Integration of transcription regulation and functional genomic data reveals lncRNA SNHG6's role in hematopoietic differentiation and leukemia.

BACKGROUND: Long non-coding RNAs (lncRNAs) are pivotal players in cellular processes, and their unique cell-type specific expression patterns render them attractive biomarkers and therapeutic targets. Yet, the functional roles of most lncRNAs remain enigmatic. To address the need to identify new druggable lncRNAs, we developed a comprehensive approach integrating transcription factor binding data with other genetic features to generate a machine learning model, which we have called INFLAMeR (Identifying Novel Functional LncRNAs with Advanced Machine Learning Resources). METHODS: INFLAMeR was trained on high-throughput CRISPR interference (CRISPRi) screens across seven cell lines, and the algorithm was based on 71 genetic features. To validate the predictions, we selected candidate lncRNAs in the human K562 leukemia cell line and determined the impact of their knockdown (KD) on cell proliferation and chemotherapeutic drug response. We further performed transcriptomic analysis for candidate genes. Based on these findings, we assessed the lncRNA small nucleolar RNA host gene 6 (SNHG6) for its role in myeloid differentiation. Finally, we established a mouse K562 leukemia xenograft model to determine whether SNHG6 KD attenuates tumor growth in vivo. RESULTS: The INFLAMeR model successfully reconstituted CRISPRi screening data and predicted functional lncRNAs that were previously overlooked. Intensive cell-based and transcriptomic validation of nearly fifty genes in K562 revealed cell type-specific functionality for 85% of the predicted lncRNAs. In this respect, our cell-based and transcriptomic analyses predicted a role for SNHG6 in hematopoiesis and leukemia. Consistent with its predicted role in hematopoietic differentiation, SNHG6 transcription is regulated by hematopoiesis-associated transcription factors. SNHG6 KD reduced the proliferation of leukemia cells and sensitized them to differentiation. Treatment of K562 leukemic cells with hemin and PMA, respectively, demonstrated that SNHG6 inhibits red blood cell differentiation but strongly promotes megakaryocyte differentiation. Using a xenograft mouse model, we demonstrate that SNHG6 KD attenuated tumor growth in vivo. CONCLUSIONS: Our approach not only improved the identification and characterization of functional lncRNAs through genomic approaches in a cell type-specific manner, but also identified new lncRNAs with roles in hematopoiesis and leukemia. Such approaches can be readily applied to identify novel targets for precision medicine.

Autosomal dominant retinitis pigmentosa with incomplete penetrance due to an intronic mutation of the PRPF31 gene.

Purpose: To identify the molecular mechanisms of the development of autosomal dominant retinitis pigmentosa (adRP) with incomplete penetrance in an Israeli Muslim Arab family. Methods: Two patients with adRP underwent a detailed ophthalmic evaluation, including funduscopic examination, visual field testing, optical coherence tomography, and electroretinography. Genetic analysis was performed using a combination of whole exome sequencing (WES) and Sanger sequencing. The pathogenicity of the identified intronic variant was evaluated in silico using several web-based tools, in vitro using a minigene-based assay, and in vivo using reverse transcription PCR analysis of lymphocyte-derived RNA. The relative abundance of alternatively spliced transcripts was evaluated using amplicon-based next-generation sequencing. The relative expression levels of PRPF31 and CNOT3 were measured using quantitative PCR (qPCR) analysis. Results: The two patients recruited in this study had childhood-onset RP, with night blindness as the initial symptom, followed by concentric restriction of the visual field. The funduscopic findings included narrowed retinal blood vessels and peripheral bone spicule pigmentation. By the third decade of life, the full-field electroretinography findings had been remarkably attenuated. In these patients, we identified a novel heterozygous intronic variant at position +5 of PRPF31 intron 11 (c.1146+5G>T). The same variant was also detected in one asymptomatic family member. Through in silico analysis, the variant was predicted to alter the splicing of intron 11. An in vitro splicing assay and a reverse transcription PCR analysis of lymphocyte-derived RNA revealed that the mutant allele yielded mainly a shorter transcript in which exon 11 was skipped. The skipping of exon 11 was expected to cause a frameshift and an aberrant truncated protein (p.Tyr359Serfs*29). The qPCR analysis revealed reduced PRPF31 expression levels in the mutation carriers, without a significant difference between the affected patient and his asymptomatic brother. We evaluated several factors that have been suggested to correlate with non-penetrance of PRPF31 mutations, including the number of cis-acting MSR1 elements adjacent to the PRPF31 core promoter, CNOT3 expression level, and CNOT3 rs4806718 single-nucleotide polymorphism. None of these factors correlated with non-penetrance in the family in this study. Conclusions: We report a novel intronic mutation in PRPF31 underlying adRP. This report expands the spectrum of pathogenic mutations in PRPF31 and further demonstrates the importance of intronic mutations. Moreover, it demonstrates the phenomenon of incomplete penetrance previously associated with PRPF31 mutations. The fact that the non-penetrance in the family in this study could not be explained by any of the known mechanisms suggests the possible contribution of a novel modifier of PRPF31 penetrance.

Retinal dystrophy as part of TTC21B-associated ciliopathy.

Background: TCC21B is a ciliary protein. The most common phenotypic features associated with TCC21B biallelic mutations are nephronophthisis and skeletal abnormalities. To date, retinal dystrophy has been reported in only one patient.Materials and Methods: Clinical evaluation included best-corrected visual acuity, cycloplegic refraction, fundus examination, fundus photography, retinal imaging by optical coherence tomography, full-field electroretinography, multifocal electroretinography, and visual evoked potentials. Genetic analysis included Whole Exome Sequencing and confirmation of the identified mutations in the patient and his parents by PCR amplification and direct sequencing.Results: A ten-year-old Caucasian male presented with nephronophthisis, high myopia and nycatalopia. Best-corrected visual acuity was preserved to 20/20 in each eye with significant myopic correction. Visual fields were constricted. Optical coherence tomography confirmed the lack of outer retinal layers in the perifoveal area on both eyes. Electroretinography confirmed significant retinal dystrophy. Whole Exome Sequencing revealed compound heterozygous mutations in the TTC21B gene.Conclusions: TTC21B is associated with ciliopathy, but retinal dystrophy is a rare finding in these patients. We report retinal dystrophy secondary to TTC21B mutations, and provide for the first time detailed clinical information of the ophthalmic phenotype.