Prevalence and Characterization of NOTCH2NLC GGC Repeat Expansions in Koreans: From a Hospital Cohort Analysis to a Population-Wide Study

Abstract

Background and objectives: GGC repeat expansions in the NOTCH2NLC gene are associated with a broad spectrum of progressive neurologic disorders, notably, neuronal intranuclear inclusion disease (NIID). We aimed to investigate the population-wide prevalence and clinical manifestations of NOTCH2NLC-related disorders in Koreans.

Methods: We conducted a study using 2 different cohorts from the Korean population. Patients with available brain MRI scans from Seoul National University Hospital (SNUH) were thoroughly reviewed, and NIID-suspected patients presenting the zigzag edging signs underwent genetic evaluation for NOTCH2NLC repeats by Cas9-mediated nanopore sequencing. In addition, we analyzed whole-genome sequencing data from 3,887 individuals in the Korea Biobank cohort to estimate the distribution of the repeat counts in Koreans and to identify putative patients with expanded alleles and neurologic phenotypes.

Results: In the SNUH cohort, among 90 adult-onset leukoencephalopathy patients with unknown etiologies, we found 20 patients with zigzag edging signs. Except for 2 diagnosed with fragile X-associated tremor/ataxia syndrome and 2 with unavailable samples, all 16 patients (17.8%) were diagnosed with NIID (repeat range: 87-217). By analyzing the Korea Biobank cohort, we estimated the distribution of repeat counts and threshold (>64) for Koreans, identifying 6 potential patients with NIID. Furthermore, long-read sequencing enabled the elucidation of transmission and epigenetic patterns of NOTCH2NLC repeats within a family affected by pediatric-onset NIID.

Discussion: This study presents the population-wide distribution of NOTCH2NLC repeats and the estimated prevalence of NIID in Koreans, providing valuable insights into the association between repeat counts and disease manifestations in diverse neurologic disorders.

Figure 1. Study Cohorts and Overall Workflow

(A) We screened the brain MRI scans of 90 undiagnosed patients with adult-onset leukoencephalopathy who showed the zigzag edging signs on DWI (SNUH cohort). As a result, 20 patients were identified, and Cas9-mediated nanopore sequencing was performed for 16 patients after excluding 2 for whom samples were unavailable and 2 patients with FXTAS. (B) We included 3,887 individuals from the Korea Biobank cohort who underwent whole-genome sequencing (Korea Biobank cohort). We investigated the NOTCH2NLC GGC repeats and identified 6 putative patients with repeat expansions. DWI = diffusion-weighted imaging.

Figure 2. Association Between NOTCH2NLC Repeats and Clinical Manifestations of Encephalitis-Like Episodes

(A) A negative association between NOTCH2NLC repeats and the occurrence of encephalitis-like episodes. The SNUH cohort of NIID patients with encephalitis-like episodes had significantly shorter NOTCH2NLC repeats than those without these events (Mann-Whitney U test, p = 0.016). (B) Changes in brain MRI findings over time in patient P04. The left and right images were acquired during encephalitis-like episodes, while the middle images were obtained during a stable stage. Two images on the left (upper, T2-FLAIR; lower, DWI) show high-signal intensities (SIs) with diffusion restriction in the left parieto-occipital lobe and multifocal confluent high SIs in both the periventricular and subcortical white matter areas. The middle and right images were taken 2 and 3 years later, respectively (upper, T2-weighted; lower, DWI). In this series of images, it is evident that leukoencephalopathy deteriorates over time, and the regions exhibiting diffusion restriction vary at each time point. DWI = diffusion-weighted imaging.

Figure 3. Exploring NOTCH2NLC GGC Repeat Expansions in the Korea Biobank Cohort

(A) Distribution of the sizes of GGC repeats in 2,737 unrelated controls (5,446 alleles). The red dashed line represents the obtained cutoff value of 63.48. The 6 outliers are colored in red. The y-axis displays the allele count value on a square root transformed scale. (B) Size comparison of the GGC repeats among various ethnicities: 2,737 Korean, 961 Asian, 8,126 White, and 282 Black populations. (C) Clustering of STR transmission patterns within the NOTCH2NLC region, which has been categorized based on the number of STRs present in the parents. The magnitude of change (∆) within the interval is indicated by the 95% confidence interval.

Figure 4. A Korean Family With NOTCH2NLC-Related Disorders Identified in the Korea Biobank Cohort

(A) Pedigree information of the KBB1 family. Both the first child (KBB1S) and the second child (KBB1P) exhibited cerebellar ataxia and Charcot-Marie-Tooth disease. The first child had more severe symptoms with rapid progression and died at the age of late 10s. (B) Brain MRI of both affected patients showed the zigzag edging sign and cerebellar atrophy. (C) Nanopore long-read sequencing for the family members. Both affected patients had NOTCH2NLC repeat expansions within the disease-associated range (KBB1S: 90; KBB1P: 109), while the father was found to possess an extremely long allele (574) that differed from the estimation by ExpansionHunter with the short-read data (57).

Figure 5. Patterns of Allele Transmissions and Methylation Within the NOTCH2NLC Promoter Region in the KBB1 Family

(A) Investigation of allele transmission based on 3 single-nucleotide variants. The allele-phasing analysis reveals that the long-expanded allele (pale blue) in the asymptomatic father was transmitted to the offspring with contraction. (B) Methylation frequencies within the NOTCH2NLC promoter region (chr1:149,390,710-149,390,901) among the KBB1 family members. The asymptomatic father (KBB1F, red) exhibits significantly higher methylation frequencies (hypermethylation) than other family members.