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.

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Criteria Specification

ClinGen Glaucoma Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for CYP1B1 Version 1.0.0

Type: Tavtigian et.al., 2020 - Bayesian adaptation of Richards et.al., 2015

Version : 1.0.0

Glaucoma VCEP

Released

11/6/2025

02:07:02

Rules for CYP1B1

Modifications to the rules following pilot phase: - PVS1_SA was added for full gene deletions (stand alone for Pathogenic classification). - PS1: Specified that this rule does not apply to initiation codons - PS3: Specified conditions for animal models of specific variants to meet PS3 if developed - PM3: Piloted the use of VUS compound heterozygous in trans. VUS were used for applying PM3 to 3 variants & contributed to changing the final classification once (from LP to P). This rule is likely to be applied more often for variants with less evidence. The VCEP has decided to keep this rule but will reassess in the future as necessary. - PM3: Specified that variants must not meet BP4 (instead of meeting PP3) - PM3: Specified that individuals with multiple VUS/LP/P variants cannot be considered as evidence of either variant when the phase is unknown or variants are in cis - PP1: Specified that compound heterozygous variants in affected segregations need to be in trans - BA1: Threshold updated with justification - BP4: Specified that the SpliceAI threshold applies to non-missense variants not meeting PVS1 or PM4

Gene: CYP1B1 (HGNC:2597) HGNC Name: cytochrome P450 family 1 subfamily B member 1 Transcripts: NM_000104.4 Disease: CYP1B1-related glaucoma with or without anterior segment dysgenesis (MONDO:0800472) 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. VCEP Specifications: Use CYP1B1 decision tree for PVS1 (Figure 2) adapted from Abou Tayoun et al.¹ CYP1B1 primary transcript (NM_000104.4) consists of 5,218 bp and encodes a 543 amino acids protein. It contains 3 exons, although only exons 2 and 3 are coding. CYP1B1 variants cause the phenotype through a loss of function (LoF) disease mechanism. NMD is predicted to be activated for nonsense or frameshift variants where the premature termination codon is prior to amino acid 330 (last 50bp of exon 2, see Figure 1). CYP1B1 is an enzyme that relies on a haem group for its activity. There is evidence supporting that the haem-binding domain (position aa460-493, located in exon 3) is vital for CYP1B1 enzymatic activity: Missense variants within this domain (e.g. R469W²,³, C470Y and S485F⁴) and truncating variants prior to this domain have no activity, even when detectable by Western blot in an overexpression system (e.g. p.E262)². Thus, truncating variants that remove this domain are expected to be null variants. Additionally, there are a number of known pathogenic truncating variants in exon 3 (e.g. R444, T404Sfs30, R355Hfs69). There are no other biologically relevant transcripts of this very small gene, therefore the options related to exons being absent from biologically relevant transcript(s) are not applicable and have been removed from the original decision tree. The Glaucoma VCEP adapted the decision tree from Abou Tayoun et al.¹ for CYP1B1 and removed options that were not applicable to the gene. Nonsense or Frameshift The haem-binding domain is considered vital for CYP1B1 enzymatic activity and the biological effect of removing the haem-binding domain is equivalent to NMD. Truncating variants that are predicted to escape NMD but remove the haem-binding domain can meet PVS1. There are no LoF variants in gnomAD after the end of haem-binding domain at residue 493, therefore the branch of the decision tree referring to variants escaping NMD in a region of unknown function with LoF variants frequent in the general population is not applicable. The final decision point in the original decision tree refers to variants that are not predicted to undergo NMD, are in a region of unknown function and LoF variants are not frequent in the general population. Since the haem-binding domain ends at aa493 and residue 489 marks the last 10% of the protein, the option related to a variant removing >10% of the protein is not applicable. If the variant is after the haem-binding domain, it is within the last 10% and PVS1_Moderate would apply as per the decision tree. Splice variants CYP1B1 only has 2 coding exons and the splice site is after the point at which NMD could occur, but more importantly, it is upstream of the haem-binding domain. There are also no in-frame exons, with codon R348 being encoded across the exon 2/3 boundary. Therefore, exon skipping or the use of a cryptic splice site preserving the reading frame is not applicable. Similar to nonsense and frameshift variants, PVS1 applies to splice variants predicting to escape NMD but removing the haem-binding domain. Deletions CYP1B1 variation causes glaucoma through LoF disease mechanism, supported by mice model Cyp1b1 deficient showing developmental eye abnormalities and phenotype similar to humans.⁵ Consequently, PVS1_stand alone (PVS1_SA) can be applied to full gene deletions. CYP1B1 has only one transcript and only 2 exons that disrupt the reading frame. There is no experimental evidence to suggest whether NMD occurs in the context of deletions in this 2 exon gene, therefore these two options have been combined. Moreover, deletion of either exon disrupts/removes the haem-binding domain because none of the exons are in frame. Similar to other truncating variants, PVS1 applies to deletions disrupting/removing the haem-binding domain. CYP1B1 variation causes glaucoma through LoF disease mechanism, supported by mice model Cyp1b1 deficient showing developmental eye abnormalities and phenotype similar to humans.⁵ Consequently, PVS1_stand alone (PVS1_SA) can be applied to full gene deletions. The deletion of exon 1 is harder to interpret as it is a non-coding exon in the 5’UTR. It would not be expected to alter reading frame, is in a region of unknown function for the protein, LoF variants in this exon are not common and it would alter <10% of the protein. Following these options on the original decision tree leads to PVS1_Moderate. The VCEP recommends downgrading this to PVS1_Supporting as the effect on the protein is not known. Duplications Similar to other truncating variants, PVS1 applies to duplications disrupting/removing the haem-binding domain and these have been combined to deletions predicted to undergo NMD under PVS1 as they lead to the same outcome. Initiation codon CYP1B1 has only 1 transcript and there are reported pathogenic variants upstream of the closest potential alternative in-frame start codon (aa132). Therefore, the option if there are no pathogenic variants upstream of the closest potential start is not applicable. The option for a different functional transcript that uses an alternative start codon has been listed, in case such a transcript is reported in the future. Stand Alone Very Strong Applies to: Nonsense/frameshifts variants predicted to undergo NMD (aa1-330) or removing the haem-binding domain (aa331-493) GT-AG 1,2 splice sites variants leading to exon skipping or use of a cryptic splice site disrupting reading frame predicted to undergo NMD (aa1-330) or removing the haem-binding domain (aa331-493) Full gene deletions and deletions removing exon 2 and/or exon 3 Duplications (≥1 exon and completely contained within the gene) disrupting the reading frame, predicted to undergo NMD (aa1-330) or removing the haem-binding domain (aa331-493), AND proven in tandem Default Point Value: 8 Modification Type: Gene-specific Strong Applies to: Duplications (≥1 exon and completely contained within the gene) disrupting the reading frame, predicted to undergo NMD (aa1-330) or removing the haem-binding domain (aa331-493), AND presumed in tandem Default Point Value: 4 Modification Type: Gene-specific,Strength Moderate Applies to: Nonsense/frameshifts variants NOT removing the haem-binding domain (aa494-Ter) GT-AG 1,2 splice sites variants leading to exon skipping or use of a cryptic splice site disrupting reading frame and NOT predicted to undergo NMD (aa1-330) or removing the haem-binding domain (aa331-493) Duplications (≥1 exon and completely contained within the gene) disrupting the reading frame after the haem-binding domain (aa4934-Ter) AND proven in tandem Initiation codon variants when there is no known alternative start codon in other transcripts and ≥1 pathogenic variant upstream of closest potential in-frame start codon Default Point Value: 2 Modification Type: Gene-specific,Strength Supporting Applies to: Deletions removing exon 1 only Duplications (≥1 exon and completely contained within the gene) disrupting the reading frame after the haem-binding domain (aa4934-Ter) AND presumed in tandem Default Point Value: 1 Modification Type: Gene-specific,Strength 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. VCEP Specifications: The combination of PP3, PM1 and PS1 should not be higher than 6 points. This rule does not apply to initiation codons Stand Alone Very Strong Strong For missense variants that do not affect splicing (SpliceAI ≤ 0.2): same amino acid change as a previously established pathogenic variant. For variants that affect splicing (SpliceAI > 0.2), refer to Table 3.⁶ Default Point Value: 4 Modification Type: Clarification Moderate For missense variants that do not affect splicing (SpliceAI ≤ 0.2): same amino acid change as a previously established likely pathogenic variant. For variants that affect splicing (SpliceAI > 0.2), refer to Table 3.⁶ Default Point Value: 2 Modification Type: Clarification,Strength Supporting For variants that affect splicing (SpliceAI > 0.2), refer to Table 3.⁶ Default Point Value: 1 Modification Type: Clarification,Strength 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. VCEP Specifications: PS2 and PM6 have been combined under PS2. See Table 2 for point system. The proposed SVI point recommendations for “phenotype consistent with gene but not highly specific” applies to PCG and “phenotype consistent with the gene but not highly specific and with high genetic heterogeneity” applies to other CYP1B1-related glaucoma phenotypes (JOAG, POAG, ASD). See Supplementary Material for phenotype list. Paternity and/or maternity of the non-carrier parent(s) need to be proven for confirmed de novo variants Both parents need to be clinically assessed and should not have a diagnosis of CYP1B1-related glaucoma phenotypes. If a parent has glaucoma or suspicious signs of glaucoma, the age at diagnosis and the phenotype should be considered before applying criteria due to the prevalence of JOAG/POAG in the population and the possibility of phenocopies. Decrease the strength of evidence by one level for de novo occurrences without an additional P/LP variant. Stand Alone Very Strong ≥ 4.0 points Default Point Value: 8 Modification Type: Disease-specific,Strength Strong ≥ 2.0 points Default Point Value: 4 Modification Type: Disease-specific Moderate ≥ 1.0 points Default Point Value: 2 Modification Type: Disease-specific,Strength Supporting ≥ 0.5 points Default Point Value: 1 Modification Type: Disease-specific,Strength Not Applicable
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. VCEP Specifications: The mechanism by which CYP1B1 variants cause the associated phenotype is a LoF mechanism. Cyp1b1-deficient mice show oxidative damage to trabecular meshwork cells, leading to apoptosis and increased IOP.²⁶,²⁷ CYP1B1 is an enzyme involved in the metabolism of different substrates, including retinol to retinal,²⁸ and steroid.³⁰ The underlying mechanism by which CYP1B1 variants cause glaucoma is currently unknown, however it is hypothesized that the reduction of the enzyme activity and protein abundance is linked to the potential role of CYP1B1 in the development and function of the eye. Although there is evidence connecting estrogen to glaucoma, the specific substrates implicated in the development of CYP1B1-associated glaucoma have not yet been characterised. Animal models of specific variants (e.g. knock in mice models) have not been reported at this stage. They would need to replicate the phenotype reported in humans (e.g. increase in IOP, degeneration of the optic nerve and/or damage to trabecular meshwork cells) in order to meet PS3. Previous functional evidence assessing the impact of CYP1B1 variants on the function of the protein have reported on reduced protein abundance, protein stability and enzymatic activity of different substrates. The Glaucoma VCEP followed the SVI recommendations from Brnich et al. when assessing functional assays to apply PS3 toward functional evidence.²⁹ The characteristics of each assay/study were extracted to assess whether they would meet minimum requirements (e.g. biological and/or technical replicates, positive and negative controls).²⁹ The methodologies were reviewed to establish which studies used the same methods and could therefore be combined for the purposes of assessing control data. The VCEP assessed all variants reported in published functional assays as part of the pilot phase to identify those classified as LB/B and LP/P without the use of functional studies. These variants was then used as validation controls to determine OddsPath as per Brnich et al.²⁹ to assess the level at which PS3 could be applied for each combination of assay/studies. Finally, studies were reviewed to assess if there were clear thresholds to differentiate abnormal vs normal readouts to distinguish LB/B from LP/P control variants. The review process included studies that measured CYP1B1 protein stability, protein abundance, and enzymatic activity for various substrates; 17β estradiol, ethoxyresorufin, retinol/retinoids, arachidonic acid and benzo[a]pyrene. The characteristics of each study reviewed is detailed in Table 5, the summary of the assays combined, validation controls, OddsPath and recommendations is available in Table 6 and the classification of variants with functional evidence in Table 7. In summary, there was quite some variability in term of study methodology. However, studies using the same methods (vectors, cell lines and detection methods) were combined when possible. There was also variability between the enzymatic activity of different substrates with the activity for each variant not always consistent between different substrates. A limited number of LB/B variants have been assessed by published studies overall, therefore PS3 could only be applied at a Supporting level at this stage. One study measured activity against benzol[a]pyrene-7R-trans-7,8-dihidrodiol to investigate the potential for increased cancer risk, due to the carcinogenic nature of this substrate produced during combustion. Although there is no known connection between this group of toxins and glaucoma, this assay tested variants observed in primary congenital glaucoma, had sufficient control variants and clearly distinguished LP/P from LB/B variants. Considering that none of the substrates assessed by the different studies have been directly implicated in the development of glaucoma, but that some studies showed clear demarcation of LB/B and LP/P validation control variants, the Glaucoma VCEP decided to apply PS3 to animal models that replicate the glaucoma phenotype as well as assays that show lack of enzymatic activity (regardless of substrate) OR low protein stability if it meets the following: It has an appropriate number of validation controls (level of evidence to be established based on validation controls and associated OddsPath). The results show clear differentiation between LP/P and LB/B variants to establish threshold (or include indeterminate results in OddsPath calculation). The assay includes both negative and positive controls as well as technical and/or biological replicates. Controls from the same general class of assays and with same methodology can be combined to calculate OddsPath. If the results from different assays are conflicting for a single variant, then the level of validation of each assay should be considered to decide whether the results from one assay can override the results from another. Ideally variants should be assessed against their background haplotype. Stand Alone Very Strong Default Point Value: 8 Strong Assays with OddsPath >18.7 as per the SVI recommendations Animal models that replicate the glaucoma phenotype Default Point Value: 4 Modification Type: Disease-specific,Gene-specific Moderate Assays with OddsPath >4.3 as per the SVI recommendations Default Point Value: 2 Modification Type: Disease-specific,Gene-specific,Strength Supporting Assays with OddsPath >2.1 as per the SVI recommendations As of 06/11/25, the following studies meet the criteria to apply PS3_supporting: Chavarria-Soley et al. 2008³¹, Pasutto et al. 2010³³ and Mammen et al. 2003³². Default Point Value: 1 Modification Type: Disease-specific,Gene-specific,Strength 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 Default Point Value: 8 Strong Default Point Value: 4 Moderate Default Point Value: 2 Supporting Default Point Value: 1 Not Applicable Comments: CYP1B1 variants cause autosomal recessive disorders associated with glaucoma. The number of probands with the variant will be addressed by PM3.
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. VCEP Specifications: Although the crystal structure of CYP1B1 has not yet been established, cytochrome P450 proteins have known conserved regions and CYP1B1 structure can be predicted by comparative modeling. CYP proteins are characterized by a highly conserved structural core divisible into α-rich and β-rich domains comprising ∼14 helices and four to six β-sheets. Their structure comprises five functional relevant regions: the haem-binding region, the meander-region, the hinge-region, residues in alpha-helices (stabilization and interaction 3D) and residues in beta-sheets.¹²,¹³ The Glaucoma VCEP analysed the presence of known pathogenic and benign variants in each of the characterized domains and helices and supporting functional evidence²,¹¹ and decided to apply PM1 to the Hinge region (aa51-61) and the haem-binding domain (aa 463-472) which are critical and well-established functional domains without benign variation. Moreover, the whole L-helix region (aa 460-493) was included as opposed to the smaller haem-binding domain as there are functional variants detected in the larger region and the whole region is important for protein function. The Meander region (aa 437-445) was excluded as containing benign variation. The G helix (aa 253-282) and I helix (aa 339-365) do not have benign variation. These are important for substrate binding and contain evaluated functional variants. PM1_Supporting applies to variants in these helices as they are less well defined in the context of CYP1B1 (as opposed to other CYPs). The combination of PP3 and PM1 should not be higher than 4 points, the combination of PP3, PM1 and PM5 should not be higher than 5 points, and the combination of PP3, PM1 and PS1 should not be higher than 6 points. Stand Alone Very Strong Strong Default Point Value: 4 Moderate Missense variants and in-frame indels in the hinge region (aa51-61) or the L-helix including the haem-binding domain (aa460-493). Default Point Value: 2 Modification Type: Gene-specific Supporting Missense variants and in-frame indels in the G helix (aa253-282) or the I helix (339-365). Default Point Value: 1 Modification Type: Gene-specific,Strength 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. VCEP Specifications: The highest allele frequency in population databases should be used. Only applies to populations of ≥ 2,000 alleles. PM2 should be used at a Supporting level as per the SVI recommendations. The Whiffin/Ware calculator¹⁴ for autosomal recessive disorders was used to obtain a population allele frequency threshold for PM2 using the lowest prevalence reported in populations (Europeans). The prevalence of PCG in European populations is among the lowest. MacKinnon et al. previously reported a disease frequency for PCG in Australia at 1/30,000.¹⁵ Dimasi et al. identified biallelic CYP1B1 variants in 22% of families with a maximum allelic contribution of 19%.¹⁶ Based on these figures and setting the penetrance at 100%, the maximum credible allele frequency calculated was 0.000515. Stand Alone Very Strong Strong Moderate Default Point Value: 2 Supporting Allele frequency ≤ 0.0005 in population databases. Default Point Value: 1 Modification Type: Gene-specific Not Applicable Comments: The current ACMG guidelines for PM2 are that variants should be absent from controls. However, carriers are expected in population databases. Additionally, population-specific contributions of CYP1B1 variants and prevalence makes it difficult to determine an adequate threshold for PM2.
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. VCEP Specifications: See Table 1 for point system for PM3. The Glaucoma VCEP decided to follow the SVI recommendations with some modifications. SVI recommendations are that variants should meet PM2 to ensure that they are rare. Since some known pathogenic variants do not meet PM2, instead the Glaucoma VCEP specified that variants assessed for PM3 must not meet BS1. Variants need to meet PM4 (for in-frame indels) or PVS1 at any strength (for null variants) and must not meet BS1 or BP4. Affected is defined as a diagnosis of PCG, ASD, JOAG or POAG (see Supplementary Material for phenotype list). 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. The variant on the other allele must have been classified following these VCEP specifications. Multiple probands from different studies can be counted if they are independent. However, the number of points given to multiple compound heterozygous cases that have the same genotype has been capped to two when the variants are not confirmed in trans to avoid counting evidence if the variants are actually in cis and inherited together. The number of points per proband if homozygosity occurrence is due to consanguinity has been decreased to 0.25 points (max points 0.5) to account for homozygous variants being present by chance in consanguineous couples. Testing of one parent (or an unaffected first-degree relative carrying one of the two variants) is sufficient to confirm in trans for compound heterozygous cases. Parental testing is not required for homozygous cases. Individuals with multiple VUS/LP/P variants cannot be considered as evidence of either variant when the phase is unknown or for multiple variants in cis. Stand Alone Very Strong ≥ 4.0 points Default Point Value: 8 Modification Type: General recommendation,Strength Strong ≥ 2.0 points Default Point Value: 4 Modification Type: General recommendation,Strength Moderate ≥ 1.0 points Default Point Value: 2 Modification Type: General recommendation Supporting ≥ 0.5 points Default Point Value: 1 Modification Type: General recommendation,Strength 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 Default Point Value: 4 Moderate Stop loss variants are not a known disease mechanism, therefore PM4 does not apply to that variant type Default Point Value: 2 Modification Type: Gene-specific Supporting Default Point Value: 1 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. VCEP Specifications: The novel change must not affect splicing (SpliceAI ≤ 0.2), must meet PP3 and have a Grantham score equal or greater than the previously established pathogenic or likely pathogenic variant. The combination of PP3, PM1 and PM5 should not be higher than 5 points. Stand Alone Very Strong Strong Same residue as 2 previously established pathogenic variants (in which case both variants must be assessed independently of PM5). Default Point Value: 4 Modification Type: Clarification,Strength Moderate Same residue as previously established pathogenic variant (assessed independently of PM5) or 2 previously established likely pathogenic variants (in which case both variants must be assessed independently of PM5). Default Point Value: 2 Modification Type: Clarification Supporting Same residue as previously established likely pathogenic variant (assessed independently of PM5). Default Point Value: 1 Modification Type: Clarification,Strength 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. VCEP Specifications: PS2 and PM6 are a combined category representing confirmed vs assumed de novo respectively. See Table 4 for point system. The proposed SVI point recommendations for “phenotype consistent with gene but not highly specific” applies to PCG and “phenotype consistent with the gene but not highly specific and with high genetic heterogeneity” applies to other CYP1B1-related glaucoma phenotypes. Paternity and/or maternity of the non-carrier parent(s) need to be proven for confirmed de novo variants Both parents need to be clinically assessed and should not have a diagnosis of CYP1B1-related glaucoma phenotypes. If a parent has glaucoma or suspicious signs of glaucoma, the age at diagnosis and the phenotype should be considered before applying criteria due to the prevalence of JOAG/POAG in the population and the possibility of phenocopies. Decrease the strength of evidence by one level for de novo occurrences without an additional P/LP variant. Stand Alone Very Strong Default Point Value: 8 Strong Default Point Value: 4 Moderate Default Point Value: 2 Supporting Default Point Value: 1 Not Applicable Comments: Refer to 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. VCEP Specifications: See Table 4 for number of segregations for each level of strength. Affected segregations are affected family members in whom compound heterozygous (in trans) or homozygous variants segregate. Affected is defined as a diagnosis of PCG, ASD, JOAG or POAG (see Supplementary Material for phenotype list). Use caution and consider phenotypes of the affected segregations, age at diagnosis and pedigree structure before applying to segregations with POAG due to the risk of phenocopy. Segregations from multiple families can be added. Unaffected family members are individuals at risk to inherit the two variants identified in the proband and are either wild-type for both variants or a heterozygous carrier for a single variant.¹⁷ The variant in trans needs to be independently assessed and classified as VUS/LP/P. Stand Alone Very Strong Strong ≥ 3 affected segregations or 2 affected segregations AND ≥ 3 unaffected segregations or 1 affected segregation AND ≥ 8 unaffected segregations. Default Point Value: 4 Modification Type: Clarification,Strength Moderate 2 affected segregations or 1 affected segregation AND ≥ 5 unaffected segregations or ≥ 10 unaffected segregations. Default Point Value: 2 Modification Type: Clarification,Strength Supporting 1 affected segregation or ≥ 5 unaffected segregations. Default Point Value: 1 Modification Type: Clarification Not Applicable
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 Default Point Value: 1 Not Applicable Comments: Although pathogenic missense variants are common in CYP1B1, the gene also has a significant amount of benign missense variants as shown by the missense constraint z score in gnomAD (z = -0.75) supporting tolerance to variation.
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. VCEP Specifications: The Glaucoma VCEP recommended using only one in silico predictor, in line with a recent study showing a lower rate of concordance when multiple software is used.¹⁹ Follow the SVI recommendations¹⁸ for the REVEL thresholds calculated for the different levels of evidence. Apply the highest level of strength met. The combination of PP3 and PM1 should not be higher than 4 points, the combination of PP3, PM1 and PM5 should not be higher than 5 points, and the combination of PP3, PM1 and PS1 should not be higher than 6 points. Stand Alone Very Strong Strong For missense variants: REVEL score of ≥ 0.932 Default Point Value: 4 Modification Type: Gene-specific,Strength Moderate For missense variants: REVEL score of 0.773-0.931 Default Point Value: 2 Modification Type: Gene-specific,Strength Supporting For missense variants: SpliceAI ≥ 0.2 OR REVEL score of 0.644-0.772 For all other variants located outside of donor/acceptor ±1,2 dinucleotide positions, when splicing assay is not available: SpliceAI ≥ 0.2 Default Point Value: 1 Modification Type: Gene-specific 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 Default Point Value: 4 Moderate Default Point Value: 2 Supporting Default Point Value: 1 Not Applicable Comments: The phenotype associated with CYP1B1 variants is not highly specific and there is genetic heterogeneity.
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. VCEP Specifications: The highest allele frequency in population databases should be used. Variant must be present in ≥ 5 alleles in any validated general continental population dataset of at least 2,000 observed alleles. The Whiffin/Ware calculator¹⁴ for autosomal recessive disorders was used to obtain a population allele frequency threshold for maximum credible allele frequency using the population with the highest ever reported genetic contribution and prevalence for the disease in a small population with founder effect (Romani people in Slovakia). The prevalence of PCG in Romani people in Slovakia was reported at 1/2,210.²¹ A single CYP1B1 variant was reported to account for all families in this population due to founder effect (penetrance, genetic and allelic contribution 100%).²⁰ Based on these figures, the maximum credible allele frequency calculated was 0.0213. However, the VCEP revised the threshold for BA1 to 0.05 based on justification below. It should be noted that populations with the highest incidences of PCG (India, Pakistan, Romani populations) are currently not well represented in gnomAD v4. Therefore, the allele frequency of some potential pathogenic variants in populations with high incidence remains uncertain. Based on gnomAD v4.1.0, only two variants currently in ClinVar have a highest allele frequency between 0.02 and 0.05: A443G (0.04963) and R368H (0.03079). There is currently insufficient evidence to classify R368H as either benign or pathogenic. It has multiple conflicting interpretations in ClinVar ranging from Benign to Pathogenic ((P (4); LP (2); VUS (11); B (1); LB (2)), highlighting the complexity of its role. LB/B classifications come from the fact that it has been considered too common in some populations with high occurrence of the phenotype (South Asian 0.0379, Middle Eastern 0.02343), while accounting for a minority of cases in Saudi Arabia²² and around 30-50% of cases in South Asia.²³,²⁴ Published evidence does not support an incomplete or low penetrance for this variant. Additionally, functional evidence (meeting criteria for PS3_Supporting) supports an impact of the variant on the protein, and computational evidence (PP3), cases, segregations (PP1_Strong) and de novo evidence (PS2_Supporting) support a pathogenic impact. An exclusion rule from BA1 is not appropriate as it would disregard its high frequency in some populations. Based on the fact that this variant would meet BA1 if the threshold was set at 0.02, and that the allele frequency of potential pathogenic variants in high incidence populations is not well represented in current population datasets available, the threshold for BA1 was set at 0.05 until more evidence becomes available to reassess it. Stand Alone Allele frequency ≥ 0.05 in population databases. Default Point Value: Not Applicable Modification Type: Clarification 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. VCEP Specifications: The highest allele frequency in population databases should be used. The variant must be present in ≥ 5 alleles in any validated general continental population dataset of at least 2,000 observed alleles. The Whiffin/Ware calculator for autosomal recessive disorders was used to obtain a population allele frequency threshold for BS1 using the population with the highest genetic contribution and prevalence for the disease in a well-defined population (Saudi Arabia).¹⁴ The prevalence of PCG in the Saudi Arabian population is among the highest. Abouelhoda et al. recently estimated the disease frequency for PCG in Saudi Arabia at 1/4,500.²⁵ Alsaif et al. published on the largest PCG cohort from Saudi Arabia: biallelic CYP1B1 variants accounted for 77% of families with a penetrance of 91% and the most common variant was p.G61E which accounted for 76% of families attributable to CYP1B1 variants.²² Based on these figures, the maximum credible allele frequency calculated was 0.01. Stand Alone Very Strong Strong Allele frequency is ≥ 0.01 (1%) in population databases. Default Point Value: -4 Modification Type: Disease-specific Moderate Default Point Value: -2 Supporting Default Point Value: -1 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 Default Point Value: -4 Moderate Default Point Value: -2 Supporting Default Point Value: -1 Not Applicable Comments: CYP1B1 variants can have an incomplete penetrance and late age of onset. Adults with known pathogenic homozygous CYP1B1 variants who had a normal eye examination have been reported.
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. VCEP Specifications: The Glaucoma VCEP agreed to follow the SVI recommendations from Brnich et al. when assessing functional assays to apply BS3 toward functional evidence.⁶ The group decided to classify all the variants reported in published functional assays without the use of functional studies in the pilot phase, find out those that are classified as LB/B and LP/P, assess if the functional evidence support the classification or not, and decide whether the functional studies would meet BS3. Stand Alone Very Strong Strong Default Point Value: -4 Moderate Default Point Value: -2 Supporting Default Point Value: -1 Not Applicable Comments: Given that normal protein abundance and stability does not rule out impact on enzymatic activity, and that normal enzymatic activity for one substrate is not indicative of other substrates, the Glaucoma VCEP decided to not apply BS3.
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-segregations with PCG, ASD or JOAG. Use caution and consider phenotypes of the affected segregations, age at diagnosis and pedigree structure before applying to non-segregations with JOAG or POAG. Default Point Value: -4 Modification Type: Clarification Moderate Default Point Value: -2 Supporting Default Point Value: -1 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 Default Point Value: -4 Moderate Default Point Value: -2 Supporting Default Point Value: -1 Not Applicable Comments: Both truncating and missense CYP1B1 variants are causative.
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 Default Point Value: -4 Moderate Default Point Value: -2 Supporting Default Point Value: -1 Not Applicable Comments: Two missense variants in cis could act synergistically or the effect of a variant occurring after a truncating variant may not be predicted.
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 Default Point Value: -4 Moderate Default Point Value: -2 Supporting Default Point Value: -1 Not Applicable Comments: CYP1B1 does not have a repetitive region without a known function.
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. VCEP Specifications: Similar to PP3, The Glaucoma VCEP decided to follow the SVI recommendations¹⁸ to apply the REVEL thresholds calculated for the different levels of evidence and to use SpliceAI for variants located outside of donor/acceptor ±1,2 dinucleotide positions.⁶ Apply the highest level of strength met. Stand Alone Very Strong Strong For missense variants: SpliceAI ≤ 0.1 AND REVEL score of ≤ 0.016 Default Point Value: -4 Modification Type: Gene-specific,Strength Moderate For missense variants: SpliceAI ≤ 0.1 AND REVEL score of 0.017-0.183 Default Point Value: -2 Modification Type: Gene-specific,Strength Supporting For missense variants: SpliceAI ≤ 0.1 AND REVEL score of 0.184-0.290 For all other variants (not meeting PVS1 or PM4) located outside of donor/acceptor ±1,2 dinucleotide positions, when splicing assay is not available: SpliceAI ≤ 0.1 Default Point Value: -1 Modification Type: Gene-specific 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 Default Point Value: -4 Moderate Default Point Value: -2 Supporting Default Point Value: -1 Not Applicable Comments: Multiple molecular diagnoses are possible and variants in different genes could have an additive effect.
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 Default Point Value: -4 Moderate Default Point Value: -2 Supporting Applies to intronic/noncoding variants outside the donor/ acceptor splice region (intronic variants at or beyond positions +7/-21) and synonymous (silent) exonic variants located outside of the first and the last 3 bases of the exon if BP4 is met. Default Point Value: -1 Modification Type: Clarification Not Applicable

Point Based Variant Classification Categories

Category	Point Ranges
Pathogenic	10
Likely Pathogenic	6 - 9
Uncertain Significance	-1 - 5
Likely Benign	-6 - -2
Benign	-7

Additional Notes : PVS1_SA is stand alone for Pathogenic

Files & Images

Table 3: PS1 code weights for variants with the same predicted splicing event as a known (likely) pathogenic variant

Table 2: Point system for PS2:

Figure 2: PVS1 decision tree: a. NMD prediction based on the premature termination codon not occurring in the 3′ most exon or the 3′-most 50bp of the penultimate exon. b. Relevant domain indicated by experimental evidence proving a critical role of the domain and/or presence of non-truncating pathogenic variants in the region. c. The role of the region in protein function is unknown, loss-of-function variants in this exon are not frequent in the general population, and the variant removes <10% of the protein.

Supplementary Material: CYP1B1-related diseases:

Table 4: Recommendations for counting segregations:

Table 1: Point system for PM3:

Figure 1: Schematic diagram of CYP1B1.: The boxes represent the 3 exons with bigger boxes for coding exons 2 & 3. The haem-binding domain (aa460-493) is represented in red. Amino acid 330 marks the site where NMD is predicted to be activated (last 50bp of exon 2). Amino acid 489 marks the last 10% of the protein. NMD: nonsense-mediated decay.

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