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Keywords: Genetic testing; Lysosomal diseases; Massively parallel sequencing; Methodology; Mutation detection

R. Owen; S.H. Rosenthal; A. Gerasimova; A. Smolgovsky; A. Buller-Burckle; F. Lacbawan

Affiliation: Quest Diagnostics, San Juan Capistrano, CA

Background: The GBA gene encodes glucocerebrosidase, a lysosomal enzyme that breaks down glucocerebroside into ceramide and glucose. Genetic variants in GBA can cause the autosomal recessive disorder Gaucher disease, the most common lysosomal storage disorder, and are strong risk factors for Parkinson’s disease. GBA analysis is complicated by the presence of a pseudogene, GBAP1, located 16 kb downstream of the GBA gene. Sanger sequencing of gene specific PCR amplicons is the most commonly used method for variant detection. However, the method is not suitable to consolidation with other analytical methods, such as next generation sequencing (NGS) when used in carrier screening for inherited disorders. Here, we aimed to design and validate a new 2-step PCR NGS library preparation method for the sequencing of GBA using an Illumina Miseq. Method: The 8 most common GBA variants included for detection were: p.N409S (N370S), c.84dupG (84GG), p.R535H (R496H), p.L483P (L444P), c.115+1G>A (IVS2+1G>A), p.V433L (V394L), p.D448H (D409H), and c.1263_1317del (del55bp). The first PCR used conventional long-range PCR to specifically amplify targets in GBA but not in GBAP1. The second PCR used nested PCR with primers containing Illumina sequencing primer binding sites which enabled addition of specimen specific barcoded sequencing adapters via concomitant fusion PCR. Sequencing ultimately was performed on the Illumina MiSeq platform. Compatibility of the method within an expanded NGS carrier screening panel was evaluated in combination with 18 other inherited diseases. Results: Method validation using 159 samples (94 positives [17 GBA positives], 65 negatives) showed 100% sensitivity and specificity. Subsequent testing using 11,778 consecutive clinical samples submitted for the validated carrier screening test showed that 2.0% (232/11,778) of cases harbored a GBA variant: 174 cases positive for N370S, 29 cases with L444P, 9 cases with R496H, 6 cases with 84GG, 6 cases with D409H, 4 cases with V394L, 3 cases with IVS2+1G>A, and 1 case with del55bp. Conclusion: This study demonstrated that the 2-step PCR NGS library construction method successfully screens for common GBA variants and can be readily consolidated for use in a comprehensive carrier screening panel.