Breakpoint mapping at nucleotide resolution in X-autosome balanced translocations associated with clinical phenotypes

Abstract

Precise breakpoint mapping of balanced chromosomal rearrangements is crucial to identify disease etiology. Ten female patients with X-autosome balanced translocations associated with phenotypic alterations were evaluated, by mapping and sequencing their breakpoints. The rearrangements' impact on the expression of disrupted genes, and inferred mechanisms of formation in each case were assessed. For four patients that presented one of the chromosomal breaks in heterochromatic and highly repetitive segments, we combined cytogenomic methods and short-read sequencing to characterize, at nucleotide resolution, breakpoints that occurred in reference genome gaps. Most of rearrangements were possibly formed by nonhomologous end joining and have breakpoints at repeat elements. Seven genes were found to be disrupted in six patients. Six of the affected genes showed altered expression, and the functional impairment of three of them were considered pathogenic. One gene disruption was considered potentially pathogenic, and three had uncertain clinical significance. Four patients presented no gene disruptions, suggesting other pathogenic mechanisms. Four genes were considered potentially affected by position effect and the expression abrogation of one of them was confirmed. This study emphasizes the importance of breakpoint-junction characterization at nucleotide resolution in balanced rearrangements to reveal genetic mechanisms associated with the patients' phenotypes, mechanisms of formation that originated the rearrangements, and genomic nature of disrupted DNA sequences.

Mechanisms of structural chromosomal rearrangement formation

“…NHEJ is considered the major mechanism responsible for the formation of balanced chromosomal alterations [ 20 , 47 ]. Moysés-Oliveira et al [ 20 ] reported mutational signatures of c-NHEJ in the breakpoints of six out of ten patients in a study of X-autosome balanced translocations.…”

Molecular Cytogenetics in the Era of Chromosomics and Cytogenomic Approaches

“…Prenatal FISH Weise and Liehr (2008) For limitations of FISH in general, there are to mention, that 1) the resolution in standard FISH is limited to kilo-to megabasepair (except for molecular combing approach), and thus exact mapping of chromosomal breakpoints needs combined approaches like, e.g., applied in Jancuskova et al (2013) or Moysés-Oliveira et al (2019), 2) disease causing gene mutations at base pair level normally cannot be accessed by FISH-one exception was recently published (Molecular Combing Section) (Nguyen et al, 2019), 3) as in cytogenetics and next-generation sequencing (NGS) for qualified evaluation and interpretation of FISH-results experienced specialists are needed, 4) corresponding to question to be studied costs of consumables may be relatively high, and 5) number of probes being applied simultaneously is limited by number of fluorochromes and software; however, recent developments proved also solutions for this (Su et al, 2020).…”

Disruption of <b><i>PCDH10</i></b> and <b><i>TNRC18</i></b> Genes due to a Balanced Translocation

“…The evolution of accurate and affordable next-generation sequencing (NGS) techniques, such as whole genome sequencing (WGS), has been fundamental to elucidate breakpoints at nucleotide resolution contrib-uting to a molecular diagnosis [Aristidou et al, 2017[Aristidou et al, , 2018Schluth-Bolard et al, 2019]. Low coverage WGS, for example, is an efficient method for the detection and characterization of balanced translocations [Liang et al, 2017], and this characterization is improved when WGS is combined with cytogenomic methods [Moysés-Oliveira et al, 2019]. A more cost-effective strategy corresponds to the sequencing of specific microdissected segments from the derivative chromosomes, targeting the genomic regions surrounding the breakpoints [Jancuskova et al, 2013].…”

“…cDNA samples were assayed in technical triplicate with standard PCR conditions of the 96-Well Fast Thermal Cycling (Thermo Fisher Scientific) and Applied Biosystems 7900HT Sequence Detection System (Thermo Fisher Scientific). A qualitative analysis was performed to assess the relative gene expression between the proband and controls as previously described [Moysés-Oliveira et al, 2019].…”