Profiling the NOTCH2NLC GGC Repeat Expansion in Parkinson's Disease in the European Population

A GGC repeat expansion in the gene Notch homolog 2 N-terminal-like C (NOTCH2NLC) is causative of neuronal intranuclear inclusion body disease (NIID). Regarding Parkinson’s disease (PD) specifically, NOTCH2NLC repeat expansions have been reported in patients with PD in the East Asian population. However, when profiled in a small number of PD cases of European ancestry enrolled in the 100 000 Genomes Project (n = 825), no enrichment of pathogenic expansion was identified. In this study, we aimed to establish the prevalence of the NOTCH2NLC GGC repeat expansion in a large number of case/control individuals to further assess if this repeat expansion is associated with the risk of PD in the European population. Among the 6595 individuals genetically screened only one individual was predicted to carry a pathogenic length GGC repeat expansion with ExpansionHunter v4.02. Oxford Nanopore long-read sequencing data was generated to validate the ExpansionHunter predicted pathogenic carrier (>66 GGC repeats). The sample was processed and sequenced using a protocol optimized for population scale long-read sequencing from frontal cortex brain tissue and repeat size was manually determined. Although ExpansionHunter predicted the GGC-repeat size to be 67, 44 to 48 were estimated via long-read sequencing, meaning the individual was an intermediate expansion carrier (Supplementary Figure S1). Further, 11 PD cases (0.36%) were predicted to carry intermediate GGC repeat expansions ranging from 40 to 62 repeats, and 15 controls (0.43%) carried intermediate GGC repeat expansions ranging from 38 to 60 repeat. Overall, no enrichment of intermediate expansions was observed in PD cases (P = 0.81) (Supplementary Table S1). However, as shown by our long-read validation and reported by other studies, although ExpansionHunter can correctly identify large expanded alleles, in some cases predicted allele length can be under/overestimated, hence experimental validation studies are needed. Because DNA was not available for the predicted intermediate expansion carriers in this present study, the role of intermediate expansions in PD warrants further study. In summary, in line with Yau and colleagues, our analysis suggests that pathogenic NOTCH2NLC GCC repeat expansions are rare and are not associated with PD in the European population. One limitation of this study is that ExpansionHunter calls were used to estimate repeat size. One of the drawbacks of using this approach is that genotyping loci that have reference allele sizes close to read length can be challenging (like in the case of NOTCH2NLC). Despite this, in their analysis of the 100 000 Genomes Project genomes Yau and colleagues demonstrate that ExpansionHunter showed high sensitivity and can successfully detect true pathogenic length NOTCH2NLC GGC repeat expansion carriers in large whole genome datasets, supporting its application as a tool for screening repeat expansion disorders. Our observation highlights the need for long-read sequencing data or repeat-primed polymerase chain reaction (PCR) in conjunction with ExpansionHunter calls to validate potential pathogenic length repeat expansion carriers to remove false positives. As the field advances, population-scale long-read sequencing datasets will allow us to unequivocally assess the role of the NOTCH2NLC GGC repeat expansion (along with other repeat expansions) in the genetics of PD.