Long-read sequencing transforms the diagnosis of congenital adrenal hyperplasia: resolving pseudogene interference and structural variations
Open accessAbstract
Background Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder primarily caused by defects in adrenal steroidogenesis. Conventional genetic methods struggle to resolve complex structural variations and pseudogene interference in key genes like CYP21A2. Our study will evaluate the efficacy of Long-Read Sequencing (LRS) as a comprehensive diagnostic tool for CAH, demonstrating its ability to simultaneously detect large structural variations, single nucleotide variants (SNVs), and small insertions or deletions. Methods Four probands with clinically diagnosed CAH underwent detailed biochemical profiling, including serum 17-hydroxyprogesterone, serum sodium and serum potassium. Genomic DNA was extracted from peripheral blood and subjected to LRS using Single-Molecule Real-Time (SMRT) Technologies (Pacifc Biosciences). A targeted panel covering the CYP21A2 and HSD3B2 genes, as well as other genes related to CAH was captured. Bioinformatic analysis included alignment with Minimap2, variant calling with Sniffles2 and Medaka, and phasing analysis to resolve pseudogene interference. Results LRS identified compound heterozygous and homozygous variants in CYP21A2 (e.g., c.293-13C > G, c.518T > A, CH-1) and novel compound heterozygous variants in HSD3B2 (c.121G > T and c.757T > G). In combination with biochemical tests, clinical manifestations, and the ACMG guidelines, these gene mutations were the cause of the patient's disease. LRS resolved pseudogene interference and provided unambiguous cis/trans phasing. Conclusion LRS is a robust diagnostic tool for CAH, offering comprehensive detection of genetic variants, including large deletions and SNVs in both cis and trans forms. Its ability to resolve pseudogenes and structural variations positions LRS as a first-tier diagnostic tool for CAH, improving accuracy, streamlining clinical workflows and ultimately benefits patients.
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LRS allows precise characterization of genome rearrangements, repetitive sequences, and regions of high sequence identity, such as the CYP21A2 locus and its highly homologous pseudogene CYP21A1P.
“By generating contiguous kilobase-scale reads, LRS allows precise characterization of genome rearrangements, repetitive sequences, and regions of high sequence identity, such as the CYP21A2 locus and its highly homologous pseudogene CYP21A1P (84, 87).”
From Evolution of molecular diagnostic strategies for 21-hydroxylase deficiency: from classical methods to advanced genomic techniques (2026) · cites this paper as [87]
The Introduction says LRS uses long reads to detect structural variations and resolve highly homologous regions, specifically identifying CYP21A2 and its pseudogene CYP21A1P. The Discussion likewise describes detection of complex structural variations. Our copy does not specifically establish precise characterization of repetitive sequences or give a kilobase-scale read length. (LLM, conf=0.84)
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“Its integration could provide complete genotyping in a single test, reducing delays and substantially lowering false-positive rates (87).”
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- Authors
- Zeng, J., Huang, X., Li, Y., Yan, T., Lou, J., Lyu, G., & Li, Y.
- Corresponding author
- Lyu, Guizhen
- Li, Yanjin
- Venue
- Frontiers in Pediatrics
- Publisher
- Frontiers Media SA
- Published
- 2025-06-27
- Volume
- 13
- Pages
- 1603819-1603819
- ISSN
- 2296-2360 (ISSN-L)
- PMC
- PMC12245889
- OpenAlex
- W4411706521
- Cited by
- 2
- References
- 43
- Citations by year
- 2026: 2
- FWCI
- 0.60
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- Frontiers in Pediatrics (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Sexual Differentiation and Disorders
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Genomics and Rare Diseases
- Health Sciences › Medicine › Endocrinology, Diabetes and Metabolism › Adrenal Hormones and Disorders
- Keywords
- congenital adrenal hyperplasia, long-read sequencing, CYP21A2, HSD3B2, structural variants, compound heterozygosity, adrenal steroidogenesis, genetic diagnosis, single-molecule real-time sequencing
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