Ubiquitous Expression of CUG or CAG Trinucleotide Repeat RNA Causes Common Morphological Defects in a Drosophila Model of RNA-Mediated Pathology

Expanded DNA repeat sequences are known to cause over 20 diseases, including Huntington’s disease, several types of spinocerebellar ataxia and myotonic dystrophy type 1 and 2. A shared genetic basis, and overlapping clinical features for some of these diseases, indicate that common pathways may contribute to pathology. Multiple mechanisms, mediated by both expanded homopolymeric proteins and expanded repeat RNA, have been identified by the use of model systems, that may account for shared pathology. The use of such animal models enables identification of distinct pathways and their ‘molecular hallmarks’ that can be used to determine the contribution of each pathway in human pathology. Here we characterise a tergite disruption phenotype in adult flies, caused by ubiquitous expression of either untranslated CUG or CAG expanded repeat RNA. Using the tergite phenotype as a quantitative trait we define a new genetic system in which to examine ‘hairpin’ repeat RNA-mediated cellular perturbation. Further experiments use this system to examine whether pathways involving Muscleblind sequestration or Dicer processing, which have been shown to mediate repeat RNA-mediated pathology in other model systems, contribute to cellular perturbation in this model

Ubiquitous CUG and CAG RNA repeat expression leads to distinct localisation in muscle nuclei.

<p>Images from cryosections hybridised with fluorescent probes to detect repeat RNA. Images are representative of observations from multiple animals and independent transgenic lines for each repeat. <b>A</b>, Schematic of the repeat expression construct, indicating the location of the repeat and probe. <b>B-E</b>, Repeat transcripts detected with a complementary repeat probe. <b>B</b>, Progeny carrying the <i>da-GAL4</i> driver alone show no signal in the nucleus. <b>C</b>, Expression of <i>4xrCUG_∼100 [line 2]</i> leads to multiple foci throughout the nucleus. <b>D, E</b>, Expression of <i>4xrCAG_∼100 [line 1]</i>, or <i>4xrCAA_∼100 [line 1]</i> leads to between one and four sites of RNA concentration (arrowheads). <b>F</b>, Schematic of the construct giving repeat expression within the context of the GFP transcript, in this case RNA is detected using a probe against the GFP sequence. <b>G-K</b>, Repeat RNA localisation when expressed within a GFP transcript. <b>G</b>, No signal is observed using the GFP complementary probe against control EV progeny. <b>H</b>, <i>4xrCUG_∼100-GFP</i> expression leads to a similar pattern of foci as in <b>C</b>. <b>I, J</b>, Expression of 4x<i>rCAG_∼100-GFP</i> or <i>4xrCAA_∼100-GFP</i> leads to a similar pattern as in <b>D</b> and <b>E</b>. <b>K</b>, Expression of a single copy of GFP, not containing a repeat, leads to the formation of a single similar site of RNA concentration.</p

Reducing Dcr-2 levels is not rate limiting for the tergite phenotype.

<p><b>A-D</b>, Proportion of progeny within each phenotype category when repeat expression is driven by <i>da-GAL4</i> alone (left column), or in the presence of the <i>Dcr-2^L811fsX</i> mutant allele such that Dicer-2 levels are reduced (right column). <b>A</b>, Wild-type control, <b>B</b>, <i>4xrCAA_∼100 [line 1]</i>, <b>C</b>, <i>4xrCUG_∼100 [line 1]</i>, <b>D</b>, <i>4xrCAG_∼100 [line 2]</i>. <b>E, F</b>, Total proportion for each genotype that shows any phenotype (‘any phenotype’ - category 2, 3 and 4) and the proportion with a strong phenotype (‘strong phenotype’ – category 3 and 4). <b>E</b>, <i>4xrCUG_∼100 [line 1]</i>, <b>F</b>, <i>4xrCAG_∼100 [line 2]</i>. None of the observed changes were statistically significant (Fisher’s exact test).</p

Reducing Mbl levels does not enhance the tergite phenotype.

<p><b>A-D</b>, Proportion of progeny within each phenotype category when repeat expression is driven by <i>da-GAL4</i> alone (left column), or in the presence of the <i>mbl^E27</i> mutant allele such that Mbl levels are reduced (right column). <b>A</b>, Wild-type control, <b>B</b>, <i>4xrCAA_∼100 [line 1]</i>, <b>C</b>, <i>4xrCUG_∼100 [line 1]</i>, <b>D</b>, <i>4xrCAG_∼100 [line 2]</i>. <b>E, F</b>, Total proportion for each genotype that shows any phenotype (‘any phenotype’ - category 2, 3 and 4) and the proportion with a strong phenotype (‘strong phenotype’ – category 3 and 4). <b>E</b>, <i>4xrCUG_∼100 [line 1]</i> shows a reduction in the proportion with any phenotype, but no change in the proportion with a strong phenotype with reduced Mbl levels. <b>F</b>, No significant effect was observed with <i>4xrCAG_∼100 [line 2]</i>. Comparisons were made using Fisher’s exact test, with significant results indicated above the proportion, where *p<0.05.</p

Tergite phenotype caused by ubiquitous expression of CAG or CUG repeat RNA in <i>Drosophila</i>.

<p><b>A</b>, wild-type flies show a regular arrangement of tergite bands along the dorsal abdomen (arrows). <b>B</b>, An example of the disrupted phenotype, whereby tergites do not fuse at all (white arrowheads), or fuse only partially (grey arrowheads). <b>C</b>, Phenotype severity was scored on a scale of 1–4, images show typical examples from each category, where category 1 is like wildtype; category 2, tergites disrupted but not split; category 3, one tergite split; and category 4, two or more tergites split. <b>D-E</b>, Graphs showing the proportion of progeny within each scoring category. <b>D</b>, <i>da-GAL4</i> driven expression of the EV control line (n = 161) and, <b>E</b>, <i>da-GAL4</i> driven expression of <i>4xrCAA_∼100</i> [line 1] (n = 148) gives no phenotype with almost all progeny like wild-type. <b>F</b>, <i>da-GAL4</i> driven expression of <i>4xrCUG_∼100 [line 1]</i> (n = 271) or <b>G</b>, <i>4xrCAG_∼100 [line 2]</i> (n = 343) gives a tergite disruption phenotype. <b>H,</b> Schematic (not to scale) showing the location of histoblast cells (black). Histoblasts proliferate and migrate to form the tergite bands (arrows). <b>I</b>, Expression within the histoblasts using <i>T155-GAL4</i> gives wild-type tergites in EV control progeny (n = 56). <b>J</b>, <i>T155-GAL4</i> expression of <i>4xrCAG_∼100</i> [line 1] (n = 25) gives a mild tergite phenotype, ***p<0.001 comparing the proportion with a phenotype in <b>I</b> and <b>J</b>.</p

Reduced Dicer-1 processing can have opposing effects on CUG or CAG RNA-mediated tergite phenotypes.

<p><b>A-D</b>, Proportion of progeny within each phenotype category for expression with <i>da-GAL4</i> alone (left column), or in the presence of the <i>Dcr-1^Q1147X</i> mutant allele such that Dcr-1 levels are reduced (right column). <b>A</b>, wildtype control, <b>B</b>, <i>4xrCAA_∼100 [line 1],. </i><b>C</b>, <i>4xrCUG_∼100 [line 1],. </i><b>D</b>, <i>4xrCAG_∼100 [line 2]. </i><b>E, F</b>, Total proportion for each genotype that shows any phenotype (‘any phenotype’ - category 2, 3 and 4) and the proportion with a strong phenotype (‘strong phenotype’ – category 3 and 4). <b>E</b>, <i>4xrCUG_∼100 [line 1]</i> shows a suppression for both measures. <b>F</b>, <i>4xrCAG_∼100</i> [line 2] shows an enhancement for both measures. Comparisons were made between the populations using Fisher’s exact test, with significant results indicated above the proportion, where *p<0.05, **p<0.01 and ***p<0.001.</p

Yield of clinically reportable genetic variants in unselected cerebral palsy by whole genome sequencing

Cerebral palsy (CP) is the most common cause of childhood physical disability, with incidence between 1/500 and 1/700 births in the developed world. Despite increasing evidence for a major contribution of genetics to CP aetiology, genetic testing is currently not performed systematically. We assessed the diagnostic rate of genome sequencing (GS) in a clinically unselected cohort of 150 singleton CP patients, with CP confirmed at >4 years of age. Clinical grade GS was performed on the proband and variants were filtered, and classified according to American College of Medical Genetics and Genomics-Association for Molecular Pathology (ACMG-AMP) guidelines. Variants classified as pathogenic or likely pathogenic (P/LP) were further assessed for their contribution to CP. In total, 24.7% of individuals carried a P/LP variant(s) causing or increasing risk of CP, with 4.7% resolved by copy number variant analysis and 20% carrying single nucleotide or indel variants. A further 34.7% carried one or more rare, high impact variants of uncertain significance (VUS) in variation intolerant genes. Variants were identified in a heterogeneous group of genes, including genes associated with hereditary spastic paraplegia, clotting and thrombophilic disorders, small vessel disease, and other neurodevelopmental disorders. Approximately 1/2 of individuals were classified as likely to benefit from changed clinical management as a result of genetic findings. In addition, no significant association between genetic findings and clinical factors was detectable in this cohort, suggesting that systematic sequencing of CP will be required to avoid missed diagnoses

Pathogenic copy number variants that affect gene expression contribute to genomic burden in cerebral palsy

Cerebral palsy (CP) is the most frequent movement disorder of childhood affecting 1 in 500 live births in developed countries. We previously identified likely pathogenic de novo or inherited single nucleotide variants (SNV) in 14% (14/98) of trios by exome sequencing and a further 5% (9/182) from evidence of outlier gene expression using RNA sequencing. Here, we detected copy number variants (CNV) from exomes of 186 unrelated individuals with CP (including our original 98 trios) using the CoNIFER algorithm. CNV were validated with Illumina 850 K SNP arrays and compared with RNA-Seq outlier gene expression analysis from lymphoblastoid cell lines (LCL). Gene expression was highly correlated with gene dosage effect. We resolved an additional 3.7% (7/186) of this cohort with pathogenic or likely pathogenic CNV while a further 7.7% (14/186) had CNV of uncertain significance. We identified recurrent genomic rearrangements previously associated with CP due to 2p25.3 deletion, 22q11.2 deletions and duplications and Xp monosomy. We also discovered a deletion of a single gene, PDCD6IP, and performed additional zebrafish model studies to support its single allele loss in CP aetiology. Combined SNV and CNV analysis revealed pathogenic and likely pathogenic variants in 22.7% of unselected individuals with CP

Aicardi Syndrome Is a Genetically Heterogeneous Disorder

Aicardi Syndrome (AIC) is a rare neurodevelopmental disorder recognized by the classical triad of agenesis of the corpus callosum, chorioretinal lacunae and infantile epileptic spasms syndrome. The diagnostic criteria of AIC were revised in 2005 to include additional phenotypes that are frequently observed in this patient group. AIC has been traditionally considered as X-linked and male lethal because it almost exclusively affects females. Despite numerous genetic and genomic investigations on AIC, a unifying X-linked cause has not been identified. Here, we performed exome and genome sequencing of 10 females with AIC or suspected AIC based on current criteria. We identified a unique de novo variant, each in different genes: KMT2B, SLF1, SMARCB1, SZT2 and WNT8B, in five of these females. Notably, genomic analyses of coding and non-coding single nucleotide variants, short tandem repeats and structural variation highlighted a distinct lack of X-linked candidate genes. We assessed the likely pathogenicity of our candidate autosomal variants using the TOPflash assay for WNT8B and morpholino knockdown in zebrafish (Danio rerio) embryos for other candidates. We show expression of Wnt8b and Slf1 are restricted to clinically relevant cortical tissues during mouse development. Our findings suggest that AIC is genetically heterogeneous with implicated genes converging on molecular pathways central to cortical development