No abstract

The key negative regulatory gene of the RAS pathway, NF1, is mutated or deleted in numerous cancer types and is associated with increased cancer risk and drug resistance. Even though women with neurofibromatosis (germline NF1 mutations) have a substantially increased breast cancer risk at a young age and NF1 is commonly mutated in sporadic breast cancers, we have a limited understanding of the role of NF1 in breast cancer. We utilized CRISPR–Cas9 gene editing to create Nf1 rat models to evaluate the effect of Nf1 deficiency on tumorigenesis. The resulting Nf1 indels induced highly penetrant, aggressive mammary adenocarcinomas that express estrogen receptor (ER) and progesterone receptor (PR). We identified distinct Nf1 mRNA and protein isoforms that were altered during tumorigenesis. To evaluate NF1 in human breast cancer, we analyzed genomic changes in a data set of 2000 clinically annotated breast cancers. We found NF1 shallow deletions in 25% of sporadic breast cancers, which correlated with poor clinical outcome. To identify biological networks impacted by NF1 deficiency, we constructed gene co-expression networks using weighted gene correlation network analysis (WGCNA) and identified a network connected to ESR1 (estrogen receptor). Moreover, NF1-deficient cancers correlated with established RAS activation signatures. Estrogen-dependence was verified by estrogen-ablation in Nf1 rats where rapid tumor regression was observed. Additionally, Nf1 deficiency correlated with increased estrogen receptor phosphorylation in mammary adenocarcinomas. These results demonstrate a significant role for NF1 in both NF1-related breast cancer and sporadic breast cancer, and highlight a potential functional link between neurofibromin and the estrogen receptor.

Cervical cancer is a common and deadly disease, especially in developing countries. We developed and implemented an interactive, tablet-based educational intervention to improve cervical cancer knowledge among women in rural Malawi. Chichewa-speaking adult women in six rural villages participated. Each woman took a pre-test, participated in the lesson, and then took a post-test. The lesson included information on cervical cancer symptoms, causes, risk factors, prevention, and treatment. Over the 6-month study period, 243 women participated. Women ranged in age from 18 to 77 years. Only 15% had education beyond primary school. Nearly half of participants (48%) had heard of cervical cancer prior to viewing the lesson. For these women, the median number of correct responses on the pre-test was 11 out of 20; after the lesson, they had a median of 18 correct responses (p<0.001). After the intervention, 93% of women indicated a desire for cervical cancer screening. Despite lack of familiarity with computers (96%), most women (94%) found the tablet easy to use. A tablet-based educational program was an effective, feasible and acceptable strategy to disseminate cervical cancer information to women with low education in rural Malawi. This method may be appropriate to distribute health information about other health topics in low-resource settings.

Neurofibromatosis type 1 (NF1) is a common genetic disorder characterized by the occurrence of nerve sheath tumors and considerable clinical heterogeneity. Some translational studies have been limited by the lack of animal models available for assessing patient-specific mutations. In order to test therapeutic approaches that might restore function to the mutated gene or gene product, we developed mice harboring NF1 patient-specific mutations including a nonsense mutation (c.2041C>T; p.Arg681*) and a missense mutation (c.2542G>C; p.Gly848Arg). The latter is associated with the development of multiple plexiform neurofibromas along spinal nerve roots. We demonstrate that the human nonsense NF1Arg681* and missense NF1Gly848Arg mutations have different effects on neurofibromin expression in the mouse and each recapitulates unique aspects of the NF1 phenotype, depending upon the genetic context when assessed in the homozygous state or when paired with a conditional knockout allele. Whereas the missense Nf1Gly848Arg mutation fails to produce an overt phenotype in the mouse, animals homozygous for the nonsense Nf1Arg681* mutation are not viable. Mice with one Nf1Arg681* allele in combination with a conditional floxed Nf1 allele and the DhhCre transgene (Nf14F/Arg681*; DhhCre) display disorganized nonmyelinating axons and neurofibromas along the spinal column, which leads to compression of the spinal cord and paralysis. This model will be valuable for preclinical testing of novel nonsense suppression therapies using drugs to target in-frame point mutations that create premature termination codons in individuals with NF1.

Tyrosinase is a key enzyme in melanin biosynthesis. Mutations in the gene encoding tyrosinase (Tyr) cause oculocutaneous albinism (OCA1) in humans. Alleles of the Tyr gene have been useful in studying pigment biology and coat color formation. Over 100 different Tyr alleles have been reported in mice, of which ≈24% are spontaneous mutations, ≈60% are radiation-induced, and the remaining alleles were obtained by chemical mutagenesis and gene targeting. Therefore, most mutations were random and could not be predicted a priori. Using the CRISPR-Cas9 system, we targeted two distinct regions of exon 1 to induce pigmentation changes and used an in vivo visual phenotype along with heteroduplex mobility assays (HMA) as readouts of CRISPR-Cas9 activity. Most of the mutant alleles result in complete loss of tyrosinase activity leading to an albino phenotype. In this study, we describe two novel in-frame deletion alleles of Tyr, dhoosara (Sanskrit for gray) and chandana (Sanskrit for sandalwood). These alleles are hypomorphic and show lighter pigmentation phenotypes of the body and eyes. This study demonstrates the utility of CRISPR-Cas9 system in generating domain-specific in-frame deletions and helps gain further insights into structure-function of Tyr gene.

Exobasidium leaf and fruit spot of blueberry (Vaccinium section Cyanococcus) is an emerging disease that has rapidly increased in prevalence throughout the southeastern USA. To determine whether this disease is caused by a new species of Exobasidium, we studied the morphology and phylogenetic relationship of the causal fungus compared with other members of the genus, including the type species E. vaccinii and other species that parasitize blueberry and cranberry (V. macrocarpon). Both scanning electron microscopy and light microscopy were used for morphological characterization. For phylogenetic analyses, we sequenced the large subunit of the rDNA (LSU) from 10 isolates collected from leaf or fruit spots of rabbiteye blueberry (V. virgatum), highbush blueberry (V. corymbosum) and southern highbush blueberry (Vaccinium interspecific hybrid) from Georgia and North Carolina and six isolates from leaf spots of lowbush blueberry (V. angustifolium) from Maine and Nova Scotia, Canada. LSU was sequenced from isolates causing red leaf disease of lowbush blueberry and red leaf spot (E. rostrupii) and red shoot (E. perenne) of cranberry. In addition, LSU sequences from GenBank, including sequences with high similarity to the emerging parasite and from Exobasidium spp. parasitizing other Vaccinium spp. and related hosts, were obtained. All sequences were aligned and subjected to phylogenetic analyses. Results indicated that the emerging parasite in the southeastern USA differs morphologically and phylogenetically from other described species and is described herein as Exobasidium maculosum. Within the southeastern USA, clustering based on host species, host tissue type (leaf or fruit) or geographic region was not detected; however, leaf spot isolates from lowbush blueberry were genetically different and likely represent a unique species.

Graphical abstractAbstractThe development of gene editing technologies, especially the CRISPR-Cas9 system, has been pivotal for understanding the functional role of proteins. Rapid and efficient genotyping methods are necessary to screen for generated mutations and streamline the isolation of homozygotes. CRISPR-Cas9 system targeting a single site in the gene typically results in small indels. Many genotyping methods utilize the heteroduplex that is formed when wild-type and mutant amplicons with small indels anneal during PCR creating a bubble due to mismatched strands. These methods include T7 endonuclease/Cel-I assay, high resolution melting (HRM) analysis, and heteroduplex mobility assay (HMA).Our protocol explains a simple, two step method of a mixing HMA (mHMA) to identify homozygous mutants, a modification of the previously published HMA. We have utilized the mHMA for screening and genotyping numerous CRISPR generated models.The mHMA method to differentiate homozygous wild type from homozygous mutant animals eliminates -DNA sequencing, even with small indels that can be difficult to discern on a gel.additional enzymatic reaction steps, such as with the T7EI/Cel-I assay.specialized equipment and analysis tools, such as with HRM analysis.

Nodal-related protein (ndr2) is a member of the transforming growth factor type β superfamily of factors and is required for ventral midline patterning of the embryonic central nervous system in zebrafish. In humans, mutations in the gene encoding nodal cause holoprosencephaly and heterotaxy. Mutations in the zebrafish, Danio rerio, ndr2 gene lead to similar phenotypes, including loss of medial floor plate, severe deficits in ventral forebrain development, and cyclopia. Alleles of the ndr2 gene have been useful in studying patterning of ventral structures of the central nervous system. Fifteen different ndr2 alleles have been reported in zebrafish, eight of which are from chemical mutagenesis, four are radiationinduced, and the remaining alleles were obtained by random insertion, gene targeting (TALEN) or unknown. Therefore, most mutation sites were random and could not be predicted a priori. Using the CRISPR-Cas9 system from Streptococcus pyogenes, we targeted distinct regions in all three exons of ndr2 and observed cyclopia in the injected (G0) embryos. We show that the use of sgRNA-Cas9 ribonucleoprotein (RNP) complexes can cause penetrant cyclopic phenotypes in injected (G0) embryos. Targeted PCR amplicon analysis using Sanger sequencing showed that most of the alleles have small indels resulting in frameshifts. The sequence information correlates with the loss of ndr2 activity. In this study, we validate multiple CRISPR targets using an in vitro nuclease assay and in vivo analysis using embryos. We describe one specific mutant allele in detail that results in the loss of the conserved terminal cysteine coding sequences. This study is another demonstration of the utility of CRISPR-Cas9 system in generating domain-specific mutations and provides further insights into structure-function of ndr2 gene.