ClinVar Genomic variation as it relates to human health

In vitro RNA and functional studies demonstrate a damaging effect with the creation of an alternative splice site causing abnormal gene splicing (Busslinger et al., 1981).; Also known as IVS-I-110G>A; This variant is associated with the following publications: (PMID: 6264391, 21797703, 25087612, 22975760, 28670940, 27979672, 31456457, 31130284, 21228398, 26372288, 1967205, 26016902, 1390250, 6895866, 6264477, 28391758, 28366028, 28667000, 14555304, 9163586, 31589614, 15609277, 9629495, 12702481, 2577233)

The heterozygous c.93-21G>A variant in HBB was identified by our study in four affected members of one family with beta thalassemia. The c.93-21G>A variant in HBB has been identified in over 160 individuals with beta thalassemia (‚Äã‚ÄãPMID: 6264477, PMID: 6264391, PMID: 24106605, PMID: 28391758, PMID: 1390250, PMID: 2200760), but has been identified in 0.03% (37/128830) of European (non-Finnish) chromosomes by the Genome Aggregation Database (gnomAD, http://gnomad.broadinstitute.org; dbSNP ID: rs35004220). Although this variant has been seen in the general population in a heterozygous state, its frequency is low enough to be consistent with a recessive carrier frequency. This variant has also been reported in ClinVar (Variation ID: 15454) and has been interpreted as pathogenic by multiple submitters. Of these 160 individuals, 138 were homozygotes (PMID: 1390250, PMID: 28391758, PMID: 24106605, PMID: 2200760), and 11 were compound heterozygotes who carried pathogenic or likely pathogenic variants in trans (PMID: 2200760, ClinVar Variation ID: 15436, ClinVar Variation ID: 15402, ClinVar Variation ID: 15457, ClinVar Variation ID: 15239; PMID: 6264391, ClinVar Variation ID: 15060; PMID: 24106605, ClinVar Variation ID: 15402, ClinVar Variation ID: 15415), which increases the likelihood that the c.93-21G>A variant is pathogenic. RT-PCR analysis of RNA from affected tissue shows evidence of altered splicing of exon 2, resulting in 19bp insertion, frameshift, and premature truncation (PMID: 16421096); altered splicing was also seen in HeLa cells (PMID: 6895866) and in a humanized mouse model (PMID: 16421096). This variant is located in the 3' splice region. Computational tools predict a splicing impact, though this information is not predictive enough to determine pathogenicity. In summary, this variant meets criteria to be classified as pathogenic for autosomal recessive beta thalassemia. ACMG/AMP Criteria applied: PS3, PM2_Supporting, PM3_VeryStrong (Richards 2015).

This sequence change falls in intron 1 of the HBB gene. It does not directly change the encoded amino acid sequence of the HBB protein. This variant is present in population databases (rs35004220, gnomAD 0.03%). This variant has been observed in individual(s) with HBB-related conditions (PMID: 1967205, 2200760, 28366028, 28391758). In at least one individual the data is consistent with being in trans (on the opposite chromosome) from a pathogenic variant. This variant is also known as IVS-I-110G>A. ClinVar contains an entry for this variant (Variation ID: 15454). Algorithms developed to predict the effect of sequence changes on RNA splicing suggest that this variant is not likely to affect RNA splicing. For these reasons, this variant has been classified as Pathogenic.

The HBB c.93-21G>A variant (rs35004220, ClinVar ID: 15454, HbVar ID: 827), also known as IVS-I-110 G>A, is reported in the literature in multiple individuals affected with beta+ thalassemia (Carrocini 2017, Hussain 2017, Jalilian 2017). This variant is one of the most common beta-thalassemia alleles in Mediterranean and Middle-Eastern countries (Kaplan 1990, HbVar database and references therein). Based on available information, this variant is considered to be pathogenic. References: Link to HbVar database: https://globin.bx.psu.edu/hbvar/hbvar.html Carrocini GCS et al. Mutational Profile of Homozygous beta-Thalassemia in Rio de Janeiro, Brazil. Hemoglobin. 2017 Jan;41(1):12-15. PMID: 28366028. Hussain A et al. Rare beta-Globin Gene Mutations in Pakistan. Hemoglobin. 2017 Mar;41(2):100-103. PMID: 28670940. Jalilian M et al. The Frequency of HBB Mutations Among beta-Thalassemia Patients in Hamadan Province, Iran. Hemoglobin. 2017 Jan;41(1):61-64. PMID: 28391758. Kaplan F et al. Beta-thalassemia genes in French-Canadians: haplotype and mutation analysis of Portneuf chromosomes. Am J Hum Genet. 1990 46(1):126-32. PMID: 1967205.

NM_000518.4(HBB):c.93-21G>A is classified as pathogenic and is associated with beta thalassemia. Sources cited for classification include the following: PMID 2200760, 1390250, 8330981, 1967205, 1390250, 2200760, 6264477, 6264391. Classification of NM_000518.4(HBB):c.93-21G>A is based on the following criteria: This is a well-established pathogenic variant in the literature that has been observed more frequently in patients with clinical diagnoses than in healthy populations. Please note: this variant was assessed in the context of healthy population screening.

This variant was identified as compound heterozygous.

Converted during submission to Pathogenic.

The variant has been observed in multiple (>3) similarly affected unrelated individuals (PMID: 28391758, 28366028, 2200760). It was observed to be in trans with the other variant (3billion dataset). The variant has been reported at least twice as pathogenic/likely pathogenic with clinical assertions and evidence for the classification (ClinVar ID: VCV000015454). It is observed at an extremely low frequency in the gnomAD v2.1.1 dataset (total allele frequency: 0.0001668). Therefore, this variant is classified as pathogenic according to the recommendation of ACMG/AMP guideline.

The c.93-21G>A variant in HBB, also known as IVS-I-110 G>A, has been reported in numerous individuals with beta-thalassemia and is one of the most common beta-thalassemia alleles in Mediterranean and Middle-Eastern countries (Kaplan 1990 PMID: 1967205, (Carrocini 2017 PMID: 28366028, Hussain 2017 PMID: 28670940, Jalilian 2017 PMID: 28391758, Baysal 1992 PMID: 1390250) https://globin.bx.psu.edu/cgi-bin/hbvar/query_vars3?mode=output&display_format=page&i=827). It has also been reported in ClinVar (Variation ID 15454) and has been identified in 14/68028 European chromosomes by gnomAD (https://gnomad.broadinstitute.org). In vitro functional studies support an impact on splicing (Busslinger 1981 PMID: 6895866). In summary, this variant meets criteria to be classified as pathogenic for autosomal recessive beta thalassemia. ACMG/AMP Criteria applied: PM2_Supporting, PS3_Moderate, PM3_very strong.

The c.93-21G>A pathogenic mutation (also known as IVS1-110G>A), located in coding intron 1 of the HBB gene, results from a G>A substitution 21 nucleotides before coding exon 2. This mutation was first described in an individual of Turkish Cypriot origin with severe thalassemia intermedia who was compound heterozygous for another pathogenic HBB mutation (Westaway D, Williamson R. Nucleic Acids Res. 1981;9:1777-1788). In a population of Turkish patients affected with beta thalassemia major, 33 individuals were homozygous for this pathogenic mutation and the allele frequency in some Turkish subpopulations is as high as 75% of disease alleles (Fettah A et al. Mediterr J Hematol Infect Dis. 2013;5(1):e2013055 and Baysal et al. Br J Haematol. 1992;81(4):607-9. ). RT-PCR studies from mRNA in both human samples and mouse models demonstrated the presence of aberrant splicing, with 90% of transcripts with abnormal splicing in homozygous mice (Vadolas J. J Biol Chem. 2006 Mar 17;281(11):7399-405). Based on the supporting evidence, c.93-21G>A is interpreted as a disease-causing mutation.

HBB: PM3:Very Strong, PM2, PP4

Based on the classification scheme VCGS_Germline_v1.3.4, this variant is classified as Pathogenic. The following criteria are met: 0102 - Loss of function is a known mechanism of disease in this gene and is associated with HBB-related hemoglobinopathies, including beta thalassemia (OMIM). Dominant negative is also a suggested mechanism (PMID: 29700171). (I) 0108 - This gene is associated with both recessive and dominant disease (OMIM). (I) 0115 - Variants in this gene are known to have variable expressivity. Variable severity has been reported in sickle cell patients carrying the same variants (PMID: 31788855). In addition, clinical severity of beta-thalassemia patients is also dependent on whether variants in HBB are heterozygous, homozygous or compound heterozygous, and the amount of residual protein that is expressed (PMID: 20301599). (I) 0217 - Non-coding variant with a suspected effect. Recombinant in vitro functional studies show this variant causes aberrant splicing resulting in a frameshift and premature termination codon, which ultimately results in severely reduced expression of beta-globin (PMID: 29700171). (SP) 0251 - This variant is heterozygous. (I) 0304 - Variant is present in gnomAD (v2) <0.01 for a recessive condition (42 heterozygotes, 0 homozygotes). (SP) 0801 - This variant has strong previous evidence of pathogenicity in unrelated individuals. This variant is one of the most common Mediterranean variants associated with beta-thalassemia and has been reported as heterozygous, homozygous, or compound heterozygous in individuals with beta-thalassemia. This variant has also been reported in individuals with sickle cell disease when in compound heterozygous with the HbS variant (PMIDs: 28667000, 28366028, 31899996, 31788855, 33851260). (SP) Legend: (SP) - Supporting pathogenic, (I) - Information, (SB) - Supporting benign