Key result
GRAde long-read sequencing identifies CYP11B1/CYP11B2 gene crossovers in ~83% of glucocorticoid-remediable aldosteronism patients.
Why the study?
Different crossover patterns of CYP11B1 and CYP11B2 chimeric genes may be associated with various clinical presentations in glucocorticoid-remediable aldosteronism, necessitating an efficient approach to identify differences between hybrid genes.
Does the GRAde long-read sequencing pipeline efficiently identify the CYP11B1/CYP11B2 chimeric form in patients with glucocorticoid-remediable aldosteronism?
Observational (n=36)
Does the GRAde long-read sequencing pipeline efficiently identify the CYP11B1/CYP11B2 chimeric form in patients with glucocorticoid-remediable aldosteronism?
The GRAde pipeline using long-read sequencing provides an efficient and precise method to identify complex CYP11B1/CYP11B2 chimeric genes in patients with glucocorticoid-remediable aldosteronism, potentially reducing false-positive diagnoses from standard PCR.
Long-read sequencing with GRAde maps CYP11B1/CYP11B2 chimeras in GRA patients; supports validation before routine diagnostic use.
BACKGROUND: Glucocorticoid-remediable aldosteronism (GRA) is a form of heritable hypertension caused by a chimeric fusion resulting from unequal crossing over between 11β-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2), which are two genes with similar sequences. Different crossover patterns of the CYP11B1 and CYP11B2 chimeric genes may be associated with a variety of clinical presentations. It is therefore necessary to develop an efficient approach for identifying the differences between the hybrid genes of a patient with GRA. RESULTS: We developed a long-read analysis pipeline named GRAde (GRA deciphering), which utilizes the nonidentical bases in the CYP11B1 and CYP11B2 genomic sequences to identify and visualize the chimeric form. We sequenced the polymerase chain reaction (PCR) products of the CYP11B1/CYP11B2 chimeric gene from 36 patients with GRA using the Nanopore MinION device and analyzed the sequences using GRAde. Crossover events were identified for 30 out of the 36 samples. The crossover sites appeared in the region exhibiting high sequence similarity between CYP11B1 and CYP11B2, and 53.3% of the cases were identified as having a gene conversion in intron 2. More importantly, there were six cases for whom the PCR products indicated a chimeric gene, but the GRAde results revealed no crossover pattern. The crossover regions were further verified by Sanger sequencing analysis. CONCLUSIONS: PCR-based target enrichment followed by long-read sequencing is an efficient and precise approach to dissecting complex genomic regions, such as those involved in GRA mutations, which could be directly applied to clinical diagnosis. The scripts of GRAde are available at https://github.com/hsu-binfo/GRAde .
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Wu et al. (2021) conducted an observational in Glucocorticoid-remediable aldosteronism (GRA) (n=36). GRAde long-read sequencing pipeline was evaluated on Identification of crossover events in the CYP11B1/CYP11B2 chimeric gene. The GRAde long-read sequencing pipeline successfully identified crossover events in the CYP11B1/CYP11B2 chimeric gene for 30 out of 36 patients with glucocorticoid-remediable aldosteronism.
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