False negatives in GBA1 sequencing due to polymerase dependent allelic imbalance

Abstract

A variant in the GBA1 gene is one of the most common genetic risk factors to develop Parkinson’s disease (PD). Here the serendipitous finding is reported of a polymerase dependent allelic imbalance when using next generation sequencing, potentially resulting in false-negative results when the allele frequency falls below the variant calling threshold (by default commonly at 30%). The full GBA1 gene was sequenced using next generation sequencing on saliva derived DNA from PD patients. Four polymerase chain reaction conditions were varied in twelve samples, to investigate the effect on allelic imbalance: (1) the primers (n = 4); (2) the polymerase enzymes (n = 2); (3) the primer annealing temperature (Ta) specified for the used polymerase; and (4) the amount of DNA input. Initially, 1295 samples were sequenced using Q5 High-Fidelity DNA Polymerase. 112 samples (8.6%) had an exonic variant and an additional 104 samples (8.0%) had an exonic variant that did not pass the variant frequency calling threshold of 30%. After changing the polymerase to TaKaRa LA Taq DNA Polymerase Hot-Start Version: RR042B, all samples had an allele frequency passing the calling threshold. Allele frequency was unaffected by a change in primer, annealing temperature or amount of DNA input. Sequencing of the GBA1 gene using next generation sequencing might be susceptible to a polymerase specific allelic imbalance, which can result in a large amount of flase-negative results. This was resolved in our case by changing the polymerase. Regions displaying low variant calling frequencies in GBA1 sequencing output in previous and future studies might warrant additional scrutiny.

Next-Generation Sequencing Analysis of GBA1: The Challenge of Detecting Complex Recombinant Alleles

“…In addition, there is evidence to suggest that the choice of polymerase used could be a factor in the accuracy of NGS variant identification. A recent study performed a large-scale screening of GBA1 based on an NGS protocol and found a significant number of false negatives due to a polymerase-dependent allelic imbalance (den Heijer et al, 2021 ). After performing a structured assessment of varied PCR conditions, they found that changing the polymerase used led to the resolution of these false negatives.…”

“…The complexity of GBA1 gene rearrangements cannot be sufficiently captured using most current NGS methods. Several recent studies using NGS technology without Sanger sequencing validation have not reported the presence of recombinant alleles, including a recent study performed on more than 3,000 PD cases (den Heijer et al, 2020). Thus, it is likely that the results presented may be an underestimation (Zampieri et al, 2017).…”

“…The use of whole-exome and whole-genome sequencing has increased the discovery rate of these variants. For example, a recent large-scale NGS strategy identified 18 novel GBA1 variants, as well as a rarely reported complex allele likely to be a Dutch founder allele (Zampieri et al, 2017;den Heijer et al, 2020). One Gaucher diagnostics laboratory relying on NGS run by Centogene (Rostock, Germany) provides an on-line list of their identified GBA1 variants (CentoLSD TM ), which now includes over 600 different GBA1 variants with differing predicted clinical significance (https://www.centogene.…”

Patient centered guidelines for the laboratory diagnosis of Gaucher disease type 1

“…Strategies to specifically analyze the GBA1 as a single gene using NGS technology have been developed [167][168][169]. Such NGS strategies allow the identification of single base pair variants and recombinant alleles (excluding the Recdelta55) with high specificity and sensitivity [167,169].…”

“…Strategies to specifically analyze the GBA1 as a single gene using NGS technology have been developed [167][168][169]. Such NGS strategies allow the identification of single base pair variants and recombinant alleles (excluding the Recdelta55) with high specificity and sensitivity [167,169]. Conversely, analysis of the GBA1 gene as part of gene panels using well designed NGS strategies that consider the presence of the pseudogene, allows only the identification of point mutations, while fail to identify both large deletions and recombinant alleles due misalignment of reads with the homologous pseudogene [170][171][172][173][174].…”