Transposons are mobile DNA segments that can disrupt gene function by inserting in or near genes. Here we show that insertional mutagenesis by the PiggyBac transposon can be used for cancer gene discovery in mice. PiggyBac transposition in genetically engineered transposon/ transposase mice induced cancers whose type (hematopoietic versus solid) and latency were dependent on the regulatory elements introduced into transposons. Analysis of 63 hematopoietic tumors revealed the unique qualities of PiggyBac for genome-wide mutagenesis and discovered many cancer genes not identified in previous retroviral or Sleeping Beauty transposon screens, including Spic, which encodes a PU.1-related transcription factor, and Hdac7, a histone deacetylase gene. PiggyBac and Sleeping Beauty have different integration preferences. To maximize the utility of the tool, we engineered 20 mouse lines to be compatible with both transposases in constitutive, tissue-or temporal-specific mutagenesis. Mice with different transposon types, copy numbers and chromosomal locations support wide applicability.Genetic screening in higher organisms has been hampered for decades by the lack of efficient insertional mutagenesis tools. Retroviruses have been used for cancer gene discovery in mice, but their application has been limited to the study of hematopoietic and mammary tumors due to viral tropism for these tissues (1). DNA transposons, which are the key insertional mutagens in lower organisms, were inactivated in vertebrate genomes millions of years ago. Only recently have new transposons been engineered to be active in mammalian cells, a development that provides opportunities for their use as genetic tools in higher organisms (2). Sleeping Beauty (SB), a TC1/mariner transposon, was reconstructed from dormant elements in fish genomes and optimized to transpose in multiple cell types (3), including mouse embryonic stem cells (4). Further improvements of SB led to its successful application for somatic mutagenesis in mice (5, 6). Another transposon, PiggyBac (PB) from the cabbage looper moth, was recently engineered to be highly active in mammalian cells (7) and has been shown to have biological properties distinct from those of SB (2, 7-9). PB can move larger DNA fragments (allowing complex transgene designs to be incorporated into the transposon) and it has a weaker tendency for local hopping in vitro (which makes it an attractive candidate for genome-wide mutagenesis). Furthermore, in contrast to SB, PB does not leave undesired footprint mutations after transposition. Finally, PB and SB have different integration preferences. (10)). All transposons possess PB and SB inverted terminal repeats (ITRs), allowing mobilization with both transposases. Promoter/enhancer elements, a splice donor, bidirectional SV40 polyAs and two splice acceptors were introduced in between the ITRs to allow gain or loss of function mutations, depending on the transposon orientation and its spatial relation to genes. Transgenic mice were generated with three va...

Purpose In the era of precision medicine, genomic characterization of blind patients is critical. Here, we evaluate the effects of comprehensive genetic analysis on the etiologic diagnosis of potentially hereditary vision loss and its impact on clinical management. Methods We studied 100 non‐syndromic and syndromic Spanish patients with a clinical diagnosis of blindness caused by alterations on the retina, choroid, vitreous and/or optic nerve. We used a next‐generation sequencing (NGS) panel (OFTALMOgenics™), developed and validated within this study, including up to 362 genes previously associated with these conditions. Results We identified the genetic cause of blindness in 45% of patients (45/100). A total of 28.9% of genetically diagnosed cases (13/45) were syndromic and, of those, in 30.8% (4/13) extraophthalmic features had been overlooked and/or not related to visual impairment before genetic testing, including cases with Mainzer‐Saldino, Bardet‐Biedl, mucolipidosis and MLCRD syndromes. In two additional cases–syndromic blindness had been proposed before, but not specifically diagnosed, and one patient with Heimler syndrome had been misdiagnosed as an Usher case before testing. 33.3% of the genetically diagnosed patients (15/45) had causative variants in genes targeted by clinical trials exploring the curative potential of gene therapy approaches. Conclusion Comprehensive genomic testing provided clinically relevant insights in a large proportion of blind patients, identifying potential therapeutic opportunities or previously undiagnosed syndromes in 42.2% of the genetically diagnosed cases (19/45).

The stereocilia of the inner ear sensory cells contain the actin-binding protein radixin, encoded by RDX. Radixin is important for hearing but remains functionally obscure. To determine how radixin influences hearing sensitivity, we used a custom rapid imaging technique to visualize stereocilia motion while measuring electrical potential amplitudes during acoustic stimulation. Radixin inhibition decreased sound-evoked electrical potentials. Other functional measures, including electrically induced sensory cell motility and sound-evoked stereocilia deflections, showed a minor amplitude increase. These unique functional alterations demonstrate radixin as necessary for conversion of sound into electrical signals at acoustic rates. We identified patients with RDX variants with normal hearing at birth who showed rapidly deteriorating hearing during the first months of life. This may be overlooked by newborn hearing screening and explained by multiple disturbances in postnatal sensory cells. We conclude radixin is necessary for ensuring normal conversion of sound to electrical signals in the inner ear.

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Objective The biallelic inheritance of an expanded intronic pentamer (AAGGG)exp in the gene encoding replication factor C subunit 1 ( RFC1) has been found to be a cause of cerebellar ataxia, neuropathy, and vestibular areflexia syndrome (CANVAS). This study describes clinical and genetic features of our patients with clinical suspicion of the syndrome. Study Design A retrospective descriptive study from an ataxia database comprising 500 patients. Setting The study was performed at the Otorhinolaryngology Department of a hospital in the north of Spain. Methods Specific genetic testing for CANVAS was performed in 13 patients with clinical suspicion of complete or incomplete syndrome. The clinical diagnosis was supported by quantitative vestibular hypofunction, cerebellar atrophy, and abnormal sensory nerve conduction testing. Results Nine of 13 (69%) patients met clinical diagnostic criteria for definite CANVAS disease. The first manifestation of the syndrome was lower limb dysesthesia in 8 of 13 patients and gait imbalance in 5 of 13. Eleven of 13 (85%) patients were carriers of the biallelic (AAGGG)exp in RFC1. Conclusion A genetic cause of CANVAS has recently been discovered. We propose genetic screening for biallelic expansions of the AAGGG pentamer of RFC1 in all patients with clinical suspicion of CANVAS, since accurate early diagnosis could improve the quality of life of these patients.

Hypothesis: Adult genetic sensorineural hearing loss (SNHL) may be underestimated. Background: The diagnosis of genetic hearing loss is challenging, given its extreme genetic and phenotypic heterogeneity, particularly in adulthood. This study evaluated the utility of next-generation sequencing (NGS) in the etiological diagnosis of adult-onset SNHL. Materials and Methods: Adults (>16 yr old) with SNHL were recruited at the Otolaryngology Department at Marqués de Valdecilla University Hospital (Spain). Environmental factors, acoustic trauma, endolymphatic hydrops, and age-related hearing loss were excluding criteria. An NGS gene panel was used, including 196 genes (OTOgenics v3) or 229 genes (OTOgenics v4) related to syndromic and nonsyndromic hearing loss. Results: Sixty-five patients were included in the study (average age at the onset of SNHL, 41 yr). Fifteen pathogenic/likely pathogenic variants considered to be causative were found in 15 patients (23% diagnostic yield) in TECTA (4), KCNQ4 (3), GJB2 (2), ACTG1 (1), COL2A1 (1), COCH (1), COCH/COL2A1 (1), STRC(1), and ABHD12 (1). Three patients had syndromic associations (20% of patients with genetic diagnosis) that had not been previously diagnosed (two Stickler type I and one polyneuropathy, hearing loss, ataxia, retinitis pigmentosa, cataract syndrome). Seven variants of unknown significance were found in COL11A1 (1), GSMDE (2), DNTM1 (1), SOX10 (1), EYA4 (1), and TECTA (1). Conclusion: NGS gene panels can provide diagnostic yields greater than 20% for adult SNHL, with a significant proportion of variant of unknown significance that could potentially contribute to increasing diagnostic output. Identifying a genetic cause enables genetic counseling, provides prognostic information and can reveal unrecognized syndromes contributing to an accurate management of their associated manifestations.

Distinguishing tumor maintenance genes from initiation, progression, and passenger genes is fundamental for developing effective cancer therapy. We engineered a Lazy Piggy screening system to first dysregulate and later restore gene expression using the Sleeping Beauty/piggyBac hybrid transposon. In vivo Lazy Piggy insertion and remobilization deplete insertions non-essential for tumor survival while enriching for maintenance insertions, uncovering potassium channels in the maintenance of medulloblastoma, the most common pediatric brain malignancy. KCNB2 is the most overexpressed potassium channel in human medulloblastoma, and Kcnb2 knockout in mice diminishes the replicative potential of medulloblastoma-propagating cells to mitigate tumor growth. Mechanistically, Kcnb2 governs potassium homeostasis to regulate plasma membrane tension-gated EGFR signaling, which drives the proliferative expansion of medulloblastoma-propagating cells. Collectively, our Lazy Piggy functional genomics reveals potassium homeostasis as a tumor maintenance essentiality and elucidates a mechanism by which potassium homeostasis integrates biomechanical and biochemical signaling to promote medulloblastoma aggression.

The stereocilia of the sensory cells in the inner ear contain high levels of the actin-binding protein radixin, encoded by the RDX gene. Radixin which is associated with mechanotransduction process such as PIP2 is known to be important for hearing but its functional role remains obscure. To determine how radixin influences hearing sensitivity, we used a custom rapid imaging technique to directly visualize stereocilia motion while measuring the amplitude of the electrical potentials produced by sensory cells during acoustic stimulation. Experiments were performed in guinea pigs, where upon blocking radixin, a large decrease in sound-evoked electrical potentials occurred. Despite this decrease other important functional measures, such as electrically induced sensory cell motility and the sound-evoked deflections of stereocilia, showed a minor amplitude increase. This unique set of functional properties alterations demonstrate that radixin is necessary to ensure that the inner ear converts sound into electrical signals at acoustic rates. Radixin is therefore a necessary and important component of the cochlear amplifier, the energy-consuming process that boosts hearing sensitivity by up to 60 dB.