X-154535277-T-C

Position:

• chrX-154535277-T-C

Variant summary

Our verdict is Benign. Variant got -10 ACMG points: 2P and 12B. PM1 BP4_Strong BA1

The NM_001360016.2(G6PD):​c.376A>G​(p.Asn126Asp) variant causes a missense change involving the alteration of a non-conserved nucleotide. The variant allele was found at a frequency of 0.0171 in 1,210,053 control chromosomes in the GnomAD database, including 2,270 homozygotes. There are 5,488 hemizygotes in GnomAD. In-silico tool predicts a benign outcome for this variant. 14/19 in silico tools predict a benign outcome for this variant. Variant has been reported in ClinVar as Conflicting classifications of pathogenicity (no stars). Another variant affecting the same amino acid position, but resulting in a different missense (i.e. N126Y) has been classified as Uncertain significance.

• Frequency:
  • Genomes: 𝑓 0.091 (1131 hom., 2718 hem., cov: 23)
  • Exomes 𝑓: 0.0096 (1139 hom. 2770 hem.)
• Consequence: G6PD NM_001360016.2 missense
• Clinical Significance: Conflicting classifications of pathogenicity criteria provided, conflicting classifications P:15 U:6 B:5 O:2
• Conservation: PhyloP100: -0.123

Genes affected

G6PD (HGNC:4057): (glucose-6-phosphate dehydrogenase) This gene encodes glucose-6-phosphate dehydrogenase. This protein is a cytosolic enzyme encoded by a housekeeping X-linked gene whose main function is to produce NADPH, a key electron donor in the defense against oxidizing agents and in reductive biosynthetic reactions. G6PD is remarkable for its genetic diversity. Many variants of G6PD, mostly produced from missense mutations, have been described with wide ranging levels of enzyme activity and associated clinical symptoms. G6PD deficiency may cause neonatal jaundice, acute hemolysis, or severe chronic non-spherocytic hemolytic anemia. Two transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Jul 2008]

ACMG classification

Verdict is Benign. Variant got -10 ACMG points.

• PM1: In a hotspot region, there are 6 aminoacids with missense pathogenic changes in the window of +-8 aminoacids around while only 2 benign, 3 uncertain in NM_001360016.2
• BP4: Computational evidence support a benign effect (MetaRNN=0.005546838).
• BA1: GnomAd4 highest subpopulation (AFR) allele frequency at 95% confidence interval = 0.308 is higher than 0.05.

Transcripts

RefSeq

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	MANE	UniProt
G6PD	NM_001360016.2	c.376A>G	p.Asn126Asp	missense_variant	5/13	ENST00000393562.10
G6PD	NM_000402.4	c.466A>G	p.Asn156Asp	missense_variant	5/13
G6PD	NM_001042351.3	c.376A>G	p.Asn126Asp	missense_variant	5/13

Ensembl

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	TSL	MANE	Appris	UniProt
G6PD	ENST00000393562.10	c.376A>G	p.Asn126Asp	missense_variant	5/13	1	NM_001360016.2	P4

Frequencies

GnomAD3 genomes AF: 0.0907 AC: 10141 AN: 111861 Hom.: 1130 Cov.: 23 AF XY: 0.0795 AC XY: 2709 AN XY: 34057

Gnomad AFR AF: 0.313

Gnomad AMI AF: 0.00

Gnomad AMR AF: 0.0381

Gnomad ASJ AF: 0.00

Gnomad EAS AF: 0.00

Gnomad SAS AF: 0.00110

Gnomad FIN AF: 0.00

Gnomad MID AF: 0.00418

Gnomad NFE AF: 0.000886

Gnomad OTH AF: 0.0673

GnomAD3 exomes AF: 0.0257 AC: 4704 AN: 183378 Hom.: 537 AF XY: 0.0161 AC XY: 1094 AN XY: 67872

Gnomad AFR exome AF: 0.319

Gnomad AMR exome AF: 0.0148

Gnomad ASJ exome AF: 0.000134

Gnomad EAS exome AF: 0.00

Gnomad SAS exome AF: 0.000734

Gnomad FIN exome AF: 0.00

Gnomad NFE exome AF: 0.000647

Gnomad OTH exome AF: 0.00993

GnomAD4 exome AF: 0.00958 AC: 10515 AN: 1098138 Hom.: 1139 Cov.: 31 AF XY: 0.00762 AC XY: 2770 AN XY: 363494

Gnomad4 AFR exome AF: 0.325

Gnomad4 AMR exome AF: 0.0168

Gnomad4 ASJ exome AF: 0.0000516

Gnomad4 EAS exome AF: 0.00

Gnomad4 SAS exome AF: 0.000609

Gnomad4 FIN exome AF: 0.00

Gnomad4 NFE exome AF: 0.000372

Gnomad4 OTH exome AF: 0.0203

GnomAD4 genome AF: 0.0907 AC: 10153 AN: 111915 Hom.: 1131 Cov.: 23 AF XY: 0.0797 AC XY: 2718 AN XY: 34121

Gnomad4 AFR AF: 0.313

Gnomad4 AMR AF: 0.0380

Gnomad4 ASJ AF: 0.00

Gnomad4 EAS AF: 0.00

Gnomad4 SAS AF: 0.000739

Gnomad4 FIN AF: 0.00

Gnomad4 NFE AF: 0.000886

Gnomad4 OTH AF: 0.0665

Alfa AF: 0.0110 Hom.: 311

Bravo AF: 0.103

TwinsUK AF: 0.000809 AC: 3

ALSPAC AF: 0.000346 AC: 1

ESP6500AA AF: 0.312 AC: 1195

ESP6500EA AF: 0.000595 AC: 4

ExAC AF: 0.0286 AC: 3471

EpiCase AF: 0.000654

EpiControl AF: 0.000652

ClinVar

Significance: Conflicting classifications of pathogenicity

Submissions summary: Pathogenic:15 Uncertain:6 Benign:5 Other:2

Revision: criteria provided, conflicting classifications

Submissions by phenotype

Anemia, nonspherocytic hemolytic, due to G6PD deficiency Pathogenic:9 Uncertain:2 Benign:2 Other:1

Uncertain significance, criteria provided, single submitter	clinical testing	Invitae	Jan 03, 2020	This sequence change replaces asparagine with aspartic acid at codon 126 of the G6PD protein (p.Asn126Asp). The asparagine residue is moderately conserved and there is a small physicochemical difference between asparagine and aspartic acid. This variant is the A+ (also known as A or A*) haplotype and it is present in population databases (rs1050829, ExAC 32%), being by far the most prevalent in the African subpopulation (PMID: 1303173). ClinVar contains an entry for this variant (Variation ID: 100055). In addition, the c.202G>A (p.Val68Met) variant and the c.376A>G (p.Asn126Asp) variant, when co-occurring in cis c.[202G>A;376A>G], is known as the G6PD A- haplotype which is present in the African populations at 0.2% (PMID: 2572288, 4359638). This variant alone has been reported to be non-deficient and not causative of disease (PMID: 26633385, 9858856, 8611726, 2321910, 1303173, 12737938). However, it has been reported in two individuals affected with drug-induced acute hemolytic anemia who were negative for the p.Val68Met variant, but these individuals could have another G6PD variant that is responsible for the observed phenotype (PMID: 27287612). ClinVar contains an entry for the G6PD A- haplotype (Variation ID: 10361). While this variant alone has been shown to have only a mild affect on enzyme activity, the c.[202G>A;376A>G] changes of the G6PD A- haplotype have been reported to act synergistically to cause dramatic reduction of the stability and enzymatic activity of the G6PD protein (PMID: 3393536, 1303173, 10734064, 6015571, 2836867, 16356170). For these reasons, this allele has been classified as Uncertain Significance. -
Pathogenic, no assertion criteria provided	clinical testing	Zotz-Klimas Genetics Lab, MVZ Zotz Klimas	Oct 30, 2023	- -
Pathogenic, no assertion criteria provided	research	Division of Human Genetics, Children's Hospital of Philadelphia	Feb 20, 2015	The heterozygous variant (c.376A>G; p.Asn126Asp) has been previously published by itself under the name of “A variant”; the resulting enzymatic activity of this variant was at 84% of wild type. This variant have been previously published as part of the same haplotype (A- variant) which results in reduced enzymatic activity to 10-23% of normal activity. -
Pathogenic, criteria provided, single submitter	clinical testing	Mendelics	May 28, 2019	- -
Uncertain significance, criteria provided, single submitter	curation	Dunham Lab, University of Washington	Aug 12, 2022	Variant found in hemizygotes with deficiency, some with anemia (PP4), but also without deficiency (BS2). Decreased activity in red blood cells (28-90%) in some hemizygotes (PS3), but normal in others (BS3). Frequency of 9.1% in gnomAD3 (BA1). -
Pathogenic, criteria provided, single submitter	clinical testing	Centogene AG - the Rare Disease Company	May 04, 2021	- -
Likely pathogenic, criteria provided, single submitter	clinical testing	Institute of Human Genetics, University of Leipzig Medical Center	Jan 12, 2022	This variant was identified as hemizygous and together with NM_001042351.3:c.202G>A._x000D_ Criteria applied: PS4, PS3_MOD, PM5_SUP -
Pathogenic, criteria provided, single submitter	clinical testing	Greenwood Genetic Center Diagnostic Laboratories, Greenwood Genetic Center	Feb 02, 2024	PS3, PS4 -
Pathogenic, criteria provided, single submitter	clinical testing	Knight Diagnostic Laboratories, Oregon Health and Sciences University	Jun 28, 2016	The c.376A>G (p.Asn126Asp) missense variant is recognized as a disease-causing variant in the G6PD gene. Functional studies demonstrate reduced enzymatic activity of the G allele compared to WT. The prevalence of the variant in affected individuals (homozygous) is increased compared with the prevalence in controls and there is familial co-segregation with disease (Bwayo et al. 2013; Manjurano et al. 2012; Maiga et al. 2014; Shah et al. 2014; Pisani et al. 2012; Vulliamy et al. 1988; Odievre et al. 2011). This variant is has been classified as pathogenic by multiple reputable clinical testing laboratories (Emory, Division of Human Genetics at Children’s Hospital of Philadelphia). Although the frequency of this variant in the African population within ExAC (http://exac.broadinstitute.org) is high it is consistent with observed and expected based on disease incidence (31.700%). In summary, this variant c.3766A>G (p.Asn126Asp) meets our criteria for a Pathogenic classification. We have confirmed this finding in our laboratory using Sanger sequencing. -
Benign, no assertion criteria provided	curation	Department Of Genetics, Sultan Qaboos University Hospital, Sultan Qaboos University	Dec 30, 2017	- -
Pathogenic, no assertion criteria provided	clinical testing	Biochemical Molecular Genetic Laboratory, King Abdulaziz Medical City	Aug 25, 2019	- -
Pathogenic, criteria provided, single submitter	clinical testing	Baylor Genetics	Jul 27, 2019	This variant was determined to be pathogenic according to ACMG Guidelines, 2015 [PMID:25741868]. -
not provided, no classification provided	phenotyping only	GenomeConnect - Invitae Patient Insights Network	-	Variant interpreted as Uncertain significance and reported on 10-30-2020 by Invitae. GenomeConnect-Invitae Patient Insights Network assertions are reported exactly as they appear on the patient-provided report from the testing laboratory. Registry team members make no attempt to reinterpret the clinical significance of the variant. Phenotypic details are available under supporting information. -
Benign, criteria provided, single submitter	research	Broad Center for Mendelian Genomics, Broad Institute of MIT and Harvard	-	The p.Asn126Asp variant, sometimes called p.Asn156Asp due to a difference in cDNA numbering, in G6PD has been reported as a polymorphism in African individuals in the literature with an allele frequency of 25% in some populations (PMID: 3393536). In vitro functional studies provide some evidence that the p.Asn126Asp variant will not impact protein function (PMID: 3393536). However, these types of assays may not accurately represent biological function. This variant has also been reported in >30% of African chromosomes, 613 hemizygotes, and 354 homozygotes by ExAC (http://gnomad.broadinstitute.org/). In summary, this variant meets criteria to be classified as benign for glucose-6-phosphate dehydrogenase deficiency. -

not provided Pathogenic:3 Uncertain:4 Benign:1

Likely benign, criteria provided, single submitter	clinical testing	ARUP Laboratories, Molecular Genetics and Genomics, ARUP Laboratories	Nov 28, 2023	- -
Pathogenic, criteria provided, single submitter	clinical testing	Institute for Clinical Genetics, University Hospital TU Dresden, University Hospital TU Dresden	Jun 23, 2022	- -
Pathogenic, criteria provided, single submitter	clinical testing	Center for Pediatric Genomic Medicine, Children's Mercy Hospital and Clinics	Aug 24, 2015	- -
Uncertain significance, criteria provided, single submitter	clinical testing	GeneDx	Nov 30, 2022	A published functional study demonstrates that N126D affects the catalytic efficiency of the enzyme and affects the overall stability of the protein when compared to wild-type G6PD (Gomez-Manzo et al., 2015); A published functional study demonstrates N126D alone showed similar catalytic activity and structural stability compared to wildtype while N126D in combination with other mutations resulted in protein structural instability and reduced catalytic activity, suggesting that the additional mutations contribute to reduced enzyme activity (Praoparotai et al., 2020); In silico analysis, which includes protein predictors and evolutionary conservation, supports that this variant does not alter protein structure/function; This variant is associated with the following publications: (PMID: 3446582, 27535533, 23144702, 2572288, 33637102, 22307442, 21931771, 1889820, 8733135, 12524354, 9452072, 21479984, 1303173, 25201310, 2836867, 3393536, 27884173, 28195434, 26990548, 27853304, 29141760, 30161219, 29072585, 32387609, 30577886, 34272389, 34426522, 33072997, 33587123, 28902532, 28939159, 34992599, 26633385) -
Likely pathogenic, criteria provided, single submitter	clinical testing	CeGaT Center for Human Genetics Tuebingen	Jul 01, 2024	G6PD: PM5, PS3:Moderate, PS4:Moderate, PP1, BP4 -
Uncertain significance, no assertion criteria provided	clinical testing	Department of Pathology and Laboratory Medicine, Sinai Health System	-	The G6PD p.N126D variant was identified in literature and is commonly known as the A+ allele. When the p.N126D variant is found on the same allele as the G6PD p.V68M variant it is known as the A- allele, and is one of the most common causes G6PD deficiency in the African population (Beutler_1989_PMID:2572288; Clark_2009_PMID:19223928). The variant was identified in dbSNP (ID: rs1050829) and ClinVar (classified as pathogenic by the Center for Pediatric Genomic Medicine, Mendelics, Knight Diagnostic Laboratories, Children's Hospital of Philadelphia and King Abdulaziz Medical City, classified as uncertain significance by GeneDx, BluePrint Genetics and Invitae, classified as likely benign by ARUP Laboratories and Illumina and classified as benign by the Derpatment of Genetics, Qaboos University Hospital Oman). The variant was identified in control databases in 6586 of 205081 chromosomes (722 homozygous; 1760 hemizygous) at a frequency of 0.03211 increasing the likelihood this could be a low frequency benign variant (Genome Aggregation Database March 6, 2019, v2.1.1). The variant was observed in the following populations: African in 6025 of 18923 chromosomes (freq: 0.3184), Latino in 417 of 28037 chromosomes (freq: 0.01487), Other in 68 of 5335 chromosomes (freq: 0.01275), South Asian in 14 of 19079 chromosomes (freq: 0.000734), European (non-Finnish) in 61 of 92560 chromosomes (freq: 0.000659) and Ashkenazi Jewish in 1 of 7669 chromosomes (freq: 0.00013), but was not observed in the East Asian or European (Finnish) populations. The p.Asn156 residue is conserved in mammals but not in more distantly related organisms however computational analyses (PolyPhen-2, SIFT, AlignGVGD, BLOSUM, MutationTaster) do not suggest a high likelihood of impact to the protein; this information is not predictive enough to rule out pathogenicity. The variant occurs outside of the splicing consensus sequence and in silico or computational prediction software programs (SpliceSiteFinder, MaxEntScan, NNSPLICE, GeneSplicer) do not predict a difference in splicing. Functional studies of the A+ allele (p.N126D variant alone), the p.V68M variant alone, and the A- allele (p.N126D + p.V68M) have demonstrated that the variants alone only cause a slight decrease in activity and enzyme yield, but show significantly decreased activity and enzyme yield when found on the same allele (A-), demonstrating a synergistic effect; therefore the A+ allele on its own is not enough to cause G6PD deficiency (Town_1992_PMID:1303173; Vulliamy_1988_PMID:3393536). In summary, based on the above information the clinical significance of this variant cannot be determined with certainty at this time. This variant is classified as a variant of uncertain significance. -
Uncertain significance, criteria provided, single submitter	clinical testing	Blueprint Genetics	Nov 02, 2018	- -
Uncertain significance, criteria provided, single submitter	clinical testing	AiLife Diagnostics, AiLife Diagnostics	Jul 02, 2021	- -

Inborn genetic diseases Pathogenic:1

Pathogenic, criteria provided, single submitter

clinical testing

Ambry Genetics

Nov 20, 2023

The c.376A>G (p.N126D) alteration is located in exon 5 (coding exon 4) of the G6PD gene. This alteration results from a A to G substitution at nucleotide position 376, causing the asparagine (N) at amino acid position 126 to be replaced by an aspartic acid (D). Based on the available evidence, this alteration is classified as pathogenic. -

Malaria, susceptibility to;C2720289:Anemia, nonspherocytic hemolytic, due to G6PD deficiency Pathogenic:1

Pathogenic, no assertion criteria provided

research

Division of Human Genetics, Children's Hospital of Philadelphia

Dec 29, 2015

- -

Anemia, nonspherocytic hemolytic, due to G6PD deficiency;C2939465:G6PD deficiency Pathogenic:1

Pathogenic, criteria provided, single submitter

research

UNC Molecular Genetics Laboratory, University of North Carolina at Chapel Hill

-

G6PD c.292G>A (p.V98M) and c.466A>G (p.N156D), often occur together in cis as part of a haplotype, referred to as the enzyme variant A- (PMID: 5448; 1303173). This variant is associated with glucose-6-phosphate dehydrogenase deficiency. -

not specified Benign:1

Likely benign, criteria provided, single submitter

clinical testing

Women's Health and Genetics/Laboratory Corporation of America, LabCorp

Apr 08, 2021

Variant summary: G6PD c.466A>G (p.Asn156Asp) results in a conservative amino acid change located in the NAD-binding domain (IPR022674) of the encoded protein sequence. Five of five in-silico tools predict a benign effect of the variant on protein function. The variant allele was found at a frequency of 0.026 in 183378 control chromosomes, predominantly at a frequency of 0.32 within the African or African-American subpopulation in the gnomAD database, including 532 homozygous females and 952 hemizygous males. These findings strongly suggest that the variant is a benign polymorphism found primarily in populations of African or African-American origin. c.466A>G has been reported in the literature in multiple individuals affected with G6PD Deficiency, however in most of these cases as part of a complex allele with another pathogenic variant, such as c.1058T>C (known as G6PD-Betica or G6PD-Betica Selma), c.292G>A (known as G6PD A-), or c.632A>T (known as G6PD-Santamaria) (e.g. Vulliamy_1988, Beutler_1989, Vulliamy_1996, Djigo_2019). These reports do not provide unequivocal conclusions about association of the variant with disease, as the variant was not identified in isolation in affected individuals. Several publications report experimental evidence evaluating an impact on protein function. The variant, c.466A>G, has been reported to have reduced affinity for substrate molecules compared to wild-type (e.g. Ramirez-Nava_2017), but retains approximately 70-85% of wild-type enzyme activity levels (e.g. Vulliamy_1988). 15 other clinical diagnostic laboratories have submitted clinical-significance assessments for this variant to ClinVar after 2014 without evidence for independent evaluation. Multiple laboratories reported the variant with conflicting assessments (benign/likely benign, n=3; uncertain significance, n=3; pathogenic, n=8; other, n=1). Based on the evidence outlined above, the variant was classified as likely benign. -

G6PD deficiency Benign:1

Likely benign, criteria provided, single submitter

clinical testing

Illumina Laboratory Services, Illumina

Jun 14, 2016

- -

G6PD A+ Other:1

other, no assertion criteria provided

literature only

OMIM

May 11, 2018

- -

Computational scores

Source: dbNSFP v4.3

Name	Calibrated prediction	Score	Prediction
AlphaMissense	Benign	0.095
BayesDel_addAF	Benign	-0.52	T
BayesDel_noAF	Benign	-0.38
CADD	Benign	1.7
DANN	Benign	0.90
DEOGEN2	Uncertain	0.69	D;D;D;.;D;.;D
FATHMM_MKL	Benign	0.34	N
LIST_S2	Benign	0.39	.;.;T;T;T;T;T
MetaRNN	Benign	0.0055	T;T;T;T;T;T;T
MetaSVM	Benign	-0.84	T
MutationAssessor	Benign	0.54	N;N;N;N;.;.;.
MutationTaster	Benign	1.0	P;P;P;P
PrimateAI	Benign	0.39	T
PROVEAN	Benign	-0.49	.;.;N;N;N;N;N
REVEL	Benign	0.27
Sift	Benign	0.24	.;.;T;T;T;T;T
Sift4G	Benign	0.43	T;.;T;T;.;.;.
Polyphen		0.0	B;B;B;.;.;.;.
Vest4		0.15
MPC		0.20
ClinPred		0.00071	T
GERP RS		0.49
Varity_R		0.28
gMVP		0.47

Splicing

Name	Calibrated prediction	Score	Prediction
SpliceAI score (max)		0.0		Details are displayed if max score is > 0.2

Find out detailed SpliceAI scores and Pangolin per-transcript scores at spliceailookup.broadinstitute.org

Publications

LitVar

Other links and lift over

dbSNP: rs1050829; hg19: chrX-153763492; COSMIC: COSV63703563; COSMIC: COSV63703563;