GGC Repeat Expansion of RILPL1 is Associated with Oculopharyngodistal Myopathy

Abstract

Objective Oculopharyngodistal myopathy (OPDM) is an adult‐onset neuromuscular disease characterized by progressive ptosis, dysarthria, ophthalmoplegia, and distal muscle weakness. Recent studies revealed that GGC repeat expansions in 5′‐UTR of LRP12, GIPC1, and NOTCH2NLC are associated with OPDM. Despite these advances, approximately 30% of OPDM patients remain genetically undiagnosed. Herein, we aim to investigate the genetic basis for undiagnosed OPDM patients in two unrelated Chinese Han families. Methods Parametric linkage analysis was performed. Long‐read sequencing followed by repeat‐primed polymerase chain reaction and amplicon length polymerase chain reaction were used to determine the genetic cause. Targeted methylation sequencing was implemented to detect epigenetic changes. The possible pathogenesis mechanism was investigated by quantitative polymerase chain reaction, immunoblotting, RNA fluorescence in situ hybridization, and immunofluorescence staining of muscle biopsy samples. Results The disease locus was mapped to 12q24.3. Subsequently, GGC repeat expansion in the promoter region of RILPL1 was identified in six OPDM patients from two families, findings consistent with a founder effect, designated as OPDM type 4. Targeted methylation sequencing revealed hypermethylation at the RILPL1 locus in unaffected individuals with ultralong expansion. Analysis of muscle samples showed no significant differences in RILPL1 mRNA or RILPL1 protein levels between patients and controls. Public CAGE‐seq data indicated that alternative transcription start sites exist upstream of the RefSeq‐annotated RILPL1 transcription start site. Strand‐specific RNA‐seq data revealed bidirectional transcription from the RILPL1 locus. Finally, fluorescence in situ hybridization/immunofluorescence staining showed that both sense and antisense transcripts formed RNA foci, and were co‐localized with hnRNPA2B1 and p62 in the intranuclear inclusions of OPDM type 4 patients. Interpretation Our findings implicate abnormal GGC repeat expansions in the promoter region of RILPL1 as a novel genetic cause for OPDM, and suggest a methylation mechanism and a potential RNA toxicity mechanism are involved in OPDM type 4 pathogenesis. ANN NEUROL 2022;92:512–526

Oculopharyngodistal myopathy: The recent discovery of an old disease

“…A dot-like sarcoplasmic deposition of p62 and small angular fibers are also common. [7][8][9][11][12][13] Ultrastructural studies have shown tubulofilamentous intranuclear inclusions of 17.3 nm in diameter in 1 OPDM1 patient, which are larger than those observed in oculopharyngeal muscular dystrophy (OPMD) or OPDM3. 9,13 In this issue of Muscle & Nerve, Shimizu and colleagues report seven Japanese patients from five families with OPDM1.…”

“…To date, the expansion of CGG repeats in the 5 0 untranslated region (5 0 UTR) of LRP12 (OPDM1), GIPC1 (OPDM2), NOTCH2NLC (OPDM3), and RILPL1 (OPDM4) have been reported to cause OPDM in Japan and China. [6][7][8][9][10][11][12][13] Among these 4 OPDM subtypes, OPDM1 is the most common OPDM subtype in Japan, accounting for 31.25% of Japanese OPDM patients, while OPDM2 is the most common OPDM subtype in China, accounting for 37.3% of Chinese OPDM…”

“…All OPDM subtypes share the pathological hallmarks of chronic myopathic changes with rimmed vacuoles and p62‐positive inclusions in myonuclei. A dot‐like sarcoplasmic deposition of p62 and small angular fibers are also common 7–9,11–13 . Ultrastructural studies have shown tubulofilamentous intranuclear inclusions of 17.3 nm in diameter in 1 OPDM1 patient, which are larger than those observed in oculopharyngeal muscular dystrophy (OPMD) or OPDM3 9,13 …”

Advances in Nucleotide Repeat Expansion Diseases: Transcription Gets in Phase

“…Repeat expansion diseases are caused by unstable expanded tri-, tetra-, penta-, hexa- or dodecanucleotides that can be located in coding or noncoding gene regions, namely 5′-untranslated regions (UTR), 3′-UTR, introns, or promoters ( Figure 1 ) [ 1 ]. In recent years, a new group of five trinucleotide repeat expansion diseases has been reported, including neuronal intranuclear inclusion disease (NIID) [ 4 , 5 ], oculopharyngeal myopathy with leukoencephalopathy 1 (OPML1) [ 4 ], and oculopharyngodistal myopathies 1–4 (OPDM1–4) [ 4 , 6 , 7 , 8 , 9 ]. Beyond the simple STR expansions, pentanucleotide pathological insertions have also been identified, with the first being the (TGGAA) n in SCA31 [ 10 ] and later the (ATTTC) n in SCA37 [ 11 ].…”

“…Many of the known repeat expansions are transcribed bidirectionally from both DNA strands [ 18 ]. More recently, bidirectional transcription has been reported for OPML1-associated gene LOC642361/NUTM2B-AS1 [ 4 ] and RILPL1 in OPDM4 [ 9 ]. The vast diversity of genes containing disease-causing repeat expansions, combined with the rapid increase in the number of pathogenic STRs that have been discovered in the last years, highlights the need for studying the molecular mechanisms triggered by these mutations.…”