Genomic and immune landscape Of metastatic pheochromocytoma and paraganglioma

The mechanisms triggering metastasis in pheochromocytoma/paraganglioma are unknown, hindering therapeutic options for patients with metastatic tumors (mPPGL). Herein we show by genomic profiling of a large cohort of mPPGLs that high mutational load, microsatellite instability and somatic copy-number alteration burden are associated with ATRX/TERT alterations and are suitable prognostic markers. Transcriptomic analysis defines the signaling networks involved in the acquisition of metastatic competence and establishes a gene signature related to mPPGLs, highlighting CDK1 as an additional mPPGL marker. Immunogenomics accompanied by immunohistochemistry identifies a heterogeneous ecosystem at the tumor microenvironment level, linked to the genomic subtype and tumor behavior. Specifically, we define a general immunosuppressive microenvironment in mPPGLs, the exception being PD-L1 expressing MAML3-related tumors. Our study reveals canonical markers for risk of metastasis, and suggests the usefulness of including immune parameters in clinical management for PPGL prognostication and identification of patients who might benefit from immunotherapy

Antisense oligonucleotide-based treatment of retinitis pigmentosa caused by USH2A exon 13 mutations

Mutations in USH2A are among the most common causes of syndromic and non-syndromic retinitis pigmentosa (RP). The two most recurrent mutations in USH2A, c.2299delG and c.2276G > T, both reside in exon 13. Skipping exon 13 from the USH2A transcript presents a potential treatment modality in which the resulting transcript is predicted to encode a slightly shortened usherin protein. Morpholino-induced skipping of ush2a exon 13 in zebrafish ush2a$^{rmc1}$ mutants resulted in the production of usherinΔexon 13 protein and a completely restored retinal function. Antisense oligonucleotides were investigated for their potential to selectively induce human USH2A exon 13 skipping. Lead candidate QR-421a induced a concentration-dependent exon 13 skipping in induced pluripotent stem cell (iPSC)-derived photoreceptor precursors from an Usher syndrome patient homozygous for the c.2299delG mutation. Mouse surrogate mQR-421a reached the retinal outer nuclear layer after a single intravitreal injection and induced a detectable level of exon skipping until at least 6 months post-injection. In conclusion, QR-421a-induced exon skipping proves to be a highly promising treatment option for RP caused by mutations in USH2A exon 13

Eyes shut homolog is important for the maintenance of photoreceptor morphology and visual function in zebrafish

<div><p>Mutations in <i>eyes shut homolog</i> (<i>EYS</i>), a gene predominantly expressed in the photoreceptor cells of the retina, are among the most frequent causes of autosomal recessive (ar) retinitis pigmentosa (RP), a progressive retinal disorder. Due to the absence of <i>EYS</i> in several rodent species and its retina-specific expression, still little is known about the exact function of EYS and the pathogenic mechanism underlying <i>EYS</i>-associated RP. We characterized <i>eys</i> in zebrafish, by RT-PCR analysis on zebrafish eye-derived RNA, which led to the identification of a 8,715 nucleotide coding sequence that is divided over 46 exons. The transcript is predicted to encode a 2,905-aa protein that contains 39 EGF-like domains and five laminin A G-like domains, which overall shows 33% identity with human EYS. To study the function of EYS, we generated a stable <i>eys</i>^{<i>rmc101/rmc101</i>} mutant zebrafish model using CRISPR/Cas9 technology. The introduced lesion is predicted to result in premature termination of protein synthesis and lead to loss of Eys function. Immunohistochemistry on retinal sections revealed that Eys localizes at the region of the connecting cilium and that both rhodopsin and cone transducin are mislocalized in the absence of Eys. Electroretinogram recordings showed diminished b-wave amplitudes in <i>eys</i>^{<i>rmc101/rmc101</i>} zebrafish (5 dpf) compared to age- and strain-matched wild-type larvae. In addition, decreased locomotor activity in response to light stimuli was observed in <i>eys</i> mutant larvae. Altogether, our study shows that absence of Eys leads to a disorganized retinal architecture and causes visual dysfunction in zebrafish.</p></div

Characterization of a stable <i>eys</i>^{<i>rmc101/rmc101</i>} zebrafish line.

<p><b>(A)</b> Sanger sequencing identified a five base pair deletion in exon 20 in <i>eys</i>^{<i>rmc101/rmc101</i>} zebrafish. <b>(B)</b> Representative gel image of RT-PCR analysis using RNA from a pool of larvae (n = 15), which shows that <i>eys</i> transcripts are present in both wild-type and <i>eys</i>^{<i>rmc101/rmc101</i>} zebrafish (upper panel). Sanger sequencing confirmed the presence of the five base pair deletion in the <i>eys</i>^{<i>rmc101/rmc101</i>} transcript (lower panel). <b>(C)</b> Protein domain structures of wild-type Eys and the truncated Eys protein that is predicted in <i>eys</i>^{<i>rmc101/rmc101</i>} zebrafish.</p

Visual motor response of zebrafish larvae.

<p><b>(A)</b> Distance moved (mm) of wild-type (blue line) and <i>eys</i>^{<i>rmc101/rmc101</i>} (red line) larvae in response to a light stimulus (dark-to-light transition at t = 50 minutes). <b>(B)</b> Comparison of difference in distance moved between wild-type and <i>eys</i>^{<i>rmc101/rmc101</i>} larvae at the dark to light transition zones. <b>(C)</b> Maximum velocity (Vmax; mm/s) of wild-type (blue line) and <i>eys</i>^{<i>rmc101/rmc101</i>} (red line) larvae in response to a light stimulus (dark-to-light transition at t = 50 minutes). <b>(D)</b> Comparison of difference in Vmax between wild-type and <i>eys</i>^{<i>rmc101/rmc101</i>} larvae at the dark to light transition zones. All experiments were done with larvae at 5 dpf (n = 120). Statistical significance (<i>p</i><0.05) is indicated with an asterisk.</p

Visual function of wild-type and <i>eys</i>^{<i>rmc101/rmc101</i>} zebrafish larvae.

<p><b>(A)</b> ERG measurements of the b-wave of wild-type (black line) and <i>eys</i>^{<i>rmc101/rmc101</i>} (red line) zebrafish larvae at 5 dpf. <b>(B)</b> ERG measurements of the a-wave of wild-type (black line) and <i>eys</i>^{<i>rmc101/rmc101</i>} (red line) zebrafish larvae at 5 dpf. <b>(C)</b> Quantification of the ERG b-wave amplitude of wild-type and <i>eys</i>^{<i>rmc101/rmc101</i>} zebrafish larvae at 5 dpf (n = 30; <i>p</i> = 0.0004). <b>(D)</b> Quantification of the ERG a-wave amplitude of wild-type and <i>eys</i>^{<i>rmc101/rmc101</i>} zebrafish larvae at 5 dpf (n = 20; <i>p</i> = 0.2324). <b>(E)</b> Optokinetic response measurements of wild-type and <i>eys</i>^{<i>rmc101/rmc101</i>} larvae at 5 dpf (n = 19).</p

Immunohistochemistry on retinal sections of wild-type and <i>eys</i>^{<i>rmc101/rmc101</i>} zebrafish.

<p><b>(A)</b> Retinal sections of wild-type and <i>eys</i>^{<i>rmc101/rmc101</i>} zebrafish at 5 dpf and 5 mpf stained with antibodies against Eys (green) and centrin (red). <b>(B)</b> BODIPY (green) staining showing disorganization of photoreceptor outer segments in <i>eys</i>^{<i>rmc101/rmc101</i>} zebrafish (5 dpf and 5 mpf) compared to age- and strain-matched wild-type zebrafish (arrows). <b>(C)</b> Retinal sections of wild-type and <i>eys</i>^{<i>rmc101/rmc101</i>} zebrafish at 5 dpf (upper panel), 2 mpf (middle panel) and 5 mpf (lower panel) stained with antibodies against rhodopsin (green) and F-actin (red). Asterisks indicate mislocalization of rhodopsin to the inner segments and synapses of photoreceptor cells. <b>(D)</b> Retinal sections of wild-type and <i>eys</i>^{<i>rmc101/rmc101</i>} zebrafish at 5 dpf, 2 mpf and 5 mpf stained with antibodies against GNAT2 (green) and F-actin (red). Arrows indicate dysmorphic outer segments in mutant zebrafish. In all images, nuclei are counterstained with DAPI (blue). INL: inner nuclear layer; ONL: outer nuclear layer; OS: outer segments. Scale: 5 μm.</p

Antisense Oligonucleotide-based Splice Correction for USH2A-associated Retinal Degeneration Caused by a Frequent Deep-intronic Mutation

International audienceUsher syndrome (USH) is the most common cause of combined deaf-blindness in man. The hearing loss can be partly compensated by providing patients with hearing aids or cochlear implants, but the loss of vision is currently untreatable. In general, mutations in the USH2A gene are the most frequent cause of USH explaining up to 50% of all patients worldwide. The first deep-intronic mutation in the USH2A gene (c.7595-2144A>G) was reported in 2012, leading to the insertion of a pseudoexon (PE40) into the mature USH2A transcript. When translated, this PE40-containing transcript is predicted to result in a truncated non-functional USH2A protein. In this study, we explored the potential of antisense oligonucleotides (AONs) to prevent aberrant splicing of USH2A pre-mRNA as a consequence of the c.7595-2144A>G mutation. Engineered 2'-O-methylphosphorothioate AONs targeting the PE40 splice acceptor site and/or exonic splice enhancer regions displayed significant splice correction potential in both patient derived fibroblasts and a minigene splice assay for USH2A c.7595-2144A>G, whereas a non-binding sense oligonucleotide had no effect on splicing. Altogether, AON-based splice correction could be a promising approach for the development of a future treatment for USH2A-associated retinitis pigmentosa caused by the deep-intronic c.7595-2144A>G mutation

Usherin defects lead to early-onset retinal dysfunction in zebrafish

Mutations in USH2A are the most frequent cause of Usher syndrome and autosomal recessive nonsyndromic retinitis pigmentosa. To unravel the pathogenic mechanisms underlying USH2A-associated retinal degeneration and to evaluate future therapeutic strategies that could potentially halt the progression of this devastating disorder, an animal model is needed. The available Ush2a knock-out mouse model does not mimic the human phenotype, because it presents with only a mild and late-onset retinal degeneration. Using CRISPR/Cas9-technology, we introduced protein-truncating germline lesions into the zebrafish ush2a gene (ush2armc1: c.2337_2342delinsAC; p.Cys780GlnfsTer32 and ush2ab1245: c.15520_15523delinsTG; p.Ala5174fsTer). Homozygous mutants were viable and displayed no obvious morphological or developmental defects. Immunohistochemical analyses with antibodies recognizing the N- or C-terminal region of the ush2a-encoded protein, usherin, demonstrated complete absence of usherin in photoreceptors of ush2armc1, but presence of the ectodomain of usherin at the periciliary membrane of ush2ab1245-derived photoreceptors. Furthermore, defects of usherin led to a reduction in localization of USH2 complex members, whirlin and Adgrv1, at the photoreceptor periciliary membrane of both mutants. Significantly elevated levels of apoptotic photoreceptors could be observed in both mutants when kept under constant bright illumination for three days. Electroretinogram (ERG) recordings revealed a significant and similar decrease in both a- and b-wave amplitudes in ush2armc1 as well as ush2ab1245 larvae as compared to strain- and age-matched wild-type larvae. In conclusion, this study shows that mutant ush2a zebrafish models present with early-onset retinal dysfunction that is exacerbated by light exposure. These models provide a better understanding of the pathophysiology underlying USH2A-associated RP and a unique opportunity to evaluate future therapeutic strategies

The ciliopathy protein CC2D2A Associates with NINL and functions in RAB8-MICAL3-regulated vesicle trafficking

Ciliopathies are a group of human disorders caused by dysfunction of primary cilia, ubiquitous microtubule-based organelles involved in transduction of extra-cellular signals to the cell. This function requires the concentration of receptors and channels in the ciliary membrane, which is achieved by complex trafficking mechanisms, in part controlled by the small GTPase RAB8, and by sorting at the transition zone located at the entrance of the ciliary compartment. Mutations in the transition zone gene CC2D2A cause the related Joubert and Meckel syndromes, two typical ciliopathies characterized by central nervous system malformations, and result in loss of ciliary localization of multiple proteins in various models. The precise mechanisms by which CC2D2A and other transition zone proteins control protein entrance into the cilium and how they are linked to vesicular trafficking of incoming cargo remain largely unknown. In this work, we identify the centrosomal protein NINL as a physical interaction partner of CC2D2A. NINL partially co-localizes with CC2D2A at the base of cilia and ninl knockdown in zebrafish leads to photoreceptor outer segment loss, mislocalization of opsins and vesicle accumulation, similar to cc2d2a-/- phenotypes. Moreover, partial ninl knockdown in cc2d2a-/- embryos enhances the retinal phenotype of the mutants, indicating a genetic interaction in vivo, for which an illustration is found in patients from a Joubert Syndrome cohort. Similar to zebrafish cc2d2a mutants, ninl morphants display altered Rab8a localization. Further exploration of the NINL-associated interactome identifies MICAL3, a protein known to interact with Rab8 and to play an important role in vesicle docking and fusion. Together, these data support a model where CC2D2A associates with NINL to provide a docking point for cilia-directed cargo vesicles, suggesting a mechanism by which transition zone proteins can control the protein content of the ciliary compartment