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

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.

Expansion of GGC Repeat in GIPC1 Is Associated with Oculopharyngodistal Myopathy.

Oculopharyngodistal myopathy (OPDM) is an adult-onset inherited neuromuscular disorder characterized by progressive ptosis, external ophthalmoplegia, and weakness of the masseter, facial, pharyngeal, and distal limb muscles. The myopathological features are presence of rimmed vacuoles (RVs) in the muscle fibers and myopathic changes of differing severity. Inheritance is variable, with either putative autosomal-dominant or autosomal-recessive pattern. Here, using a comprehensive strategy combining whole-genome sequencing (WGS), long-read whole-genome sequencing (LRS), linkage analysis, repeat-primed polymerase chain reaction (RP-PCR), and fluorescence amplicon length analysis polymerase chain reaction (AL-PCR), we identified an abnormal GGC repeat expansion in the 5' UTR of GIPC1 in one out of four families and three sporadic case subjects from a Chinese OPDM cohort. Expanded GGC repeats were further confirmed as the cause of OPDM in an additional 2 out of 4 families and 6 out of 13 sporadic Chinese individuals with OPDM, as well as 7 out of 194 unrelated Japanese individuals with OPDM. Methylation, qRT-PCR, and western blot analysis indicated that GIPC1 mRNA levels were increased while protein levels were unaltered in OPDM-affected individuals. RNA sequencing indicated p53 signaling, vascular smooth muscle contraction, ubiquitin-mediated proteolysis, and ribosome pathways were involved in the pathogenic mechanisms of OPDM-affected individuals with GGC repeat expansion in GIPC1. This study provides further evidence that OPDM is associated with GGC repeat expansions in distinct genes and highly suggests that expanded GGC repeat units are essential in the pathogenesis of OPDM, regardless of the genes in which the expanded repeats are located.

Genetic origin of sporadic cases and RNA toxicity in neuronal intranuclear inclusion disease.

BACKGROUND: GGC repeat expansion in NOTCH2NLC has been recently linked to neuronal intranuclear inclusion disease (NIID) via unknown disease mechanisms. Herein, we explore the genetic origin of the sporadic cases and toxic RNA gain-of-function mechanism in NIID. METHODS: Multiple genetic screenings were performed on NIID individuals and their available family members. Methylation status of blood DNA, NOTCH2NLC mRNA level from muscle biopsies and RNA foci from skin biopsies of NIID individuals or asymptomatic carriers were evaluated and compared. RESULTS: In two sporadic NIID families, we identified two clinically and pathologically asymptomatic fathers carrying large GGC repeat expansion, above 300 repeats, with offspring repeat numbers of 172 and 148, respectively. Further evaluation revealed that the GGC repeat numbers in the sperm from two asymptomatic fathers were only 63 and 98, respectively. The CpG island in NOTCH2NLC of the asymptomatic carriers was hypermethylated, and accordingly, the NOTCH2NLC mRNA levels were decreased in the asymptomatic fathers. GGC repeat expansion RNA formed RNA foci and sequestered RNA binding proteins into p62 positive intranuclear inclusions in NIID individuals but not in the control or asymptomatic carrier. CONCLUSION: Our study suggested the GGC repeat expansion in NOTCH2NLC might have a disease-causing number ranging from ~41 to ~300 repeats. The contraction of GGC repeat expansion in sperm could be a possible mechanism for the paternal-biased origin in some sporadic or recessive inherited NIID individuals. The toxic RNA gain-of-function mechanism was identified to be involved in the pathogenicity of this disease.

The GGC repeat expansion in NOTCH2NLC is associated with oculopharyngodistal myopathy type 3.

Oculopharyngodistal myopathy (OPDM) is an adult-onset neuromuscular disease characterized by progressive ocular, facial, pharyngeal and distal limb muscle involvement. Trinucleotide repeat expansions in LRP12 or GIPC1 were recently reported to be associated with OPDM. However, a significant portion of OPDM patients have unknown genetic causes. In this study, long-read whole-genome sequencing and repeat-primed PCR were performed and we identified GGC repeat expansions in the NOTCH2NLC gene in 16.7% (4/24) of a cohort of Chinese OPDM patients, designated as OPDM type 3 (OPDM3). Methylation analysis indicated that methylation levels of the NOTCH2NLC gene were unaltered in OPDM3 patients, but increased significantly in asymptomatic carriers. Quantitative real-time PCR analysis indicated that NOTCH2NLC mRNA levels were increased in muscle but not in blood of OPDM3 patients. Immunofluorescence on OPDM muscle samples and expressing mutant NOTCH2NLC with (GGC)69 repeat expansions in HEK293 cells indicated that mutant NOTCH2NLC-polyglycine protein might be a major component of intranuclear inclusions, and contribute to toxicity in cultured cells. In addition, two RNA-binding proteins, hnRNP A/B and MBNL1, were both co-localized with p62 in intranuclear inclusions in OPDM muscle samples. These results indicated that a toxic protein gain-of-function mechanism and RNA gain-of-function mechanism may both play a vital role in the pathogenic processes of OPDM3. This study extended the spectrum of NOTCH2NLC repeat expansion-related diseases to a predominant myopathy phenotype presenting as OPDM, and provided evidence for possible pathogenesis of these diseases.

Deciphering Oligodendrocyte Lineages in the Human Fetal Central Nervous System Using Single-Cell RNA Sequencing.

Oligodendrocytes form myelin sheaths and wrap axons of neurons to facilitate various crucial neurological functions. Oligodendrocyte progenitor cells (OPCs) persist in the embryonic, postnatal, and adult central nervous system (CNS). OPCs and mature oligodendrocytes are involved in a variety of biological processes such as memory, learning, and diseases. How oligodendrocytes are specified in different regions in the CNS, in particular in humans, remains obscure. We here explored oligodendrocyte development in three CNS regions, subpallium, brainstem, and spinal cord, in human fetuses from gestational week 8 (GW8) to GW12 using single-cell RNA sequencing. We detected multiple lineages of OPCs and illustrated distinct developmental trajectories of oligodendrocyte differentiation in three CNS regions. We also identified major genes, particularly transcription factors, which maintain status of OPC proliferation and promote generation of mature oligodendrocytes. Moreover, we discovered new marker genes that might be crucial for oligodendrocyte specification in humans, and detected common and distinct genes expressed in oligodendrocyte lineages in three CNS regions. Our study has demonstrated molecular heterogeneity of oligodendrocyte lineages in different CNS regions and provided references for further investigation of roles of important genes in oligodendrocyte development in humans.

A large pedigree study confirmed the CGG repeat expansion of RILPL1 Is associated with oculopharyngodistal myopathy.

BACKGROUND: Oculopharyngodistal myopathy (OPDM) is an autosomal dominant adult-onset degenerative muscle disorder characterized by ptosis, ophthalmoplegia and weakness of the facial, pharyngeal and limb muscles. Trinucleotide repeat expansions in non-coding regions of LRP12, G1PC1, NOTCH2NLC and RILPL1 were reported to be the etiologies for OPDM. RESULTS: In this study, we performed long-read whole-genome sequencing in a large five-generation family of 156 individuals, including 21 patients diagnosed with typical OPDM. We identified CGG repeat expansions in 5'UTR of RILPL1 gene in all patients we tested while no CGG expansion in unaffected family members. Repeat-primed PCR and fluorescence amplicon length analysis PCR were further confirmed the segregation of CGG expansions in other family members and 1000 normal Chinese controls. Methylation analysis indicated that methylation levels of the RILPL1 gene were unaltered in OPDM patients, which was consistent with previous studies. Our findings provide evidence that RILPL1 is associated OPDM in this large pedigree. CONCLUSIONS: Our results identified RILPL1 is the associated the disease in this large pedigree.

Detection of Structural Variations and Fusion Genes in Breast Cancer Samples Using Third-Generation Sequencing.

Background: Structural variations (SVs) are common genetic alterations in the human genome that could cause different phenotypes and diseases, including cancer. However, the detection of structural variations using the second-generation sequencing was limited by its short read length, which restrained our understanding of structural variations. Methods: In this study, we developed a 28-gene panel for long-read sequencing and employed it to Oxford Nanopore Technologies and Pacific Biosciences platforms. We analyzed structural variations in the 28 breast cancer-related genes through long-read genomic and transcriptomic sequencing of tumor, para-tumor, and blood samples in 19 breast cancer patients. Results: Our results showed that some somatic SVs were recurring among the selected genes, though the majority of them occurred in the non-exonic region. We found evidence supporting the existence of hotspot regions for SVs, which extended our previous understanding that they exist only for single nucleotide variations. Conclusion: In conclusion, we employed long-read genomic and transcriptomic sequencing to identify SVs from breast cancer patients and proved that this approach holds great potential in clinical application.