Classification of c.293-7C>G in the CYP21A2 gene: Contributor to Congenital Adrenal Hyperplasia or Simply a Passenger Variant?
Laboratory Genetics and Genomics
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Primary Categories:
- Laboratory Genetics
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Secondary Categories:
- Laboratory Genetics
Introduction
Congenital adrenal hyperplasia (CAH) is an autosomal recessive genetic condition that affects adrenal hormone production. In over 90% of cases, CAH is caused by 21-hydroxylase deficiency, encoded by CYP21A2 (NM_000500.7). The disease phenotype ranges from altered secondary sex characteristics to salt wasting due to varying amounts of residual enzyme activity.
Case Presentation
CYP21A2 full gene sequencing and deletion/duplication testing was ordered on a 4-year-old female of Ashkenazi Jewish decent. No phenotype was provided for this child. Both parents were positive for CYP21A2 variants on carrier screening at an outside laboratory. Her mother carried c.844G>T (p.Val282Leu), while her father carried a common 30kb deletion.
Diagnostic Workup
Multiplex ligation-dependent probe amplification (MLPA) revealed two copies of CYP21A2, indicating our patient did not inherit her father’s 30kb deletion. Sanger sequencing showed p.Val282Leu, and a second variant, c.293-7C>G. In some cases, phasing can be resolved; however, the methods used did not allow for phasing of these variants for this individual.
Treatment and Management
Parental testing was recommended to resolve phasing and confirm affected versus carrier status.
Outcome and Follow-Up
Our laboratory classifies the c.293-7C>G variant as likely pathogenic.
The c.293-7C>G variant is considered a variant of uncertain significance (VUS) in ClinVar by two laboratories. It has been reported in the literature and observed multiple times by our laboratory in cis with p.Val282Leu. Classification of this variant is challenging, as most observations are in cis with p.Val282Leu, a pathogenic variant associated with 20-50% residual enzyme activity and a non-classic phenotype. c.293-7C>G has an allele frequency of 0.11% in the Ashkenazi Jewish population (0.003% overall). Literature reports and our internal experience suggest that when c.[293-7C>G;844G>T] is detected in trans with a variant causing minimal (<1%) or no residual enzyme activity, a salt-wasting phenotype is observed, a phenotype not expected for p.Val282Leu alone. Thus, c.293-7C>G may further decrease enzyme activity. Splice AI predicted loss of the splice acceptor (score 0.26), a similar effect to the common pathogenic variant, c.293-13C>G, which is associated with salt-wasting. Furthermore, a literature report described an individual with a salt-wasting phenotype who was homozygous for c.293-7C>G alone, and a functional studyexpressing c.293-7C>G in a CYP21A2 minigene in cultured mammalian cells suggested that the variant impaired usage of the intron 2 acceptor splice site resulting in intron retention and no activity.
Discussion
The 2015 Standards and Guidelines for variant interpretation are challenging to use when two variants impacting enzyme activity are identified in cis, which happens in genes like CYP21A2 that undergo pseudogene conversions. The PM3 criterion can be applied for recessive disorders when a variant is detected in trans with a pathogenic variant, while the BP2 criterion may be applied when a variant is observed in cis with a pathogenic variant. Deciding which criteria to apply depends, in part, on the patient phenotype, which is not always available in a reference laboratory setting. Published case studies, functional studies, residual enzyme activity, and patient phenotype can be helpful to determine whether two variants in cis have a more pronounced impact than the more common variant alone.
Conclusion
This case was challenging due to variant pathogenicity and phasing uncertainty. Most observations of c.293-7C>G are in cis with p.Val282Leu, so c.293-7C>G has been previously classified as a VUS. However, given the more severe phenotype described in the literature and observed internally in multiple patients, functional studies, and Splice AI prediction, we classify this variant as likely pathogenic. Furthermore, this case exemplifies the importance of comprehensive CYP21A2 analysis when counseling patients in the preconception and prenatal settings, as the c.293-7C>G was not assessed on the carrier screening test.
Congenital adrenal hyperplasia (CAH) is an autosomal recessive genetic condition that affects adrenal hormone production. In over 90% of cases, CAH is caused by 21-hydroxylase deficiency, encoded by CYP21A2 (NM_000500.7). The disease phenotype ranges from altered secondary sex characteristics to salt wasting due to varying amounts of residual enzyme activity.
Case Presentation
CYP21A2 full gene sequencing and deletion/duplication testing was ordered on a 4-year-old female of Ashkenazi Jewish decent. No phenotype was provided for this child. Both parents were positive for CYP21A2 variants on carrier screening at an outside laboratory. Her mother carried c.844G>T (p.Val282Leu), while her father carried a common 30kb deletion.
Diagnostic Workup
Multiplex ligation-dependent probe amplification (MLPA) revealed two copies of CYP21A2, indicating our patient did not inherit her father’s 30kb deletion. Sanger sequencing showed p.Val282Leu, and a second variant, c.293-7C>G. In some cases, phasing can be resolved; however, the methods used did not allow for phasing of these variants for this individual.
Treatment and Management
Parental testing was recommended to resolve phasing and confirm affected versus carrier status.
Outcome and Follow-Up
Our laboratory classifies the c.293-7C>G variant as likely pathogenic.
The c.293-7C>G variant is considered a variant of uncertain significance (VUS) in ClinVar by two laboratories. It has been reported in the literature and observed multiple times by our laboratory in cis with p.Val282Leu. Classification of this variant is challenging, as most observations are in cis with p.Val282Leu, a pathogenic variant associated with 20-50% residual enzyme activity and a non-classic phenotype. c.293-7C>G has an allele frequency of 0.11% in the Ashkenazi Jewish population (0.003% overall). Literature reports and our internal experience suggest that when c.[293-7C>G;844G>T] is detected in trans with a variant causing minimal (<1%) or no residual enzyme activity, a salt-wasting phenotype is observed, a phenotype not expected for p.Val282Leu alone. Thus, c.293-7C>G may further decrease enzyme activity. Splice AI predicted loss of the splice acceptor (score 0.26), a similar effect to the common pathogenic variant, c.293-13C>G, which is associated with salt-wasting. Furthermore, a literature report described an individual with a salt-wasting phenotype who was homozygous for c.293-7C>G alone, and a functional studyexpressing c.293-7C>G in a CYP21A2 minigene in cultured mammalian cells suggested that the variant impaired usage of the intron 2 acceptor splice site resulting in intron retention and no activity.
Discussion
The 2015 Standards and Guidelines for variant interpretation are challenging to use when two variants impacting enzyme activity are identified in cis, which happens in genes like CYP21A2 that undergo pseudogene conversions. The PM3 criterion can be applied for recessive disorders when a variant is detected in trans with a pathogenic variant, while the BP2 criterion may be applied when a variant is observed in cis with a pathogenic variant. Deciding which criteria to apply depends, in part, on the patient phenotype, which is not always available in a reference laboratory setting. Published case studies, functional studies, residual enzyme activity, and patient phenotype can be helpful to determine whether two variants in cis have a more pronounced impact than the more common variant alone.
Conclusion
This case was challenging due to variant pathogenicity and phasing uncertainty. Most observations of c.293-7C>G are in cis with p.Val282Leu, so c.293-7C>G has been previously classified as a VUS. However, given the more severe phenotype described in the literature and observed internally in multiple patients, functional studies, and Splice AI prediction, we classify this variant as likely pathogenic. Furthermore, this case exemplifies the importance of comprehensive CYP21A2 analysis when counseling patients in the preconception and prenatal settings, as the c.293-7C>G was not assessed on the carrier screening test.