Phenotype Driven Analysis of Whole Genome Sequencing Identifies Deep Intronic Variants that Cause Retinal Dystrophies by Aberrant Exonization.

Purpose: To demonstrate the effectiveness of combining retinal phenotyping and focused variant filtering from genome sequencing (GS) in identifying deep intronic disease causing variants in inherited retinal dystrophies. Methods: Affected members from three pedigrees with classical enhanced S-cone syndrome (ESCS; Pedigree 1), congenital stationary night blindness (CSNB; Pedigree 2), and achromatopsia (ACHM; Pedigree 3), respectively, underwent detailed ophthalmologic evaluation, optical coherence tomography, and electroretinography. The probands underwent panel-based genetic testing followed by GS analysis. Minigene constructs (NR2E3, GPR179 and CNGB3) and patient-derived cDNA experiments (NR2E3 and GPR179) were performed to assess the functional effect of the deep intronic variants. Results: The electrophysiological findings confirmed the clinical diagnosis of ESCS, CSNB, and ACHM in the respective pedigrees. Panel-based testing revealed heterozygous pathogenic variants in NR2E3 (NM_014249.3; c.119-2A>C; Pedigree 1) and CNGB3 (NM_019098.4; c.1148delC/p.Thr383Ilefs*13; Pedigree 3). The GS revealed heterozygous deep intronic variants in Pedigrees 1 (NR2E3; c.1100+1124G>A) and 3 (CNGB3; c.852+4751A>T), and a homozygous GPR179 variant in Pedigree 2 (NM_001004334.3; c.903+343G>A). The identified variants segregated with the phenotype in all pedigrees. All deep intronic variants were predicted to generate a splice acceptor gain causing aberrant exonization in NR2E3 [89 base pairs (bp)], GPR179 (197 bp), and CNGB3 (73 bp); splicing defects were validated through patient-derived cDNA experiments and/or minigene constructs and rescued by antisense oligonucleotide treatment. Conclusions: Deep intronic mutations contribute to missing heritability in retinal dystrophies. Combining results from phenotype-directed gene panel testing, GS, and in silico splice prediction tools can help identify these difficult-to-detect pathogenic deep intronic variants.

Structural genome analysis in cultivated potato taxa.

KEY MESSAGE: Twelve potato accessions were selected to represent two principal views on potato taxonomy. The genomes were sequenced and analyzed for structural variation (copy number variation) against three published potato genomes. The common potato (Solanum tuberosum L.) is an important staple crop with a highly heterozygous and complex tetraploid genome. The other taxa of cultivated potato contain varying ploidy levels (2X-5X), and structural variations are common in the genomes of these species, likely contributing to the diversification or agronomic traits during domestication. Increased understanding of the genomes and genomic variation will aid in the exploration of novel agronomic traits. Thus, sequencing data from twelve potato landraces, representing the four ploidy levels, were used to identify structural genomic variation compared to the two currently available reference genomes, a double monoploid potato genome and a diploid inbred clone of S. chacoense. The results of a copy number variation analysis showed that in the majority of the genomes, while the number of deletions is greater than the number of duplications, the number of duplicated genes is greater than the number of deleted ones. Specific regions in the twelve potato genomes have a high density of CNV events. Further, the auxin-induced SAUR genes (involved in abiotic stress), disease resistance genes and the 2-oxoglutarate/Fe(II)-dependent oxygenase superfamily proteins, among others, had increased copy numbers in these sequenced genomes relative to the references.

Retrotransposon insertion as a novel mutational event in Bardet-Biedl syndrome.

BACKGROUND: Bardet-Biedl syndrome (BBS) is an autosomal recessive pleiotropic disorder of the primary cilia that leads to severe visual loss in the teenage years. Approximately 80% of BBS cases are explained by mutations in one of the 21 identified genes. Documented causative mutation types include missense, nonsense, copy number variation (CNV), frameshift deletions or insertions, and splicing variants. METHODS: Whole genome sequencing was performed on a patient affected with BBS for whom no mutations were identified using clinically approved genetic testing of the known genes. Analysis of the WGS was done using internal protocols and publicly available algorithms. The phenotype was defined by retrospective chart review. RESULTS: We document a female affected with BBS carrying the most common BBS1 mutation (BBS1: Met390Arg) on the maternal allele and an insertion of a ~1.7-kb retrotransposon in exon 13 on the paternal allele. This retrotransposon insertion was not automatically annotated by the standard variant calling protocols used. This novel variant was identified by visual inspection of the alignment file followed by specific genome analysis with an available algorithm for transposable elements. CONCLUSION: This report documents a novel mutation type associated with BBS and highlights the importance of systematically performing transposon detection analysis on WGS data of unsolved cases.

Construction of Benzo[c]carbazoles and Their Antitumor Derivatives through the Diels-Alder Reaction of 2-Alkenylindoles and Arynes.

The direct assembly of benzo[c]carbazole derivatives via the Diels-Alder reaction of arynes and easily accessible 2-alkenylidoles was reported. By employing different aryne precursor loads, 6,7-dihydrobenzo[c]carbazoles or aryl-substituted 7,11b-dihydrobenzo[c]carbazoles could be controllably generated in good to excellent yields under a nitrogen atmosphere. On the other hand, when the reaction was conducted under oxygen, oxidated/aromatized product benzo[c]carbazoles could be generated directly with high selectivity and efficiency in a one-step manner. Interestingly, the benzo[c]carbazole-5-carboxamide amidation derivatives of the above products showed good antitumor activities. The inhibitory effect of these molecules against cancer cells was also described.

Fast-switching bistable cholesteric intensity modulator.

A fast-switching bistable optical intensity modulator is demonstrated. Using a dual-frequency cholesteric liquid crystal, the direct switching is achieved from the scattering focal conic state to the transparent long-pitch planar state. In comparison with the bistable cholesteric devices proposed previously, our device, characterized by its capability of direct two-way transitions between the two bistable states, possesses a very short transition time from the focal conic state to the planar state as short as 10 ms. No voltage has to be applied to sustain the optical states, making the device low energy consuming. Potential applications of this device are addressed.