Abstract
BackgroundGlucocorticoid-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.ResultsWe 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.ConclusionsPCR-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.
Highlights
Glucocorticoid-remediable aldosteronism (GRA) is a form of herit‐ able 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
The challenge of GRA chimeric form identification Previous studies have suggested that a variety of CYP11B1/CYP11B2 chimeric forms are associated with different clinical presentations [5, 6]
Variety of CYP11B1/CYP11B2 chimeric forms in GRA samples We collected 36 samples from patients who were diagnosed with GRA based on clinical practice guidelines, which was confirmed by using the long-range polymerase chain reaction (PCR) technique to reveal the chimeric genes
Summary
Glucocorticoid-remediable aldosteronism (GRA) is a form of herit‐ able 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. GRA is caused by a chimeric gene resulting from a nonhomologous crossing-over event on chromosome 8q24.3, between the 11β‐hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2) genes This chimeric enzyme contains the promoter of CYP11B1 at the 5′ end and the coding sequences from CYP11B2 at the 3′ end. Because of the relatively high error rate of nanopore sequencing, if the bases aligned to discriminating sites did not exactly match either CYP11B1 or CYP11B2, we considered them to be sequencing or PCR errors and did not include them in the fusion plot This fusion plot provides the intuitive and interpretable chimeric form for each sample. There were no fusion patterns in the fusion plots for Sample no
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