6-32039081-C-G

Position:

• chr6-32039081-C-G

Variant summary

Our verdict is Likely pathogenic. Variant got 9 ACMG points: 9P and 0B. PP3 PP5_Very_Strong

The NM_000500.9(CYP21A2):​c.293-13C>G variant causes a splice polypyrimidine tract, intron change involving the alteration of a non-conserved nucleotide. The variant allele was found at a frequency of 0.00216 in 151,236 control chromosomes in the GnomAD database, including 1 homozygotes. In-silico tool predicts a benign outcome for this variant. Variant has been reported in ClinVar as Likely pathogenic (★★).

• Frequency:
  • Genomes: 𝑓 0.0022 (1 hom., cov: 29)
  • Exomes 𝑓: 0.0024 (6 hom.)
  • Failed GnomAD Quality Control
• Consequence: CYP21A2 NM_000500.9 splice_polypyrimidine_tract, intron
• Clinical Significance: Pathogenic/Likely pathogenic criteria provided, multiple submitters, no conflicts P:33 O:1
• Conservation: PhyloP100: -0.233

Genes affected

CYP21A2 (HGNC:2600): (cytochrome P450 family 21 subfamily A member 2) This gene encodes a member of the cytochrome P450 superfamily of enzymes. The cytochrome P450 proteins are monooxygenases which catalyze many reactions involved in drug metabolism and synthesis of cholesterol, steroids and other lipids. This protein localizes to the endoplasmic reticulum and hydroxylates steroids at the 21 position. Its activity is required for the synthesis of steroid hormones including cortisol and aldosterone. Mutations in this gene cause congenital adrenal hyperplasia. A related pseudogene is located near this gene; gene conversion events involving the functional gene and the pseudogene are thought to account for many cases of steroid 21-hydroxylase deficiency. Two transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Jul 2008]

ACMG classification

Verdict is Likely_pathogenic. Variant got 9 ACMG points.

• PP3: Splicing scoreres supports a deletorius effect: Scorers claiming Pathogenic: max_spliceai. No scorers claiming Uncertain. No scorers claiming Benign.
• PP5: Variant 6-32039081-C-G is Pathogenic according to our data. Variant chr6-32039081-C-G is described in ClinVar as [Likely_pathogenic]. Clinvar id is 12155.Status of the report is criteria_provided_multiple_submitters_no_conflicts, 2 stars. Variant chr6-32039081-C-G is described in Lovd as [Pathogenic].

Transcripts

RefSeq

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	MANE	Protein	UniProt
CYP21A2	NM_000500.9	c.293-13C>G		splice_polypyrimidine_tract_variant, intron_variant		ENST00000644719.2	NP_000491.4
CYP21A2	NM_001368143.2	c.-126C>G		5_prime_UTR_variant	3/10		NP_001355072.1
CYP21A2	NM_001368144.2	c.-126C>G		5_prime_UTR_variant	2/9		NP_001355073.1
CYP21A2	NM_001128590.4	c.203-13C>G		splice_polypyrimidine_tract_variant, intron_variant			NP_001122062.3

Ensembl

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	TSL	MANE	Protein	Appris	UniProt
CYP21A2	ENST00000644719.2	c.293-13C>G		splice_polypyrimidine_tract_variant, intron_variant			NM_000500.9	ENSP00000496625	P1

Frequencies

GnomAD3 genomes AF: 0.00216 AC: 327 AN: 151120 Hom.: 1 Cov.: 29

Gnomad AFR AF: 0.00182

Gnomad AMI AF: 0.00

Gnomad AMR AF: 0.00158

Gnomad ASJ AF: 0.00461

Gnomad EAS AF: 0.00138

Gnomad SAS AF: 0.00272

Gnomad FIN AF: 0.000669

Gnomad MID AF: 0.00318

Gnomad NFE AF: 0.00263

Gnomad OTH AF: 0.00289

GnomAD3 exomes AF: 0.00226 AC: 501 AN: 221616 Hom.: 0 AF XY: 0.00242 AC XY: 289 AN XY: 119232

Gnomad AFR exome AF: 0.00155

Gnomad AMR exome AF: 0.00204

Gnomad ASJ exome AF: 0.00371

Gnomad EAS exome AF: 0.00210

Gnomad SAS exome AF: 0.00242

Gnomad FIN exome AF: 0.000831

Gnomad NFE exome AF: 0.00261

Gnomad OTH exome AF: 0.00146

GnomAD4 exome Data not reliable, filtered out with message: AS_VQSR AF: 0.00245 AC: 3525 AN: 1440550 Hom.: 6 Cov.: 74 AF XY: 0.00237 AC XY: 1691 AN XY: 714484

Gnomad4 AFR exome AF: 0.00151

Gnomad4 AMR exome AF: 0.00199

Gnomad4 ASJ exome AF: 0.00378

Gnomad4 EAS exome AF: 0.00247

Gnomad4 SAS exome AF: 0.00279

Gnomad4 FIN exome AF: 0.00107

Gnomad4 NFE exome AF: 0.00249

Gnomad4 OTH exome AF: 0.00248

GnomAD4 genome AF: 0.00216 AC: 327 AN: 151236 Hom.: 1 Cov.: 29 AF XY: 0.00207 AC XY: 153 AN XY: 73856

Gnomad4 AFR AF: 0.00182

Gnomad4 AMR AF: 0.00158

Gnomad4 ASJ AF: 0.00461

Gnomad4 EAS AF: 0.00138

Gnomad4 SAS AF: 0.00272

Gnomad4 FIN AF: 0.000669

Gnomad4 NFE AF: 0.00263

Gnomad4 OTH AF: 0.00286

Alfa AF: 0.000598 Hom.: 9188

TwinsUK AF: 0.00324 AC: 12

ALSPAC AF: 0.00234 AC: 9

ExAC AF: 0.00205 AC: 247

ClinVar

Significance: Pathogenic/Likely pathogenic

Submissions summary: Pathogenic:33 Other:1

Revision: criteria provided, multiple submitters, no conflicts

Submissions by phenotype

Classic congenital adrenal hyperplasia due to 21-hydroxylase deficiency Pathogenic:21 Other:1

Pathogenic, criteria provided, single submitter	clinical testing	Illumina Laboratory Services, Illumina	Jan 15, 2019	The CYP21A2 c.293-13C>G variant is an intronic variant. Across a selection of the available literature, this variant has been reported in a homozygous state in at least 13 individuals with congenital adrenal hyperplasia due to 21-hydroxylase deficiency and in a compound heterozygous state in at least 48 patients (Speiser et al. 1992; Yoo et al. 2013; Hong et al. 2015; Mohamed et al. 2015). In several families, presumably unaffected parents were identified to be heterozygous carriers of the c.293-13C>G variant. This variant is reported at a frequency of 0.003896 in the Ashkenazi Jewish population from the Genome Aggregation Database. Functional studies in COS-7 cells showed absent CYP21A2 activity for this variant (Higashi et al. 1988). Further, the c.293-13C>G variant was shown to result in abnormal splicing, producing a frameshift which results in premature truncation of the protein. Based on the collective evidence, the c.293-13C>G variant is pathogenic for congenital adrenal hyperplasia due to 21-hydroxylase deficiency. -
Pathogenic, no assertion criteria provided	research	Medical Laboratory Center, Huzhou Maternal and Child Health Hospital	-	- -
Pathogenic, criteria provided, single submitter	clinical testing	Mendelics	May 28, 2019	- -
Pathogenic, criteria provided, single submitter	clinical testing	Breakthrough Genomics, Breakthrough Genomics	Oct 27, 2020	This variant is a well-established pathogenic variant and reported as the most frequent nondeletional mutation found in patients with classic congenital adrenal hyperplasia due to 21-hydroxylase deficiency [PMID: 20301350, 1644925, 2845408, 25041270]. The variant has been reported to cause aberrant splicing of intron 2 resulting in a shift in the translational reading frame [PMID: 20301350, 2845408, 1869518]. -
Pathogenic, criteria provided, single submitter	clinical testing	Provincial Medical Genetics Program of British Columbia, University of British Columbia	Dec 28, 2023	- -
Pathogenic, criteria provided, single submitter	clinical testing	Baylor Genetics	May 03, 2023	- -
Pathogenic, criteria provided, single submitter	clinical testing	3billion	May 22, 2022	The variant is observed at an extremely low frequency in the gnomAD v2.1.1 dataset (total allele frequency: 0.220%). However, frequency data for this variant in the general population cannot be distinguished from that of the (CYP21P) pseudogene, and are therefore uninformative in assessment of variant pathogenicity. In silico tools predict the variant to alter splicing and produce an abnormal transcript (SpliceAI: 0.75). The variant has been reported to be in trans with a pathogenic variant as either compound heterozygous or homozygous in at least 2 similarly affected unrelated individuals (PMID: 24904866, 25630015, 26206692). Therefore, this variant is classified as pathogenic according to the recommendation of ACMG/AMP guideline. -
not provided, no classification provided	phenotyping only	GenomeConnect, ClinGen	-	Variant interpreted as Pathogenic and reported on 11-12-2018 by Lab or GTR ID 500068. GenomeConnect assertions are reported exactly as they appear on the patient-provided report from the testing laboratory. GenomeConnect staff make no attempt to reinterpret the clinical significance of the variant. -
Pathogenic, criteria provided, single submitter	clinical testing	Institute of Human Genetics, University of Leipzig Medical Center	Jul 21, 2021	This variant was identified as compound heterozygous with NM_000500.9:c.?_939+50del. -
Likely pathogenic, criteria provided, single submitter	clinical testing	Genomics England Pilot Project, Genomics England	-	- -
Pathogenic, no assertion criteria provided	curation	GeneReviews	Aug 29, 2013	- -
Pathogenic, no assertion criteria provided	literature only	OMIM	Aug 01, 2003	- -
Pathogenic, no assertion criteria provided	curation	GeneReviews	Aug 29, 2013	- -
Likely pathogenic, criteria provided, single submitter	clinical testing	Center for Genomic Medicine, King Faisal Specialist Hospital and Research Center	Apr 04, 2024	- -
Pathogenic, no assertion criteria provided	clinical testing	Zotz-Klimas Genetics Lab, MVZ Zotz Klimas	Nov 24, 2023	- -
Pathogenic, criteria provided, single submitter	clinical testing	Victorian Clinical Genetics Services, Murdoch Childrens Research Institute	Jul 16, 2023	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 congenital adrenal hyperplasia, due to 21-hydroxylase deficiency (MIM#201910) and nonclassic type hyperandrogenism, due to 21-hydroxylase deficiency (MIM#201910). (I) 0106 - This gene is associated with autosomal recessive disease. (I) 0210 - Splice site variant proven to affect splicing of the transcript with a known effect on protein sequence. RNA studies have shown that this variant causes aberrant splicing which is predicted to cause a frameshift and create a premature termination codon (PMID: 2845408). (SP) 0252 - This variant is homozygous. (I) 0304 - Variant is present in gnomAD (v3) <0.01 for a recessive condition (325 heterozygotes, 1 homozygote). (SP) 0508 - In silico predictions for abnormal splicing are conflicting. (I) 0801 - This variant has strong previous evidence of pathogenicity in unrelated individuals. This has been classified as pathogenic by mulitple clinical laboratories in ClinVar and is very well reported in both compound heterozygous and homozygous states in individuals with congenital adrenal hypoplasia (PMIDs: 35355919, 31586465, 32289882). (SP) 1209 - This variant has been shown to be both maternally and paternally inherited (biallelic) (by trio analysis). (I) Legend: (SP) - Supporting pathogenic, (I) - Information, (SB) - Supporting benign -
Pathogenic, no assertion criteria provided	clinical testing	Lifecell International Pvt. Ltd	-	This variant present in Intron 2 of the CYP21A2 gene c.293-13A/C>G (NM_000500.7). This variant was observed in a proband with increased level of 17-OHP enzyme (>303.7nmol/L) which was screened for advanced newborn screening with confirmatory genetic reflex testing at lifecell diagnostics. This variant has a minor allele frequency of 0.1988% and 0.2261% in 1000 genomes and gnomAD Exomes databases respectively. This variant is characterized by the substitution of A or C nucleotide at 13 bp before the end of intron 2 to G, and activates a cryptic upstream 3' splice acceptor site and causing aberrant splicing (New et al., 2013)This variant has been reported for 21-hydroxylase deficiency (Billerbeck AE et al., 2002 PMID: 12213891, Higashi et al., 1988 PMID: 2845408, Speiser et al., 1992 PMID: 1644925). -
Pathogenic, criteria provided, single submitter	clinical testing	Fulgent Genetics, Fulgent Genetics	Oct 31, 2018	- -
Pathogenic, criteria provided, single submitter	clinical testing	Institute of Human Genetics Munich, Klinikum Rechts Der Isar, TU München	Oct 26, 2017	- -
Pathogenic, criteria provided, single submitter	clinical testing	Myriad Genetics, Inc.	Dec 20, 2019	NM_000500.7(CYP21A2):c.293-13C>G is classified as pathogenic in the context of congenital adrenal hyperplasia and is associated with the classic form of the disease. Sources cited for classification include the following: PMID 1644925 and 2845408. Classification of NM_000500.7(CYP21A2):c.293-13C>G 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. -
Pathogenic, criteria provided, single submitter	clinical testing	Women's Health and Genetics/Laboratory Corporation of America, LabCorp	Mar 15, 2023	Variant summary: CYP21A2 c.293-13C>G alters a non-conserved nucleotide located close to a canonical splice site and therefore could affect mRNA splicing, leading to a significantly altered protein sequence. Several computational tools predict a significant impact on normal splicing: Two predict the variant weakens a 3' acceptor site, and one predicts the variant abolishes a 3' acceptor site. At least one publication reports experimental evidence that this variant affects mRNA splicing (e.g., Higashi_1988). The variant allele was found at a frequency of 0.0023 in 221616 control chromosomes (gnomAD v2.1, Exomes cohort), however, this allele frequency data may be unreliable due to reported possible pseudogene overlap. c.293-13C>G has been reported in the literature in numerous homozygous and compound heterozygous individuals affected with Congenital Adrenal Hyperplasia/Salt Wasting, presenting with salt-wasting, simple virilizing, and non-classical phenotypes (e.g., Wilson_2007, New_2013). These data indicate that the variant is very likely to be associated with disease. At least one publication reports experimental evidence evaluating an impact on protein function, finding that COS cells transfected with the variant displayed undetectable steroid 21-hydroxylase activity (e.g., Higashi_1988). Nineteen ClinVar submitters (evaluation after 2014) have cited the variant, with eighteen submitters classifying the variant as pathogenic and only one submitter classifying the variant as uncertain significance. Based on the evidence outlined above, the variant was classified as pathogenic. -
Pathogenic, criteria provided, single submitter	curation	Laboratory of Medical Genetics, National & Kapodistrian University of Athens	Feb 01, 2024	- -

not provided Pathogenic:8

Pathogenic, criteria provided, single submitter	clinical testing	Revvity Omics, Revvity	Mar 20, 2023	- -
Pathogenic, criteria provided, single submitter	clinical testing	Greenwood Genetic Center Diagnostic Laboratories, Greenwood Genetic Center	Feb 05, 2021	PS3, PS4 -
Pathogenic, criteria provided, single submitter	clinical testing	Labcorp Genetics (formerly Invitae), Labcorp	Jan 26, 2024	This sequence change falls in intron 2 of the CYP21A2 gene. It does not directly change the encoded amino acid sequence of the CYP21A2 protein. The frequency data for this variant in the population databases (gnomAD) is considered unreliable due to the presence of homologous sequence, such as pseudogenes or paralogs, in the genome. This variant has been observed in individual(s) with classic salt-wasting, simple virilizing, and non-classic congenital adrenal hyperplasia due to 21-hydroxylase deficiency (PMID: 20080860, 30995443). 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 IVS2-13A/C>G, I2G, c.293-13A/C>G, In2G. ClinVar contains an entry for this variant (Variation ID: 12155). 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. -
Pathogenic, criteria provided, single submitter	clinical testing	Al Jalila Children’s Genomics Center, Al Jalila Childrens Speciality Hospital	Dec 17, 2022	- -
Pathogenic, criteria provided, single submitter	clinical testing	Athena Diagnostics	Mar 28, 2023	Frequency data for this variant in the general population cannot be distinguished from that of the CYP21P pseudogene, and are therefore uninformative in assessment of variant pathogenicity (Genome Aggregation Database (gnomAD), Cambridge, MA (URL: http://gnomad.broadinstitute.org)). In some published literature, this variant is referred to by nucleotide 655 or 656, and also called the intron 2 G, I2G, or I2 splice variant. In multiple individuals, this variant has been seen in trans with other recessive pathogenic variants in CYP21A2, suggesting this variant is also pathogenic. Assessment of experimental evidence suggests this variant results in abnormal protein function. This variant leads to aberrant RNA splicing and the resulting enzyme has little or no activity (PMID: 2845408). -
Pathogenic, criteria provided, single submitter	clinical testing	Eurofins Ntd Llc (ga)	Jul 24, 2015	- -
Pathogenic, criteria provided, single submitter	clinical testing	Quest Diagnostics Nichols Institute San Juan Capistrano	Mar 28, 2023	The CYP21A2 c.293-13C>G variant (also known as IVS2-13A/C>G or Intron 2G) has been reported in the published literature to disrupt normal splicing of the CYP21A2 mRNA (PMID: 2845408 (1988)) and is usually associated with classic CAH, either simple virilizing or salt wasting (PMIDs: 1644925 (1992), 12213891 (2002), 12788880 (2003), 12915679 (2003), 18381579 (2008), 31586465 (2020), 32289882 (2020), 32959514 (2020)). The frequency of this variant in the general population, 0.0037 (36/9714 chromosomes (Genome Aggregation Database, http://gnomad.broadinstitute.org)), is consistent with pathogenicity. Based on the available information, this variant is classified as pathogenic. -
Pathogenic, criteria provided, single submitter	clinical testing	Clinical Genetics Laboratory, Skane University Hospital Lund	Aug 18, 2022	- -

Adrenal hyperplasia Pathogenic:1

Pathogenic, criteria provided, single submitter

clinical testing

New York Genome Center

Jan 08, 2020

- -

Inborn genetic diseases Pathogenic:1

Pathogenic, criteria provided, single submitter

clinical testing

Ambry Genetics

Apr 22, 2016

- -

See cases Pathogenic:1

Pathogenic, criteria provided, single submitter

clinical testing

Institute of Human Genetics, University Hospital Muenster

Oct 19, 2018

ACMG categories: PS3,PM2,PM3,PP1,PP4,PP5 -

Congenital adrenal hyperplasia Pathogenic:1

Pathogenic, criteria provided, single submitter

clinical testing

Human Genome Sequencing Center Clinical Lab, Baylor College of Medicine

Jun 18, 2020

This c.293-13C>G variant is among the most frequent pathogenic variants in patients with classic congenital adrenal hyperplasia due to 21-hydroxylase deficiency [OMIM 613815.0006]. It causes premature splicing of the intron and a shift in the translational reading frame [PMID 15146390, 25525159]. This variant has been detected in 206 heterozygous and 2 homozygous individuals from the ExAC population database (http://exac.broadinstitute.org/variant/16-3293310-A-G) and in affected patients at the homozygous state in our internal database. This variant is classified as pathogenic. Homozygosity for this variant is also considered medically actionable. -

Computational scores

Source: dbNSFP v4.3

Name	Calibrated prediction	Score	Prediction
BayesDel_addAF	Benign	-0.54	T
BayesDel_noAF	Benign	-0.56
CADD	Benign	1.7
DANN	Benign	0.38
Eigen	Benign	-1.4
Eigen_PC	Benign	-1.5
FATHMM_MKL	Benign	0.0080	N
LIST_S2	Benign	0.23	T
MetaRNN	Benign	0.0066	T
MetaSVM	Benign	-1.0	T
MutationTaster	Benign	1.0	N;N
PROVEAN	Benign	0.10	N
REVEL	Benign	0.044
Sift	Benign	1.0	T
Sift4G	Pathogenic	0.0	D
MVP		0.25
ClinPred		0.0033	T
GERP RS		-0.59

Splicing

Name	Calibrated prediction	Score	Prediction
SpliceAI score (max)		0.75		Details are displayed if max score is > 0.2
DS_AL_spliceai		0.75		Position offset: 13

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

Publications

LitVar

Other links and lift over

dbSNP: rs6467; hg19: chr6-32006858;