Long-Read Sequencing

Comprehensive Analysis of Congenital Adrenal Hyperplasia (CACAH)

Comprehensive Coverage of CYP21A2-TNXB, Resolving Pseudogene Interference

The Diagnostic Gaps

Congenital Adrenal Hyperplasia (CAH) is characterized by deficiencies in several enzymes in the adrenal steroidogenesis pathway, resulting in impaired cortisol synthesis.¹ Over 90% of CAH cases are 21-hydroxylase deficiency (21-OHD), which can be clinically classified into three types: classic salt-wasting, classic simple-virilizing, and non-classic CAH.²

While most cases of classic CAH are detected early via biochemical newborn screening, it faces significant diagnostic gaps in milder cases. Biochemical assays have false-positive rates between 0.4% and 9.3%³, and about 20% of simple-virilizing CAH cases are missed in newborn screening⁴. Furthermore, non-classic CAH is frequently misdiagnosed due to mild or nonspecific symptoms like infertility and hyperandrogenism.

CAH Types

Frequency

Genes

21-hydroxylase deficiency

(21-OHD)

90-95%

CYP21A2

11β-Hydroxylase deficiency

5-8%

CYP11B1

17α-Hydroxylase deficiency

CYP17A1

3β-Hydroxysteroid dehydrogenase type 2 deficiency

HSD3B2

Lipoid CAH

Rare

STAR

POR deficiency

Rare

POR

SCC deficiency

Rare

CYP11A1

Information derived from reference 2

A Novel Approach with LRS

Comprehensive Analysis of Spinal Congenital Adrenal Hyperplasia (CACAH) offers the most complete analysis for all CAH classifications. Because genotype directly correlates with clinical phenotype in >90% of CAH cases⁵, precise genetic testing is vital for patient management and carrier screening.

Routine assays (MLPA, Sanger, NGS) often fail to distinguish the functional CYP21A2-TNXB from its pseudogene, CYP21A2P-TNXA. They struggle to detect frequent CYP21A2-TNXB mutations: recombination, conversions, and CNVs.

CACAH utilizes LRS to accurately map the entire locus for 7 CAH genes. It identifies precise breakpoints and separates functional genes from pseudogenes, making it the superior choice for comprehensive CAH genotyping.

Proven Superiority

Studies demonstrate that CACAH proband testing outperforms the routine trio assays (Trio-NGS + Trio-MLPA)⁶

From concordant results, CACAH upgraded the resolution (from exon-level down to precise nucleotide breakpoints) for CYP21A2-TNXB/CYP21A2P-TNXA recombination
Corrected discordant results in 14.58% of probands, including previously missed variants, contiguous SNVs previously miscalled as deletion

In a retrospective study group (96 probands and 191 family members from 95 families)⁶

When considering our send-out sequencing services:

Consultation: Contact our team for the most current test specifications.
Sample Preparation: Check sample types and shipment requirements to ensure high-quality results. Please check your local export regulations and logistics partners.
Submission: Contact Xcelom when placing an order. Include the completed Test Request and Consent Form, along with any required documents.

Sample Requirements

Peripheral Blood: 2 mL in EDTA tube

Dried Blood Spot (DBS): 3 spots, ≥ 8mm diameter each

Long-fragment gDNA

'**For prenatal cases, please include maternal sample for STR to exclude maternal contamination.

Transport Conditions

2-8℃, arrive within 72 hours

Testing Scope

Basic panel

Detects 514 Pathogenic (P) /Likely Pathogenic (LP) variants, including:

CYP21A2-TNXB: 12 deletions, 273 SNVs/InDels, and 12 CNVs
CYP11B1: 1 deletion and 79 SNVs/InDels
CYP17A1: 55 SNVs/InDels
HSD3B2: 36 SNVs/InDels
STAR: 46 SNVs/InDels

Comprehensive panel

Detects P/LP/some VUS SNVs/InDels, selected large intragenic deletions in 7 genes (CYP21A2, CYP11B1, CYP17A1, HSD3B2, STAR, POR, and CYP11A1), and specific CNVs in the CYP21A2

Prenatal Comprehensive panel

Detects P/LP SNVs/InDels, selected large intragenic deletions in 7 genes (CYP21A2, CYP11B1, CYP17A1, HSD3B2, STAR, POR, and CYP11A1), and specific CNVs in the CYP21A2

Turnaround Time (TAT)

15 working days

Xcelom Limited and Berry Genomics provide an end-to-end turnkey solution for labs wishing to conduct this test in-house.

Laboratory Setup: Full consultation
Workflow Integration: SOPs, reagents, and staff training
Bioinformatics: Complete software suite for sample management and automated report generation

Platform

PacBio Sequel IIe, Vega and Revio system

Sample Type

gDNA from dried blood spot, blood, buccal swab, and amniotic fluid

Test per Batch

Max. 288/ 144 Tests per SMRT Cell (Mixed Batching Supported)

Operation Time

~ 69 hours (Hand on time: ~10.5 hours)

Automation

Supported

Coverage

5/ 7 genes associated with CAH

The provided time is based on the PacBio Sequel IIe system and may vary across different labs and systems.

Resources

CACAH Brochure

References:

1. El-Maouche D, Arlt W, Merke DP. Congenital adrenal hyperplasia. Lancet. 2017;390(10108):2194-2210.

2. Krone N, Arlt W. Genetics of congenital adrenal hyperplasia. Best Pract Res Clin Endocrinol Metab. 2009;23(2):181-192.

3. Hayashi GY, Carvalho DF, de Miranda MC, et al. Neonatal 17-hydroxyprogesterone levels adjusted according to age at sample collection and birthweight improve the efficacy of congenital adrenal hyperplasia newborn screening. Clin Endocrinol (Oxf). 2017;86(4):480-487.

4. Gidlöf S, Wedell A, Guthenberg C, von Döbeln U, Nordenström A. Nationwide neonatal screening for congenital adrenal hyperplasia in sweden: a 26-year longitudinal prospective population-based study. JAMA Pediatr. 2014;168(6):567-574.

5. Finkielstain GP, Chen W, Mehta SP, et al. Comprehensive genetic analysis of 182 unrelated families with congenital adrenal hyperplasia due to 21-hydroxylase deficiency. J Clin Endocrinol Metab. 2011;96(1):E161-E172

6. Wang Y, Zhu G, Li D, et al. High clinical utility of long-read sequencing for precise diagnosis of congenital adrenal hyperplasia in 322 probands. Hum Genomics. 2025;19(1):3.