A pilot study on the clinical utility of blocker displacement amplification to detect rare alleles in mosaicism

Abstract

Background: Mosaicism refers to two or more different populations of cells with different genotypes co-existed within an individual. Previous literatures estimated the percentage of mosaic cases in rare diseases to be 4-13%, with a much lower percentage locally at 2%. Detection of rare mutant alleles were challenging due to their presence in low level and sequence homology with the much more abundant wildtype. This study aimed to pilot test the blocker displacement amplification (BDA) method previously reported by Wu et. al. (2017) for the efficient detection of rare single nucleotide variant (SNV) as well as rare exonic deletions. Methods: BDA acted as a rare allele enrichment assay by incorporating a blocker that has an exact complementary sequence with the wildtype. Sequence with the rare mutant allele would subsequently manifest a mismatch bubble between the template-blocker duplex, increasing the favourability of primer displacing the blocker that eventually lead to the enrichment of rare allele. We designed respective blockers and primer pairs to perform BDA for two patients with rare diseases that have apparent de novo mutations: one child with an heterozygous TSC2: c.4493+1G>A variant and one child with an heterozygous deletion of exons 10 to 12 in SPTAN1 gene. We modified the existing BDA method to incorporate a gap-polymerase chain reaction (GAP-PCR) to detect exonic deletion. Results: Preliminary results from gel electrophoresis and/or sanger sequencing showed that BDA selectively amplified the mutant alleles in all trio samples and there was currently no evidence of parental mosaicisms in both cases by using peripheral blood samples. A known positive mosaic case shall be included in the future to further validate the results. Based on these preliminary results, investigations using germ cells (sperm/egg) would be needed to completely rule out parental mosaicism since mosaicism could exist exclusively in germ cells in some rare occasions. Carrying a hidden mutation in the germ cells will have an impact on recurrence and reproductive risk for the next pregnancies, hence have a great importance in family planning. Conclusion: This pilot study evaluated the possible usage of BDA to detect rare SNV and rare exonic deletions. The next step would be to identify more subjects, collect appropriate sample type to test BDA and cross validate the results with existing methods such us deep sequencing/digital polymerase chain reaction (dPCR). BDA could be an economical alternative for the screening of rare mutant alleles when comparing to deep sequencing, which costs 300 fold more, as well as dPCR which costs 3 fold more. Additional application of BDA includes noninvasive cancer recurrence monitoring or any other areas that require discovery and profiling of rare alleles.