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Characterization of FMR1 Repeat Expansion and Intragenic Variants by Indirect Sequence Capture

Traditional methods for the analysis of repeat expansions, which underlie genetic disorders, such as fragile X syndrome (FXS), lack single-nucleotide resolution in repeat analysis and the ability to characterize causative variants outside the repeat array. These drawbacks can be overcome by long-rea...

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Autores principales: Grosso, Valentina, Marcolungo, Luca, Maestri, Simone, Alfano, Massimiliano, Lavezzari, Denise, Iadarola, Barbara, Salviati, Alessandro, Mariotti, Barbara, Botta, Annalisa, D’Apice, Maria Rosaria, Novelli, Giuseppe, Delledonne, Massimo, Rossato, Marzia
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8504923/
https://www.ncbi.nlm.nih.gov/pubmed/34646309
http://dx.doi.org/10.3389/fgene.2021.743230
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author Grosso, Valentina
Marcolungo, Luca
Maestri, Simone
Alfano, Massimiliano
Lavezzari, Denise
Iadarola, Barbara
Salviati, Alessandro
Mariotti, Barbara
Botta, Annalisa
D’Apice, Maria Rosaria
Novelli, Giuseppe
Delledonne, Massimo
Rossato, Marzia
author_facet Grosso, Valentina
Marcolungo, Luca
Maestri, Simone
Alfano, Massimiliano
Lavezzari, Denise
Iadarola, Barbara
Salviati, Alessandro
Mariotti, Barbara
Botta, Annalisa
D’Apice, Maria Rosaria
Novelli, Giuseppe
Delledonne, Massimo
Rossato, Marzia
author_sort Grosso, Valentina
collection PubMed
description Traditional methods for the analysis of repeat expansions, which underlie genetic disorders, such as fragile X syndrome (FXS), lack single-nucleotide resolution in repeat analysis and the ability to characterize causative variants outside the repeat array. These drawbacks can be overcome by long-read and short-read sequencing, respectively. However, the routine application of next-generation sequencing in the clinic requires target enrichment, and none of the available methods allows parallel analysis of long-DNA fragments using both sequencing technologies. In this study, we investigated the use of indirect sequence capture (Xdrop technology) coupled to Nanopore and Illumina sequencing to characterize FMR1, the gene responsible of FXS. We achieved the efficient enrichment (> 200×) of large target DNA fragments (~60–80 kbp) encompassing the entire FMR1 gene. The analysis of Xdrop-enriched samples by Nanopore long-read sequencing allowed the complete characterization of repeat lengths in samples with normal, pre-mutation, and full mutation status (> 1 kbp), and correctly identified repeat interruptions relevant for disease prognosis and transmission. Single-nucleotide variants (SNVs) and small insertions/deletions (indels) could be detected in the same samples by Illumina short-read sequencing, completing the mutational testing through the identification of pathogenic variants within the FMR1 gene, when no typical CGG repeat expansion is detected. The study successfully demonstrated the parallel analysis of repeat expansions and SNVs/indels in the FMR1 gene at single-nucleotide resolution by combining Xdrop enrichment with two next-generation sequencing approaches. With the appropriate optimization necessary for the clinical settings, the system could facilitate both the study of genotype–phenotype correlation in FXS and enable a more efficient diagnosis and genetic counseling for patients and their relatives.
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spelling pubmed-85049232021-10-12 Characterization of FMR1 Repeat Expansion and Intragenic Variants by Indirect Sequence Capture Grosso, Valentina Marcolungo, Luca Maestri, Simone Alfano, Massimiliano Lavezzari, Denise Iadarola, Barbara Salviati, Alessandro Mariotti, Barbara Botta, Annalisa D’Apice, Maria Rosaria Novelli, Giuseppe Delledonne, Massimo Rossato, Marzia Front Genet Genetics Traditional methods for the analysis of repeat expansions, which underlie genetic disorders, such as fragile X syndrome (FXS), lack single-nucleotide resolution in repeat analysis and the ability to characterize causative variants outside the repeat array. These drawbacks can be overcome by long-read and short-read sequencing, respectively. However, the routine application of next-generation sequencing in the clinic requires target enrichment, and none of the available methods allows parallel analysis of long-DNA fragments using both sequencing technologies. In this study, we investigated the use of indirect sequence capture (Xdrop technology) coupled to Nanopore and Illumina sequencing to characterize FMR1, the gene responsible of FXS. We achieved the efficient enrichment (> 200×) of large target DNA fragments (~60–80 kbp) encompassing the entire FMR1 gene. The analysis of Xdrop-enriched samples by Nanopore long-read sequencing allowed the complete characterization of repeat lengths in samples with normal, pre-mutation, and full mutation status (> 1 kbp), and correctly identified repeat interruptions relevant for disease prognosis and transmission. Single-nucleotide variants (SNVs) and small insertions/deletions (indels) could be detected in the same samples by Illumina short-read sequencing, completing the mutational testing through the identification of pathogenic variants within the FMR1 gene, when no typical CGG repeat expansion is detected. The study successfully demonstrated the parallel analysis of repeat expansions and SNVs/indels in the FMR1 gene at single-nucleotide resolution by combining Xdrop enrichment with two next-generation sequencing approaches. With the appropriate optimization necessary for the clinical settings, the system could facilitate both the study of genotype–phenotype correlation in FXS and enable a more efficient diagnosis and genetic counseling for patients and their relatives. Frontiers Media S.A. 2021-09-27 /pmc/articles/PMC8504923/ /pubmed/34646309 http://dx.doi.org/10.3389/fgene.2021.743230 Text en Copyright © 2021 Grosso, Marcolungo, Maestri, Alfano, Lavezzari, Iadarola, Salviati, Mariotti, Botta, D’Apice, Novelli, Delledonne and Rossato. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Genetics
Grosso, Valentina
Marcolungo, Luca
Maestri, Simone
Alfano, Massimiliano
Lavezzari, Denise
Iadarola, Barbara
Salviati, Alessandro
Mariotti, Barbara
Botta, Annalisa
D’Apice, Maria Rosaria
Novelli, Giuseppe
Delledonne, Massimo
Rossato, Marzia
Characterization of FMR1 Repeat Expansion and Intragenic Variants by Indirect Sequence Capture
title Characterization of FMR1 Repeat Expansion and Intragenic Variants by Indirect Sequence Capture
title_full Characterization of FMR1 Repeat Expansion and Intragenic Variants by Indirect Sequence Capture
title_fullStr Characterization of FMR1 Repeat Expansion and Intragenic Variants by Indirect Sequence Capture
title_full_unstemmed Characterization of FMR1 Repeat Expansion and Intragenic Variants by Indirect Sequence Capture
title_short Characterization of FMR1 Repeat Expansion and Intragenic Variants by Indirect Sequence Capture
title_sort characterization of fmr1 repeat expansion and intragenic variants by indirect sequence capture
topic Genetics
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8504923/
https://www.ncbi.nlm.nih.gov/pubmed/34646309
http://dx.doi.org/10.3389/fgene.2021.743230
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