ClinVar Genomic variation as it relates to human health

Variant summary: HBB c.93-22_95del25 is located in a canonical splice-site and is predicted to affect mRNA splicing resulting in a significantly altered protein due to either exon skipping, shortening, or inclusion of intronic material. Several computational tools predict a significant impact on normal splicing: five predict the variant abolishes a 3 prime acceptor site, which has been confirmed by a functional study (Orkin_1983). This variant was absent in 251322 control chromosomes (gnomAD). c.93-22_95del25 has been reported in the literature in individuals affected with Beta Thalassemia (Orkin_1983, Adekile_2015). These data indicate that the variant is likely to be associated with disease. Three clinical diagnostic laboratories have submitted clinical-significance assessments for this variant to ClinVar after 2014 without evidence for independent evaluation. All laboratories classified the variant as pathogenic. Based on the evidence outlined above, the variant was classified as pathogenic.

PVS1, PS3, PM2, PP4, PP5

Canonical splice site variant in a gene for which loss-of-function is a known mechanism of disease; Not observed at significant frequency in large population cohorts (gnomAD); This variant is associated with the following publications: (PMID: 6308558, 35467101, 6190800)

This variant results in the deletion of part of exon 2 (c.93-22_95del) of the HBB gene. It is expected to disrupt RNA splicing. Variants that disrupt the donor or acceptor splice site typically lead to a loss of protein function (PMID: 16199547), and loss-of-function variants in HBB are known to be pathogenic (PMID: 23637309). This variant is not present in population databases (gnomAD no frequency). This variant has been observed in individual(s) with beta-thalassemia (PMID: 6190800, 20437613, 23637309, 26076396). This variant is also known as IVS-1, 25bp del. ClinVar contains an entry for this variant (Variation ID: 15442). Studies have shown that this variant alters HBB gene expression (PMID: 6190800). For these reasons, this variant has been classified as Pathogenic.

The HBB c.93-22_95del variant (also known as IVS-1 25 bp del, HbVarID: 974, rs193922563) is reported in the literature in several individuals with beta-thalassemia (Adekile 2015, Miri-Moghaddam 2016, Orkin 1983, HbVar database). This variant was found in three siblings of one family, all of whom also carried an additional pathogenic variant (Adekile 2015). This variant is also reported in ClinVar (Variation ID: 15442). This variant is absent from the Genome Aggregation Database, indicating it is not a common polymorphism. This variant deletes 25 nucleotides and abolishes the canonical splice acceptor site of intron 1, which is likely to disrupt gene function. Indeed, RNA analyses of cells expressing this variant show a lack of properly spliced transcripts (Orkin 1983). Based on available information, this variant is considered to be pathogenic. References: Link to HbVar database: https://globin.bx.psu.edu/hbvar/menu.html Adekile AD et al. Clinical and Molecular Characteristics of Non-Transfusion-Dependent Thalassemia in Kuwait. Hemoglobin. 2015;39(5):320-6. PMID: 26076396. Miri-Moghaddam E et al. Molecular Characterization of ß-Thalassemia Intermedia in Southeast Iran. Hemoglobin. 2016 Jun;40(3):173-8. PMID: 27117567. Orkin SH et al. Inactivation of an acceptor RNA splice site by a short deletion in beta-thalassemia. J Biol Chem. 1983 Jun 25;258(12):7249-51. PMID: 6190800.

The c.93-22_95del25 variant in HBB has been reported in individuals affected with Beta Thalassemia (Orkin 1983 PMID: 6190800, Adekile 2015 PMID: 26076396, Al Gazali 2010 PMID: 20437613). It has also been reported in ClinVar (Variation ID 15415) and was absent from large population studies. This deletion encompasses the canonical splice site (+/- 1,2) and is predicted to cause altered splicing leading to an abnormal or absent protein. Loss of function of the HBB gene is an established disease mechanism in autosomal recessive beta thalassemia. In summary, this variant meets criteria to be classified as pathogenic for autosomal recessive beta thalassemia. ACMG/AMP Criteria applied: PM2_Supporting, PVS1, PM3.

The c.93-22_95del25 pathogenic mutation, located at the boundary of intron 1 and coding exon 2 of the HBB gene, results from a deletion of 25 nucleotides between nucleotide positions 93-22 and 95. This alteration is predicted to disrupt the canonical splice acceptor site sequence and result in the deletion of three nucleotides from coding exon 2. This alteration was reported in an Asian Indian individual with beta-thalassemia and was shown to abolish the normal splicing when expressed in HeLa cells (Orkin SH et al. J. Biol. Chem., 1983 Jun;258:7249-51). In addition to the clinical data presented in the literature, alterations that disrupt the canonical splice donor site are typically deleterious in nature, this alteration is interpreted as a disease-causing mutation.

The variant is not observed in the gnomAD v2.1.1 dataset. This variant was predicted to alter splicing and result in a loss or disruption of normal protein function. Multiple pathogenic loss-of-function 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: VCV000015442). Therefore, this variant is classified as Pathogenic according to the recommendation of ACMG/AMP guideline.

The invariant splice acceptor variant c.93-22_95del in HBB gene has been observed in compound heterozygous state in multiple individuals with beta-thalassemia Adekile et. al., 2015; Thein SL, 2013. This variant is also known as IVS-1, 25bp del. Studies have shown that this variant alters HBB gene expression Orkin et. al., 1983. The c.93-22_95del variant is novel not in any individuals in gnomAD Exomes and 1000 Genomes. This variant has been reported to the ClinVar database as Pathogenic by multiple submitters. This variant results in the deletion of part of exon 2 c.93-22_95del of the HBB gene. It is expected to disrupt RNA splicing. Variants that disrupt the donor or acceptor splice site typically lead to a loss of protein function Baralle et. al., 2005, and loss-of-function variants in HBB are known to be pathogenic Thein SL, 2013. For these reasons, this variant has been classified as Pathogenic.