Repeat expansion scanning of the NOTCH2NLC gene in patients with multiple system atrophy

Abstract

Objective: Trinucleotide GGC repeat expansion in the 5'UTR of the NOTCH2NLC gene has been recognized as the pathogenesis of neuronal intranuclear inclusion disease (NIID). Previous studies have described that some NIID patients showed clinical and pathological similarities with multiple system atrophy (MSA). This study aimed to address the possibility that GGC repeat expansion in NOTCH2NLC might be associated with some cases diagnosed as MSA.

Methods: A total of 189 patients with probable or possible MSA were recruited to screen for GGC repeat expansion in NOTCH2NLC by repeat-primed PCR (RP-PCR). In addition, long-read sequencing (LRS) was performed for all patients with RP-PCR-positive expansion, five patients with RP-PCR-negative expansion, and five controls on the Nanopore platform. Skin biopsies were performed on two patients with GGC expansion.

Results: Five of 189 patients (2.6%) were found to have GGC expansion in NOTCH2NLC. LRS results identified that the five patients had GGC expansion between 101 and 266, but five patients with RP-PCR-negative expansion and five controls had GGC expansion between 8 and 29. Besides the typical symptoms and signs of MSA, patients with GGC expansion might have longer disease duration, severe urinary retention, and prominent cognitive impairment. In the skin samples from the patients with GGC expansion, typical p62-postive but alpha-synuclein-negative intranuclear inclusions were found in fibroblasts, adipocyte and ductal epithelial cells of sweat glands.

Conclusion: Trinucleotide GGC repeat expansion in NOTCH2NLC could be observed in patients with clinically diagnosed MSA. Adult-onset NIID should be considered as a differential diagnosis of MSA.

Figure 1

Repeat‐primed PCR was used to identify the repeat expansion in the NOTCH2NLC gene. The long saw‐tooth curves indicated that the numbers of GGC in the five patients did exceed a value of at least 100 repeat expansions, A to E stand for patient 1 to patient 5, respectively. The sequencing map showed a single peak wave without saw‐tooth pattern in a healthy control subject (F).

Figure 2

Nanopore long‐read sequencing (LRS) verified the GGC repeat expansion of NOTCH2NLC. LRS revealed 12 reads of repeats between 18 and 24, and 7 reads of repeats between 112 and 138 in patient 1(A). LRS showed 14 reads of repeats between 11 and 16, and 7 reads of repeats between 140 and 155 in patient 2 (B). LRS exhibited 21 reads of repeats between 22 and 28, and 6 reads of repeats between 118 and 135 in patient 3 (C). LRS identified 11 reads of repeats between 12 and 16, and 10 reads of repeats between 101 and 126 in patient 4 (D). LRS found 26 reads of repeats between 9 and 23, and 5 reads of repeats between 210 and 266 in patient 5 (E). LRS only showed 21 reads of repeats between 18 and 26 in a control (F).

Figure 3

Magnetic resonance imaging representative in this group of MSA patients. Cerebellar atrophy and hot cross bun sign were indicated in a MSA‐C patient without GGC expansion (A–C). Putamen atrophy and lineal T2 high intensity of the lateral margin of the putamen were observed in a MSA‐P patient without GGC expansion (D–F). White matter lesions (WML) suggesting chronic ischemic arteriopathy were notable in a MSA‐C patient without GGC expansion (G–I). White matter lesions and cortical atrophy were found in a MSA‐C patient with GGC expansion (J–L).

Figure 4

Skin biopsy of two MSA patients with GGC repeat expansion. HE stain revealed typical eosinophilic intranuclear inclusions in the nuclei of fibroblasts and ductal epithelial cells of sweat glands (A). Magnification of the rectangle in A (B, arrow). These eosinophilic inclusions in fibroblasts and ductal epithelial cells of sweat glands (C, arrow) and fat cells (D, arrow) were p62 positive, but alpha‐synuclein negative (E). Electron microscopy revealed a pile of round‐halo filamentous materials in the center of the nucleus (F).