Molecular insights into the mechanism of nonrecurrent
            <i>F8</i>
            structural variants: Full breakpoint characterization and bioinformatics of DNA elements implicated in the upmost severe phenotype in hemophilia A

Abelleyro, Miguel Martín; Radic, Claudia Pamela; Marchione, Vanina Daniela; Waisman, Karen; Tetzlaff, Tomás; Neme, Daniela; Rossetti, Liliana Carmen; Brasi, Carlos Daniel de

doi:10.1002/humu.23977

Cited by 9 publications

(29 citation statements)

References 47 publications

(75 reference statements)

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“…In order to understand the molecular mechanisms leading to these Xq28 duplications, the characterization of breakpoints at the nucleotide level was required. Though many F8 deletion breakpoints have been sequenced, 25–27 sequencing duplication junctions has proved more challenging. LR‐PCR has been successfully used to characterize 10 F8 intragenic tandem duplications 17 .…”

Section: Discussionmentioning

confidence: 99%

Complete characterisation of two new large Xq28 duplications involving F8 using whole genome sequencing in patients without haemophilia A

et al. 2021

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Introduction Depending on the location of insertion of the gained region, F8 duplications can have variable clinical impacts from benign impact to severe haemophilia A phenotype. Aim To characterize two large Xq28 duplications involving F8 incidentally detected by chromosome microarray analysis (CMA) in two patients presenting severe intellectual disability but no history of bleeding disorder. Methods Whole genome sequencing (WGS) was performed in order to characterize the two large Xq28 duplications at nucleotide level. Results In patient 1, a 60–73 kb gained region encompassing the exons 23–26 of F8 and SMIM9 was inserted at the int22h‐2 locus following a non‐homologous recombination between int22h‐1 and int22h‐2. We hypothesized that two independent events, micro‐homology‐mediated break‐induced replication (MMBIR) and break‐induced replication (BIR), could be involved in this rearrangement. In patient 2, the CMA found duplication from 101 to 116‐kb long encompassing the exons 16–26 of F8 and SMIM9. The WGS analysis identified a more complex rearrangement with the presence of three genomic junctions. Due to the multiple micro‐homologies observed at breakpoints, a replication‐based mechanism such as fork stalling and template switching (FoSTeS) was greatly suspected. In both cases, these complex rearrangements preserved an intact copy of the F8. Conclusion This study highlights the value of WGS to characterize the genomic junction at the nucleotide level and ultimately better describe the molecular mechanisms involved in Xq28 structural variations. It also emphasizes the importance of specifying the structure of the genomic gain in order to improve genotype‐phenotype correlation and genetic counselling.

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Section: Discussionmentioning

confidence: 99%

Complete characterisation of two new large Xq28 duplications involving F8 using whole genome sequencing in patients without haemophilia A

et al. 2021

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“…For this purpose, we used several practical approaches, including SNP-array, WGS and customized PCRs. Followed by a bioinformatic approach, screening the surroundings of the SVs' breakpoints for the identification of DNA DSB stimulator motifs, which have been found statistically significantly more frequent at breakpoint junctions than expected by chance according to Abelleyro et al (2020) (Abelleyro et al, 2020).…”

Section: Discussionmentioning

confidence: 99%

“…On the other hand, we want to highlight the important role that secondary structures must be exerting in the origin of SVs, given that we found them in 7/8 breakpoints. Its implication for DSB formation is suggested by the fact that if we randomly select 50pb throughout the genome it is very likely that they form secondary structures, but if we simulate random breakpoints and analyze 25bp at each side of it, the chances of finding secondary structures involving them are greatly reduced (Abelleyro et al, 2020).…”

Section: Discussionmentioning

confidence: 99%

“…These elements constitute a heterogeneous group of sequences with different lengths (3-18bp), that may act as stimulators for DSB, triggering an incorrect DNA repair/DNA replication leading to non-allelic recombination. The study was performed using the Human Reference Genome (GRCh38) [NC 000023.11: 31641233-32372273 downloaded 5-Sep-2018 from the NCBI website, www.ncbi.nlm.nih.gov/] and was based on a recently reported strategy (Abelleyro et al, 2020). For this analysis, DSB stimulation motifs that showed significant Expected values (E-values <0.05) in random points from the referred study were considered (Abelleyro et al, 2020 (Jurka, 1997).…”

Section: Deletion and Duplication Breakpoints Bioinformatic Analysismentioning

confidence: 99%

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A Multi-Technique Molecular Analysis of a DMD Family With Two Independent Mutational Events and a Manifesting Pregnant Woman. Bioinformatic Analysis of Molecular Scars at Breakpoint Junctions and Hypothesization of the Underlying Molecular Mechanisms.

Luce

Abelleyro

Carcione

et al. 2020

Preprint

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Our work depicts a familial Duchenne muscular dystrophy case with a complex structural variant (cxSV) and a manifesting pregnant woman. Were our aims to provide molecular diagnosis and hypothesize mechanisms underlying the origin of the cxSV. We implemented a multi-technique approach including MLPA, STRs-segregation, AR-assay, SNP-array, WGS and a bioinformatic algorithm for identification of double strand breaks (DSB) stimulator motifs. We established the carrier status of the prenatal sample and explained its mother s symptomatology by skewed X-chromosome inactivation. Furthermore, an ancestral familial ex38-43 duplication plus a de novo ex45-54 deletion was revealed in the proband, who carried the cxSV in a recombinant maternal X-chromosome. Characterization of cxSV s breakpoints junction and its surrounding sequences allowed us to identify DSB stimulator motifs. The replication-dependent "Fork Stalling and Template Switching" mechanism was predicted to be the most likely scenario for the duplication s origin. Whilst, the de novo deletion could arise from a germline event of inter-chromosome non-allelic recombination involving the "Non-Homologous End Joining" mechanism. The multi-technique strategy enabled precise diagnosis, accurate genetic assessment and widen the understanding of the molecular mechanisms involved in SVs' generation. Finally, the further comprehension of the occurrence of DMD variants, favors the development of new gene therapy strategies.

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“…Human Gene Mutation Database (HGMD) has subdivided genomic deletions into microdeletions (1-20 bp) and gross deletions (>20 bp) (Stenson et al, 2020), but this distinction was originally made fairly arbitrarily for reasons of practical utility rather than for any cogent biological reason. Many studies (Carvalho & Lupski, 2016;Keute et al, 2020;Maranchie et al, 2004;Sahoo et al, 2006) have suggested the involvement of different mechanisms in the formation of microdeletions and gross deletions including nonhomologous end-joining (NHEJ), microhomology-mediated end-joining (MMEJ), non-allelic homologous recombination (NAHR), retrotransposon-mediated mechanisms, and replication-based errors including fork stalling and template switching (FoSTeS) and microhomology-mediated break-induced replication (MMBIR) (Abelleyro et al, 2020;Eckelmann et al, 2020;Bauters et al, 2008;Carvalho et al, 2009;Férec et al, 2006;Gadgil et al, 2020;Hu et al, 2019;Lee et al, 2007;Marey et al, 2016;Summerer et al, 2018;J. Vogt et al, 2014;Zhang et al, 2009Zhang et al, , 2010.…”

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confidence: 99%

Distinct sequence features underlie microdeletions and gross deletions in the human genome

Stenson

Ball

et al. 2022

Human Mutation

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Microdeletions and gross deletions are important causes (~20%) of human inherited disease and their genomic locations are strongly influenced by the local DNA sequence environment. This notwithstanding, no study has systematically examined their underlying generative mechanisms. Here, we obtained 42,098 pathogenic microdeletions and gross deletions from the Human Gene Mutation Database (HGMD) that together form a continuum of germline deletions ranging in size from 1 to 28,394,429 bp. We analyzed the DNA sequence within 1 kb of the breakpoint junctions and found that the frequencies of non-B DNA-forming repeats, GC-content, and the presence of seven of 78 specific sequence motifs in the vicinity of pathogenic deletions correlated with deletion length for deletions of length ≤30 bp.Further, we found that the presence of DR, GQ, and STR repeats is important for the formation of longer deletions (>30 bp) but not for the formation of shorter deletions (≤30 bp) while significantly (χ 2 , p < 2E−16) more microhomologies were identified flanking short deletions than long deletions (length >30 bp). We provide evidence to support a functional distinction between microdeletions and gross deletions. Finally, we propose that a deletion length cut-off of 25-30 bp may serve as an objective means to functionally distinguish microdeletions from gross deletions.

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Molecular insights into the mechanism of nonrecurrent F8 structural variants: Full breakpoint characterization and bioinformatics of DNA elements implicated in the upmost severe phenotype in hemophilia A

Cited by 9 publications

References 47 publications

Complete characterisation of two new large Xq28 duplications involving F8 using whole genome sequencing in patients without haemophilia A

Complete characterisation of two new large Xq28 duplications involving F8 using whole genome sequencing in patients without haemophilia A

A Multi-Technique Molecular Analysis of a DMD Family With Two Independent Mutational Events and a Manifesting Pregnant Woman. Bioinformatic Analysis of Molecular Scars at Breakpoint Junctions and Hypothesization of the Underlying Molecular Mechanisms.

Distinct sequence features underlie microdeletions and gross deletions in the human genome

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