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).