ClinVar Genomic variation as it relates to human health

The p.Thr383IlefsX13 (or p.Thr383fsX) (NM_019098.4 c.1148delC) variant in CNGB3 has been reported in >100 individuals with achromatopsia and was the most common CNGB3 variant identified in patients with this disease (Kohl 2003, Sundin 2010) . The reported patients were either homozygous or compound heterozygous with ano ther CNGB3 variant. This variant has been identified in 0.3% (187/66554) of Euro pean chromosomes by the Exome Aggregation Consortium (ExAC, http://exac.broadins titute.org; dbSNP rs397515360). Please note that for diseases with recessive inh eritance, clinical variability, or reduced penetrance pathogenic variants may be present at a low frequency in the general population. This variant is predicted to cause a frameshift, which alters the protein?s amino acid sequence beginning at position 383 and leads to a premature termination codon 13 amino acids downs tream. This alteration is then predicted to lead to a truncated or absent protei n. Loss of function of the CNGB3 gene is an established disease mechanism in ind ividuals with achromatopsia. In summary, this variant meets our criteria to be c lassified as pathogenic for achromatopsia in an autosomal recessive manner based upon its segregation in affected individuals and predicted impact on protein fu nction.

This variant was classified as: Pathogenic. The following ACMG criteria were applied in classifying this variant: PVS1,PS3,PM3,PP4,PP5.

The variant is observed at an extremely low frequency in the gnomAD v2.1.1 dataset (total allele frequency: 0.175%). Frameshift: predicted to result in a loss or disruption of normal protein function through nonsense-mediated decay (NMD) or protein truncation. Multiple pathogenic variants are reported downstream of the variant. The variant has been reported at least twice as pathogenic with clinical assertions and evidence for the classification (ClinVar ID: VCV000005225). Therefore, this variant is classified as pathogenic according to the recommendation of ACMG/AMP guideline.

Clinical significance based on ACMG v2.0

The CNGB3 c.1148delC (p.Thr383IlefsTer13) variant results in a frameshift and is predicted to result in a premature termination of the protein. The p.Thr383IlefsTer13 variant has not been reported in the literature in individuals with Stargardt disease but is described as the most common pathogenic variant accounting for over 70% of all CNGB3 disease-causing alleles and approximately 40% of all achromatopsia-associated alleles (Wiszniewski et al 2007; Aboshiha et al. 2016). Across a selection of the available literature the p.Thr383IlefsTer13 variant is found in a total of 406 patients including 112 in a homozygous state, 41 in a compound heterozygous state, and eight in a heterozygous state (Sundin et al. 2000; Nishiguchi et al. 2005; Kohl et al. 2005; Wiszniewski et al. 2007). The p.Thr383IlefsTer13 variant showed segregation with disease. The variant was absent from 146 controls but is reported at a frequency of 0.00339 in the European American population of the Exome Sequencing Project. Wiszniewski et al. (2007) used haplotype analysis to demonstrate a founder effect among individuals of European ancestry for the variant thus explaining the high frequency. Functional studies showed that the p.Thr383IlefsTer13 variant resulted in a gain-of-function with enhanced channel activity and an increased sensitivity to cell death compared to wild type (Bright et al. 2005; Liu et al. 2013). Expression studies in Xenopus oocytes demonstrated that the p.Thr383IlefsTer13 variant failed to produce a CNGB3 subunit sufficient for normal cone photoreceptor function, and is essentially a null variant (Peng et al. 2003). Due to the potential impact of frameshift variants and the collective evidence from the literature, the p.Thr383IlefsTer13 variant is classified as a pathogenic variant for CNGB3-related disorders. This variant was observed by ICSL as part of a predisposition screen in an ostensibly healthy population.

This variant was identified as homozygous

Based on the classification scheme VCGS_Germline_v1.3.4, this variant is classified as Pathogenic. Following criteria are met: 0103 - Loss of function and gain of function are known mechanisms of disease in this gene and are associated with achromatopsia 3 (MIM#262300) (PMIDs: 16379026, 23805033). (I) 0106 - This gene is associated with autosomal recessive disease. (I) 0201 - Variant is predicted to cause nonsense-mediated decay (NMD) and loss of protein (premature termination codon is located at least 54 nucleotides upstream of the final exon-exon junction). (SP) 0251 - This variant is heterozygous. (I) 0304 - Variant is present in gnomAD (v2) <0.01 for a recessive condition (489 heterozygotes, 2 homozygotes). The high prevalence of this variant is due to a founder effect in European populations (PMID: 17265047). (SP) 0701 - Other NMD predicted variants comparable to the one identified in this case have very strong previous evidence for pathogenicity (DECIPHER). (SP) 0801 - This variant has strong previous evidence of pathogenicity in unrelated individuals. This variant has been observed in many individuals with achromatopsia in both homozygous and compound heterozygous states, and is thought to account for over 70% of all CNGB3-related achromatopsia cases (ClinVar, PMIDs: 25770143, 17265047). (SP) 1206 - This variant has been shown to be paternally inherited in a research setting. (I) Legend: (SP) - Supporting pathogenic, (I) - Information, (SB) - Supporting benign

Frameshift variant predicted to result in protein truncation or nonsense mediated decay in a gene for which loss-of-function is a known mechanism of disease; Published functional studies demonstrate that if a truncated CNGB3 protein is produced, then the resultant CNG channel complex leads to abnormal cone photoreceptor function (Peng et al., 2003; Liu et al., 2013); This variant is associated with the following publications: (PMID: 15657609, 17265047, 28041643, 22975760, 12815043, 16379026, 23805033, 20079539, 10888875, 28929832, 28341476, 29053603, 28746191, 29769798, 30609409, 30337596, 30190494, 30718709, 30418171, 31980526, 32036094, 32581362, 33546218, 32860008, 15712225, 28166811, 14757870, 34426522, 33851411, 32531858, 33562422, 33737949, 32037395)

Variant summary: CNGB3 c.1148delC (p.Thr383IlefsX13) results in a premature termination codon, predicted to cause a truncation of the encoded protein or absence of the protein due to nonsense mediated decay, which are commonly known mechanisms for disease. This variant is a commonly known pathogenic variant. The variant allele was found at a frequency of 0.0017 in 250592 control chromosomes in the gnomAD database, including 2 homozygotes. c.1148delC has been reported in the literature in multiple individuals affected with Achromatopsia (eg. Michaelides_2004, Thiadens_2009, etc). 24 clinical diagnostic laboratories have submitted clinical-significance assessments for this variant to ClinVar after 2014 without evidence for independent evaluation. All labs classified the variant as pathogenic/likely pathogenic. Based on the evidence outlined above, the variant was classified as pathogenic.

This sequence change creates a premature translational stop signal (p.Thr383Ilefs*13) in the CNGB3 gene. It is expected to result in an absent or disrupted protein product. Loss-of-function variants in CNGB3 are known to be pathogenic (PMID: 28795510). This variant is present in population databases (rs397515360, gnomAD 0.3%), and has an allele count higher than expected for a pathogenic variant. This premature translational stop signal has been observed in individual(s) with achromatopsia (PMID: 10888875, 10958649, 15657609, 15712225, 17265047). 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: 5225). For these reasons, this variant has been classified as Pathogenic.

The c.1148delC (p.T383Ifs*13) alteration, located in exon 10 (coding exon 10) of the CNGB3 gene, consists of a deletion of one nucleotide at position 1148, causing a translational frameshift with a predicted alternate stop codon after 13 amino acids. This alteration is expected to result in loss of function by premature protein truncation or nonsense-mediated mRNA decay. Based on data from gnomAD, this alteration has an overall frequency of 0.18% (493/281708) total alleles studied. The highest observed frequency was 0.28% (71/25066) of European (Finnish) alleles. This is a common, recurrent mutation identified in multiple unrelated patients with achromatopsia in both the homozygous and compound heterozygous state (Sundin, 2000; Kohl, 2005; Wiszniewski, 2007; Mayer, 2017). The nucleotide position is prone to deletion and represents a mutational hotspot (Kohl, 2000). Based on the available evidence, this alteration is classified as pathogenic.

CNGB3: PVS1, PM2, PM3:Supporting, PS3:Supporting

Undetermined rare ocular disorder with frequency of less than eight patients

The CNGB3 c.1148delC variant is predicted to result in a frameshift and premature protein termination (p.Thr383Ilefs*13). This variant has been reported many times as causative for autosomal recessive achromatopsia (see for examples Kohl et al. 2005. PubMed ID: 15657609; Nishiguchi et al. 2005. PubMed ID: 15712225; Wiszniewski et al. 2007. PubMed ID: 17265047). This variant is reported in 0.28% of alleles in individuals of European (Finnish) descent and with a global allele frequency of 0.18% in gnomAD, indicating this variant is relatively common. Frameshift variants in CNGB3 are expected to be pathogenic. Given the evidence, we interpret this variant as pathogenic.