Neuropathy target esterase activity defines phenotypes among PNPLA6 disorders.

Biallelic pathogenic variants in the PNPLA6 gene cause a broad spectrum of disorders leading to gait disturbance, visual impairment, anterior hypopituitarism and hair anomalies. PNPLA6 encodes neuropathy target esterase (NTE), yet the role of NTE dysfunction on affected tissues in the large spectrum of associated disease remains unclear. We present a systematic evidence-based review of a novel cohort of 23 new patients along with 95 reported individuals with PNPLA6 variants that implicate missense variants as a driver of disease pathogenesis. Measuring esterase activity of 46 disease-associated and 20 common variants observed across PNPLA6-associated clinical diagnoses unambiguously reclassified 36 variants as pathogenic and 10 variants as likely pathogenic, establishing a robust functional assay for classifying PNPLA6 variants of unknown significance. Estimating the overall NTE activity of affected individuals revealed a striking inverse relationship between NTE activity and the presence of retinopathy and endocrinopathy. This phenomenon was recaptured in vivo in an allelic mouse series, where a similar NTE threshold for retinopathy exists. Thus, PNPLA6 disorders, previously considered allelic, are a continuous spectrum of pleiotropic phenotypes defined by an NTE genotype:activity:phenotype relationship. This relationship, and the generation of a preclinical animal model, pave the way for therapeutic trials, using NTE as a biomarker.

Restoration of RPGR expression in vivo using CRISPR/Cas9 gene editing.

Mutations in the gene for Retinitis Pigmentosa GTPase Regulator (RPGR) cause the X-linked form of inherited retinal degeneration, and the majority are frameshift mutations in a highly repetitive, purine-rich region of RPGR known as the OFR15 exon. Truncation of the reading frame in this terminal exon ablates the functionally important C-terminal domain. We hypothesized that targeted excision in ORF15 by CRISPR/Cas9 and the ensuing repair by non-homologous end joining could restore RPGR reading frame in a portion of mutant photoreceptors thereby correcting gene function in vivo. We tested this hypothesis in the rd9 mouse, a naturally occurring mutant line that carries a frameshift mutation in RPGRORF15, through a combination of germline and somatic gene therapy approaches. In germline gene-edited rd9 mice, probing with RPGR domain-specific antibodies demonstrated expression of full length RPGRORF15 protein. Hallmark features of RPGR mutation-associated early disease phenotypes, such as mislocalization of cone opsins, were no longer present. Subretinal injections of the same guide RNA (sgRNA) carried in AAV sgRNA and SpCas9 expression vectors restored reading frame of RPGRORF15 in a subpopulation of cells with broad distribution throughout the retina, confirming successful correction of the mutation. These data suggest that a simplified form of genome editing mediated by CRISPR, as described here, could be further developed to repair RPGRORF15 mutations in vivo.

Targeted deletion of an NRL- and CRX-regulated alternative promoter specifically silences FERM and PDZ domain containing 1 (Frmpd1) in rod photoreceptors.

Regulation of cell type-specific gene expression is critical for generating neuronal diversity. Transcriptome analyses have unraveled extensive heterogeneity of transcribed sequences in retinal photoreceptors because of alternate splicing and/or promoter usage. Here we show that Frmpd1 (FERM and PDZ domain containing 1) is transcribed from an alternative promoter specifically in the retina. Electroporation of Frmpd1 promoter region, -505 to +382 bp, activated reporter gene expression in mouse retina in vivo. A proximal promoter sequence (-8 to +33 bp) of Frmpd1 binds to neural retina leucine zipper (NRL) and cone-rod homeobox protein (CRX), two rod-specific differentiation factors, and is necessary for activating reporter gene expression in vitro and in vivo. Clustered regularly interspaced short palindromic repeats/Cas9-mediated deletion of the genomic region, including NRL and CRX binding sites, in vivo completely eliminated Frmpd1 expression in rods and dramatically reduced expression in rod bipolar cells, thereby overcoming embryonic lethality caused by germline Frmpd1 deletion. Our studies demonstrate that a cell type-specific regulatory control region is a credible target for creating loss-of-function alleles of widely expressed genes.

Maturation arrest in early postnatal sensory receptors by deletion of the miR-183/96/182 cluster in mouse.

The polycistronic miR-183/96/182 cluster is preferentially and abundantly expressed in terminally differentiating sensory epithelia. To clarify its roles in the terminal differentiation of sensory receptors in vivo, we deleted the entire gene cluster in mouse germline through homologous recombination. The miR-183/96/182 null mice display impairment of the visual, auditory, vestibular, and olfactory systems, attributable to profound defects in sensory receptor terminal differentiation. Maturation of sensory receptor precursors is delayed, and they never attain a fully differentiated state. In the retina, delay in up-regulation of key photoreceptor genes underlies delayed outer segment elongation and possibly mispositioning of cone nuclei in the retina. Incomplete maturation of photoreceptors is followed shortly afterward by early-onset degeneration. Cell biologic and transcriptome analyses implicate dysregulation of ciliogenesis, nuclear translocation, and an epigenetic mechanism that may control timing of terminal differentiation in developing photoreceptors. In both the organ of Corti and the vestibular organ, impaired terminal differentiation manifests as immature stereocilia and kinocilia on the apical surface of hair cells. Our study thus establishes a dedicated role of the miR-183/96/182 cluster in driving the terminal differentiation of multiple sensory receptor cells.

Prickle1 is expressed in distinct cell populations of the central nervous system and contributes to neuronal morphogenesis.

Development of axons and dendrites constitutes a critical event in neuronal maturation and seems to require signaling through the planar cell polarity (PCP) pathway. Mutations in components of the PCP pathway lead to a spectrum of neurological phenotypes and disorders. For example, a missense mutation in Prickle 1 (Pk1) is associated with progressive myoclonus epilepsy (PME) in humans, and its reduced gene dosage increases sensitivity to induced seizure in mice. In an effort to unravel the role of the PCP pathway in mammalian neuronal development, we examined the expression of Pk1 in the central nervous system (CNS) using in situ hybridization (ISH) in combination with a genetic knock-in approach. We show that Pk1 transcripts are detected in the postmitotic cells of the subplate and cortical plate during mid- and late stages of cortical neurogenesis. In adult brain, Pk1 is expressed in distinct neuronal and glial cell populations, with dynamic formation of dendrites and glial processes during development. Of all the cell types in the mature retina, the highest expression of Pk1 is detected in cholinergic amacrine neurons. Knockdown of Pk1 by shRNA or dominant-negative constructs causes reduced axonal and dendritic extension in hippocampal neurons. Similarly, Pk1 knockdown in neonatal retina leads to defects in inner and outer segments and axon terminals of photoreceptors. Our studies implicate Pk1 function in axonal-dendritic development associated with the maturation of CNS neurons.

Neuropathy target esterase activity predicts retinopathy among PNPLA6 disorders.

Biallelic pathogenic variants in the PNPLA6 gene cause a broad spectrum of disorders leading to gait disturbance, visual impairment, anterior hypopituitarism, and hair anomalies. PNPLA6 encodes Neuropathy target esterase (NTE), yet the role of NTE dysfunction on affected tissues in the large spectrum of associated disease remains unclear. We present a clinical meta-analysis of a novel cohort of 23 new patients along with 95 reported individuals with PNPLA6 variants that implicate missense variants as a driver of disease pathogenesis. Measuring esterase activity of 46 disease-associated and 20 common variants observed across PNPLA6 -associated clinical diagnoses unambiguously reclassified 10 variants as likely pathogenic and 36 variants as pathogenic, establishing a robust functional assay for classifying PNPLA6 variants of unknown significance. Estimating the overall NTE activity of affected individuals revealed a striking inverse relationship between NTE activity and the presence of retinopathy and endocrinopathy. This phenomenon was recaptured in vivo in an allelic mouse series, where a similar NTE threshold for retinopathy exists. Thus, PNPLA6 disorders, previously considered allelic, are a continuous spectrum of pleiotropic phenotypes defined by an NTE genotype:activity:phenotype relationship. This relationship and the generation of a preclinical animal model pave the way for therapeutic trials, using NTE as a biomarker.

Retinal Development and Pathophysiology in Kcnj13 Knockout Mice.

Purpose: We constructed and characterized knockout and conditional knockout mice for KCNJ13, encoding the inwardly rectifying K+ channel of the Kir superfamily Kir7.1, mutations in which cause both Snowflake Vitreoretinal Degeneration (SVD) and Retinitis pigmentosa (RP) to further elucidate the pathology of this disease and to develop a potential model system for gene therapy trials. Methods: A Kcnj13 knockout mouse line was constructed by inserting a gene trap cassette expressing beta-galactosidase flanked by FRT sites in intron 1 with LoxP sites flanking exon two and converted to a conditional knockout by FLP recombination followed by crossing with C57BL/6J mice having Cre driven by the VMD2 promoter. Lentiviral replacement of Kcnj13 was driven by the EF1a or VMD2 promoters. Results: Blue-Gal expression is evident in E12.5 brain ventricular choroid plexus, lens, neural retina layer, and anterior RPE. In the adult eye expression is seen in the ciliary body, RPE and choroid. Adult conditional Kcnj13 ko mice show loss of photoreceptors in the outer nuclear layer, inner nuclear layer thinning with loss of bipolar cells, and thinning and disruption of the outer plexiform layer, correlating with Cre expression in the overlying RPE which, although preserved, shows morphological disruption. Fundoscopy and OCT show signs of retinal degeneration consistent with the histology, and photopic and scotopic ERGs are decreased in amplitude or extinguished. Lentiviral based replacement of Kcnj13 resulted in increased ERG c- but not a- or b- wave amplitudes. Conclusion: Ocular KCNJ13 expression starts in the choroid, lens, ciliary body, and anterior retina, while later expression centers on the RPE with no/lower expression in the neuroretina. Although KCNJ13 expression is not required for survival of the RPE, it is necessary for RPE maintenance of the photoreceptors, and loss of the photoreceptor, outer plexiform, and outer nuclear layers occur in adult KCNJ13 cKO mice, concomitant with decreased amplitude and eventual extinguishing of the ERG and signs of retinitis pigmentosa on fundoscopy and OCT. Kcnj13 replacement resulting in recovery of the ERG c- but not a- and b-waves is consistent with the degree of photoreceptor degeneration seen on histology.

Creating Knockin Alleles in Mouse Embryonic Stem Cells by CRISPR/Cas9-Mediated Homologous Recombination Without Drug Selection.

The rapidly evolving CRISPR/Cas9-mediated genome editing provides the convenience of genome manipulation directly in mouse zygotes for a number of genomic manipulations; but knockins of large insertions prove to be relatively inefficient at least with double-strand DNA as targeting constructs. Here, we describe an alternative approach to the direct genome editing in mouse zygotes by generating knockin alleles in mouse embryonic stem cells first with CRIPSR-mediated homologous recombination. Our results show this approach is efficient and requires no drug selection in mouse embryonic stem cells as in classic gene targeting. As the result, knockin alleles across many target loci are created in mouse embryonic stem cells and readily transmitted through germline. The knockin alleles created in ES cells can also serve as valuable tools for in vitro stem cell differentiation.

From tissue phenotype to proteotype: sensitive protein identification in microdissected tumor tissue.

Correlation of disease phenotype with protein profile (proteotype) is a significant challenge for biomedical research. The main obstacles have been the need to insure sufficient quantities of pure protein sample, the reproducibility of protein display, and rapid and accurate protein identification. We present a modified approach that combines enhanced detection sensitivity with tissue microdissection from frozen primary renal cancer tissues of different histological subtypes, followed by 2D gel analysis and protein identification with MALDI mass spectrometry. We obtained reliable and highly consistent results in phenotypically similar tumors of each individual subtype by performing strict morphological control of the analyzed tumor cells without physical or chemical alteration of the frozen tissue samples. By application of non-oxidizing silver staining, proteins were resolved and identified with high levels of specificity and sensitivity. This new combination of techniques allows not only for sensitive identification of specific protein patterns that correspond to a histological tumor phenotype, but also for identification of specific disease-associated protein targets.

Protein patterns and proteins that identify subtypes of glioblastoma multiforme.

Glioblastoma multiforme (GBM) has been subdivided into two types based on clinical and genetic findings: primary tumors, which arise de novo, and secondary tumors, which progress from lower grade gliomas to GBMs. To analyse this dichotomy at the protein level, we employed selective tissue microdissection to obtain pure populations of tumor cells, which we studied using two-dimensional protein gel electrophoresis (2-DGE) and protein sequencing of select target proteins. Protein patterns were analysed in a blinded manner from the clinical and genetic data. 2-DGE clearly identified two distinct populations of tumors. 2-DGE was reproducible and reliable, as multiple samples analysed from the same patient gave identical results. In addition, we isolated and sequenced 11 proteins that were uniquely expressed in either the primary or the secondary GBMs, but not both. We demonstrate that specific proteomic patterns can be reproducibly identified by two-dimensional gel electrophoresis from limited numbers of selectively procured, microdissected tumor cells and that two patterns of GBMs, primary versus secondary, previously distinguished by clinical and genetic differences, can be recognized at the protein level. Proteins that are expressed distinctively may have important implications for the diagnosis, prognosis, and treatment of patients with GBM.

Multilineage potential of homozygous stem cells derived from metaphase II oocytes.

Human stem cells derived from human fertilized oocytes, fetal primordial germ cells, umbilical cord blood, and adult tissues provide potential cell-based therapies for repair of degenerating or damaged tissues. However, the diversity of major histocompatibility complex (MHC) antigens in the general population and the resultant risk of immune-mediated rejection complicates the allogenic use of established stem cells. We assessed an alternative approach, employing chemical activation of nonfertilized metaphase II oocytes for producing stem cells homozygous for MHC. By using F1 hybrid mice (H-2-B/D), we established stem cell lines homozygous for H-2-B and H-2-D, respectively. The undifferentiated cells retained a normal karyotype, expressed stage-specific embryonic antigen-1 and Oct4, and were positive for alkaline phosphatase and telomerase. Teratomatous growth of these cells displayed the development of a variety of tissue types encompassing all three germ layers. In addition, these cells demonstrated the potential for in vitro differentiation into endoderm, neuronal, and hematopoietic lineages. We also evaluated this homozygous stem cell approach in human tissue. Five unfertilized blastocysts were derived from a total of 25 human oocytes, and cells from one of the five hatched blastocysts proliferated and survived beyond two passages. Our studies demonstrate a plausible "homozygous stem cell" approach for deriving pluripotent stem cells that can overcome the immune-mediated rejection response common in allotransplantation, while decreasing the ethical concerns surrounding human embryonic stem cell research.