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

Comprehensive short and long read sequencing analysis for the Gaucher and Parkinson’s disease-associated GBA gene

“…This allowed us to provide the first large-scale data on CNVs, and to evaluate the frequency of all classes of GBA variants in PD and LBD with greater accuracy than before. Reciprocal recombinants in particular are likely missed in PD studies 16 , including a recent targeted short-read study which detected none in 3402 patients 8 , although one study using exome data with qPCR validation reported CNGs in 1.2% of PD and 0.7% of controls 22 . In non-diseased individuals, we noted that CNGs were more common in those with “African” ancestry, with greater copy number variability.…”

“…Current sequencing approaches to characterise GBA have major pitfalls, and to date, no single approach has fully resolved recombinants 6 . The correct alignment of short reads when there is a highly similar pseudogene is intrinsically problematic, and GBA is challenging in exome and whole-genome sequencing (WGS) 8 – 10 , containing camouflaged regions 11 . Moreover, the reliability of the standard WGS secondary analysis pipelines such as the Genome Analysis Toolkit best practice workflow 12 has not been formally assessed.…”

Newborn screening for Gaucher disease in Japan

“…The exonic sequence homology between GBA and GBAP1 is more than 86%, increasing enhancing the likelihood of homologous recombination. Several different recombinant alleles and their crossover sites have been reported [ [21] , [25] ] (Supplemental data 2). The presence of the highly homologous pseudogene makes analysis of the GBA gene challenging.…”

Long-read single molecule real-time (SMRT) sequencing of GBA1 locus in Gaucher disease national cohort from Argentina reveals high frequency of complex allele underlying severe skeletal phenotypes: Collaborative study from the Argentine Group for Diagnosis and Treatment of Gaucher Disease

“…Comprehensive GBA1 genotyping remains challenging [ 10 , 11 , 25 , 26 ], due to the vast multiplicity of disease mutations and highly homologous pseudogene in proximity, that harbors numerous disease mutations. Several variants normally present in the GBA1P sequence, when they occur in GBA1 , generally cause severe forms of GD, e.g., L444P, complex alleles with multiple pseudogene variants in tandem, D409H, and 55 bp deletion, etc.…”

“…Moreover, this standard approach does not allow phasing of the pathogenic variants. Next generation sequencing (NGS) platforms [ 27 ], utilize short read lengths (from 25 bp to 400 bp), also inadequate for precise identification of GBA1 variants, especially for complex alleles and structural variants (SVs) [ 11 , 26 ].…”