ClinVar Genomic variation as it relates to human health

Variant classified as Uncertain Significance - Favor Benign. The p.Arg368His var iant in CYP1B1 has been reported in the homozygous or compound heterozygous stat e in several individuals of varying ethnic backgrounds with primary congenital g laucoma (Bejjani 2000, Reddy 2003, Chitsazian 2007, de Melo 2015, Rauf 2016, Yan g 2017) and functional studies suggest an impact to enzyme function (Choudhary 2 008, Pasutto 2010, Mookherjee 2012, Kabra 2017). However, this variant has also been identified in the homozygous or compound heterozygous state in at least 8 unaffected individuals (Bejjani 2000, Alsaif 2018) and has been identified in 3. 1% (939/30622) of South Asian chromosomes, 2.2% (222/10030) of Ashkenazi Jewish chromosomes, and 13 homozygotes in the Genome Aggregation Database (gnomAD, http ://gnomad.broadinstitute.org). It was also identified at an allele frequency of 1.6% in a Saudi Arabian population cohort and 2.4% in a Qatari population cohort (Alsaif 2018, Fakhro 2016). These allele frequencies are higher than the maximu m expected allele frequency for a pathogenic variant in the CYP1B1 gene. In summ ary, the clinical significance of the p.Arg368His variant is uncertain due to co nflicting evidence. ACMG/AMP criteria applied: PM3_VeryStrong, PS3_Moderate, PP3 , BA1, BS2.

Our patient is a girl, heterozygous for a maternally inherited pathogenic missense variant (NM_000104.3:c.1103G>A; p.Arg368His) in the CYP1B1 gene, whose mutations are the most common cause of primary congenital glaucoma (Qashqai et al., 2018). However, this pathogenic variant alone is not sufficient to explain her phenotype, as glaucoma due to CYP1B1 mutations is typical of an autosomal recessive inheritance (Reis et al., 2016).

This variant was classified as: Uncertain significance. The following ACMG criteria were applied in classifying this variant: PS1,PP3.

Variant summary: CYP1B1 c.1103G>A (p.Arg368His) results in a non-conservative amino acid change in the encoded protein sequence. Five of five in-silico tools predict a damaging effect of the variant on protein function. The variant allele was found at a frequency of 0.0058 in 247034 control chromosomes, predominantly at a frequency of 0.03 within the South Asian subpopulation in the gnomAD database, including 13 homozygotes. The observed variant frequency within South Asian control individuals in the gnomAD database is approximately 7-fold of the estimated maximal expected allele frequency for a pathogenic variant in CYP1B1 causing Primary Congenital Glaucoma phenotype (0.0043), strongly suggesting that the variant is a benign polymorphism found primarily in populations of South Asian origin. c.1103G>A has been reported in the literature in individuals affected with Primary Congenital Glaucoma, primary open angle glaucoma, or anterior segment developmental anomalies including glaucoma, as a biallelic genotype with reduced penetrance (e.g. Bejjani_2000, Tanwar_2009, Kaur_2018, Patel_2019, Zavarzadeh_2022, Lin_2024). One study examining the variant in a large cohort of affected individuals and their unaffected family members found the variant was only associated with a penetrance of 23% in the compound heterozygous and homozygous states and that the variant had a similar or reduced frequency in their affected cohort compared to other ethnically similar cohorts of unaffected individuals (Alsaif_2019). Therefore, these reports do not provide unequivocal conclusions about association of the variant with Primary Congenital Glaucoma. Several publications report experimental evidence evaluating an impact on protein function, with the most pronounced variant effect resulting in <10% of normal activity (e.g. Choudhary_2008, Pasutto_2010, Mookherjee_2012, Banerjee_2016). The following publications have been ascertained in the context of this evaluation (PMID: 29556725, 27243976, 10655546, 18622259, 30788381, 38219857, 23028769, 19643970, 30653986, 19536304, 36239105). ClinVar contains an entry for this variant (Variation ID: 7739). Based on the evidence outlined above, the variant was classified as uncertain significance.

Across a selection of the available literature, the CYP1B1 c.1103G>A (p.Arg368His) missense variant has been reported in a total of 78 individuals with a spectrum of eye disorders. Within this group, the p.Arg368His variant was identified in a homozygous state in 31 individuals, in a compound heterozygous state in 17 individuals, and in a heterozygous state in 30 individuals. Primary congenital glaucoma was the most commonly described phenotype, however other forms of childhood-onset and adulthood-onset glaucoma, as well as Peter's anomaly, Rieger's anomaly, and a case of micropthalmia with iris and fundal colobomas were also reported (Bejjani et al. 2000; Panicker et al. 2002; Vincent et al. 2002; Reddy et al. 2003; Acharya et al. 2006; Chavarria-Soley et al. 2006; Vincent et al. 2006; Chitsazian et al. 2007; Dimasi et al. 2007; Kumar et al. 2007; Suri et al. 2009; Tanwar et al. 2009; Pasutto et al. 2010; Azmanov et al. 2011; Prokudin et al. 2014). The p.Arg368His variant was also found in a homozygous state in two unaffected individuals and in a compound heterozygous state in ten self-reported unaffected individuals (Bejjani et al. 2000; Suri et al. 2009). The p.Arg368His variant was reported in a heterozygous state in five of 1030 controls and is also reported at a frequency of 0.030660 in the South Asian population of the Exome Aggregation Consortium. This database also includes thirteen homozygotes. In vitro functional studies showed that the variant demonstrated reduced enzymatic activity (Pasutto et al. 2010; Mookherjee et al. 2012). Expression of the variant protein in E. coli resulted in approximately one-sixth of the amount of stable protein compared to wild type and severely reduced metabolism of all substrates tested (Choudhary et al. 2008). While the p.Arg368His variant shows a strong association with disease, the presence of the variant in unaffected individuals in a homozygous and compound heterozygous state, and the high frequency in the population database suggests the variant results in greatly reduced disease penetrance. However, based on the high number of patient cases and functional data, the p.Arg368His variant is classified as pathogenic for CYP1B1-related disorders. This variant was observed by ICSL as part of a predisposition screen in an ostensibly healthy population.

The p.Arg368His variant in CYP1B1 has been identified in at least 13 individuals with primary congenital glaucoma and in multiple unaffected individuals (PMID: 10655546, 19793111, 25091052, 19643970), but has also been identified in >2% of South Asian chromosomes and 10 homozygotes by ExAC (http://gnomad.broadinstitute.org/). In vitro functional studies provide some evidence that the p.Arg368His variant may impact protein function (PMID: 23028769, 19643970). However, these types of assays may not accurately represent biological function. In summary, the clinical significance of this variant is uncertain.

Based on the classification scheme VCGS_Germline_v1.3.4, this variant is classified as a VUS-3B. Following criteria are met: 0102 - Loss of function is a known mechanism of disease in this gene and is associated with congenital, juvenile, or adult onset primary open anglet glaucoma 3A (MIM#231300) and anterior segment dysgenesis 6, multiple subtypes (MIM#617315). (I) 0106 - This gene is associated with autosomal recessive disease. (I) 0112 - The condition associated with this gene has incomplete penetrance (PMID: 29556725). (I) 0200 - Variant is predicted to result in a missense amino acid change from arginine to histidine. (I) 0252 - This variant is homozygous. (I) 0306 - Variant is present in gnomAD (v2) >=0.03 and <0.05 for a recessive condition (1442 heterozygotes, 13 homozygotes). (SB) 0309 - An alternative amino acid change at the same position has been observed in gnomAD (v3) (31 heterozygotes, 0 homozygotes). (I) 0501 - Missense variant consistently predicted to be damaging by multiple in silico tools or highly conserved with a major amino acid change. (SP) 0604 - Variant is not located in an established domain, motif, hotspot or informative constraint region. (I) 0702 - Other missense variants comparable to the one identified in this case have strong previous evidence for pathogenicity. These alternative missense changes (p.Arg368Cys, p.Arg368Leu) have been reported in several homozygous individuals with congenital glaucoma (PMID: 29142762, PMID: 25580891, PMID: 17591938, PMID: 18852424, PMID: 26997785). (SP) 0808 - Previous reports of pathogenicity for this variant are conflicting. This variant has been classified once a likely benign, and more commonly as either a VUS or as pathogenic with incomplete penetrance. It has been reported in many homozygous and compound heterozygous individuals with various eye disorders, where publications recognise it as one of the most common variants in Iranian populations (ClinVar, LOVD, PMID: 32510024). However, a recent study observed the variant more commonly in a control cohort than in affected individuals, and concluded it’s unlikely to be disease causing (PMID: 29556725). (I) 1002 - This variant has moderate functional evidence supporting abnormal protein function. Transfected HEK293 cells have been demonstrated to have significantly reduced retinol and steroid enzyme activity and protein expression (PMID: 27243976, PMID: 19643970). (SP) 1208 - Inheritance information for this variant is not currently available in this individual. (I) Legend: (SP) - Supporting pathogenic, (I) - Information, (SB) - Supporting benign

Identified in the heterozygous and homozygous state in multiple individuals with primary congenital glaucoma in published literature, however, it has also been observed in the heterozygous and homozygous state in multiple unaffected individuals (Bejjani et al., 2000; Afzal et al., 2018; Gupta et al., 2018); Published functional studies demonstrate a damaging effect with lower 17 beta-estradiol metabolizing activity comparing to wild type, although the stability was similar to wild type (Choudhary et al., 2008; Mookherjee et al., 2012; Banerjee et al., 2016); In silico analysis supports that this missense variant has a deleterious effect on protein structure/function; This variant is associated with the following publications: (PMID: 25091052, 19236111, 12036985, 16735994, 15475877, 27243976, 27508083, 19204079, 21081970, 30788381, 24281366, 18622259, 23028769, 19793111, 24123366, 26489929, 16688110, 16490498, 11980847, 11774072, 10655546, 26094658, 25333069, 19643970, 27535533, 28620713, 28384041, 14507861, 29556725, 30479709, 29522511, 17591938, 17718864, 30108387, 30270463, 29168043, 19536304, 30609409, 30653986, 20827438, 30479704, 34426522, 15037581, 33726816, 35085548)

This sequence change replaces arginine, which is basic and polar, with histidine, which is basic and polar, at codon 368 of the CYP1B1 protein (p.Arg368His). This variant is present in population databases (rs79204362, gnomAD 3%), including at least one homozygous and/or hemizygous individual. This missense change has been observed in individual(s) with glaucoma (PMID: 10655546, 11980847, 15037581, 19744731, 30108387; Invitae). In at least one individual the data is consistent with being in trans (on the opposite chromosome) from a pathogenic variant. It has also been observed to segregate with disease in related individuals. ClinVar contains an entry for this variant (Variation ID: 7739). Advanced modeling of protein sequence and biophysical properties (such as structural, functional, and spatial information, amino acid conservation, physicochemical variation, residue mobility, and thermodynamic stability) has been performed at Invitae for this missense variant, however the output from this modeling did not meet the statistical confidence thresholds required to predict the impact of this variant on CYP1B1 protein function. Experimental studies have shown that this missense change affects CYP1B1 function (PMID: 18622259, 19643970, 23028769, 27243976). This variant disrupts the p.Arg368 amino acid residue in CYP1B1. Other variant(s) that disrupt this residue have been determined to be pathogenic (Invitae). This suggests that this residue is clinically significant, and that variants that disrupt this residue are likely to be disease-causing. For these reasons, this variant has been classified as Pathogenic.

The missense c.1103G>A p.Arg368His variant in CYP1B1 gene has been reported in multiple individuals affected with primary congenital glaucoma Kaur et al. 2018; Bashir et al. 2018. Functional studies have revealed a significant decrease in the relative enzyme activity of CYP1B1 due to the p.Arg368His variant supporting moderately abnormal protein function with transfected HEK293 cells leading to significantly reduced retinol and steroid enzyme activity and protein expression Pasutto et al. 2010; Banerjee et al. 2016. This variant is reported to be segregating with the disease in the related individuals Bashir et al. 2018. This variant is the most common variant in Indian populations, and is also present in multiple healthy individuals from the control population Bashir et al. 2018. The p.Arg368His variant is reported with an allele frequency of 0.6% 1438 heterozygotes and 13 homozygotes in the gnomAD exomes database. This variant has been reported to the ClinVar database as Pathogenic / Uncertain Significance multiple submissions. The amino acid change p.Arg368His in CYP1B1 is predicted as conserved by GERP++ and PhyloP across 100 vertebrates. The amino acid Arg at position 368 is changed to a His changing protein sequence and it might alter its composition and physico-chemical properties. For these reasons, this variant has been classified as a Variant of Uncertain Significance VUS.

CYP1B1: PS3:Moderate, BS1

The CYP1B1 c.1103G>A variant is predicted to result in the amino acid substitution p.Arg368His. This variant has been reported in the heterozygous and homozygous states in individuals with primary congenital glaucoma (Pasutto et al. 2010. PubMed ID: 19643970; Bejjani et al. 2000. PubMed ID: 10655546). A non-penetrant phenotype has been observed in a family homozygous for this variant (Bejjani et al. 2000. PubMed ID: 10655546). Additionally, digenic inheritance has been reported in an individual who was heterozygous for this variant and a pathogenic variant in the MYOC gene, which has also been associated with glaucoma (Vincent et al. 2002. PubMed ID: 11774072). Potential digenic inheritance has also been reported in two families who were heterozygous for the CYP1B1 c.1103G>A variant as well as a variant in the TEK gene, which has been associated with primary congenital glaucoma (Kabra et al. 2017. PubMed ID: 28620713). In functional studies, the p.Arg368His amino acid change resulted in substantially reduced estradiol and retinol metabolizing activity (Banerjee et al. 2016. PubMed ID: 27243976). Notably, other substitutions of the same amino acid (p.Arg368Cys and p.Arg368Leu) have been documented causative for glaucoma (HGMD - Human Gene Mutation Database). The CYP1B1 c.1103G>A variant has been registered in public databases with allele frequencies up to ~3%, including 13 homozygotes, which is likely too high to be consistent with highly penetrant pathogenic variant. This variant is also registered in the ClinVar database with conflicting interpretations of pathogenicity (https://www.ncbi.nlm.nih.gov/clinvar/variation/7739/). At this time, the clinical significance of this variant is uncertain due to the absence of conclusive functional and genetic evidence.

Variant interpreted as Pathogenic and reported on 07/06/2015 by Lab or GTR ID 1006. GenomeConnect assertions are reported exactly as they appear on the patient-provided report from the testing laboratory. GenomeConnect staff make no attempt to reinterpret the clinical significance of the variant.