This gene sequencing panel includes both sequencing and deletion/duplication (CNV) analysis for all coding regions of the CYP21A2 gene. Analysis is performed utilizing either LR-PCR followed by NGS sequencing or MLPA. All variants are classified according to American College of Genetics and Genomics (ACMG) guidelines.

21-hydroxylase deficiency is a disease that causes excessive androgen production in the adrenal glands. There are three distinct types of 21-hydroxylase deficiency: salt wasting, simple virilizing, and non-classic. The salt wasting form of the disease is the most severe with an age of onset typically in the fetal or neonatal periods and have symptoms of excessive loss of sodium in the urine, poor feeding, weight loss, dehydration, vomiting, ambiguous genitalia in females, small testses in males, an early growth spurt, shorter adult height, decreased ferility, hirsutism in females, irregular menstruation, and male pattern baldness. The simple virilizing form has an age of onset in the fetal or neonatal period, with symptoms of anbiguous genitalia in females, small testes in males, an early growth spurt, shorter adult height, decreased ferility, hirsutism in females, irregular menstruation, and male pattern baldness. The non-classic form has an age of onset in adolesence or adulthood with symptoms of hirsuitism in females, male pattern baldness, irregular menstruation, decreased fertility, early beard growth and small testes in males. The incidence of the salt wasting and simple virilizing forms for 21-hydroxylase deficiency is estimated to be ~ 1 in 15,000. The incidence of the non-classic form of 21-hydroxylase deficiency is estimated to be ~ 1 in 1000.

CYP21A2 long-range PCR was performed to capture the genomic sequences for the real CYP21A2 gene from this individual’s genomic DNA to prevent the pseudogene from being co-amplified. Next-generation sequencing (NGS) was performed on an Illumina system with 100 base pair paired-end reads. A base is considered to have sufficient coverage at 20X and an exon is considered fully covered if all coding bases plus three nucleotides of flanking sequence on either side are covered at 20X or more. Low coverage regions, if any, are limited to ~1% or less of the nucleotides in the test unless a pathogenic variant explaining the phenotype is discovered. A list of these regions is available upon request. Alignment to the human reference genome (hg19) is performed and annotated variants are identified in the targeted region. Variants are called at a minimum coverage of 8X and an alternate allele frequency of 20% or higher. Indels and Single nucleotide variants (SNVs) are confirmed by Sanger sequence analysis before reporting at the director's discretion. This assay cannot detect variants in regions of the exome that are not covered, such as deep intronic, promoter, and enhancer regions or long repetitive regions. Copy number variation (CNV) analysis was assessed using MLPA. This analysis cannot determine the location or orientation of a duplication. This assay is not designed to detect mosaicism; possible cases of mosaicism may be investigated at the discretion of the laboratory director.