Systematic Detection of Pathogenic Alu Element Insertions in NGS-Based Diagnostic Screens: TheBRCA1/BRCA2Example

Abstract

Pathogenic Alu element insertions are rarely reported, whereas their occurrence is expected to be much higher. Alu containing alleles are usually out-competed during the PCR process and consequently undetectable with the classical screening methods. However, with the introduction of the next generation sequencing (NGS) technology in the diagnostic field, new opportunities are emerging. NGS data for a particular genomic region can be seen as the summation of all the individual sequences (reads) obtained for that region and no longer as the mean of this sum as it is the case for traditional Sanger sequencing. Because each single read covering that region is expected to be generated from a different template molecule, the presence of one single mutant read must theoretically be sufficient to identify the mutation. However, generation and identification of mutant reads bearing Alu insertions remains challenging and several wet/dry bench parameters need to be optimized. Hereby we present the proof of principle of a NGS-based mutation screening procedure allowing the detection of inherited Alu insertions within any predefined sequence by investigating 2 cases: c.1739_1740insAlu in BRCA1 and c.156_157insAlu in BRCA2.

AluY-mediated germline deletion, duplication and somatic stem cell reversion inUBE2Tdefines a new subtype of Fanconi anemia

“…Alu-mediated deletions have been identified in other genetic disorders ( 59 , 60 ) and also in other DNA repair/FA genes such as BRCA2 and FANCA ( 61 – 64 ). These homology-mediated rearrangements/deletions are increasingly recognized as an important mechanism for introducing variations and causing mutations in the human genome ( 59 , 65 ).…”

Large Genomic Rearrangements of BRCA1 and BRCA2 among Patients Referred for Genetic Analysis in Galicia (NW Spain): Delimitation and Mechanism of Three Novel BRCA1 Rearrangements

“…However, the optimization of next generation sequencing standard protocols for detection of Alu element rearrangements have resulted in false positive reads [32]. The shortcoming of MLPA is that it does not identify LGR breakpoints, as is necessary for recognition of recurrent rearrangements, for inference of the molecular mechanisms of rearrangement, and for rapid analysis of a proband's relatives.…”

Transposable Elements in Human Cancer: Causes and Consequences of Deregulation

“…However, TE insertion also targets genes that are involved in the DNA repair pathway. The breast cancer susceptibility gene BRCA2 has been affected by multiple TE insertions in cancers [12,98]. In addition, the APC gene is also disrupted by multiple TE insertions ( Alu and LINE1 ) and is suggested to play a role in colorectal carcinogenesis [13,99].…”

“…In addition, rearrangements due to Alu insertion and the presence of Alu in the MLH1 and MLH2 proteins are associated with hereditary non-polyposis colorectal cancer [92]. Recombination events due to the high density of Alu elements within the BRCA1 gene are associated with the important deregulation of genomic integrity in breast cancer [12,98]. Alu repeats have also contributed significantly to chromosomal translocations, including BCR/ABL rearrangement in chronic myelogeneous leukemia [126].…”