ClinVar Genomic variation as it relates to human health

This variant in exon 9 of the G6PD gene results in the amino acid substitution from glutamic acid to lysine at codon 347 (p.Glu347Lys) with the sequence change of c.1039G>A (NM_000402.4). This variant was observed in a proband with a decreased level of G6PD enzyme (3.1 U/dl) which was screened for advanced newborn screening with confirmatory genetic reflex testing at Lifecell diagnostics. There is a small physicochemical difference between glutamic acid and lysine. The observed variant is not present in both the 1000 Genomes and gnomAD databases. The reference base is conserved across the species and in-silico predictions by Polyphen and SIFT are damaging. This is a Class III variant associated with moderate G6PD deficiency (10-60% enzyme activity) and hemolysis with stressors only. According to the previous studies this variant is also known as G6PD Kerala, G6PD Kalyan, G6PD Kerala Kalyan, G6PD Jamnaga, and G6PD Rohini. This variant has previously been reported for Glucose-6- phosphate dehydrogenase (G6PD) deficiency by Islam MT et al., 2018. The Missense Variants Z-Score for this variant is 2.60. Missense Variants Z-Score is produced by the Exome Aggregation Consortium (60,706 adult humans) by computing a signed Z score for the deviation of observed counts from the expected number. Positive Z scores indicate increased constraint (intolerance to variation) and therefore that the gene had fewer missense variants than expected. This variant is a prevalent G6PD variant in the South Asian population, typically associated with partial reduction of enzyme activity (Ahluwalia A et al. 1992; PMID: 1303182), (Islam MT et al. 2018; PMID: 30097005), ( Ninokata A et al, 2006; PMID: 16528451), ( Sukumar S et al. 2004; PMID: 15315792), ( Sarker SK et al. 2016; PMID: 27880809), (Minucci A et al, 2012; PMID: 22293322).

The G6PD c.1039G>A (p. Glu347Lys) missense variant, which is also commonly reported as c.949G>A (p.Glu317Lys) and G6PD Kerala-Kalyan, and less commonly reported as G6PD Jamnagar or Rohini, is one of the most common disease-causing variants in South Asian populations. The p.Glu347Lys variant has been estimated to account for between 1.1% and 24.5% of disease-causing alleles in the Indian population (Sukumar et al. 2004; Devendra et al. 2020). This variant has also been reported in affected individuals of Bangladeshi, Burmese, and Thai descent (Nuchprayoon et al. 2008; Sarker et al. 2016). The p.Glu347Lys variant is typically associated with glucose-6-phosphate dehydrogenase enzyme activity levels between 10% - 60% of wild type and is considered a class III variant according the WHO classification system for G6PD variants (Minucci et al. 2012; Harcke et al. 2019). The p.Glu347Lys variant is reported at a frequency of 0.01138 in the South Asian population of the Genome Aggregation Database. This allele frequency is high but is consistent with the disease prevalence estimates. Although the consequences of p.Glu347Lys have not been characterized experimentally, the Glu347 residue is known to interact with Lys275, which is the site of the another disease-causing variant, p.Lys275Asn or G6PD Bangkok. This interaction forms a salt bridge between subunits B and C of the tetrameric structure in wild-type G6PD (Boonyuen et al. 2017). Based on the collective evidence and application of the ACMG criteria, the p. Glu347Lys variant is classified as pathogenic for glucose-6-phosphate dehydrogenase deficiency.

The hemizygous, maternally inherited c.949G>A p.(Glu317Lys) variant identified in the G6PD gene substitutes an evolutionarily conserved glutamic acid with Lysine at position 317/516 (exon 9/13) in the dimer interface region [PMID: 31294066] of the encoded protein. This variant is also called c.1039G>Ap.(Glu347Lys) based on transcript NM_000402.4, and is reported in ClinVar [ClinVar ID:10401] as Pathogenic. This variant is observed in 322 alleles (0.04% minor allele frequency with 6 homozygotes and 151 hemizygotes) in population databases (gnomAD v2.1.1 and v3.1.2, TOPMed Freeze 8, All of Us) including in 242 alleles(1.07% minor allele frequency with 1 homozygote and 148 hemizygotes) in South Asian subpopulation (gnomAD v2.1.1 and v3.1.2(non-Topmed)). In silico predictions are in favor of damaging effect for p.(Glu317Lys) (REVEL = 0.648). The p.(Glu317Lys) variant in G6PD has previously been reported in individuals with G6PD deficiency and is also known as G6PD Kerala, G6PD Kalyan, G6PD Kerala Kalyan, G6PD Jamnagar, and G6PD Rohini in the literature [(PMID: 15996881, 1303182, 30097005,16528451, 15315792, 27880809, 27535533, 33069889]. The c.949G>A p.(Glu317Lys) variant in G6PD is reported as Class III variant according to WHO classification and associated with moderate G6PD deficiency (10-60% enzyme activity) and hemolysis with stressors only [PMID:15996881,22293322]. Based on available evidence this maternally inherited hemizygous c.949G>A p.(Glu317Lys) variant identified in the G6PD gene is classified as Pathogenic.

This sequence change replaces glutamic acid, which is acidic and polar, with lysine, which is basic and polar, at codon 317 of the G6PD protein (p.Glu317Lys). This variant is present in population databases (rs137852339, gnomAD 1.1%), and has an allele count higher than expected for a pathogenic variant. This variant is a prevalent G6PD variant in the South Asian population, typically associated with partial reduction of enzyme activity (PMID: 1303182, 30097005, 16528451, 15315792, 30097005, 27880809, 27535533). By WHO classification, this is a Class III variant associated with moderate G6PD deficiency (10-60% enzyme activity) and hemolysis with stressors only (PMID: 22293322). This variant is also known as G6PD Kerala, G6PD Kalyan, G6PD Kerala–Kalyan, G6PD Jamnaga, and G6PD Rohini. ClinVar contains an entry for this variant (Variation ID: 10401). 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 G6PD protein function. For these reasons, this variant has been classified as Pathogenic.

Based on the classification scheme VCGS_Germline_v1.3.4, 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 G6PD deficient haemolytic anemia (favism) (MIM#300908). (I) 0109 - This gene is associated with X-linked recessive disease. (I) 0200 - Variant is predicted to result in a missense amino acid change from glutamic acid to lysine. (I) 0251 - Variant is heterozygous. (I) 0305 - Variant is present in gnomAD >=0.01 and <0.03 for a recessive condition (v2: 94 heterozygotes, 1 homozygote, 132 hemizygotes). (I) 0502 - Missense variant with conflicting in silico predictions and uninformative conservation. (I) 0600 - Variant is located in the annotated G6PD C-terminal domain (DECIPHER). (I) 0705 - No comparable variants have previous evidence for pathogenicity. (I) 0801 - Strong previous evidence of pathogenicity in unrelated individuals. This variant has previously been classified as a G6PD Class III variant and is known as the Kerala-Kalyan variant. Individuals with this variant have moderate to mild G6PD enzyme activity (WHO recommendations; G6PD database; PMID: 22293322; 30097005, 31833391). (SP) 1206 - This variant has been shown to be paternally inherited (by trio analysis). (I) Legend: (SP) - Supporting pathogenic, (I) - Information, (SB) - Supporting benign

G6PD: PP1:Strong, PS4, PS3:Moderate

Variant found in unrelated hemizygotes with G6PD deficiency but no other symptoms (PS4_M, PP4). Also segregates with deficiency in multiple brothers, and heterozygous sister has slightly decreased activity (PP1). Decreased activity in red blood cells of hemizygotes (19-52%) (PS3). Modeling predicts disruption of function (PP3). Reported as pathogenic by multiple clinical testing groups (PP5). Post_P 0.994 (odds of pathogenicity 1517, Prior_P 0.1).

The amino acid Glu at position 347 is changed to a Lys changing protein sequence and it might alter its composition and physico-chemical properties. This variant is a prevalent G6PD variant in the South Asian population, typically associated with partial reduction of enzyme activity (Ahluwalia A et al, Islam MT et al). By WHO classification, this is a Class III variant associated with moderate G6PD deficiency (10-60% enzyme activity) and hemolysis with stressors only (Minucci A et al). This variant is also known as G6PD Kerala, G6PD Kalyan, G6PD Kerala–Kalyan, G6PD Jamnagar, and G6PD Rohini in the literature. The p.Glu347Lys variant in reported with the allele frequency of 0.1111% in gnomAD Exomes and is novel (not in any individuals) in1000 Genomes. This variant has been reported in Clinvar as Conflicting interpretations of pathogenicity - pathogenic/ uncertain significance. In silico tools predict the variant to be tolerated. The residue is conserved across species. The amino acid change p.Glu347Lys in G6PD is predicted as conserved by GERP++ and PhyloP across 100 vertebrates. For these reasons, this variant has been classified as Pathogenic.

In silico analysis supports that this missense variant has a deleterious effect on protein structure/function; Class III variant according to the WHO classification system, associated with moderate G6PD deficiency (10-60% enzyme activity) and hemolysis with stressors only (PMID: 22293322); Previously described as G6PD Kerala, G6PD Kalyan, G6PD Kerala-Kalyan, G6PD Jamnagar, and G6PD Rohini (PMID: 1303182, 15996881); This variant is associated with the following publications: (PMID: 32036089, 30097005, 36508454, 34551338, 34620237, 33069889, 20713184, 27880809, 27535533, 17233850, 1303182, 15996881, 22293322, 36681081, 38312194, 38488835)