Gene discovery in inherited retinal diseases using whole genome sequencing and autozygome based analysis

Vision is the most developed of the human senses. Our eyes can be considered as the portal through which we gain knowledge, we communicate, as well as we appreciate all the beauty of the world. The goal of this work is to better understand the molecular etiology underlying rare Mendelian cases of retinal degeneration and create knowledge about this disease’s mechanisms, in view of future developments of therapeutic interventions. In our approach, we couple the use of whole-genome, whole-exome, and RNA-sequencing, along with bench side basic research, in order to assess and better understand the functional consequences that identified mutations elicit on normal molecular and physiological processes. From the beginning of this PhD, my training focused on bioinformatics and specifically on methods of analysis of next generation sequencing data. Provided that my background is that of a molecular biologist, I could in fact develop all my projects from A to Z, by performing in silico genomic data analysis and by implementing it into experimental research approaches. The main results of my work consist in novel disease-gene associations, in particular for syndromic ciliopathies causing loss of vision and male infertility. In the projects on the TTLL5 and ARL2BP genes we link two structurally similar, yet functionally different, ciliary organelles - the photoreceptor sensory cilium and the sperm flagellum – associating vision and the reproductive system. Another novel disease-gene association is discussed in the third project, where we bring evidences for the contribution of a disruptive structural variant in the NMNAT1 gene to a rare, severe, and unique form of syndromic Leber congenital amaurosis. Finally, in the fourth project, we perform a whole-genome sequencing investigation of unresolved, negative exomes, and we identify new structural variants in the EYS and CNGA1 genes, as well as a large chromosomal rearrangement leading to uniparental isodysomy

Whole exome sequencing and homozygosity mapping reveals genetic defects in consanguineous Iranian families with inherited retinal dystrophies

Acknowledgements This research was funded by the Swiss National Science Foundation (Grant #176097 to CR). We would like to express gratitude to the patients and all their family members that participated in this study for their valuable cooperation and participation.Peer reviewedPublisher PD

Whole exome sequencing in 17 consanguineous Iranian pedigrees expands the mutational spectrum of inherited retinal dystrophies

Funding Information: We would like to thank all of the participating families. We are also grateful to the Swiss Confederation for the award of a PhD fellowship to AUR, to Mashhad University of Medical Sciences for supporting part of the work, in the framework of the PhD thesis of AS, to the Swiss National Science Foundation for grant # 176097 to CR, and to the Fondation Guillaume Gentil for support to ASF.Peer reviewedPublisher PD

Acr-23 encodes a Monepantel-sensitive channel in Caenorhabditis elegans

Monepantel is a member of the recently identified class of anthelmintics known as the amino-acetonitrile derivatives (AADs). Monepantel controls all major gastro-intestinal nematodes in sheep including those that are resistant to the classical anthelmintics. Previous studies have shown that the Caenorhabditis elegans acr-23 and the Haemonchus contortus Hco-mptl-1 genes may be prominent targets of monepantel. With this discovery it became possible to investigate the mode of action of monepantel in nematodes at the molecular level. In the present study, we show that a C. elegans mutant acr-23 strain is fully rescued by expressing the wild-type acr-23 gene. Moreover, we present a new mutant allele, and characterize acr-23 alleles genetically. We also show that acr-23 is expressed in body wall muscle cells, and provide therefore a possible explanation for the paralysis caused by monepantel. Furthermore, genetic evidence suggests that the chaperone RIC-3 is required for expression of full monepantel resistance. Finally, we present reconstitution of the C. elegans ACR-23 receptor in Xenopus laevis oocytes and provide direct evidence of its modulation by monepantel. Conversely, co-injection of the chaperone RIC-3 had no impact for channel reconstitution in X. laevis oocytes. These results reinforce the involvement of the ACR-23 family in the mode of action of monepantel and advance our understanding of this new class of anthelmintics

An Alu-mediated duplication in NMNAT1, involved in NAD biosynthesis, causes a novel syndrome, SHILCA, affecting multiple tissues and organs

We investigated the genetic origin of the phenotype displayed by three children from two unrelated Italian families, presenting with a previously unrecognized autosomal recessive disorder that included a severe form of spondylo-epiphyseal dysplasia, sensorineural hearing loss, intellectual disability and Leber congenital amaurosis (SHILCA), as well as some brain anomalies that were visible at the MRI. Autozygome-based analysis showed that these children shared a 4.76 Mb region of homozygosity on chromosome 1, with an identical haplotype. Nonetheless, whole-exome sequencing failed to identify any shared rare coding variants, in this region or elsewhere. We then determined the transcriptome of patients' fibroblasts by RNA sequencing, followed by additional whole-genome sequencing experiments. Gene expression analysis revealed a 4-fold downregulation of the gene NMNAT1, residing indeed in the shared autozygous interval. Short- and long-read whole-genome sequencing highlighted a duplication involving 2 out of the 5 exons of NMNAT1 main isoform (NM_022787.3), leading to the production of aberrant mRNAs. Pathogenic variants in NMNAT1 have been previously shown to cause non-syndromic Leber congenital amaurosis (LCA). However, no patient with null biallelic mutations has ever been described, and murine Nmnat1 knockouts show embryonic lethality, indicating that complete absence of NMNAT1 activity is probably not compatible with life. The rearrangement found in our cases, presumably causing a strong but not complete reduction of enzymatic activity, may therefore result in an intermediate syndromic phenotype with respect to LCA and lethality

Choline concentration dependence.

<p>A) Current traces from a choline concentration response curve obtained from a <i>Xenopus</i> oocyte expressing ACR-23 receptors. The bars indicate the time period of choline perfusion. Choline concentrations are indicated above the bars. B) Current amplitude measured in <i>Xenopus</i> oocytes expressing a wild-type ACR-23 receptor (filled circles) or a mutant ACR-23 receptor (filled squares) after choline perfusion. The mutant ACR-23 subunit corresponding to allele <i>acr-23(cb101)</i> bears a D112N substitution in the extracellular loop. Mean ± SD of experiments carried out with 4–5 oocytes from two batches are shown.</p

ACR-23 protein during <i>C.elegans </i> development.

<p>A) L2 larva expressing a <i>acr-23::gfp</i> transgene. Twisted morphology is caused by the transformation marker <i>rol-6</i>(<i>su1006)</i>. B) Same worm expressing <i>myo-3::mCherry</i>. C) and D) Expression in the adult. Bar, 50 µm. E) Analysis of ACR-23::GFP expression in response to monepantel after 24 hours of exposure. Loading and general protein expression were tested with α-tubulin and LET-512, respectively.</p

Monepantel is a direct agonist of ACR-23 channel.

<p>A) Current traces obtained from a <i>Xenopus</i> oocyte expressing ACR-23 upon perfusion with monepantel. The bars indicate the time period of monepantel perfusion. Monepantel concentrations are indicated above the bars. B) Averaged monepantel current amplitudes measured with monepantel (filled circles), monepantel sulfone (open circles) and with monepantel (filled triangles) and monepantel sulfone (open triangles) supplemented with 0.3 mM choline. One point is in brackets as the measured current reached the linear limit of the amplifier (20 µA), preventing a precise measurement. The effect of monepantel on mutant ACR-23(<i>cb101</i>) is shown by the filled squares. Mean ± SEM of experiments carried out with 3–6 oocytes from two batches are shown.</p

Effects of monepantel on <i>C. elegans</i> and rescue of mutant <i>acr-23</i>.

<p>A) Adult N2 hermaphrodite with hatched embryos on 60 µM monepantel. L1 and L2 larvae are paralyzed and coiled (carcass of mother is outlined). B) Higher resolution of an arrested L2 larva. The germline is outlined. C) <i>acr-23</i>(<i>cb27</i>) animals carrying an extrachromosomal array containing wild-type <i>acr-23</i> grown on standard NGM. Transgenic animals have a rolling phenotype (red arrowhead; non transgenic worms are denoted by a black arrowhead in C and D). D) In presence of monepantel, only non-rolling siblings are mobile and fertile. Transgenic animals are immobilized and dying or dead. Panels C) and D) show plates with the progeny of one single rolling parent incubated for 6 days at 20°C. E) Phenotypes of homozygotic and heterozygotic <i>acr-23(cb27). </i><i>eDf43</i> is a deficiency that spans <i>acr-23</i> and neighboring genes. Depicted adult worms are one heterozygote, with dominant pharyngeal GFP expression (<i>nT1g</i>), and one non-glowing <i>acr-23(cb27)</i> homozygote, both grown on 20 µM monepantel. White arrows indicate all embryos present in the uterus. Similar results were obtained with <i>acr-23(ok2804)/nT1g</i> heterozygotes (not shown). Asterisks indicate the head. Bar, 50 µm. F) <i>acr-23</i> mutants make fewer reversals. The rate of reversal events, where worms shift from forward to backward locomotion or vice versa, was measured in starved worms crawling on food-free NGM plates at 20°C. <i>n</i> is the number of movies analyzed, each tracking 15 to 60 individuals over at least 1 minute. *, P-value = 6.4E-5 versus N2 by Student's T-test (two-tailed). <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003524#s3" target="_blank">Results</a> are expressed as mean with s.e.m as error bars.</p

Effect of <i>ric-3</i> on the response to monepantel.

<p>A) Dose response to monepantel. The average number of progeny reaching adulthood after 3 days is plotted versus the concentration of monepantel. <i>ric-3(md158)</i> mutants are partially resistant to low doses of monepantel. B) <i>ric-3(md158)</i> adult grown on 1 µM monepantel (bottom) compared to a sibling grown in absence of the drug (top). The reduced length is certainly caused by muscle contraction due to monepantel. White arrows and arrowheads point at the heads and vulvae, respectively. C) ACR-23::GFP protein levels in wild-type (lanes 1,2) and <i>ric-3</i> (lanes 3,4) animals after 2 hours of exposure to 10 µM monepantel (lanes 2,4). α-Tubulin was used to verify equal loading. D) Growth of wild-type and mutant strains on monepantel. Light gray (0 µM), dark gray (20 µM) and black (50 µM) bars represent the growth rate after 9 days of culture. In the absence of monepantel, wild-type (N2) worms grow much faster than any of the mutants tested. Genotypes are indicated. Values for growth rates are defined in the methods section (1, poor growth; 5, robust growth). <i>acr-23</i> single mutants grew to level 5 in less than 5 days, whereas <i>ric-3; acr-23</i> double mutants reached level 2 in absence of monepantel, and level 4 in presence of the drug. E) Growth rates after 9 days on the inactive R-enantiomer of monepantel. F) <i>ric-3(md158); acr-23(cb27)</i> adult grown in absence of monepantel. G) Adult <i>ric-3(md158); acr-23(cb27)</i> sibling grown on 20 µM monepantel. In both panels (F and G), one germline arm is outlined. Arrowheads and arrows point at oocytes and early embryos, respectively. The outlined germline arm shows robust production of gametes (panel G). Scale bar, 50 µm.</p