The gene CYP21A2, mapped to the RCCX module in 6p21.3, is responsible for the 21-hydroxylase deficiency (21OHD). In this work, we used Oxford Nanopore Technology (ONT) Long Reads (LR) sequencing to analyze samples from a large Argentinian cohort of 21OHD. Our goal was to gain additional information about the GVs involved in the rearrangements, to obtain higher resolution for the breakpoints of converted alleles and to retrieve new data of TNX and CYP21A1P genes. A total of 34 samples were sequenced in 2 amplicons of 8.5 Kb covering the centromeric and telomeric RCCX modules. The number of GVs found varied between 17-106 and all expected pathogenic GVs and new ones were obtained with the LR sequencing workflow developed. From the 21 alleles with macroconverted or chimeric genes containing the promoter of the CYP21A1P, we uncovered 5 of them lacking GVs commonly expected to be involved in 21OHD rearrangements. Importantly, we reclassified one patient with a deletion that had been overlooked with the current diagnostic technologies applied. These new findings change the genetic counseling for the patient and his family related to the presence of a carrier allele for Congenital Adrenal Hyperplasia and Ehlers-Danlos Syndrome (CAH-X). Furthermore, by addressing the study of the centromeric and the telomeric RCCX modules, we found 19 GVs for CYP21A1P and 29 GVs for TNXA not previously described in Latin American populations or not reported in any of the consulted databases. This study may represent the first one applying ONT LR in clinical studies in Latin America, highlighting the importance of LR sequencing as a high-resolution method of diagnosis and better cost-effective balance. It allows us to better understand the diversity of the RCCX modules and to gain knowledge of the molecular pathogenesis of the disease.

The authors have declared no competing interest.

This work was supported by grants from the University of Buenos Aires (PIDAE 2020 and 2022, LD), from the National Agency for the Promotion of Science and Technology (ANPCyT, PICT-2021-CAT-II-00018, LK), and from the Administracion Nacional de Laboratorios e Institutos de Salud (FOCANLIS 2022, NRU:2022-5, MT).

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Yes

The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

The protocol was approved and gave by the ethics committee of the Administracion Nacional de Laboratorios e Institutos de Salud (A.N.L.I.S.), Buenos Aires, Argentina

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Yes

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All data produced in the present study are available upon reasonable request to the authors