The Japan Registry for Adult Subjects of Spinal Muscular Atrophy (jREACT-SMA): Protocol for a Longitudinal Observational Study

BackgroundSpinal muscular atrophy (SMA) is an autosomal recessive genetic neuromuscular disorder with progressive muscle weakness and atrophy, mainly caused by lower motor neuron degeneration resulting from decreased levels of the survival motor neuron protein. Recently, 3 disease-modifying therapies for SMA (nusinersen, onasemnogene abeparvovec, and risdiplam) were approved in Japan that are expected to improve the prognosis of patients with SMA. Long-term clinical follow-up of adult patients treated with disease-modifying therapies and the natural history of SMA are essential to assess the real-world effectiveness of available treatments. Until recently, nusinersen was the only treatment option for patients with SMA in Japan; however, because Japanese approval of nusinersen was based on global clinical trials in infants and children aged 0-15 years with SMA, the effectiveness of nusinersen in adult patients has not been fully assessed in Japan. In addition, longitudinal clinical data of adult patients have not been systematically collected in Japan. ObjectiveThis longitudinal observational study of adult patients with SMA who have been diagnosed with 5q-SMA in Japan aims to gain a better understanding of the natural history of SMA, as well as the long-term effectiveness of disease-modifying therapies. Here, we describe the protocol for the study. MethodsThe Japan Registry for Adult Subjects of Spinal Muscular Atrophy (jREACT-SMA) study is a longitudinal (prospective and retrospective) observational study with a 60-month prospective follow-up being conducted at 19 investigational sites using the newly established jREACT-SMA registry. Patients aged ≥18 years with genetically confirmed 5q-SMA were planned to be enrolled in the registry from December 2020 to May 2022. The planned enrollment was 100 patients. The protocol was approved on September 28, 2020 (approval 2020-0289) by the ethical review committee of Nagoya University. Registration, demographics, genetic diagnosis, motor functions, patient-reported outcomes/quality-of-life outcomes, and other clinical data have been or will be collected. ResultsAs of May 2022, 113 patients had been enrolled, and the completion of patient registration has been extended from May 2022 to December 2022. Data at registration and during the follow-up period were and will be prospectively collected at least once a year until November 2025 (maximum 60 months). Data analyses will be conducted when all data have been collected. Results are expected to be available in 2026 and the study is expected to be completed by March 2027. ConclusionsThis jREACT-SMA study will provide longitudinal prospective follow-up data in adult patients with SMA in Japan, including data on the natural history of the disease and data on the long-term effectiveness of disease-modifying therapies. Trial RegistrationUniversity Hospital Medical Information Network Center Clinical Trials Registry UMIN000042015; https://rctportal.niph.go.jp/en/detail?trial_id=UMIN000042015 International Registered Report Identifier (IRRID)DERR1-10.2196/3887

Argonaute2 and LaminB modulate gene expression by controlling chromatin topology

<div><p><i>Drosophila</i> Argonaute2 (AGO2) has been shown to regulate expression of certain loci in an RNA interference (RNAi)-independent manner, but its genome-wide function on chromatin remains unknown. Here, we identified the nuclear scaffolding protein LaminB as a novel interactor of AGO2. When either AGO2 or LaminB are depleted in Kc cells, similar transcription changes are observed genome-wide. In particular, changes in expression occur mainly in active or potentially active chromatin, both inside and outside LaminB-associated domains (LADs). Furthermore, we identified a somatic target of AGO2 transcriptional repression, <i>no hitter</i> (<i>nht</i>), which is immersed in a LAD located within a repressive topologically-associated domain (TAD). Null mutation but not catalytic inactivation of <i>AGO2</i> leads to ectopic expression of <i>nht</i> and downstream spermatogenesis genes. Depletion of either AGO2 or LaminB results in reduced looping interactions within the <i>nht</i> TAD as well as ectopic inter-TAD interactions, as detected by 4C-seq analysis. Overall, our findings reveal coordination of AGO2 and LaminB function to dictate genome architecture and thereby regulate gene expression.</p></div

AGO2 and LaminB attenuate transcription genome-wide.

<p>A) Western blot analysis showing knockdown efficiencies for each protein analyzed. Tubulin is included as loading control. B) Changes in neuRNA levels upon depletion of AGO2. Statistically significant changes include 710 up-regulated genes (red) and 195 down-regulated genes (blue). (For AGO2 rescue experiments see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007276#pgen.1007276.s001" target="_blank">S1 Fig</a>). Genes containing an AGO2 peak at their promoter are additionally colored orange. FET <i>p</i>-values and odds ratios indicate significance of association between affected genes and presence of AGO2 peak at promoters. C) Scatterplot comparing neuRNA-seq profiles from AGO2 and LaminB knockdowns. Pearson’s R corresponds to correlation coefficient of the two profiles. FET <i>p</i>-values and odds ratios indicate significance of the association between coordinately up-regulated (466, red) or down-regulated (76, blue) genes in both knockdowns. D) Scatterplot comparing neuRNA-seq profiles from Dcr-2 and AGO2 knockdowns. Genes with undetermined log2 fold change (due to zero counts in one condition) are fixed to a minimal value of -1.5 so they can be visually represented on the plots. Genes with zero counts in both conditions are not displayed. E) Scatterplot comparing neuRNA-seq profiles from Dcr-2 and LaminB knockdowns. F) Immunofluorescence analysis for AGO2 and LaminB in mock, AGO2- and LaminB-depleted cells. AGO2 (green, Liu antibody), LaminB (red), and DAPI staining (blue) are shown. Scale bar represents 14 μm.</p

AGO2 and LaminB attenuate transcription in active chromatin and additional sites across the genome.

<p>A) Heatmap showing enrichment and depletion of neuRNA-seq up-regulated genes in AGO2-, LaminB, both- or Dcr-2-depleted cells across different chromatin features such as chromatin domains (LADs and DPSs), chromatin colors, transcription start sites (TSS), and transcription polyadenylation site (TPS). The color scale indicates the log₂ odds ratio of the Fisher's exact tests, where negative (blue) indicates depletion and positive (red) indicates enrichment. B) Heatmap showing enrichment and depletion of neuRNA-seq down-regulated genes in AGO2-, LaminB, both- or Dcr-2-depleted cells with respect to chromatin features. C) Heatmap showing enrichment and depletion of AGO2 and Pol II ChIP-seq peaks with respect to chromatin features. Colormap represents the log₂ fold change as reported by the Genomic Association Test (GAT). D) Representative screenshot of co-up-regulated genes in AGO2 KD and LaminB KD embedded within a LAD with TSS located in active RED chromatin. The red and blue bars below the neuRNA-seq signals correspond to significant differences (up and down, respectively) relative to mock-treated cells. Black bars at the bottom of each screenshot correspond to AGO2 peaks. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007276#pgen.1007276.s002" target="_blank">S2 Fig</a>.</p

AGO2 and LaminB control chromatin topology of the <i>nht</i> TAD as well as surrounding regions.

<p>A) 3C analysis between the <i>nht</i> promoter as anchor (salmon bar) and different sites located within or at LAD/TAD borders. The <i>y</i>-axis represents the relative interaction frequency, and the <i>x</i>-axis shows genomic coordinates. LADs are shown in grey. TADs, AGO2 peaks, and 3C primers are shown as black bars. Testis-expressed genes are highlighted in green. Error bars correspond to standard deviation of four experiments. Significant comparisons relative to mock are highlighted with asterisks (Student’s t-test). B) Chromosome-wide view of 4C-seq signal using the <i>nht</i> promoter as anchor (green triangle), LADs (grey bars), TAD borders, and AGO2 peaks (black bars). The y-axis corresponds to normalized counts per window. Sites showing statistically significant differences in interaction (FDR<0.05) relative to mock-treated cells upon AGO2 or LaminB depletion are shown as rectangles under the corresponding track with decreased (blue) and increased (red) interaction indicated. Red triangle highlights a distant TAD containing the <i>bgm</i> gene that significantly increases its interaction with the <i>nht</i> promoter upon depletion of LaminB. Blue lines indicate zoomed-in regions. C) Representative maximal projections of images for dual DNA-FISH using a probe that recognizes the <i>nht</i> TAD (green) and an adjacent TAD (termed <i>bgm</i>, red) in mock, AGO2- and LaminB-depleted cells. Percentage of colocalization between both probes is indicated on right. Statistically significant value relative to mock is highlighted with an asterisk (LaminB KD, p<2.2e-16 Chi-squared test). Nuclei were stained with DAPI. Scale bar represents 16 μm. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007276#pgen.1007276.s004" target="_blank">S4 Fig</a>.</p

<i>prp16</i> has a defect in chromosome segregation.

<p>(A) <i>prp16-2</i> is hypersensitive to TBZ. Serially diluted cells indicated on the left of the panel were spotted on MMAU plates without TBZ (-TBZ) or with 10 μg/ml TBZ (+TBZ), and then incubated at 33°C for 5 days. <i>Δago1</i> was spotted as a control; this strain has a defect in RNAi-induced formation of centromeric heterochromatin. (B) <i>prp16-2</i> yields lagging chromosomes. Cells were double-stained with DAPI (red in the merged image) and antibody against tubulin (TAT1, green). Arrows indicate the lagging chromosome. The graph under the pictures shows the frequency of lagging chromosomes in late-anaphase cells in 972 (WT), <i>prp16-2</i>, or <i>Δdcr1</i>. Cells from three independent cultures at 33°C were analyzed. More than 50 late-anaphase cells were assessed for lagging chromosomes in each culture.</p

Deletion of the upstream region affects pre-mRNA splicing and H3K9 dimethylation.

<p>(A) Schematic representation of <i>dg</i> fragments containing serial deletions in the upstream region. Constructs were fused to the <i>nmt1</i> promoter and terminator (gray arrows and boxes, respectively). Numbers on the left represent the lengths of intron-containing <i>dg</i> DNA fragments and the Act1/<i>dg</i> chimeric fragment ligated to the <i>nmt1</i> promoter and terminator. The 5’-long construct consists of the upstream region (570 bp), <i>dg</i> intron (138 bp), and downstream region (106 bp). The Act1/<i>dg</i> construct consists of the actin gene fragment (442 bp) and the <i>dg</i>-short fragment (390 bp). (B) Splicing of <i>dg</i> ncRNAs transcribed from serially deleted fragments was examined by RT-PCR (left panel) and qRT-PCR (right panel) using plasmid-specific primers. Products specific for spliced <i>dg</i> ncRNA were amplified using spliced product-specific primers. The ratios of the spliced products to the total transcripts (spliced and unspliced products) were calculated and graphed. <i>Act1</i> mRNA was analyzed as a loading control. No bands were detected in samples without the reverse transcription reaction. (C) ChIP analysis of H3K9me2 at the <i>dg</i> region was performed with the H3K9me2 antibody and strains possessing the indicated plasmids. qPCR was performed using plasmid-specific primers. (D) Splicing of the Act1/<i>dg</i> chimeric transcript was analyzed by RT-PCR (upper panel) and qRT-PCR (lower panel). Splicing of 5’-long and <i>dg</i>-short transcripts were also examined as controls.</p

Removal of the intron from the <i>dg</i> transcript impairs the formation of heterochromatin.

<p>(A) Schematic representation of the minichromosomes. The intron-containing minichromosome (Full; pH-cc2) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006606#pgen.1006606.ref011" target="_blank">11</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006606#pgen.1006606.ref030" target="_blank">30</a>] contains the cc2 (central domain) region, <i>ura4</i>^<i>+</i> gene, <i>sup3-e</i> gene, and a 2.1 kb <i>Kpn</i>I–<i>Kpn</i>I fragment derived from the <i>otr dg</i> region. The intron-less minichromosome (Less) lacks the <i>dg</i> intron. The arrow indicates the position of the minichromosome-specific reverse primer. (B) ChIP analyses of H3K9me2 (left panel) and Swi6p (right panel) at the <i>dg</i> sequences on the minichromosomes in the indicated strains. Quantitative PCR was performed using minichromosome-specific primers.</p