Fig 2: Methylation and expression at the NOTCH2NLC locus. (A and B) Methylation status across the expanded GGC repeat region was determined using LRS data from three affected individuals (Patients F1-III8, F1-III10 and S1), 10 normal control subjects, and four NIID patients. (A) There were no significant differences between three affected individuals (Patients F1-III8, F1-III10 and S1) and 10 normal controls in methylation. (B) There were no significant differences in methylation between three affected individuals (Patients F1-III8, F1-III10 and S1) and four NIID patients. (C and D) Methylation analysis of the promoter region of expanded GGC repeats in the 5'UTR of the NOTCH2NLC gene in blood. Methylation levels were significantly increased in three asymptomatic carriers (Patients F-II5, F-II7 and father of Patient S2), compared with OPDM3 patients (Patients F-III8, S1 and S3) and controls (F-II2, F-II3 and mother of Patient S2). M = methylated; U = unmethylated. Ratio of methylation level of NOTCH2NLC to that of unmethylation level (M/U) was used to represent the relative methylation status. (E and F) Analysis of transcriptional levels of NOTCH2NLC mRNA in normal control subjects, OPDM3 patients and asymptomatic carriers by RT-qPCR in peripheral blood or muscle samples. No significant difference was detected between control subjects and OPDM3 patients (Patients F1-III8, F1-III10, S1 and S3), while asymptomatic carriers showed significantly lower NOTCH2NLC mRNA levels in peripheral blood (*P < 0.05) (E). The transcriptional levels of NOTCH2NLC mRNA were in an increasing trend in muscle samples (F) from two OPDM3 patients (Patients F1-III10 and S2).

Fig 3: Validation of GGC repeat expansions and repeat sizes in NOTCH2NLC among OPDM3 patients and normal control subjects. (A) Representative results of RP-PCR analysis showing GGC repeat expansions in Patients F1-III8, F1-III10 and S1. No repeat expansions were shown in one unaffected member Patient F1-III9. Experiments were conducted three times with reproducible results. LRS data showed estimated GGC repeat count of NOTCH2NLC reached more than 100 in Patients F1-III8 (B) and S1 (C) but no more than 25 in the unaffected member Patient F1-II8 (D). (E) Frequency distribution of GGC repeat units in NOTCH2NLC ranging from 6 to 26 among 109 normal control subjects. DNA samples from normal controls were amplified by NOTCH2NLC-specific primers and revealed by fragment analysis. Heterozygosity rate in normal controls was 77.98%. (F) Pie chart for the percentages of disease-causing gene mutations and unknown gene mutations in a cohort of Chinese OPDM patients. Disease-causing genes, trinucleotide repeat expansions in the 5'UTRs of LRP12, GIPC1 and NOTCH2NLC, accounted for 4.17%, 50% and 16.67% of cases in 24 unrelated Chinese OPDM patients, respectively, whereas unknown genetic cause accounted for 29.17% of cases in this cohort.

Fig 4: MRI and pathological changes of the OPDM3 patients. (A–F) Muscle MRI of OPDM3 patients showed fatty infiltration of lower limb muscles, with the distal muscles (B, D and F calf level) more severely affected than the proximal muscles (A, C and E thigh level). (A and B) Muscle MRI of Patient F1-III10: moderate weakness with a disease duration of 6 years, ambulatory without support. (C and D) Muscle MRI of Patient F1-III8, moderate weakness with a disease duration of 9 years, ambulatory without support. (E and F) Muscle MRI of Patient S1: severe weakness with a disease duration of 11 years, ambulatory without support. (G–N) Brain MRIs of Patients S2 (G and H), S1 (I and J), F1-III8 (K and L), and S3 (M and N). (G) Hyperintense linear lesions in corticomedullary junctions in a diffuse weight image (DWI). (H) Severely diffuse leukoencephalopathy on T2-weighted fluid-attenuated inversion recovery (T2-FLAIR). (I–L) Mild white matter signal abnormalities on T2-FLAIR in periventricular white matter and splenium of corpus callosum after 18 years (Patient S1) and 15 years (Patient F1-III8) of the disease, respectively. (M and N) Normal brain MRIs on T2-FLAIR. (O) Haematoxylin and eosin, and (P) modified Gomori trichrome (mGT) stainings of muscle sections from Patient F1-III8, showing dystrophic change with variation in fibre size and endomysial fibrosis, and fibres with rimmed vacuoles (marked by arrow and shown at higher magnification). The intramuscular peripheral nerves were well myelinated (arrowhead) (P). (Q) Electron microscopy of muscle tissue from Subject F1-III10 revealed various myelin figures and autophagic vacuoles containing osmiophilic deposits and amorphous materials. (R and S) Electron microscopy of muscle sample from Patient S2 showed intranuclear inclusions contained filamentous aggregates (marked by arrowhead and shown at higher magnification). (T) Haematoxylin and eosin staining of skin sections from Patient S1 showed intranuclear inclusions in the fibroblasts (marked by arrow and shown at higher magnification). (U) Electron microscopy of skin sample from Patient S1 showed intranuclear inclusions. (V) Immunofluorescence on skin sections from Patient S1 showing p62 positive intranuclear inclusions in the sweat gland (marked by arrow and shown at higher magnification). Scale bars = 50 µm (O and T), 100 µm (P), 2 µm (Q), 0.5 µm (R, S and U) and 25 µm (V).

Fig 5: Validation of GGC repeat expansions in the NOTCH2NLC gene among three index patients by RP-PCR. Three index patients showed a saw-tooth pattern of the repeat expansion in the NOTCH2NLC.