The CGG repeat expansion in RILPL1 is associated with oculopharyngodistal myopathy type 4

Abstract

Recent studies indicate that CGG repeat expansions in LRP12, GIPC1, and NOTCH2NLC are associated with oculopharyngodistal myopathy (OPDM) types 1, 2, and 3, respectively. However, some clinicopathologically confirmed OPDM cases continue to have unknown genetic causes. Here, through a combination of long-read whole-genome sequencing (LRS), repeat-primed polymerase chain reaction (RP-PCR), and fluorescence amplicon length analysis PCR (AL-PCR), we found that a CGG repeat expansion in the 5' UTR of RILPL1 is associated with familial and simplex OPDM type 4 (OPDM4). The number of repeats ranged from 139 to 197. Methylation analysis indicates that the methylation levels in RILPL1 were unaltered in OPDM4 individuals. Analyses of muscle biopsies suggested that the expanded CGG repeat might be translated into a toxic poly-glycine protein that co-localizes with p62 in intranuclear inclusions. Moreover, analyses suggest that the toxic RNA gain-of-function effects also contributed to the pathogenesis of this disease. Intriguingly, all four types of OPDM have been found to be associated with the CGG repeat expansions located in 5' UTRs. This finding suggests that a common pathogenic mechanism, driven by the CGG repeat expansion, might underlie all cases of OPDM.

Keywords: CGG repeat expansion; RILPL1; co-regulation; intranuclear inclusion; long-read whole-genome sequencing; oculopharyngodistal myopathy; polyG disease.

Figure 1

Muscle MRI and myopathological changes of the OPDM4-affected individuals (A) Pedigrees of family 1 and OPDM-affected simplex individual 1. OPDM-affected individuals are indicated by filled symbols. The individual with essential tremor but not OPDM is indicated by a half-filled symbol. All numbered individuals had available blood DNA. (B–G) Different extents of fatty infiltration of lower-limb muscles by muscle MRI in F1-III-1 (B–D) and S1 (E–G). (H–K) Variation in fiber size and rimmed vacuoles (marked by an arrow) on muscle biopsy in S1 (H and I) compared with the control (J and K). (L–O) Intranuclear filamentous inclusions (L and M) and various myelin figures and autophagic vacuoles (N and O) of muscle tissue upon electron microscopy in F1-II-2 (L) and S1 (M–O). (H and J) H&E staining; (I and K) mGT staining. Scale bars represent 20 μm (H–K), 500 nm (L), 1 μm (M), and 2 μm (N and O).

Figure 2

Identification of the CGG repeat expansion and repeat sizes in RILPL1 by LRS in OPDM-affected individuals (A)The Integrative Genomics Viewer showed that three lanes carried the RILPL1 CGG repeat expansion (chr12: 124,018,267–124,018,302, hg19 version), as opposed to the normal allele, in in F1-III-1 and S1. (B–D) LRS data showed an estimated CGG repeat count of RILPL1 of more than 100 in OPDM-affected individuals F1-III-1 (B) and S1 (C) but no more than 40 in the controls (D).

Figure 3

Validation of the CGG repeat expansion and repeat sizes in RILPL1 by RP-PCR and AL-PCR in OPDM-affected individuals (A) Schematic representation of RILPL1. The disease-associated repeat expansion (red boxes) was identified in the 5′ UTR. The primer set was designed for RP-PCR analysis to identify the CGG repeat expansion (red lines and arrows). (B) Representative results of RP-PCR analysis showing CGG repeat expansion in RILPL1 in S1. No CGG repeat expansion was found in the unaffected control. Experiments were conducted three times with reproducible results. (C) Representative results of AL-PCR analysis showing the repeat count of expanded CGG in RILPL1 in S1. The number of repeats was multiplied by 3 bp per repeat. No CGG repeat expansion was detected in the unaffected control. (D) Fragment analysis showed that the frequency distribution of CGG repeat units in RILPL1 ranged from 9 to 16 among 200 normal controls. (E) There was no significant correlation between the number of CGG repeats and the age at onset in 11 Chinese OPDM4-affected individuals (r = 0.3382, p = 0.3091, n = 11). (F) Pie chart for the percentages of disease-causing gene mutations and unknown gene mutations in three cohorts of Chinese OPDM-affected individuals. Trinucleotide repeat expansions in the 5′ UTRs of LRP12, GIPC1, NOTCH2NLC, and RILPL1 accounted for 3.9%, 37.3%, 13.7%, and 21.6% of disease cases in 51 unrelated Chinese OPDM-affected individuals, respectively, whereas unknown genetic causes accounted for 23.5% of disease cases in this cohort.

Figure 4

Methylation status analysis and immunoblot on muscle biopsy samples (A) Methylation status across the expanded CGG repeat region in RILPL1 from whole-blood DNA was determined with LRS data from four OPDM4-affected individuals (F1-III-1, S1, S2, and S3) and 10 healthy individuals. No significant difference in methylation was detected between affected individuals and healthy individuals. (B) Quantification of methylation levels between OPDM4-affected individuals and healthy individuals. (C) Methylation status between expanded alleles and non-expanded alleles in four OPDM4-affected individuals (F1-III-1, S1, S2 and S3). No significant difference in methylation was detected between expanded and non-expanded alleles. (D) Quantification of methylation levels between expanded alleles and non-expanded alleles. (E) Immunoblot analysis of RILPL1 in three OPDM4-affected individuals with expanded CGG repeats in RILPL1 and seven age-matched control subjects. GAPDH was used as a loading control. Experiments were conducted thrice with reproducible results. (F) Quantification of relative RILPL1 abundance in each group; no significant difference was observed between OPDM4 affected individuals and controls. (G) Immunoblot analysis of RILPL1 in 12 control subjects at different ages; these included four control subjects under 25 years old, five control subjects 25 to 50 years old, and three control subjects over 50 years old. GAPDH was used as a loading control. Experiments were conducted thrice with reproducible results. (H) Quantification of relative RILPL1 abundance in each group; no significant difference was observed among control individuals at different ages. Data were analyzed with a Student’s t test; ns, not significant.

Figure 5

Immunofluorescence and RNA FISH on muscle biopsy samples (A) Immunofluorescence showing glycine and p62 co-localized in the intranuclear inclusions of OPDM4-affected individuals (F1-II-2 and S1) but not in the control individual (higher-magnification images of representative co-localization were shown in the insets of the corresponding panels). The scale bar represents 25 μm. (B) Probes 1 and 2 were complementary to the adjacent downstream and upstream regions of repeat expansions in RILPL1 mRNA. (C) RNA FISH combined with immunofluorescence indicated that expanded repeats in RILPL1 mRNA formed RNA foci (labeled by probe 1 or probe 2) and were co-localized with MBNL1 and p62 in the intranuclear inclusions of OPDM4-affected individuals (F1-II-2 and S1) but not in the control individual (higher-magnification images of representative co-localization are shown in the insets of the corresponding panels). The scale bar represents 25 μm.