ClinVar Genomic variation as it relates to human health

The c.27dupG (p.Ser10Valfs*14) frameshift variant in the HBB gene is a well-known variant associated with autosomal recessive Beta-Thalassemia (Baysal, 2005; Sinha et al., 2009; Al-Gazali et al., 2010; Ankala et al., 2015). This variant is predicted to cause a protein termination in exon 1 (out of a total of 3 exons in the coding sequence). Frameshift variants have been described in the HBB gene in several affected individuals and are, therefore, a common mechanism of disease. This variant is located upstream of two heme binding sites and two 2,3-biphosphoglycerate binding sites, and thus, the truncated protein would lack these important binding sites. This c.27dupG has been reported at low frequency in the control population databases (Exome Sequencing Project [ESP] = NA, 1000 Genomes = NA, and ExAC = 0.206%). In silico algorithms predict the resulting transcript will be targeted for the Nonsense Mediated Decay (NMD) Pathway. Therefore, this collective evidence supports the classification of the c.27dupG (p.Ser10Valfs*14) as a recessive Pathogenic variant for Beta-Thalassemia.

Converted during submission to Pathogenic.

This sequence change creates a premature translational stop signal (p.Ser10Valfs*14) in the HBB gene. It is expected to result in an absent or disrupted protein product. Loss-of-function variants in HBB are known to be pathogenic (PMID: 23637309). This variant is present in population databases (rs35699606, gnomAD 0.2%). This premature translational stop signal has been observed in individual(s) with autosomal recessive beta thalassemia (PMID: 9949622, 27263053, 28391758, 28635337). This variant is also known as CD8/9 (+G), codon 8/9 (+G), FSC 8/9 (+G), and c.27_28insG. ClinVar contains an entry for this variant (Variation ID: 36308). Algorithms developed to predict the effect of sequence changes on RNA splicing suggest that this variant may disrupt the consensus splice site. For these reasons, this variant has been classified as Pathogenic.

The HBB c.27dup; p.Ser10ValfsTer14 variant (also known as Ser9fs when numbered from the mature protein or as Codons 8/9 (+G), rs35699606, HbVar ID: 786) has been reported in individuals with beta (0) thalassemia, both in the homozygous state and in trans to another pathogenic variant (Jalilian 2017, Kazazian 1984, Muhammad 2017, HbVar database and references therein). Consistent with reports that it is prevalent in affected individuals of South Asian descent (Jalilian 2017, Kazazian 1984, Muhammad 2017), this variant is found in the South Asian population with an overall allele frequency of 0.2% (62/30614 alleles) in the Genome Aggregation Database. This variant causes a frameshift by duplicating a single nucleotide, so it is predicted to result in a truncated protein or mRNA subject to nonsense-mediated decay. Based on available information, this variant is considered to be pathogenic. References: Link to HbVar database: https://globin.bx.psu.edu/hbvar/hbvar.html 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. Kazazian HH Jr et al. Molecular characterization of seven beta-thalassemia mutations in Asian Indians. EMBO J. 1984 Mar;3(3):593-6. PMID: 6714226. Muhammad R et al. Population-Based Genetic Study of Beta-Thalassemia Mutations in Mardan Division, Khyber Pakhtunkhwa Province, Pakistan. Hemoglobin. 2017 Mar;41(2):104-109. PMID: 28635337.

HBB: PM3:Very Strong, PVS1, PM2:Supporting

The HBB c.27dupG (p.Ser10ValfsTer14) variant results in a frameshift and is predicted to result in premature termination of the protein. The p.Ser10ValfsTer14 variant, also known as cd8/9+G, is a well-described pathogenic variant. Across a selection of the available literature the p.Ser10ValfsTer14 variant has been reported in at least 229 disease alleles in individuals with HBB-related disorders, including in at least 38 patients in a homozygous state and 30 patients in a compound heterozygous state. The zygosity of the remaining alleles is not known (Kazazian et al. 1984; Villegas et al. 1998; Ansari et al. 2011; Hoppe et al 2013; El-Shanshory et al. 2014; Yasmeen et al. 2016). The variant is reported at frequencies from 1% to 47% depending on the ethnic background but is more common in the South Asian and Middle Eastern populations (Lahiry et al. 2008; Ansari et al. 2011). Control data are unavailable for the p.Ser10ValfsTer14 variant, which is reported at a frequency of 0.00206 in the South Asian population of the Exome Aggregation Consortium. Based on the potential impact of frameshift variants and the supporting evidence from the literature, the p.Ser10ValfsTer14 variant is classified as pathogenic for HBB-related disorders. This variant was observed by ICSL as part of a predisposition screen in an ostensibly healthy population.

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; This variant is associated with the following publications: (PMID: 22975760, 27453201, 28635337, 6714226, 8537236, 9949622, 28391758, 27263053)

NM_000518.4(HBB):c.27dupG(S10Vfs*14) is classified as pathogenic in the context of Hb beta chain-related hemoglobinopathy; it is associated with beta thalassemia and is classified as a beta-zero variant. Sources cited for classification include the following: PMID 22110956, 9949622, 24369358, 6714226, 16821247 and 21509314. Classification of NM_000518.4(HBB):c.27dupG(S10Vfs*14) is based on the following criteria: The variant causes a premature termination codon that is expected to be targeted by nonsense-mediated mRNA decay and is reported in individuals with the relevant phenotype. Please note: this variant was assessed in the context of healthy population screening.

Viafet Genomics Laboratory has identified this variant in one homozygous and three compound heterozygous individuals affected with Beta-Thalassemia. In addition, this variant was reported in 26 individuals, as part of Carrier Screening testing, who were not affected with this condition.

The variant is observed at an extremely low frequency in the gnomAD v2.1.1 dataset (total allele frequency: 0.025%). 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: VCV000036308 / PMID: 6714226). Therefore, this variant is classified as pathogenic according to the recommendation of ACMG/AMP guideline.

The frameshift duplication NM_000518.5 (HBB):c.27dupG (p.Ser10Valfs*14) has been reported to ClinVar as Pathogenic with a status of (2 stars) criteria provided, multiple submitters, no conflicts (Variation ID 36308 as of 2022-06-02). The p.Ser10Valfs*14 variant is observed in 62/30,614 (0.2025%) alleles from individuals of gnomAD South Asian background in gnomAD. The p.Ser10Valfs*14 variant is novel (not in any individuals) in 1kG. This variant is predicted to cause loss of normal protein function through protein truncation caused a frameshift mutation. The frame shifted sequence continues 14 residues until a stop codon is reached. This variant is a frameshift variant which occurs in an exon of HBB upstream of where nonsense mediated decay is predicted to occur. This variant has been previously classified as pathogenic, indicating that the region is critical to protein function. There are 83 downstream pathogenic loss of function variants, with the furthest variant being 123 residues downstream of this variant. This indicates that the region is critical to protein function. The p.Ser10Valfs*14 vari ant is a loss of function variant in the gene HBB, which is intolerant of Loss of Function variants, as indicated by the presence of existing pathogenic loss of function variant NP_000509.1:p.M1L and 61 others. This premature translational stop signal has been observed in individual(s) with autosomal recessive beta thalassemia and loss of function in HBB is known to be pathogenic (Muhammad R et al., 2017). For these reasons, this variant has been classified as Pathogenic.

The frame shift (p.Ser10ValfsTer14) variant has been observed in numerous individuals and families affected with Thalassemia-beta, dominant inclusion-body and has been reported as a prevalent disease-associated variant in several populations (Muhammad et. al., 2017; Villegas et. al., 1998, Jalilian et. al., 2017). The p.Ser10ValfsTer14 variant is novel (not in any individuals) in 1000 Genomes and has allele frequency of 0.002508% in gnomAD database. This variant has been reported to the ClinVar database as Pathogenic. This variant causes a frameshift starting with codon Serine 10, changes this amino acid to Valine residue, and creates a premature Stop codon at position 14 of the new reading frame, denoted p.Ser10ValfsTer14. It is predicted to cause loss of normal protein function through protein truncation. Loss of function variants have been previously reported to be disease causing. For these reasons, this variant has been classified as Pathogenic. In the absence of another reportable variant, the molecular diagnosis of beta thalassemia is not confirmed.

The p.Ser10Valfs*14 variant in HBB is a well-known pathogenic variant associated with autosomal recessive beta-Thalassemia (selected references Kazazian 1984 PMID: 6714226, Villegas 1998 PMID: 9949622, Jalilian 2017 PMID: 283917585). This variant has been reported in ClinVar (Variation ID 36308) and was identified in 16/4826 South Asian chromosomes by gnomAD (https://gnomad.broadinstitute.org). This variant is predicted to cause a frameshift, which alters the protein’s amino acid sequence beginning at position 10 and leads to a premature termination codon 14 amino acids downstream. This alteration is then predicted to lead to a truncated 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_Strong.

Based on the classification scheme VCGS_Germline_v1.3.5, this variant is classified as Pathogenic. 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) 0204 - Variant is predicted to result in a truncated protein (premature termination codon is located within the first 102 nucleotides of the coding sequence and is predicted to escape nonsense-mediated decay). (SP) 0252 - This variant is homozygous. (I) 0304 - Variant is present in gnomAD <0.01 (v4: 206 heterozygotes, 2 homozygotes). (SP) 0701 - Other truncation variants comparable to the one identified in this case has very strong previous evidence for pathogenicity (DECIPHER). (SP) 0801 - This variant has very strong previous evidence of pathogenicity in unrelated individuals. This variant has been reported as pathogenic by multiple clinical laboratories in ClinVar. This variant has also been observed in multiple unrelated homozygous and compound heterozygous individuals with beta-thalassemia, including at least four individuals who are compound heterozygous for this variant and c.92+5G>C (PMID: 27263053). (SP) Legend: (SP) - Supporting pathogenic, (I) - Information, (SB) - Supporting benign

The HBB c.27dupG variant is predicted to result in a frameshift and premature protein termination (p.Ser10Valfs*14). This variant (which also may be referred to as c.27_28insG) has been previously reported to be causative for beta-thalassemia (Kazazian et al. 1984. PubMed ID: 6714226; Giardine et al. 2014. PubMed ID: 24137000; Jalilian et al. 2017. PubMed ID: 28391758; Muhammad et al. 2017. PubMed ID: 28635337). This variant is reported in 0.20% of alleles in individuals of South Asian descent in gnomAD. Frameshift variants in HBB are expected to be pathogenic. This variant is interpreted as pathogenic.

The HBB c.27dupG (NP_000509.1:p.Ser10fs) is a beta0 mutation. The variant is a frameshift mutation leads to premature protein termination. When this variant present in homozygous or in compound heterozygous with other pathogenic HBB mutation leads to severe type of thalassemia. Patients needing monthly transfusion, often presented with hepatosplenomegaly, Iron overload. The frequency of the variant in thalassemia patient in Eastern India is 0.39 % as per our multicentric project - A Genetic Diagnostic Algorithm Based Study for Thalassemia in Northern and Eastern Indian Populations, Funded by Dept. of Biotechnology , Govt of India [Project No. BT/PR26461/MED/12/821/2018]