3, PM4, PM5, PP3_Moderate, PP4_Moderate) AND ≥ 4 Supporting (PS1_Supporting, PS3_Supporting, PM2_Supporting, PM3_Supporting, PM4_Supporting, PM5_Supporting, PP3, PP4)

1 Strong (PVS1_Strong, PS1, PS2, PM3_Strong) AND 2 Moderate (PVS1_Moderate, PS1_Moderate, PS2_Moderate, PS3_Moderate, PM1, PM3, PM4, PM5, PP3_Moderate, PP4_Moderate)

1 Very Strong (PVS1) AND 1 Supporting (PM2_Supporting)

Benign

≥ 2 Strong (BS1, BS2, BS4, BP7_Strong)

1 Stand Alone (BA1)

Likely Benign

1 Strong (BS1, BS2, BS4, BP7_Strong) AND 1 Supporting (BS3_Supporting, BP4, BP7)

≥ 2 Supporting (BS3_Supporting, BP4, BP7)

Files & Images

Appendix 2_PVS1_Decision Tree_IDUA: IDUA-specific modifications have been made to the decision tree that was provided by the SVI.

Appendix 3_Functional assays: Describes published in vitro functional assays for IDUA variants expressed in a heterologous cell type; thresholds are assay-specific; not to be used for enzyme assay in patient samples.

Appendix 5_PP1 guidance_IDUA:

Appendix 1_PVS1 strength_IDUA: Provides details on the molecular consequence of loss of any exon within the IDUA gene, taking into account the number of nucleotides and amino acids that are critical to the function of the gene product.

Appendix 4_PM3 points system_IDUA: